Exploring Pharmacological Mechanisms of Xuefu Zhuyu...
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Research ArticleExploring Pharmacological Mechanisms of Xuefu ZhuyuDecoction in the Treatment of Traumatic Brain Injury viaa Network Pharmacology Approach
Yuanyuan Zhong1 Jiekun Luo 1 Tao Tang 1 Pengfei Li1 Tao Liu12 Hanjin Cui1
YangWang 13 and Zebing Huang 3
1 Institute of Integrative Medicine Xiangya Hospital Central South University Changsha 410008 China2Department of Gerontology Traditional Chinese Medicine Hospital Affiliated to XinjiangMedical University Urumqi 830000China3Department of Infectious Disease Hunan Key Laboratory of Viral Hepatitis Xiangya Hospital Central South UniversityChangsha 410008 China
Correspondence should be addressed to YangWang wangyang xy87csueducn and ZebingHuang huangabing0330csueducn
Received 22 June 2018 Accepted 17 September 2018 Published 4 October 2018
Academic Editor Darren R Williams
Copyright copy 2018 Yuanyuan Zhong et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
Objectives Xuefu Zhuyu decoction (XFZYD) a traditional Chinesemedicine (TCM) formula has been demonstrated to be effectivefor the treatment of traumatic brain injury (TBI)However the underlying pharmacologicalmechanisms remain unclearThis studyaims to explore the potential action mechanisms of XFZYD in the treatment of TBI and to elucidate the combination principle ofthis herbal formula Methods A network pharmacology approach including ADME (absorption distribution metabolism andexcretion) evaluation target prediction known therapeutic targets collection network construction and molecule docking wasused in this study Results A total of 119 bioactive ingredients from XFZYD were predicted to act on 47 TBI associated specificproteins which intervened in several crucial pathological processes including apoptosis inflammation antioxidant and axongenesis Almost each of the bioactive ingredients targeted more than one protein The molecular docking simulation showedthat 91 pairs of chemical components and candidate targets had strong binding efficiencies The ldquoJunrdquo ldquoChenrdquo and ldquoZuo-Shirdquoherbs from XFZYD triggered their specific targets regulation respectively Conclusion Our work successfully illuminates theldquomulticompounds multitargetsrdquo therapeutic action of XFZYD in the treatment of TBI by network pharmacology with moleculedocking methodThe present workmay provide valuable evidence for further clinical application of XFZYD as therapeutic strategyfor TBI treatment
1 Introduction
Traumatic brain injury (TBI) is a major cause of deathand disability [1] At least 10 million severe TBIs result indeath or hospitalization annually worldwide [2] Approx-imately 17 million Americans sustain a TBI each yearleading to over 14 million emergency department visits275 000 hospital admissions and 50 000 deaths that con-tribute to one-third of all injury-related deaths [3] In theEuropean Union alone an estimated 15 million hospitaladmissions and 57000 deaths annually attribute to TBI[4] In China TBI-related mortality remains a high levelranging from 1299 to 1706 per population of 100 000
persons [5]Thus TBI has afforded huge social and economicburden
TBI is a diverse group of sterile injuries induced byprimary and secondary mechanisms that give rise to celldeath inflammation and neurologic dysfunction in patientsof all demographics [6 7]The primary injury is caused by themechanical stress or shear force on tissues with no therapeu-tic agents [8] The secondary injury includes a wide varietyof processes like activation of inflammatory and immuneresponse [9 10] calciumoverload [11] glutamate toxicity [12]and mitochondrial dysfunction [13] among others Currentguidelines for the management of the secondary injury areprimarily supportive including the emphasis on surveillance
HindawiEvidence-Based Complementary and Alternative MedicineVolume 2018 Article ID 8916938 20 pageshttpsdoiorg10115520188916938
2 Evidence-Based Complementary and Alternative Medicine
(ie intracranial pressure) and the preventive measures toreduce morbidity and mortality [14] Despite the fact thatdetailed medicines contain free-radical scavengers antago-nists of N-methyl-D-aspartate and calcium channel blockers[15] the results of the controlled clinical trials of these drugsare disappointing [16] Neuroscientists and doctors tend tosearch for potential novel drugs from traditional Chinesemedicine (TCM) library to treat TBI [17]
TCM is a comprehensive medicinal system that has beenused in clinical practice for thousands of years and playsan important role in the health maintenance for people allover the world [18 19] The validated curative effects of TCMmake it a feasible alternative therapeutic agent for diseasetreatment Xuefu Zhuyu decoction (XFZYD) a representa-tive TCM formula was first recorded in Correction of Errorsin Medical Works by Qing-ren Wang XFZYD consists of11 crude herbs Persicae Semen (Tao ren) Carthami Flos(Hong hua) Radix Paeoniae Rubra (Chi shao) ChuanxiongRhizoma (Chuan xiong) Achyranthis Bidentatae Radix (Niuxi) Angelicae Sinensis Radix (Dang gui) Rehmannia glutinosaLibosch (Sheng di huang) Platycodon Grandiforus (Jie geng)Aurantii Fructus (zhi qiao) Radix Bupleuri (chai hu) andlicorice (Gan cao)Themain chemicals from XFZYD includeflavonoids organic acids terpenoids and steroidal saponins[20ndash22] The formula has been proven reliable and effectivefor curing various diseases including unstable angina pectoris[23 24] coronary artery disease [25] thromboembolic stroke[26] ischemic stroke [27] and TBI The therapeutic agent ofXFZYD is to promote blood circulation and remove bloodstasis according to the TCM theory Several randomizedcontrolled clinical trials and animal experiments have showeddefinite therapeutic effects of XFZYD for the treatment ofTBI [28ndash31] Recent researches demonstrate that XFZYDprovides neuroprotection via anti-inflammatory pathway andcognitive improvement through synaptic regulation [32 33]However merely these evidences to explain the multipletherapeutic mechanisms of TCM for TBI treatment areunavailable Because the effects of TCM are always contro-versial in terms of their abstract theory unclear basis com-plex interactions between various ingredients and complexinteractive biological systems [25] it is essential to developan advanced technique to deeply uncover the synthesizedpharmacological effects of XFZYD in the treatment of TBI
With the development of TCM modernization networkpharmacology has become a novel method to elucidate themulti-druggable targets effects of TCM [34] TCM networkpharmacology first proposed by Shao Li [35] makes itfeasible to understand the effective constituents and targetsof the herbs from TCM formula This analytical methodintegrates bioinformatics systems biology and polypharma-cology and further utilizes network analysis to imply themultiple actions of drugs across multiple scales ranging frommolecularcellular to tissueorganism levels [36 37] Coin-ciding with the holistic and systemic characteristics of TCMnetwork pharmacology is expected to bridge the gap betweenTCM and modern medicine [25] Previous researches haveclarified the scientific basis and systematic features of herbalmedicine to treat diseases through network pharmacologysuch asQing-Luo-Yin andMa-HuangDecoction etc [38 39]
In the present work we explored the pharmacologicalmechanisms of XFZYD acting on TBI via a network pharma-cology approach Network analyses and molecular dockingmethod were used to reveal candidate drug targets relatedto TBI Target analysis suggested that XFZYD regulatedseveral key biological processes of TBI development suchas apoptosis inflammation blood coagulation and axongenesis These processes contributed to the clarifying of themolecular mechanisms of XFZYD for TBI treatment Thiswill help to improve the effectiveness and specificity of TCMclinical usage (Figure 1 depicts a flowchart of the entireresearch procedure)
2 Methods
21 Database Construction The chemical ingredients of11 herbs in XFZYD were screened from Traditional Chi-nese Medicine Systems Pharmacology database (TCMSPhttplspnwueducntcmspphp) [40] As a chemically ori-ented herbal encyclopedia TCMSP can provide comprehen-sive information about herb ingredients including chemicalstructural data oral bioavailability drug targets and theirrelationships with diseases as well as the biological or physio-logical consequences of drug actions involving drug-likenessintestinal epithelial permeability and aqueous solubility [40]The structures of these compoundswere saved asmol2 formatfor further analysis Discovery studio 25 was employed tooptimize these molecules with a Merck molecular force field(MMFF) All detailed information about these ingredients isprovided in Table S1
22 Pharmacokinetic Prediction Due to the disadvantagesof biological experiments as being time-consuming and ofhigh cost identification of ADME (absorption distributionmetabolism and excretion) properties by in silico toolshas now become a necessary paradigm in pharmaceuticalresearch In this study 2 ADME-related models includingthe evaluation of oral bioavailability (OB) and drug-likeness(DL) were employed to identify the potential bioactivecompounds of XFZYD
Oral bioavailability (OB) one of the most importantpharmacokinetic parameters represents the speed of a drugof becoming available to the body and the eventuallyabsorbed extent of the oral dose [41] which is particularly sig-nificant in drug discovery of TCM formost oral Chinese herbformulas Poor OB is indeed the main reason responsible forthe unsuccessful development of compounds into therapeuticdrugs in drug screening cascades Here a reliable in silicomodel OBioavail 11 [42] which integrates the metabolism(P450 3A4) and transport (P-glycoprotein) information wasemployed to calculate the OB values of herbal ingredientsIn this study OBge30 (a suggested criterion by TCMSPdatabase) was regarded as one threshold for screening pos-sible candidate drugs presently while 2 compounds withOB le 30 were also taken into consideration due to theirtherapeutic effects according to literatures such as amygdalinand hydroxysafflor yellow A [43 44]
Drug-likeness (DL) is a qualitative profile used in drugdesign to evaluate whether a compound is chemically suitable
Evidence-Based Complementary and Alternative Medicine 3
TCMSP database Molecule information ofcompounds in XFZYD
Candidate compounds profile
Candidate targets profileCandidate targets overlap analysisamong three group of herbs
Candidate compounds overlapanalysis among three group of herbs
HB-cC-cT network
Known therapeuticproteins for TBI
TBI specific proteins
OB DL
STRING
Overlap analysis of targets betweenXFZYD and TBI specific proteins
Cytoscape 340
TTD amp OMIM
HB-pC-pT network TBI specific PPInetwork
Molecule dockingGO and KEGG
pathway analysis
Binding mode between potentialtargets and compounds in XFZYD
Biological significance of potentialtargets of XFZYD on TBI
Synergistic effects ofTCM formula
HPRDTarget prediction
Figure 1 A schematic diagram of the network pharmacology-based strategies for determining the pharmacological mechanisms of the herbalformula XFZYD on TBI
for drug and how drug-like a molecule is with respect toparameters affecting its pharmacodynamic and pharmacoki-netic profiles which ultimately impacts its ADME properties[45] In this study the drug-likeness (DL) index (see (1))using the Tanimoto coefficient [46] was computed for eachcompound in XFZYD
119879 (119883119884) =119883 sdot 119884
1198832 + 1198842 minus 119883 sdot 119884(1)
where X represents the molecular descriptors of herb com-pounds and Y is the average molecular properties of all com-pounds in Drugbank database (httpwwwdrugbankca)Compounds with DL ge018 (average value for Drugbank)were selected as bioactive compounds in XFZYD
In summary compounds with OB ge30 and DLge018were selected for subsequent research and others wereexcluded The criteria used here were mainly for (1) extract-ing information from the herbs as much as possible with theleast number of components and (2) explaining the obtainedmodel by the reported pharmacological data
23 Target Prediction To obtain the molecular targets ofthese active ingredients an in-house developedmodel SysDTbased on Random Forest (RF) and Support Vector Machine(SVM) methods [47] was proposed which efficiently inte-grated large-scale information on chemistry genomics andpharmacology This approach shows impressive performanceof prediction for drug-target interactions with a concordanceof 8283 a sensitivity of 8133 and a specificity of 9362
respectively [40] UniProtKB (httpwwwuniprotorg) wasemployed to obtain the standard name of the predicted targetproteins
24 TBI-Specific Protein Collecting The known therapeutictarget proteins of TBI were screened fromTherapeutic TargetDatabase (TTD available httpbiddnusedusggroupcjttd)TTD is a publicly accessible database which provides com-prehensive information about the known therapeutic proteinand nucleic acid targets described in the literature thetargeted disease conditions the pathway information and thecorresponding drugsligands directed at each of these targets[48] We also searched for Online Mendelian Inheritancein Man database (OMIM available httpomimorg) toget proteins related to TBI OMIM catalogues all knowndiseases with a genetic component and then possibly linksthem to the relevant genes in the human genome andprovides references for further research and tools for genomicanalysis of a catalogued gene [49] Then the proteinsacquired from both databases were used as hub proteinsand submitted to Human protein Reference Database [50](HPRD available httpwwwhprdorg) and STRING [51](httpsstring-dborg) to generate the proteins interactingwith these hub proteins HPRD is a database containingcurated proteomic information pertaining to human pro-teins The human protein-protein interaction (PPI) data onHPRD (Release 9) consists of 39240 interactions among 9617genesThe STRINGdatabase provides both experimental andpredicted interaction information providing a probabilisticassociation confidence score
4 Evidence-Based Complementary and Alternative Medicine
25 Molecule Docking The LibDock algorithm based on theCHARMm Force Field in Discovery Studio (DS) 25 wasused in this study to evaluate the potential molecular bindingmode between bioactive compounds and putative targetsThecrystal structure of the target proteins of XFZYD for treatingTBI was downloaded from the RCSB Protein Data Bank(wwwrcsborg) The 3D chemical structures of bioactivecompounds were downloaded from PubMed Compounddatabase or TCMSP database and subjected to minimize theenergy by using molecular mechanics-2 (MM2) force fieldThe protein preparation protocol was used before dockingsuch as inserting missing atoms in incomplete residuesremoving water and protonating titratable residues The lig-and preparation protocol was employed before docking suchas removing duplicates enumerating isomers and generating3D conformations The protein-ligand docking active sitewas defined by the location of the original ligand All otherdocking and consequent scoring parameters were kept attheir default settings The compound was considered to be apotentially active ingredient if the LibDock score was higherthan the original ligand
26 Network Construction and Analysis Network construc-tion was performed as follows
(1) The herbs candidate compounds and candidate tar-gets of XFZYD were used to construct an herb-candidate compound-candidate target (HB-cC-cT)network
(2) The PPI data obtained above was used to establish theTBI-specific protein interaction network
(3) Herbs potential compounds and putative targetsfrom XFZYD for treating TBI were used to builda herb-potential compounds-potential targets (HB-pC-pT) network
(4) Potential targets and the biology process they partici-pate in were used to construct the pT-F network
(5) Compounds and targets through molecule dockingvalidating were used to build a compound-target (C-T) network
All networks were generated and analyzed by Cytoscape 340[52] an open source of bioinformatic package for biologicalnetwork analysis and visualization Two topological param-eters including degree and betweenness were calculated forthe obtained networks which disclose the significance ofa node
27 Gene Ontology (GO) and Pathway Enrichment Analy-sis The functional enrichment tool DAVID [53] (DAVIDhttpsdavidncifcrfgov) ver 68) was used to calculateboth the KEGG pathway and GO biological processes (BP)enrichment
28 Statistical Analysis All data were expressed as mean plusmnstandard deviation (SD) The molecule descriptors data wereanalyzed by one-way ANOVA The criterion for statisticalsignificance was p lt 005 Statistical analyses were conductedusing the SPSS 240
3 Results
TCM an experience-based medicine has been widely usedfor thousands of years It has accumulated abundant clinicalexperience forming a comprehensive and unique medicalsystem [35] The complexity of the phytochemical compo-nents makes it extremely difficult to illustrate the actionmechanisms of XFZYD from a molecule or system levelAs a chief mean of treating diseases clinically generallyTCM doctors prescribe formula based on the principle ofldquoJun-Chen-Zuo-Shirdquo ldquoJunrdquo (monarch) treats the main causeor primary symptoms of the disease ldquoChenrdquo (minister)enhances the actions of ldquoJunrdquo or treats the accompanyingsymptoms ldquoZuordquo (adjuvant) not only reduces or eliminatesthe possible toxic effects of the Jun or Chen but also treatsthe accompanying symptoms ldquoShirdquo (guide) helps to deliveror guide the other herbs to the target organs [18] Accordingto the unique feature of TCM ourwork tried to perform ldquoJun-Chen-Zuo-Shirdquo based system study to clarify the multiplemechanisms of XFZYD in the treatment of TBI
31 Herbal Ingredient Comparison and Target Predictionof XFZYD We obtained 162 components originated fromXFZYD Of these compounds 160 chemicals that were inaccord with standard requirements were searched from theTCMSP database The other 2 components amygdalin andhydroxysafflor yellow A were taken into consideration fortheir obvious pharmacological action as well The detailedinformation of these compounds is showed in Table S1Persicae Semen (Tao ren) and Carthami Flos (Hong hua)the Jun (monarch) herbs of XFZYD contained 36 bioactivecomponents which accounted for 22 of the 162 chemicalsRadix Paeoniae Rubra (Chi shao) Chuanxiong Rhizoma(Chuan xiong) andAchyranthis Bidentatae Radix (Niu xi) theChen (minister) herbs contained 31 bioactive componentswhich accounted for 19 of the 162 chemicals AngelicaeSinensis Radix (Dang gui) Rehmannia glutinosa Libosch(Sheng di huang) Platycodon Grandiforus (Jie geng) AurantiiFructus (zhi ke) Radix Bupleuri (chai hu) and licorice (Gancao) the Zuo-Shi (adjuvant and guide) herbs of XFZYDcontained 109 bioactive components which accounted for67 of the 162 chemicals Ingredients from these herbswere compared based on the 6 important drug-associateddescriptors including molecular weight (MW) number ofhydrogen-bond donors (nHdon) number of hydrogen-bondacceptors (nHacc) partition coefficient between octanol andwater (AlogP) oral bioavailability (OB) and drug-likeness(DL)The distributions of the 6 descriptors of the ingredientsfrom the three groups are shown in Table 1 and Figure 2 Wefoundno significant differences in the values ofMW(p=016)nHdon (p=032) nHacc (p=061) and AlogP (p=082) amongthe 3 groups However the average OB value of compoundsfrom the march herbs is 5559plusmn2391 which was significantlydifferent from the Chen herbs (OB=4564plusmn1327 P=0004)and the Zuo-Shi herbs (OB=4877plusmn1505 P=0021) Thefollowing 2 groups had no significant difference in OBvalue (P=0272) As for DL the Chen herbs revealed thehighest DL index (053plusmn023) which displayed significantdifference from the Zuo-Shi herbs (044plusmn019 p=0012) while
Evidence-Based Complementary and Alternative Medicine 5
Table 1 Comparison of molecular properties among the Jun Chen and Zuo-Shi herbs
INDEX MW nHdon nHacc AlogP OB DL(mean plusmn SD) (mean plusmn SD) (mean plusmn SD) (mean plusmn SD) (mean plusmn SD) (mean plusmn SD)
Jun herbs 38028 (9423) 265 (226) 511 (321) 337 (436) 5559 (2391) 049 (018)Chen herbs 38180 (9160) 218 (195) 55 (347) 314 (315) 4564 (1327) 053 (023)Zuo-shi herbs 35745 (8822) 261 (160) 56 (249) 344 (185) 4877 (1505) 044 (019)OB oral bioavailability MW molecular weight DL drug-likeness AlogP partition coefficient between octanol and water nHacc number of hydrogen-bondacceptors nHdon number of hydrogen-bond donors
Table 2 Top 10 candidate compounds according to 2 centrality indicators
Compounds Degree Compounds Betweennessquercetin 153 quercetin 035875838kaempferol 65 naringenin 008768253luteolin 48 kaempferol 007665228wogonin 46 luteolin 0057362047-Methoxy-2-methyl isoflavone 44 baicalein 005542898beta-sitosterol 41 beta-sitosterol 0041007baicalein 40 wogonin 003968963formononetin 39 Stigmasterol 003586788naringenin 39 nobiletin 003057461isorhamnetin 38 formononetin 003051437
showing nodifferencewith the Junherbs (049plusmn018 p=0333)Figure 3(a) indicated that 5 bioactive compounds wereshared by the Jun Chen and Zuo-Shi herbs One compoundoverlapped between the Jun andChen herbs while there were2 compounds shared by the Chen and Zuo-Shi herbs Onecompound overlapped between the Jun and Zuo-Shi herbs
32 Target Prediction of XFZYD A total of 285 potentialtargets from the 162 compounds were generated using thetarget prediction model The amounts of potential targets hitby the Jun Chen and Zuo-Shi drugs were 217 218 and 261respectively The detailed data of the targets is shown in TableS2 As depicted in Figure 3(b) there was a significant targetoverlap among the 3 groups (189 candidate targets) but lessoverlap between the Jun andChenherbs (9 candidate targets)The number of targets shared by the Jun and Zuo-Shi herbswas 14 while 10 targets were overlapped between the Chenand Zuo-Shi herbs
33 HB-cC-cT Network Construction and Analysis We nextestablished aHB-cC-cT network through network analysis toilluminate the relationship among the herbs candidate com-pounds and candidate targets (Figure 3(c)) This networkconsisted of 485 nodes (11 herbs 162 candidate compoundsand 285 candidate targets) and 2585 edges A herb (triangle)and cC (square) are connected if the compound is containedin this herb and the edges between cC and cT represent theinteraction The size of nodes is proportional to the valueof degree The larger size of the node means more phar-macologically important Two centrality indicators degreeand betweenness identify the important nodes within thenetwork Different centralities reflect different importanceof nodes in a network from different angles Interestingly
both of the two types of centrality indicators uniformlyconfirmed themost important 10 candidate compounds fromXFZYD and the top 10 targets anchored by XFZYD (Tables2 and 3) Figure 3(c) demonstrated that licorice possessedthe largest degree (88) compared with other herbs originatedfrom XFZYD This implicated that it contained the mostbioactive compounds (88) including quercetin (Mol 148degree=153) kaempferol (Mol 108 degree=65) 7-Methoxy-2-methyl isoflavone (Mol 33 degree=44) formononetin (Mol63 degree=39) naringenin (Mol 133 degree=39) isorham-netin (Mol 105 degree=38) medicarpin (Mol 131 degree=35)and licochalcone a (Mol 113 degree=33) followed byPersicae Semen (degree=19) Carthami Flos (degree=18)Achyranthis Bidentatae Radix (degree=17) Radix PaeoniaeRubra (degree=14) Radix Bupleuri (degree=12) ChuanxiongRhizoma (degree=6) Aurantii Fructus (degree=4) Platy-codon Grandiforus (degree=4) Rehmannia glutinosa Libosch(degree=3) andAngelicae Sinensis Radix (degree=2) PersicaeSemen (degree=19) and Carthami Flos (degree=18) the Junherbs from XFZYD possessed 29 specific compounds and5 unique target proteins including ALB CTNNB1 MMP10LYZ andNFKB1 Twenty-three Chen-specific potential com-pounds targeted 10 specific proteins including CD14 LBPNR3C1 BBC3 TEP1 PRKCD FN1 PDE10A GSTA1 andGSTA2 The Zuo-Shi herbs possessed the largest number ofspecific compounds (101) and 48 unique proteins such asHTR3A ADRA1D PYGM OLR1 CHRM5 RXRB STAT3MAPK10 OPRD1 and MAPK3 There were 189 proteinsanchored by the Jun Chen and Zuo-Shi drugs
Among the 162 candidate compounds quercetin hadthe largest value of degree (153) implicating its critical rolein XFZYD The four herbs namely Carthami Flos (Jun)Achyranthis Bidentatae Radix (Chen) and licorice and
6 Evidence-Based Complementary and Alternative Medicine
50
40
30
20
10
0
50
40
40
30
20
20
10
0
0
10 30 50 70 90 110
60
60
60
01 02 03 04 05 06 07 08
225 275 325 375 425 475 525 575 625 minus6minus5minus4minus3minus2minus1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
minus2 minus1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
70
minus1 1 3 5 7 9 11 13 15 17
perc
entage
s (
)
perc
entage
s (
)50
40
30
20
10
0
perc
entage
s (
)
perc
entage
s (
)
40
20
0
60
perc
entage
s (
)50
40
30
20
10
0
perc
entage
s (
)
OB
MW
nHdon
DL
nHacc
Distribution
Distribution
Distribution
Distribution
Distribution
Distribution
AlogP
Jun herbsChen herbsZuo-Shi herbs
Jun herbsChen herbsZuo-Shi herbs
Jun herbsChen herbsZuo-Shi herbs
Jun herbsChen herbsZuo-Shi herbs
Jun herbsChen herbsZuo-Shi herbs
Jun herbsChen herbsZuo-Shi herbs
Figure 2 The profile distributions of six important molecular properties for all ingredients from the Jun Chen and Zuo-Shi herbs OBOral bioavailability MW molecular weight DL drug-likeness AlogP partition coefficient between octanol and water nHacc number ofhydrogen-bond acceptors nHdon number of hydrogen-bond donors
Evidence-Based Complementary and Alternative Medicine 7
Jun herbs(29)
Zuo-
Shi h
erbs
(101
)
Chen herbs
(23)
1
2
15
(a)
Jun herbs(5)
Zuo-
Shi h
erbs
(48)
Chen herbs
(10)
9
10
14189
(b)
20Mol 1200000
MMMol 152Mol 15Mol 1521Mol 15Mol 15
Mol 8888
Mol 29Mol 2Mol 2Mol 2Mol 29
Mol 3Mol 3Mol 35Mol 3535
1Mol 121Mol 121Mol 122
Mol 124Mol 124Mol 1Mol 124Mol 124Mol 124
MMol 10Mol 1Mol 10M 00MMMM
MMol 73Mol 77333222
ABATABATA
MAPK3MAPKMAPK
UGT1A1GT1UGT1A1
GOT1GOTT1
SREBF1REBFSREBF1
RXRBRXRBRXRB
CYP19A1CYP19A1YP19A
PLB1PLBPLB1
FASLGFASLGASL
BCHEBCHHE
HSD3B1HSD3
MTTPMTT
CES1CES
TIMP1TIMP
ATP5BATP5
MAPK10MAPK 0
BADBAD
CHRM5CHRM5
EPHBEPHBPHB2
HSD3B2HSD3B2SD3B
SOAT2OATSOAT2
FASNFASNFASN
MT-ND6MT-N
HTR3AHTR3HTR3APLA2G4APLA2G4AA2G
PKIAAIA
ADH1AADHAD
AKR1C1KR1AKR1C1
CREB1CREBB1
HMGCRMGHMGCR
SOAT1OAT
ABCC1ABCC
OPRD1OPRDOPRD1
VCPVCP
LDLRLDLR
ADRA1DDRAA1D
NFATC1FATCFATCATCFATCA
EGLN1GLNGLNGLNGLN
FABP5ABP5ABPABPABPABP5
CYCSCYCSCYCSCYCSYCSFOSL1OSLOSLOSLOSL
ALOX12LOXLOXOXLOX
TDRD7DRDDDRDDRDDRDAPODAPODAPOPOAPOD
NNNNOXNOXNOXNOX5NOXX
Mol 40
NFKB1NFKBFKBFKBFKBLYZLYZLYZZLYZ CTNNB1TNNBTNNNNNN MMP10MMPMPMPMMP1ALBALBALBALBALB
3Mol 433MMol 28Mol 28Mol 2Mol 28
HSPA5SPASPASPA5HSPA5AERBB2ERBB2ERBBRBBB2ERBB2
HSPB1HSPBSPBSPBHSPB1HSPB1B
NQO1NQO1NQONQO1NQONQO1TOP1TOP1TOP1TOPTOPT
PORPPORPORPORPORR ACPPACPPACPPACPPCPP
SLC2ALC2A4LC2A4C2A4LC2A4LC2A4LC
SERPINE1SERPINE1PINRPINERPINPIN
JUNJUNNJUNNN
PTGER3PTGER3TGERTGERGERTGER
COL1A1OL1AOL1AOL1AA1CO
SSLPISLPSLPISLPISLPII
CCCALCCC MMMMMMSS
MMMMP1MMPMMPMMPTNFTNFTNFFTNF
CCND1CCNDCCNDCNDCNDCCND
SMDPSMDPSMDPSMD3PSMD3PSMDNFNFNFFFNF
ELK 1ELKELK LK ELK ELK CCL2CCL2CCLCCL2CCL2CCL2
MMol 14MMol 14Mol 1447
MMol 15MMol 1Mol 115Mol 15M
Mol 122Mol 122Mol 122MMol 12Mol 122222
MMol 24Mol 2Mol 2Mol 2444
Mol 78MMol 78Mol 7Mol 7Mol 788
Mol 38MMMMol 3Mol 3338
Mol 154Mol 154MMol 15ol 1ol 155M
Mol 67MMol 6Mol 6Mol Mol 67Mol 677766
Mol 64MMol 64Mol 6Mol 6Mol 64
Mol 48Mol 48MMMol 4Mol 4448M 4
PPPPARAPARPARPAR
PCOLCEPCOLCECOLCOLCCOLC
SPP1SPP1SPP1PP1SPPP1 AKR1C3AKR1C3KR1CKR1CKR1CKR
NNNOSNN 2222 DUOXDUOXDUOXDUOX2DUOXDUOX2
MCL1MCL1MCLMCLMCLMCL
PARP1ARP1ARPARPPARP1PEEMPOMPOMPOMPOM DDDUDUDUDDU
NGASYNGASYNGAP1NGAPNGAAP1RARAARARA
BAXXXXB X ESR1
GSTM2GSTM2GSTMSTMSTMGSTMSHSAAHSAAHSA1AHSA1AHSAHSAA GABRAGABRA2GABRA2GABRAGABRAGABRAMM2MMM2
CHRM2CHRM2CHRMHRMHRMCHRM2
VDRVDRVDRRVDRVDRARGABRA1GABRA1GABRAA11GABRA1G
IL888IL88
PPPPRSPRSPRSSSSSSSPPARDPARDPARPARDPARDPARDSELESELESELESELEEE
COL3A1COL3A1OL3AOL3AOL3AA1OL3ARB11111
CYP1A2222222 NCOANCOANCOANCOANCOANCOA22222211111 RRRRRR
Quercetin
Mol 9444444
Mol 99
Mol 63
Mol 75555
Mol 37Mol 37Mol 37Mol 37MolMol 33Mol 37MolM
Mol 158
Mol 1Mol 10Mol 1Mol 10000
Mol 49Mol 4Mol 4Mol 4Mol 44Mol 494Mol 4
Mol 6Mol 6666M 66
MMol 60Mol 60Mol 6MM
Mol 101Mol 101Mol 101Mol 10011
Mol 12Mol 12Mol 12Mol 1Mol 12221
Mol 11MMol 1155Mol 11555M6060660
Mol 85Mol 85Mol 85MMol 88558554MMol 114Mol 114444M
Mol 116Mol 116Mol 116MMol 1ol 1Mol 11616M 1
Mol 9000
Mol 100Mol 1000Mol 106MMMol 106Mol 10Mol 1066Mol 322 Mol 30Mol 30Mol 30000MM
Mol 140MMol 14MMol 14Mol 1440Mol 14
PlatycodPlatycodatycodPlatycodP ononoGrandifondifoGrandGrand rususus
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Mol 84
Mol 23 Mol 130Mol 13Mol 13Mol 11Mol 130Mol 13Mol 1MMol 13
Mol 11Mol 1Mol 1Mol 11Mol 11
0Mol 200000
Mol 34Mol 3Mol 3MolMol 3344Mol 3Mol 134
Mol 151
Mol 126Mol 126Mol 12666
Mol 8111AurantiiAurantAuranAurantiitFructusructusFructuructuFr
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Mol 444
MMol 82Mol 8Mol 8222Mol 88
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Mol 142Mol 1422
Mol 36
0Mol 80Mol 88Mol 80M 80
Mol 277Mol 135Mol 13555M 555
Mol 141MMol 144Mol 14111
Mol 104Mol 104MMMol 10Mol 104044M
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21Mol 21MMol 221111
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Mol 118MMol 11Mol 1Mol 11Mol 1Mol 11ol
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M l 149MMol 1449Mol 149Mol 149
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Mol 89Mol 125MMol 1ol 1Mol 12MMMol 1MM
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Mol 72MolMol 7Mol 7MolMol 72M 7M Mol 933Mol 112Mol 112Mol 112Mol 1122M
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Mol 86Mol 86Mol 8Mol 8Mol 866686
Mol 161Mol 161Mol 161Mol 16Mol 16161MMo
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Mol 97Mol 162MMol 16Mol 1Mol 1ol 111M 1
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Mol 128Mol 128Mol 1ol 1Mol 121MolMol 199Mol 117Mol 117777
Mol 744444Mol 77
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Mol 42MMMol 42M
MMMol 3Mol 3
Mol 137Mol 13Mol 13ol 13Mol 13Mol 13733
Mol 59Mol 59olMol 5Mol 59oMol 5Mol 559
Mol 129lMol 12ol 12ol 12Mol 129o 2ol 12o
Mol 62Mol 62Mol 6Mol 6Mol 6Mol 62Mol 6Mol 6ol
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CXCL11CXCL11XCL1XCL1XCLXCL1p53p53p53p
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PLATPLATPLAPLATPLATL
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CHEKCHEKCHEKHEK2HEK2HEK
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HK2HK2HK2HK2HK2HK2
RUNX2UNXUNXUNXRUNX2UNXHSF1HSF1HSF1HSF1HSF1HSF1
IGFBP3GFBPGFBPFBPIGFBP3
STAT1T1TAT1TATSTAT1TATT
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PRSS3RSSSIRT1SIRTGATMGAT OLR1OLRAPOBAPOPYGMPYGM
TGFB1GFBGFBGFBGFBTGFB1F10 MMP9MMPMMPMMPP9PNFE2L2FE2LFE2LFE2LNFE2L2FE2L2L
Mol 160Mol 44Mol 44ol 4Mol 444Mol 444ol 4ol 4ol 44
Mol 55MoMol 5Mol 5Mol 555Mol 55Mol 5Mol 5MoMol 5
MMMol 132
MMMol 57
CRPCRPCRPCRPPHH
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CXCL10CXCL10XCXCLCXCLCXCL IGG
CLDN4LDN4LDN4LDN4ABABABAAB RRAHRAHRBBBIB
RARRAF1RAF1RAF11KKKKKKaemKKKKK pffefeferereerf referololol
CASP9CCASPCASP9PMDM2MDMMMDMMDMMDM2MDMMDM
BMAOBMAOBMAOBMAOBMAOBINSRINSRINSRINSRINSRRRNCOA1NCOA11NCOA1
HTR2AHTR2HTR2HTR222A
CHRMCHRMCHRM3MM3BCL2LCL2CL2L1LCL2BCL2L1111 CHCCHCCCCH
BCL2222BCL2CASP8CASP8CASP8CASP8CASP
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BaicalBaicalBaBaicalcacacalicaBB calcaac ininininininninninnCHUCHUKHUKCHUKHUKUKH AAAAAA
EGFREGFRGFREGFREGFR
NFKBIAFKBIFKBFKBNFKBIAFKBI
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APPAPAPPAPPPAPPAPPA
CASP7CASPCASPASPCASPCASPPP IL4IL444L4L44
NUF2NUFNUFNUFNUFNUFNUFNUFN
CDK4CDKCDKCDKCDKCDKCD
XIAPXIAPXIAXIAXIAXIAX MEMEMETMETMETMEETMMME
ADCY2ADCYDCYDCDCYDCD
Mol 127
NNR3C2NR3C2NR3CNR3C2N
DPP4
DCAF5DCACAFDCAF55DCAFAKR1B1AKR1BKKR1BKR1BKR1BCTRB1CTRBTRBCTRBCTRB
LTA4HLTA4LTA4TA4LTA4LTA4H4
XDHXDHXDHHHXDH
PTGS2
Mol 51Mol 51Mol 5MMolMolMol 5Mol 5MMol 5
Mol 145Mol 14Mol 14ol 1ol 1ol 1Mol 1Mo
Mol 146MMMMool 1ol 11461
Mol 143Mol 14Mol 14Mool 1ol 11Mol 1411
Mol 70MMMolMol 7Mol 77Mol 70Mol 7Mol 7
Mol 71Mol 7Mol Mol 777Mol 71Mol 7Mol
Mol 25Mol 25MMMol Mol 22Mol 2
Mol 79Mol 7Mol 7Mol Mol 7Mol 779MM 7
Mol 26Mol 26MMol 2Mol Mol 22Mol 2M 22
CA22222222ADH1BADHADHDHADH1ADH1DH KCNMA1CNMCNMCNMCNMKCNMAKCNMCGABRA6GABRAABRAABRAABRAGABRAGA A6GA RAGRIA2GRIAGRIAGRIAGRIAAAA2GRIA
RASSFRASSF1ASSFASSFASSF
Mol 31MMol 3Mol 3Mol 3311M 3Mol 98Mol 98Mol 98MMol 9Mol 999Mol 98MM
GSTP1GSTP1GSTPGSTPGSTPGSTPF33333
Mol 69Mol 6MMol 6Mol 6Mol 66Mol 69Mol 69M 66
VCAM1VCAMCAMCAMVCAM1C
MAPK8MAPK8MAPKMAPMAPKM
CYP3A4CYP3A4YP3AYP3AYP3AY
Mol 68Mol 68MMol 6Mol 6Mol 66MMol 68Mol 6MMol 16Mol 16Mol 1Mol 1Mol Mol 1Mol 16
MMMMol 6MMol 66Mol 666Mo
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Mol 9566Mol 65Mol 6Mol 6MMol MolMol 6M 666M
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IGF2IGF2IGF2IGF2F
FOSFOSFOSFOSSFOSFOSSSS
CCNB1CCNBCNBCNBCCNB1CCNB1CDK1CDK1CDKCDKCDKCC
PRKCBPRKCBRKCRKCRKCR CB
CHRM1CHRM11CHRM11
HHHHSP9HHHH 0ABABAB2B2B2B2PPPPPPPPPPPPTOP2A ACACACACACACACACAACACAACACA
PRKACA
TOP2TOP2
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CAV1CCAVCAVCAV1V1CAV1
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ADRB1ADRBRBADRBADR
ADRADRADRA2A2AA2A
SOD1SSODSOD1D11
NOS33
PLAUPLAUPLAAUUPLAUPRXRA
PGR
HHMOX11MOXMOXHMOX
GSTM1GSTM1GSTMGSTMSTMAAA
HIF1AHIF1HIF1HIF11AHIF1A
FOSL2OSLOSLOSL22FO
ALOX5ALOXALOXALOXALOX5
VEGFAVEGFAVEGFAVEGFAV FFA
PON1PONPONPONN1PON
PIK3CGPIK3CGGG
SULT1E1SULT1E1ULT1ELT1LT1GAGAABRAGABRA3AGG
AKT1AKTT1AKT11A
CHRMCHRMCHRMCHRM4CHRM4RMM
MAOAMAOAMAOAMAOMAOCD40LGCD40LGCD40LGD40LGCD40LGCD40LBRA3RAAABRA3CHRCHHHCHR
ADRB2
(c)
Figure 3 HB-cC-cT network of XFZYD (a) and (b) The distribution of different candidate compounds and targets in the network (red theJun herbs-specific cC (a) and cT (b) aqua the Chen herbs-specific cC (a) and cT (b) periwinkle the Zuo-Shi herbs-specific cC (a) and cT(b) claybank common cC (a) and cT (b) between the Jun and Chen herbs blue common cC (a) and cT (b) between the Jun and Zuo-Shiherbs green common cC (a) and cT (b) between the Chen and Zuo-Shi herbs purple common cC (a) and cT (b) among the 3 group ofherbs (c) The triangles with circle backgrounds represent the herbs (HB) the squares and circles represent the candidate compounds (cC)and candidate targets (cT) The red triangles squares and circles represent corresponding HB cC and cT in the Jun herbs the same is toaqua representing the Chen herbs and periwinkle representing the Zuo-Shi herbs The claybank squares and circles represent correspondingcC and cT overlap between the Jun and Chen herbs the same is to blue representing the overlap between the Jun and Zuo-Shi herbs and thegreen representing the overlap between the Chen and Zuo-Shi herbs The purple squares and circles represent the corresponding cC and cTshared by the 3 group of herbs The size of the node is proportional to the value of degree
Radix Bupleuri (Zuo-Shi) contained quercetin It targeted149 bioactive proteins PTGS2 (degree=126) HSP90AB2P(degree=85) AR (degree=81) NCOA2 (degree=75) PRSS1(degree=68) PTGS1 (degree=67) PPARG (degree=66)and F10 (degree=60) were predicted as the major candidatetargets of quercetin followed by kaempferol (degree=65)which was contained by Carthami Flos (Jun) AchyranthisBidentatae Radix (Chen) and Radix Bupleuri licorice(Zuo-Shi) It targeted 61 bioactive proteins includingPTGS2 (degree=126) HSP90AB2P (degree=85) CALM(degree=81) AR (degree=81) NOS2 (degree=76) andNCOA2 (degree=75) Quercetin stigmasterol kaempferol
baicalin and beta-sitosterol existed in 3 the Jun Chenand Zuo-Shi drugs demonstrating crucial roles of thesecomponents The 5 ingredients in the Jun Chen and Zuo-Shiherbs targeted 186 bioactive proteins which accounted for65 targets of XFZYD Baicalein existed in the Jun andChen herbs Luteolin existed in the Jun and Zuo-Shi herbsSpinasterol and sitosterol existed in the Chen and Zuo-Shiherbs 126 bioactive compounds targeted PTGS2 followedby ESR1 (88) HSP90AB2P (85) AR (81) CALM (81) NOS2(76) NCOA2 (75) PRSS1 (68) PTGS1 (67) and PPARG(66) As depicted in Figure 3(c) these target proteins wereanchored by ingredients in the 3 group drugs
8 Evidence-Based Complementary and Alternative Medicine
Table 3 Top 10 target proteins of XFZYD according to 2 centrality indicators
Proteins Degree Proteins BetweennessPTGS2 126 PTGS2 0128936ESR1 88 NCOA2 0060806HSP90AB2P 85 HSP90AB2P 0049647AR 81 PRKACA 0046484CALM 81 PTGS1 004365NOS2 76 AR 0030252NCOA2 75 PRSS1 0025575PRSS1 68 ESR1 0022212PTGS1 67 PPARG 0021403PPARG 66 PGR 0020685Note the centrality indicators identify the important nodes within the network Higher degree centrality and betweenness centrality indicate greaterimportance
The analysis of the network revealed that quercetin (Mol148) kaempferol (Mol 108) luteolin (Mol 127) wogonin (Mol160) 7-Methoxy-2-methyl (Mol 33) and beta-sitosterol (Mol41) were predicted as the major active compounds of XFZYDTheproteins including F2 NOS2 PTGS1 PTGS2 and CALMwere predicted as essential pharmacological proteins for thetherapeutic effects of XFZYD
34 Analyses on TBI Based Specific Protein Interaction Net-work Network biology integrated with different kinds ofdata including physical or functional networks and diseasegene sets is used to interpret humandiseases Protein-proteininteraction networks (PPI) are fundamental to understandthe cellular organizations biological processes and proteinfunctions [54] From the systematic perspective the analysisof TBI-related PPI will improve the understanding of thecomplicated molecular pathways and the dynamic processesunderlying TBI We screened the TBI-specific genes andprotein targets using Online Mendelian Inheritance in Mandatabase (OMIM) and Therapeutic Target Database (TTD)Figure 4 indicated that 21 TBI-specific genesproteins wereacquired Further these hub-proteins were submitted toHPRD and STRING to establish the TBI-specific proteininteraction network The detailed information of the TBI-specific proteins is shown in Table S3 The results suggestedthat the network consisted of 489 nodes and 5738 edges(Figure 4(a)) We obtained top 10 TBI-related proteinsaccording to 2 centrality indicators generated and summa-rized in Table 4 Interestingly we found that the node withhigher betweenness tends to possess larger degree (Fig-ure 5) Network topology analysis showed that protoonco-gene tyrosine-protein kinase Src (SRC degree=164 between-ness=0059) RAC-alpha serinethreonine-protein kinase(AKT1 degree=157 betweenness=0045) Serum albumin(ALB degree=153 betweenness=0069) Epidermal growthfactor receptor (EGFR degree=139 betweenness=0053)and Amyloid beta A4 protein (APP degree=134 between-ness=0072) contributed to the essential role in the patho-physiology of TBI All of these indicated that the top mutualtarget proteins performed various beneficial functions to treatTBI at the molecular level For example SRC is activatedfollowing engagement of many different classes of cellular
receptors It participates in signal pathways that controla diverse spectrum of biological activities including genetranscription immune response cell adhesion cell cycleprogression apoptosis migration and transformation [55]SRC can result in blood-brain barrier (BBB) disruptionand brain edema at the acute stage the inhibition of SRCfamily kinases can protect hippocampal neurons and improvecognitive function after TBI [56] AKT1 regulates manyprocesses including metabolism proliferation cell survivalgrowth and angiogenesis [57] The PI3KAKTPTEN path-way has been shown to play a pivotal role in neuroprotectionenhancing cell survival by stimulating cell proliferation andinhibiting apoptosis after TBI [58] ALB is the main proteinof plasma and can be a biomarker to predict outcome ofTBI [59] Its main function is the regulation of the colloidalosmotic pressure of blood We found that it may participatein pathological process of TBI
KEGG pathway analysis was also used to determine thefunctions of proteins Table 5 describes the top 10 significantlyenriched KEGG pathways These pathways play crucial rolesin pathophysiology process which have also been widelydiscussed in existing literature For instanceMAPK signalingpathway can promote pathological axonal death throughtriggering a local energy deficit [60] Neurotrophin signalingthrough Trk receptors regulates cell survival proliferationthe fate of neural precursors axon and dendrite growth andpatterning and the expression and activity of functionallyimportant proteins such as ion channels and neurotransmit-ter receptors [61] Engagement of cells with the extracellularmatrix (ECM) proteins is crucial for various biological pro-cesses including cell adhesion differentiation and apoptosiscontributing to maintenance of tissue integrity and woundhealing [62]The 47 TBI-specific proteins targeted byXFZYD(yellow and green) were further discussed below
35 HB-pC-pT Network pT-F Network Construction andMolecule Docking Analyses of XFZYD for the Treatment ofTBI To investigate the therapeutic mechanisms of XFZYDfor the treatment of TBI a HB-pC-pT network of XFZYDfor treating TBI was built (Figure 6) 47 TBI-specific targetproteins (circles) were targeted by 119 potential compounds(squares) from XFZYD (Figure 6) Detailed information
Evidence-Based Complementary and Alternative Medicine 9
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HADHB
TP53BP2
DRD1
ST13
PRPF40A
TRAIP
KIDINS220
GRK6
MMP17
LTBR
EIF6
PGAM1
CHD3
PRSS3
APBB3
CHRNA7
KRT10
RIMS1
CRMP1
PDIK1L
SORBS3
SYMPK
GCN1L1
RNF19A
HOMER3
GRK4
JARID2
MYLK3
EFS
CRB1
HSD17B10
TST
HOMER2
PHC3
TM2D1 FICD
PEG3
CABLES1
LGALS2
ITIH1
HOXB2
TAF4FLOT1
BABAM1
CFH
HIP1R
LTBMYL4
NCOR1
CASP4
NF1
AP4E1
CLCA2
IGDCC4
NECAB3
CXorf27
SH3GLB1
CTCF
PLCG2
(a)
STAT1
EGFR
FN1
PLAT
SOD1
PRKCA CDK1
GSK3B
ALB APP
PPP3CA
IFNG
BBC3
SCN5A
KCNMA1
MAP2
ACHE
MAPK3F2
PTPN1
NOS3 BCL2
CAV1
CTNNB1
PRKCB
TGFB1CASP8
TNF
AKT1
F7
MAPK1LDLR
PRKCD
SLC6A3
DRD1
CASP3
CHRNA7
PRSS3
TP53BP2
CASP7
EPHB2
SYNGAP1
CALM1
CTSD
BACE1
ADRA1B
(b)
Figure 4 TBI-related protein interaction network (a) 21 hub proteins (red and green) were identified through the analysis of TherapeuticTargetDatabase (TTD) aswell asOnlineMendelian Inheritance inMan (OMIM) 4 overlapped protein targets (green)were obtained between21 hub proteins in TBI and candidate targets of XFZYD Periwinkle proteins fromHPRD and STRING analyses were not targeted by XFZYD(b) 47 candidate protein targets of XFZYD were screened for treating TBI Yellow candidate protein targets of XFZYD The size of nodes isproportional to the value of degree
10 Evidence-Based Complementary and Alternative Medicine
Table 4 Top 10 proteins of TBI specific proteins according to 2 centrality indicators
Proteins Degree Proteins BetweennessSRC 164 APP 0071814AKT1 157 ALB 0069343ALB 153 SRC 0058568EGFR 139 EGFR 0052938APP 134 AKT1 0045466GAPDH 131 HTT 0037164HSP90AA1 108 GAPDH 0034549BCL2 106 HSP90AA1 0027596MAPK1 105 CTNNB1 0021759HRAS 105 GNB1 0019492Note the centrality indicators identify the important nodes within the network Higher degree centrality and betweenness centrality indicate greaterimportance
Table 5 Top 10 significantly enriched KEGG pathways in TBI-specific proteins
Pathway ID Pathway description Gene count FDR4010 MAPK signaling pathway 44 299E-235200 Pathways in cancer 43 113E-184722 Neurotrophin signaling pathway 41 101E-344151 PI3K-Akt signaling pathway 40 111E-154510 Focal adhesion 39 292E-225205 Proteoglycans in cancer 39 410E-215010 Alzheimer s disease 34 124E-204015 Rap1 signaling pathway 33 769E-174014 Ras signaling pathway 33 646E-164020 Calcium signaling pathway 31 505E-17
0
001
002
003
004
005
006
007
008
0 20 40 60 80 100 120 140 160 180
Betw
eenn
ess
Degree
APP
ALB SRCEGFR
AKT1HTT
Figure 5 Relationship between degree and betweenness centralityin the TBI-specific protein interaction network
for the 119 potential compounds is shown in Table S4Similarly 5 pharmacologically active ingredients in the JunChen and Zuo-Shi groups including quercetin stigmasterolkaempferol baicalin and beta-sitosterol anchored 33 TBI-specific proteins such as CALM SCN5A F2 ACHE F7ADRA1B NOS3 BCL2 CASP3 and AKT1 11 Jun-specificcompounds targeted 2 specific targets (ALB CTNNB1) while12 Chen-specific compounds anchored 3 unique proteins(PRKCD FN1 and BC3) The Zuo-Shi herbs possessed thelargest number of active compounds (89) and targeted 5
unique proteins including EPHB2 BACE1 LDLR MAPK3and PRSS3 GSK3Bwas the common target between theChenandZuo-Shi drugs KCNMA1 CASP7 andAPPwere targetedby the Jun and Zuo-Shi drugs
The top 10 candidate compounds and targets to treat TBIwere showed in Tables 6 and 7 Formost of active compoundsfrom XFZYD each component hit more than one targetTable 6 demonstrated that quercetin had the highest numberof targets (degree =76) followed by kaempferol (degree=43) beta-sitosterol (degree =35) stigmasterol (degree =19)luteolin (degree=18) baicalein (degree=15) 7-Methoxy-2-methyl isoflavone (degree=12) wogonin (degree=12)nobiletin (degree=11) and naringenin (degree=9) Forinstance quercetin (3310158404101584057-pentahydroxyflavone) is anaturally occurring flavonoid commonly found in fruits andvegetables It regulates multiple biological pathways elicitinginduction of apoptosis as well as inhibiting angiogenesisand proliferation [63 64] Quercetin can attenuate neuronalautophagy and apoptosis in rat traumatic brain injury modelvia activation of PI3KAkt signaling pathway [65] It has alsobeen reported to have a protective ability against oxidativestress and mutagenesis in normal cells [66 67] Kaempferol(3 41015840 5 7 tetrahydroxy flavone) is a yellow-colored flavonoidthat is widely distributed in many botanical families[68] It has been shown to possess a variety of biologicalcharacteristics including effects of anti-inflammatory[69] antioxidative [70] tumor growth inhibition [71] and
Evidence-Based Complementary and Alternative Medicine 11
Mol 22Mol 33
Mol 29Mol 32 Mol 23Mol 20
Mol 97Mol 106
Mol 104Mol 100Mol 94
Mol 63Mol 90
Mol 87
Mol 91Mol 61
Mol 19
Mol 109
Mol 161
Mol 118
Mol 111
Mol 158
Mol 114
Mol 149
Mol 141
Mol 152
Mol 14
CASP3
BCL2
NOS3
SCN5A
F7
ADRA1B
ACHE
F2
Mol 4
Mol 49
Mol 127
APP CASP7
Mol 92
Mol 54
Mol 6
Mol 36
KCNMA1
Mol 27
Mol 13Mol 105Mol 140
Mol 17
Mol 39
Mol 2
Mol 21
Mol 12
Mol 142
Mol 126
Mol 135
Mol 121
Mol 119
Mol 124
Mol 120
Mol 117
Mol 133
Mol 131
Mol 134
PlatycodonGrandiforus
Aurantii Fructus
Licorice
Radix Bupleuri
RehmanniaglutinosaLibosch
AngelicaeSinensis Radix
Mol 8
Mol 73
Mol 75
Mol 77
Mol 80
Mol 7
Mol 82
Mol 76
Mol 74
Mol 60
Mol 46
Mol 150
Mol 151
Mol 112
Mol 115
Mol 11
Mol 116
Mol 113
Mol 110
Mol 1
Mol 9Mol 101Mol 10Mol 93
Mol 103Mol 89 Mol 107
Mol 96PRSS3 LDLRMAPK3 BACE1 EPHB2
Mol 35Mol 81
Mol 86
Mol 85
Mol 84
Mol 88
Mol 83Mol 30
Mol 137
ChuanxiongRhizoma
Mol 3
Mol 57
Mol 139
Achyranthis
Bidentatae
RadixMol 102
CASP8
IGHG1AKT1p53
CHRNA7
Mol 40
SLC6A3 PTPN1
SOD1MAPK1
Mol 38
Mol 95
Carthami Flos
Mol 24
Mol 78 Persicae Semen
Mol 122
Mol 25
Mol 98
TGFB1
GSK3B
PRKCA
Radix Paeoniae Rubra
Mol 52
Mol 42
Mol 62
Mol 138
EGFR
STAT1 PPP3CAPLAT
Stigmasterol
MAP2
Beta-sistosterol Quercetin
Kaempferol
SYNGAP1
CALM
Baicalin
PRKCB
CAV1
CTSD
TNF
DRD1
CDK1
IFNG
FN1BBC3 PRKCD
Mol 132
Mol 160
Mol 58
Mol 67
Mol 69 Mol 43ALB
Mol 64CTNNB1
xiongixioChuanxC nxnxioaamaRhihihizoma
Achyranthisiistntnt
aeeaeBidenBidennntatae
RadixadixR diR di
oniae nianiaiaonRadix PaeoPaePRadix P eeoeonRRRRubra dondonddoodPlatycolatycPlatyycPl ycoyccoPlatPlatycoPlatyccocod
rususrusususruGranG anGranGranGranGrGGranndndiforu
ructusuctuc sructusuctusructusruAurantiirA ntaA ntiitiirantAurantintiurantAur iiii Fru
cecececececececeecLiLLiLLiLiLLLLLLLLiLiLiLLiLLLiLicoric
leuririeuuurieurileurleurieurieurieurieurileRadix BBRadRad Bdix Bdixx R i Bdi BBBadix Radix BBuple
RehmanniaRe mRe m nnRehmanniamReRehmhmannnniamanniaRehmehmanniRR nosasasasaosaosglutilutglutgluulutiglgl titinos
LiboschLibLLibo hschib chLiboschboscLLib chschLibos
icaecaecacaeicaeicAngelAnAngeAngelAnAng lAAngeelielicdix dix dixdixdSinensinensSS nennensSinSSiSinensSinensSine sissis Rad
FloslololFlhamaCartharthhaamami Fl
menmennnnmicae Scae SSem
Figure 6 HB-pC-pT network of XFZYD for treating TBI The triangles with circle backgrounds represent the herbs (HB) the squares andcircles represent the potential compounds (pC) and targets (pT)The red triangles squares and circles represent corresponding HB pC andpT in the Junherbs the same is to aqua representing theChen herbs and periwinkle representing the Zuo-Shi herbsThe claybank squares andcircles represent corresponding pC and pT shared by the Chen and Zuo-Shi herbs the same is to blue representing the overlap between theJun and Zuo-Shi herbs and the green representing the overlap between the Chen and Zuo-Shi herbsThe purple squares and circles representthe corresponding pC and pT shared by the 3 groups of herbs
alleviating insulin resistance in type 2 diabetic rats [72]Beta-sitosterol (BS) is a vegetable-derived compound foundin various plants and is suggested to modulate the immunefunction inflammation and pain levels by controlling theproduction of inflammatory cytokines [73]
For targets analysis CALM possesses the largest degree(degree =84) followed by GSK3B (degree =61) SCN5A(degree = 59) F2 (degree = 43) ACHE (degree=28)F7 (degree=28) ADRA1B (degree=28) NOS3 (degree=20)BCL2 (degree=18) and CASP3 (degree=18) which demon-strated their crucial therapeutic effects for treating TBI Forinstance CALM possess an essential position in calciumsignaling pathway and is related to morphological changesmigration proliferation and secretion of cytokines andreactive oxygen species ofMicroglial cells [74]The activationof CaMKII120572 major isoform of Ca2+calmodulin-dependentprotein kinase (CaMK) in brain is directly associated withthe production of proinflammatory cytokines such as TNF-120572and IL-1120573 [75] GSK-3120573 is a serinethreonine-protein kinasewhich is abundant in the central nervous system (CNS)particularly in neurons [76] It can control gene transcrip-tion axonal transport and cytoskeletal dynamics in growthcones [77] The inhibition of GSK-3120573 attenuates apoptotic
signals and prevents neuronal death [78] There is increasingevidence that prothrombin (F2) and its active derivativethrombin are expressed locally in the central nervous systemBeside the central role in the coagulation cascade the gener-ation of thrombin leads to receptor mediated inflammatoryresponses cell proliferationmodulation cell protection andapoptosis [79 80] The role in brain injury depends upon itsconcentration as higher amounts cause neuroinflammationand apoptosis while lower concentrations might even becytoprotective [81]
Direct tissue damage aswell as hypoxic-ischemic increas-ing anaerobic glycolysis of the brain tissue results in theATP-stores depletion and failure of energy-dependent mem-brane ion pumps especially for voltage-dependent Ca2+ andNa+-channels Accumulated Ca2+ activates lipid peroxidasesproteases and phospholipases and caspases at the sametime increasing the intracellular concentration of free fattyacids and free radicals leading to necrosis or apoptosis ofneurocyte [82] At the same time the activation of residentglial cells microglia and astrocytes and the infiltration ofblood leukocytes secrete various immune mediators elicitedinflammatory responses which subsequently intersect withadjacent pathological cascades including oxidative stress
12 Evidence-Based Complementary and Alternative Medicine
Table 6 Top 10 potential candidate compounds of XFZYD for treating TBI according to 2 centrality indicators
Compound Degree Compound Betweennessquercetin 76 quercetin 01682179kaempferol 43 kaempferol 005501511beta-sitosterol 35 wogonin 005141376Stigmasterol 19 beta-sitosterol 004451294luteolin 18 naringenin 003251738baicalein 15 beta-carotene 002977217-Methoxy-2-methyl isoflavone 12 nobiletin 002787992wogonin 12 7-Methoxy-2-methyl isoflavone 002096439nobiletin 11 luteolin 002090223naringenin 9 baicalein 001453488
Table 7 Top 10 potential targets of XFZYD for treating TBI according to 2 centrality indicators
Targets Degree Targets BetweennessCALM 84 CALM 021452046GSK3B 61 SCN5A 011441591SCN5A 59 GSK3B 009553896F2 43 F2 00689171ACHE 28 BCL2 002651903F7 28 CASP3 002372836ADRA1B 28 F7 002032647NOS3 20 ACHE 001845996BCL2 18 ADRA1B 001704505CASP3 18 NOS3 00166699
excitotoxicity or reparative events including angiogenesisscarring and neurogenesis [83] Function analysis of the 47target proteins regulated by XFZYD was mainly associatedwith core pathophysiology process of TBI (Figure 7) 47 targetproteins were connected with 16 key process related to TBIMost of the targets have one or more links to other biolog-ical process such as apoptosis cell proliferation superoxideanion generation nitric oxide biosynthetic process responseto calcium ion I-kappaB kinaseNF-kappaB signaling andregulation of inflammation The above analysis implied themultifunction character of these target proteins regulated byXFZYD Of these target proteins 25 proteins (53 of the 47)such as TGFB1 EGFR CAV1 MAPK1 PRKCB and AKT1were responsible for regulating the apoptosis process 17 forblood coagulation 14 for cell proliferation and axon genesisand 12 for hypoxia andMAPK cascade We found that TGFB1was the crucial protein because it participated in 9 biologicalprocesses related to TBI such as apoptosis blood coagulationcell proliferation and MAPK cascade followed by EGFR (8)CAV1 (7) MAPK1 (6) PRKCB (6) and AKT1 (6)
Overall these observations strongly support the evidencethat the generated HB-pCminuspT network and pT-F networkhave important roles in treating TBI further validating thedrug targeting approach
Molecule docking was used to further validate the bind-ing mode between candidate compounds and their targetproteins We found that 18 TBI-specific target proteins inter-acted with 91 candidate compounds from XFZYD (Figure 8)
Other 29 target proteins were not discussed for the lackof proper protein crystal structure The detailed moleculedocking results are shown in Table S5The 6 essential proteinsincluding GSK3B AKT1 CDK1 F2 NOS3 and ACHEwere used to elucidate the exact binding mode (Figure 9)Quercetin was located within the binding cavity of AKT1 andCDK1 (Figures 9(a) and 9(b) and S1A B) Four conventionalhydrogen bonds were formed between quercetin and AKT1by interacting with the key amino acids including ILE-290 THR-211 and SER 205 Additionally 120587-120587 interactionsbetween quercetin and TRP-90 were found in the activesite which helped the stabilization of the compound at thebinding site (Figure 9(a) S1A) Figure 9(b) and S1B suggestedthat five conventional hydrogen bonds (LEU-83 ASP-146and LYS-33) and 120587-120587 interaction (PHE-80) were formedbetween quercetin and CDK1 The GSK3B-FA complexes(Figure 9(c) S1C) were stabilized by 6 hydrogen-bondinginteractions between FA and LYS-85 GLU-97 TYR-134 andARG-141 Glyasperin Bmainly bonds to F2 through hydrogenbonds by interacting with the key amino acids includingGLY-193 SER-195 and GLY-219 (Figure 9(d) S1D) andan edge-to-face 120587 minus 120587 interaction was also observed withTYR-228 The GLN-247 GLU-351 and GLY-355 from theactive site pocket of NOS3 participated in the hydrogen-bondformationwith 1-Methoxyphaseollidin (Figure 9(e) S1E)The(-)-Medicocarpin formed a total of 6 hydrogen bonds withSER-293 PHE-295 ARG-296 and TRY-341 in the active siteof ACHE (Figure 9(f) S1F) Besides an edge-to-face 120587 minus 120587
Evidence-Based Complementary and Alternative Medicine 13
KCNMA1
P53
IFNG
PPP3CA
CASP8
SCN5A
I-kappaB kinase NF-kappaB signaling
Regulation of inflammation
Response to calcium ion
TGFB1
TNF
PRKCA
CHRNA7
CAV1
STAT1
MAPK cascadeAKT1
BCL2
APP
EGFR
MAPK1
Angiogenesis
SYNGAP1
MAP2
PTPN1
Axonogenesis
GSK3B
EPHB2
CDK1
CASP7FN1
MAPK3
CTNNB1
F2
Autophagy
Cell proliferation
BACE1
SLC6A3
DRD1
ADRA1BACHE
CALMCASP3
PLAT
Apoptotic process
NOS3
PRKCB
Blood coagulation
Response to hypoxia
Superoxide anion generation
Nitric oxide biosynthetic process
Astrocyte activation
Amyloid-beta regulation
Microglial cell activation
LDLR
PRSS3
F7
PRKCD
SOD1ALB
BBC3
Figure 7 pT-F network of XFZYD for treating TBI 16 biological processes (red square) the 47 target proteins (periwinkle circle) of XFZYDparticipate in for treating TBI
interactionwas also observedwith TYR-337 From the resultshydrogen-bonding and edge-to-face 120587 minus 120587 interactions playkey roles in the proteinminusligand recognition and stabilitywhich may be helpful in determining the inhibitor activitiesAnd the C-T network confirmed the potential therapeuticeffects of the candidate compounds from XFZYD to treatTBI through interacting with the relevant proteins The com-putational analysis further elucidated the accurate moleculemechanisms between active compounds and targets
4 Discussion
Traumatic brain injury (TBI) is a growing public healthproblem worldwide and is a leading cause of death anddisability [84] Although major progress has been made in
understanding the pathophysiology of this injury this hasnot yet led to substantial improvements in outcome by alack of treatments which have proven successful during phaseIII trials for modern medicine [85 86] TCM rooted inthousands of years of history may offer an alternative or acomplementary strategy for the treatment of TBI XFZYDa representative formula in TCM has been used for yearsto treat TBI in China and has been demonstrated to beeffective in clinical practice However its ldquomulticomponentsrdquoand ldquomultitargetsrdquo features make it much difficult to decipherthemolecular mechanisms of XFZYD in the treatment of TBIfrom a systematic perspective if employing routine methods
In the present study a network pharmacology-basedmethod was employed to elucidate the pharmacologicalmechanisms of XFZYD to treat TBI according to the drug
14 Evidence-Based Complementary and Alternative Medicine
Figure 8 C-T network through molecule docking validating 119 potential compounds (green triangles) interacting with 18 potential targets(yellow circles) of XFZYDThe size of the nodes is proportional to the value of degree
combination principle of TCM We first proposed a newmodeling system combining OB and DL screening multipledrug targets prediction and validation network constructionand molecule docking to probe the efficiency of a typicalTCM formula XFZYD for the treatment of TBI The 11herbs from XFZYD possessed 162 bioactive compounds andtargeted 285 proteins There were 5 compounds and 189
target proteins overlapped among the Jun Chen and Zuo-Shigroup Furthermore 47 TBI-specific proteins were targetedby 119 (73) bioactive compounds from XFZYD Similarly 5common compounds and 33 (70) common target proteinsamong the 3 groups of drugs were observed Most of thebioactive ingredients targeted more than one protein The 47target proteins regulated several essential pathophysiological
Evidence-Based Complementary and Alternative Medicine 15
(a) (b) (c)
(d) (e) (f)
Figure 9 Hydrogen-bonding networks within the binding site of the compoundminustarget complexes obtained from molecule docking(a) AKT1-quercetin (b) CDK1-quercetin (c) GSK3B-FA (d) F2-glyasperin B (e) NOS3-1-Methoxyphaseollidin and (f) ACHE-(-)-Medicocarpin The molecules are presented as ball and stick models Active site amino acid residues are represented as lines Dotted bluelines in these pictures represent hydrogen bonds with distance unit of A Other O and N atoms are colored as red and blue respectively
processes of TBI that referred to apoptosis inflammationcell proliferation superoxide anion generation nitric oxidebiosynthetic process response to calcium ion etc The aboveanalysis reveals that the synergistic action mechanisms ofXFZYDmay be (1) bioactive compounds overlapping amongthe different group of herbs (2) specific bioactive com-pounds from different groups of herbs targeting the sameproteins (3) specific bioactive compounds from differentgroups of herbs targeting different proteins which participatein the same pathophysiological process of the disease Toa certain degree the 5 compounds including quercetinstigmasterol kaempferol baicalin and beta-sitosterol playedessential role in XFZYD for TBI treatment MAPK3MAPK1AKT1 PRKCA TNF PRKCB EGFR BCL2 GSK3B CASP3PPP3CA and NOS3 were the main target proteins regulatedby XFZYD in the treatment of TBI
Interestingly Beta-carotene (Mol 43) from Carthami Flos(the Jun herb) specifically regulated 120573-Catenin (CTNNB1)and played critical role for curing TBI The 120573-Catenin is acritical downstream component of the Wnt pathway whichplays essential role in the regulation of mammalian neuraldevelopment [87] In vitro and in vivo studies demonstrate
that the Wnt120573-catenin pathway regulates the proliferationand differentiation of neural progenitor cells [88] Neuronaldifferentiation is induced by overexpression of 120573-cateninor the pharmacological inhibition of GSK3120573 (the phospho-rylating enzyme of 120573-catenin) [89 90] This pathway alsopromotes blood vessel formation during vascular develop-ment as well as the vascular repair process after TBI [91]In addition wogonin (Mol 160) from Achyranthis BidentataeRadix (the Chen herb) specifically targeted fibronectin (FN1)Bcl-2-binding component 3 (BBC3) and Protein KinaseC delta type (PRKCD) FN1 an important component ofthe extracellular matrix (ECM) environment promotes cellmigration neurite outgrowth and synapse formation dur-ing neural development [92] It aggregates in the injuredbrain and plays a neuroprotection role through antiapoptosisand anti-inflammation ways following TBI [93 94] BBC3namely p53 upregulated modulator of apoptosis (PUMA) iscritical for the p53-dependent apoptosis pathway which playsan important role in hippocampal neuronal loss and associ-ated cognitive deficits [95] PRKCD one of PKC isoformsactivates signal transduction pathways involved in neuronalregeneration [96] synaptic transmissionplasticity [97] and
16 Evidence-Based Complementary and Alternative Medicine
activation of apoptosis processes [98] as well as higher brainfunctions such as learning andmemory [99]The activators ofPKCare effective for the treatment of TBI [100] FurthermoreMitogen-activated protein kinase 3 (MAPK3) Beta-secretase1 (BACE1) Ephrin type-B receptor 2 (EphB) and low-densitylipoprotein receptor (LDLR) were specifically targeted bythe ingredients in the Zuo-Shi herbs Naringenin (Mol133) targeted LDLR MAPK3 while euchrenone (Mol 60)anchored BACE1 and nobiletin (Mol 134) targeted EphB2MAPK3 is an essential component of the MAP kinase signaltransduction pathway It has been appreciated recently thatthe ERK12 cascade plays a fundamental role in synapticplasticity and memory [101] BACE1 is responsible for pro-duction of A120573 from amyloid precursor protein (APP) A120573can cause cell death activate inflammatory pathways [102]and prime proapoptotic pathways for activation by otherinsults [103] The blocking of BACE1 can ameliorate motorand cognitive deficits and reduce cell loss after experimentalTBI in mice [104] EphB is localized to synaptic sites inhippocampal neurons [105] The interaction between EphBand NMDA receptors regulates excitatory synapse formation[106] LDLR acts as an important receptor that facilitatesbrain A120573 clearance and inhibits amyloid deposition [107]and then ameliorates Alzheimerrsquos disease neuropathologyafter TBI [108] The analysis above indicates the ldquoJunrdquoldquoChenrdquo and ldquoZuo-Shirdquo herbs from XFZYD trigger theirspecific targets regulation respectively for the therapeuticeffects
XFZYD is a very famous traditional Chinese formula inpromoting qi circulation and removing blood stasis accord-ing to TCM theory However several limited researches havedemonstrated its efficacy for treating TBI such as anti-inflammatory and synaptic regulation [30 31] which arein accord with our study However previous studies merelypartially deciphered the molecule mechanism of XFZYD fortreating TBI This study reports 119 bioactive compounds inXFZYD that target 47 TBI-specific proteins such as MAPK3MAPK1 AKT1 PRKCA TNF PRKCB and EGFR Theseproteins regulate several crucial pathophysiological processesof TBI such as apoptosis inflammation blood coagulationand axon genesis Our study demonstrates that the therapeu-tic actions of XFZYD refer to ldquomulticompoundsrdquo ldquomultitar-getsrdquo features rather than only the improvement of bloodcirculation With the help of molecule docking methodwe further validate the interactions between bioactive com-pounds and potential targets of XFZYD The hydrogen-bonding and edge-to-face 120587 minus120587 interactions play key roles inthe proteinminusligand recognition and stability This provides avaluable reference for further experimental investigations ofbioactive ingredients and therapeutic targets of XFZYD fortreating TBI
5 Conclusion
Our work successfully illuminates the efficiency of XFZYDfor the treatment of TBI as well as herb combination ruleof TCM formula Network pharmacology with moleculedocking method confirms the ldquomulticompounds multitar-getsrdquo therapeutic actions of XFZYD in the treatment of TBI
The present work may provide valuable evidence for furtherclinical application of XFZYD for treating TBI
Abbreviations
TBI Traumatic brain injuryXFZYD Xuefu Zhuyu decoctionTCM Traditional Chines medicineDL Drug-likenessOB Oral bioavailabilityHB-cC-cT Herb-candidate compound-candidate
targetHB-pC-pT Herb-potential compound-potential targetCALM CalmodulinGSK3B Glycogen synthase kinase-3 betaSCN5A Sodium channel protein type 5 subunit
alphaF2 ProthrombinACHE AcetylcholinesteraseGO Gene OntologyKEGG Kyoto Encyclopedia of Genes and
Genomes
Data Availability
The data used to support the findings of this study areavailable from the corresponding author upon request
Conflicts of Interest
The authors declare that they have no conflicts of interest
Authorsrsquo Contributions
YangWang and Zebing Huang participated in the conceptionand design of the study YuanyuanZhong YangWang ZebingHuang Jiekun Luo Tao Tang and Pengfei Li acquired andanalyzed the data Yuanyuan Zhong Tao Liu and HanjinCui drafted and revised the manuscript The correspondingauthor and all of the authors have read and approved the finalsubmitted manuscript
Acknowledgments
This work was supported by the National Natural ScienceFoundation of China (Nos 81673719 81874409 81603670and 81803948) Project funded byChina Postdoctoral ScienceFoundation (Nos 2016M600639 and 2017T100614)
Supplementary Materials
Table S1 162 bioactive compounds in XFZYD Table S2 targetproteins of XFZYD Table S3 TBI-specific proteins Table S4119 potential compounds of XFZYD for treating TBI TableS5 docking result of 18 target proteins with 91 potential com-pounds Fig S1 the exact bindingmode between active ingre-dients and protein targets obtained from molecule docking(A) AKT1-quercetin (B) CDK1-quercetin (C) GSK3B-FA
Evidence-Based Complementary and Alternative Medicine 17
(D) F2-glyasperin B (E) NOS3-1-Methoxyphaseollidin and(F)ACHE-(-)-Medicocarpin (Supplementary Materials)
References
[1] L Yang Y Chu D Tweedie et al ldquoPost-trauma administrationof the pifithrin-120572 oxygen analog improves histological andfunctional outcomes after experimental traumatic brain injuryrdquoExperimental Neurology vol 269 pp 56ndash66 2015
[2] J A Langlois W Rutland-Brown and M M Wald ldquoTheepidemiology and impact of traumatic brain injury a briefoverviewrdquo The Journal of Head Trauma Rehabilitation vol 21no 5 pp 375ndash378 2006
[3] H A Alsulaim B J Smart A O Asemota et al ldquoConsciousstatus predictsmortality among patientswith isolated traumaticbrain injury in administrative datardquo The American Journal ofSurgery vol 214 no 2 pp 207ndash210 2017
[4] M Majdan D Plancikova A Maas et al ldquoYears of life lost dueto traumatic brain injury in Europe A cross-sectional analysisof 16 countriesrdquo PLoS Medicine vol 14 no 7 p e1002331 2017
[5] P Cheng P Yin P Ning et al ldquoTrends in traumatic braininjury mortality in China 2006ndash2013 A population-basedlongitudinal studyrdquo PLoS Medicine vol 14 no 7 Article IDe1002332 2017
[6] M V Russo and D B McGavern ldquoInflammatory neuroprotec-tion following traumatic brain injuryrdquo Science vol 353 no 6301pp 783ndash785 2016
[7] WWang H Li J Yu et al ldquoProtective Effects of ChineseHerbalMedicine Rhizoma drynariae in Rats After Traumatic BrainInjury and Identification of Active Compoundrdquo MolecularNeurobiology vol 53 no 7 pp 4809ndash4820 2016
[8] M Das S Mohapatra and S S Mohapatra ldquoNew perspectiveson central and peripheral immune responses to acute traumaticbrain injuryrdquo Journal of Neuroinflammation vol 9 p 236 2012
[9] J W Finnie ldquoNeuroinflammation Beneficial and detrimentaleffects after traumatic brain injuryrdquo Inflammopharmacologyvol 21 no 4 pp 309ndash320 2013
[10] T Cheng W Wang Q Li et al ldquoCerebroprotection of flavanol(minus)-epicatechin after traumatic brain injury viaNrf2-dependentand -independent pathwaysrdquo Free Radical Biology amp Medicinevol 92 pp 15ndash28 2016
[11] W Young ldquoRole of calcium in central nervous system injuriesrdquoJournal of Neurotrauma vol 9 Suppl 1 pp S9ndashS25 1992
[12] R Bullock A Zauner J S Myseros et al ldquoEvidence forProlonged Release of Excitatory Amino Acids in Severe HumanHead Trauma Relationship to Clinical Eventsrdquo Annals of theNew York Academy of Sciences vol 765 no 1 pp 290ndash297 1995
[13] A T Mazzeo A Beat A Singh and M R Bullock ldquoTherole of mitochondrial transition pore and its modulation intraumatic brain injury and delayed neurodegeneration afterTBIrdquo Experimental Neurology vol 218 no 2 pp 363ndash370 2009
[14] S Gennai A Monsel Q Hao et al ldquoCell-Based therapy fortraumatic brain injuryrdquo British Journal of Anaesthesia vol 115no 2 pp 203ndash212 2015
[15] J Ghajar ldquoTraumatic brain injuryrdquo The Lancet vol 356 no9233 pp 923ndash929 2000
[16] D J Loane and A I Faden ldquoNeuroprotection for traumaticbrain injury translational challenges and emerging therapeuticstrategiesrdquoTrends in Pharmacological Sciences vol 31 no 12 pp596ndash604 2010
[17] Y Wang X Fan T Tang et al ldquoRhein and rhubarb simi-larly protect the blood-brain barrier after experimental trau-matic brain injury via gp91phox subunit of NADPH oxidaseROSERKMMP-9 signaling pathwayrdquo Scientific Reports vol 6p 37098 2016
[18] D-X Kong X-J Li and H-Y Zhang ldquoWhere is the hopefor drug discovery Let history tell the futurerdquo Drug DiscoveryTherapy vol 14 no 3-4 pp 115ndash119 2009
[19] F Cheung ldquoTCM made in Chinardquo Nature vol 480 no 7378pp S82ndashS83 2011
[20] C Fu Z Xia Y Liu et al ldquoQualitative analysis of majorconstituents from Xue Fu Zhu Yu Decoction using ultra highperformance liquid chromatographywith hybrid ion trap time-of-flight mass spectrometryrdquo Journal of Separation Science vol39 no 17 pp 3457ndash3468 2016
[21] H-J Zhang and Y-Y Cheng ldquoAn HPLCMS method for iden-tifying major constituents in the hypocholesterolemic extractsof Chinese medicine formula lsquoXue-Fu-Zhu-Yu decoctionrsquordquoBiomedical Chromatography vol 20 no 8 pp 821ndash826 2006
[22] L Zhang L Zhu YWang et al ldquoCharacterization and quantifi-cation of major constituents of Xue Fu Zhu Yu by UPLC-DAD-MSMSrdquo Journal of Pharmaceutical and Biomedical Analysisvol 62 pp 203ndash209 2012
[23] X Yang X Xiong G Yang and J Wang ldquoChinese patentmedicine Xuefu Zhuyu capsule for the treatment of unstableangina pectoris a systematic review of randomized controlledtrialsrdquo Complementary Therapies in Medicine vol 22 no 2 pp391ndash399 2014
[24] J Wang X Yang F Chu et al ldquoThe effects of Xuefu Zhuyuand Shengmai on the evolution of syndromes and inflammatorymarkers in patients with unstable angina pectoris after percuta-neous coronary intervention a randomised controlled clinicaltrialrdquoEvidence-BasedComplementary andAlternativeMedicinevol 2013 Article ID 896467 9 pages 2013
[25] Q Zhang H Yu J Qi et al ldquoNatural formulas and the natureof formulas Exploring potential therapeutic targets based ontraditional Chinese herbal formulasrdquo PLoS ONE vol 12 no 2p e0171628 2017
[26] J LeeWHsu T Yen et al ldquoTraditional ChinesemedicineXue-Fu-Zhu-Yu decoction potentiates tissue plasminogen activatoragainst thromboembolic stroke in ratsrdquo Journal of Ethnophar-macology vol 134 no 3 pp 824ndash830 2011
[27] Y-C Shen C-K Lu K-T Liou et al ldquoCommon and uniquemechanisms of Chinese herbal remedies on ischemic strokemice revealed by transcriptome analysesrdquo Journal of Ethnophar-macology vol 173 pp 370ndash382 2015
[28] F Xue-hai G Yan and L Jian-tao ldquoTherapeutic effect of XiefuZhuyu decoction joint brain protein hydrolysat injection intraumatic brain injury and its effect on cerebrospinal fluid ofET-1rdquo Chinese Journal of Biochemical Pharmaceutics no 02 pp55ndash58 2017
[29] L Shuxiang W Jingchun C Jie et al ldquoEffect of Xuefu Zhuyudecoction on the neural functional recovery and living abilityin patients with craniocerebral injuryrdquo Modern Journal ofIntegrated Traditional Chinese andWesternMedicine no 04 pp350ndash352 2015
[30] L Zhong W Ning and W Dong-pi ldquoModel Study of theTherapy of Replenishing Qi and Activating Blood Circulationon Protecting the Brain Impairment Caused by Hypoxic -ischemic Encephalopathy in SD Ratsrdquo Journal of TraditionalChinese Medicine vol 07 pp 1552ndash1555 2011
18 Evidence-Based Complementary and Alternative Medicine
[31] M Sun X-P Zhan C-Y Jin J-Z Shan S Xu and Y-LWang ldquoclinical observation on treatment of post-craniocerebraltraumatic mental disorder by integrative medicinerdquo ChineseJournal of Integrative Medicine vol 14 no 2 pp 137ndash141 2008
[32] Z Xing Z Xia W Peng et al ldquoXuefu Zhuyu decoction atraditional Chinese medicine provides neuroprotection in arat model of traumatic brain injury via an anti-inflammatorypathwayrdquo Scientific Reports vol 6 Article ID 20040 2016
[33] J Zhou T Liu H Cui et al ldquoXuefu zhuyu decoction improvescognitive impairment in experimental traumatic brain injuryvia synaptic regulationrdquo Oncotarget vol 8 no 42 2017
[34] S Li ldquoExploring traditional chinese medicine by a novel thera-peutic concept of network targetrdquo Chinese Journal of IntegrativeMedicine vol 22 no 9 pp 647ndash652 2016
[35] S Li and B Zhang ldquoTraditional Chinese medicine networkpharmacology theory methodology and applicationrdquo ChineseJournal of Natural Medicines vol 11 no 2 pp 110ndash120 2013
[36] A L Hopkins ldquoNetwork pharmacology the next paradigm indrug discoveryrdquoNature Chemical Biology vol 4 no 11 pp 682ndash690 2008
[37] Y Li C Han J Wang et al ldquoInvestigation into the mechanismof Eucommia ulmoides Oliv based on a systems pharmacologyapproachrdquo Journal of Ethnopharmacology vol 151 no 1 pp452ndash460 2014
[38] Y Yao X Zhang Z Wang et al ldquoDeciphering the combinationprinciples of Traditional Chinese Medicine from a systemspharmacology perspective based on Ma-huang DecoctionrdquoJournal of Ethnopharmacology vol 150 no 2 pp 619ndash638 2013
[39] Bo Zhang Xu Wang and Shao Li ldquoAn Integrative Platform ofTCM Network Pharmacology and Its Application on a HerbalFormula Qing-Luo-Yinrdquo Evidence-Based Complementary andAlternativeMedicine vol 2013 Article ID 456747 12 pages 2013
[40] J Ru P Li J Wang et al ldquoTCMSP a database of systemspharmacology for drug discovery from herbal medicinesrdquoJournal of Cheminformatics vol 6 no 1 p 13 2014
[41] J V Turner D J Maddalena and S Agatonovic-KustrinldquoBioavailability Prediction Based on Molecular Structure for aDiverse Series of Drugsrdquo Pharmaceutical Research vol 21 no 1pp 68ndash82 2004
[42] X XuW Zhang CHuang et al ldquoAnovel chemometricmethodfor the prediction of human oral bioavailabilityrdquo InternationalJournal of Molecular Sciences vol 13 no 6 pp 6964ndash6982 2012
[43] A Zhang W Pan J Lv and H Wu ldquoProtective Effect ofAmygdalin on LPS-Induced Acute Lung Injury by InhibitingNF-120581B and NLRP3 Signaling Pathwaysrdquo Inflammation vol 40no 3 pp 745ndash751 2017
[44] X Wei H Liu X Sun et al ldquoHydroxysafflor yellow A protectsrat brains against ischemia-reperfusion injury by antioxidantactionrdquo Neuroscience Letters vol 386 no 1 pp 58ndash62 2005
[45] W PWalters andM A Murcko ldquoPrediction of lsquodrug-likenessrsquordquoAdvanced Drug Delivery Reviews vol 54 no 3 pp 255ndash2712002
[46] Y Yamanishi M Kotera M Kanehisa and S Goto ldquoDrug-target interactionprediction from chemical genomic and phar-macological data in an integrated frameworkrdquo Bioinformaticsvol 26 no 12 pp i246ndashi254 2010
[47] H Yu J Chen X Xu et al ldquoA systematic prediction ofmultiple drug-target interactions from chemical genomic andpharmacological datardquo PLoS ONE vol 7 no 5 Article IDe37608 2012
[48] X Chen Z L Ji and Y Z Chen ldquoTTD therapeutic targetdatabaserdquo Nucleic Acids Research vol 30 no 1 pp 412ndash4152002
[49] A Hamosh A F Scott J S Amberger C A Bocchini andV AMcKusick ldquoOnline Mendelian Inheritance in Man (OMIM) aknowledgebase of human genes and genetic disordersrdquo NucleicAcids Research vol 33 pp D514ndashD517 2005
[50] T S Keshava Prasad RGoel K Kandasamy et al ldquoHuman pro-tein reference databasemdash2009 updaterdquo Nucleic Acids Researchvol 37 no 1 pp D767ndashD772 2009
[51] A Franceschini D Szklarczyk S Frankild et al ldquoSTRING v91protein-protein interaction networks with increased coverageand integrationrdquoNucleic Acids Research vol 41 no 1 pp D808ndashD815 2013
[52] P Shannon A Markiel O Ozier et al ldquoCytoscape a softwareEnvironment for integratedmodels of biomolecular interactionnetworksrdquo Genome Research vol 13 no 11 pp 2498ndash25042003
[53] G Dennis Jr B T Sherman D A Hosack et al ldquoDAVIDDatabase for Annotation Visualization and IntegratedDiscov-eryrdquo Genome Biology vol 4 no 5 p P3 2003
[54] L Yang X Zhao and X Tang ldquoPredicting disease-related pro-teins based on clique backbone in protein-protein interactionnetworkrdquo International Journal of Biological Sciences vol 10 no7 pp 677ndash688 2014
[55] S MThomas and J S Brugge ldquoCellular functions regulated bySRC family kinasesrdquo Annual Review of Cell and DevelopmentalBiology vol 13 pp 513ndash609 1997
[56] D Z Liu F R Sharp K C Van et al ldquoInhibition of Src familykinases protects hippocampal neurons and improves cognitivefunction after traumatic brain injuryrdquo Journal of Neurotraumavol 31 no 14 pp 1268ndash1276 2014
[57] B D Manning and A Toker ldquoAKTPKB Signaling Navigatingthe Networkrdquo Cell vol 169 no 3 pp 381ndash405 2017
[58] Y Kitagishi and S Matsuda ldquoDiets involved in PPAR andPI3KAKTPTEN pathway may contribute to neuroprotectionin a traumatic brain injuryrdquoAlzheimerrsquos ResearchampTherapy vol5 no 5 p 42 2013
[59] D Chen L Bao S-Q Lu and F Xu ldquoSerum albumin andprealbuminpredict the poor outcomeof traumatic brain injuryrdquoPLoS ONE vol 9 no 3 Article ID e93167 2014
[60] J Yang Z Wu N Renier et al ldquoPathological axonal deaththrough aMapk cascade that triggers a local energy deficitrdquoCellvol 160 no 1-2 pp 161ndash176 2015
[61] E J Huang and L F Reichardt ldquoTrk receptors roles in neuronalsignal transductionrdquoAnnual Review of Biochemistry vol 72 pp609ndash642 2003
[62] J W Lee and R Juliano ldquoMitogenic signal transductionby integrin- and growth factor receptor-mediated pathwaysrdquoMolecules and Cells vol 17 no 2 pp 188ndash202 2004
[63] C S Yang J M Landau M T Huang and H L NewmarkldquoInhibition of carcinogenesis by dietary polyphenolic com-poundsrdquo Annual Review of Nutrition vol 21 pp 381ndash406 2001
[64] A Murakami H Ashida and J Terao ldquoMultitargeted cancerprevention by quercetinrdquoCancer Letters vol 269 no 2 pp 315ndash325 2008
[65] G Du Z Zhao Y Chen et al ldquoQuercetin attenuates neuronalautophagy and apoptosis in rat traumatic brain injury modelvia activation of PI3KAkt signaling pathwayrdquo NeurologicalResearch vol 38 no 11 pp 1ndash8 2016
Evidence-Based Complementary and Alternative Medicine 19
[66] Q Cai R O Rahn and R Zhang ldquoDietary flavonoidsquercetin luteolin andgenistein reduce oxidativeDNAdamageand lipid peroxidation and quench free radicalsrdquoCancer Lettersvol 119 no 1 pp 99ndash107 1997
[67] G A Bongiovanni E A Soria and A R Eynard ldquoEffectsof the plant flavonoids silymarin and quercetin on arsenite-induced oxidative stress in CHO-K1 cellsrdquo Food and ChemicalToxicology vol 45 no 6 pp 971ndash976 2007
[68] H Li L Yang Y Zhang et al ldquoKaempferol inhibits fibroblastcollagen synthesis proliferation and activation in hypertrophicscar via targeting TGF-beta receptor type Irdquo Biomedicine ampPharmacotherapy = Biomedecine amp Pharmacotherapie vol 83pp 967ndash974 2016
[69] K P Devi D S Malar S F Nabavi et al ldquoKaempferol andinflammation From chemistry to medicinerdquo PharmacologicalResearch vol 99 pp 1ndash10 2015
[70] M Zhou H Ren J Han W Wang Q Zheng and DWang ldquoProtective effects of kaempferol against myocardialischemiareperfusion injury in isolated rat heart via antioxidantactivity and inhibition of glycogen synthase kinase-3120573rdquo Oxida-tive Medicine and Cellular Longevity vol 2015 p 481405 2015
[71] C-F Lee J-S Yang F-J Tsai et al ldquoKaempferol inducesATMp53-mediated death receptor and mitochondrial apop-tosis in human umbilical vein endothelial cellsrdquo InternationalJournal of Oncology vol 48 no 5 pp 2007ndash2014 2016
[72] C Luo H Yang C Tang et al ldquoKaempferol alleviates insulinresistance via hepatic IKKNF-120581B signal in type 2 diabetic ratsrdquoInternational Immunopharmacology vol 28 no 1 pp 744ndash7502015
[73] A B Awad and C S Fink ldquoPhytosterols as anticancer dietarycomponents evidence and mechanism of actionrdquo Journal ofNutrition vol 130 no 9 pp 2127ndash2130 2000
[74] K Farber and H Kettenmann ldquoFunctional role of calciumsignals for microglial functionrdquoGlia vol 54 no 7 pp 656ndash6652006
[75] Y Lu Y Gu X Ding et al ldquoIntracellular Ca2+ homeostasisand JAK1STAT3 pathway are involved in the protective effectof propofol on BV2 microglia against hypoxia-induced inflam-mation and apoptosisrdquo PLoS ONE vol 12 no 5 p e01780982017
[76] K Leroy and J-P Brion ldquoDevelopmental expression and local-ization of glycogen synthase kinase-3120573 in rat brainrdquo Journal ofChemical Neuroanatomy vol 16 no 4 pp 279ndash293 1999
[77] C-M Liu E-M Hur and F-Q Zhou ldquoCoordinating geneexpression and axon assembly to control axon growth Potentialrole ofGSK3 signalingrdquo Frontiers inMolecularNeuroscience vol3 p 3 2012
[78] D A E Cross A A Culbert K A Chalmers L Facci S DSkaper and A D Reith ldquoSelective small-molecule inhibitors ofglycogen synthase kinase-3 activity protect primary neuronesfrom deathrdquo Journal of Neurochemistry vol 77 no 1 pp 94ndash102 2001
[79] V S Ossovskaya and NW Bunnett ldquoProtease-activated recep-tors contribution to physiology and diseaserdquo PhysiologicalReviews vol 84 no 2 pp 579ndash621 2004
[80] M Steinhoff J Buddenkotte V Shpacovitch et al ldquoProteinase-activated receptors transducers of proteinase-mediated signal-ing in inflammation and immune responserdquoEndocrine Reviewsvol 26 no 1 pp 1ndash43 2005
[81] H Krenzlin V Lorenz S Danckwardt O Kempski and BAlessandri ldquoThe importance of thrombin in cerebral injury and
diseaserdquo International Journal of Molecular Sciences vol 17 no1 2016
[82] M J McGinn and J T Povlishock ldquoPathophysiology of Trau-matic Brain InjuryrdquoNeurosurgery Clinics of North America vol27 no 4 pp 397ndash407 2016
[83] T Woodcock and M C Morganti-Kossmann ldquoThe role ofmarkers of inflammation in traumatic brain injuryrdquo Frontiersin Neurology vol 4 article 18 2013
[84] F Tanriverdi H J Schneider G Aimaretti B E Masel F FCasanueva and F Kelestimur ldquoPituitary dysfunction after trau-matic brain injury A clinical and pathophysiological approachrdquoEndocrine Reviews vol 36 no 3 pp 305ndash342 2015
[85] J V Rosenfeld A I Maas P Bragge M C Morganti-Kossmann G T Manley and R L Gruen ldquoEarly managementof severe traumatic brain injuryrdquoThe Lancet vol 380 no 9847pp 1088ndash1098 2012
[86] B M Aertker S Bedi and C S Cox ldquoStrategies for CNS repairfollowing TBIrdquo Experimental Neurology vol 275 no 3 pp 411ndash426 2016
[87] K Adachi Z Mirzadeh M Sakaguchi et al ldquo120573-catenin signal-ing promotes proliferation of progenitor cells in the adultmousesubventricular zonerdquo Stem Cells vol 25 no 11 pp 2827ndash28362007
[88] Y Hirabayashi and Y Gotoh ldquoStage-dependent fate determina-tion of neural precursor cells in mouse forebrainrdquo NeuroscienceResearch vol 51 no 4 pp 331ndash336 2005
[89] S Ding T Y H Wu A Brinker et al ldquoSynthetic smallmolecules that control stem cell faterdquo Proceedings of theNationalAcadamy of Sciences of the United States of America vol 100 no13 pp 7632ndash7637 2003
[90] N Israsena M Hu W Fu L Kan and J A Kessler ldquoThepresence of FGF2 signaling determines whether 120573-cateninexerts effects on proliferation or neuronal differentiation ofneural stem cellsrdquo Developmental Biology vol 268 no 1 pp220ndash231 2004
[91] A Salehi A Jullienne M Baghchechi et al ldquoUp-regulation ofWnt120573-catenin expression is accompanied with vascular repairafter traumatic brain injuryrdquo Journal of Cerebral Blood Flow ampMetabolism vol 38 no 2 pp 274ndash289 2017
[92] L F Reichardt and K J Tomaselli ldquoExtracellular matrixmolecules and their receptors Functions in neural develop-mentrdquoAnnual Review of Neuroscience vol 14 pp 531ndash570 1991
[93] R M Gibson S E Craig L Heenan C Tournier and M JHumphries ldquoActivation of integrin 12057251205731 delays apoptosis ofNtera2 neuronal cellsrdquoMolecularandCellularNeuroscience vol28 no 3 pp 588ndash598 2005
[94] C C Tate A J Garcıa andMC LaPlaca ldquoPlasma fibronectin isneuroprotective following traumatic brain injuryrdquoExperimentalNeurology vol 207 no 1 pp 13ndash22 2007
[95] C Culmsee and M P Mattson ldquop53 in neuronal apoptosisrdquoBiochemical and Biophysical Research Communications vol 331no 3 pp 761ndash777 2005
[96] M S Geddis and V Rehder ldquoThe phosphorylation state ofneuronal processes determines growth cone formation afterneuronal injuryrdquo Journal of Neuroscience Research vol 74 no2 pp 210ndash220 2003
[97] M L Craske M Fivaz N N Batada and T Meyer ldquoSpines andneurite branches function as geometric attractors that enhanceprotein kinase C actionrdquoThe Journal of Cell Biology vol 170 no7 pp 1147ndash1158 2005
20 Evidence-Based Complementary and Alternative Medicine
[98] A Muscella C Vetrugno L G Cossa et al ldquoApoptosis by[Pt(OOrsquo-acac)(gamma-acac)(DMS)] requires PKC-deltamedi-ated p53 activation in malignant pleural mesotheliomardquo PloSone vol 12 no 7 Article ID e0181114 2017
[99] J S Bonini W C Da Silva L R M Bevilaqua J H MedinaI Izquierdo and M Cammarota ldquoOn the participation ofhippocampal PKC in acquisition consolidation and reconsol-idation of spatial memoryrdquo Neuroscience vol 147 no 1 pp 37ndash45 2007
[100] O Zohar R Lavy X Zi et al ldquoPKC activator therapeutic formild traumatic brain injury in micerdquo Neurobiology of Diseasevol 41 no 2 pp 329ndash337 2011
[101] J David Sweatt ldquoTheneuronalMAPkinase cascade a biochem-ical signal integration system subserving synaptic plasticity andmemoryrdquo Journal ofNeurochemistry vol 76 no 1 pp 1ndash10 2001
[102] Y Matsuoka M Picciano B Maleste et al ldquoInflammatoryresponses to amyloidosis in a transgenic mouse model ofAlzheimerrsquos diseaserdquo The American Journal of Pathology vol158 no 4 pp 1345ndash1354 2001
[103] L Esposito L Gan G-Q Yu C Essrich and L MuckeldquoIntracellularly generated amyloid-120573 peptide counteracts theantiapoptotic function of its precursor protein and primesproapoptotic pathways for activation by other insults in neu-roblastoma cellsrdquo Journal of Neurochemistry vol 91 no 6 pp1260ndash1274 2004
[104] D J Loane A Pocivavsek C E-H Moussa et al ldquoAmyloidprecursor protein secretases as therapeutic targets for traumaticbrain injuryrdquo Nature Medicine vol 15 no 4 pp 377ndash379 2009
[105] R Torres B L Firestein H Dong et al ldquoPDZ proteins bindcluster and synaptically colocalize with Eph receptors and theirephrin ligandsrdquo Neuron vol 21 no 6 pp 1453ndash1463 1998
[106] M B Dalva M A Takasu M Z Lin et al ldquoEphB receptorsinteract with NMDA receptors and regulate excitatory synapseformationrdquo Cell vol 103 no 6 pp 945ndash956 2000
[107] J M Basak P B Verghese H Yoon J Kim and D MHoltzman ldquoLow-density lipoprotein receptor represents anapolipoprotein E-independent pathway of A120573 uptake anddegradation by astrocytesrdquoThe Journal of Biological Chemistryvol 287 no 17 pp 13959ndash13971 2012
[108] L Yao X Gu Q Song et al ldquoNanoformulated alpha-mangostinameliorates Alzheimerrsquos disease neuropathology by elevatingLDLR expression and accelerating amyloid-beta clearancerdquoJournal of Controlled Release vol 226 pp 1ndash14 2016
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Evidence-Based Complementary andAlternative Medicine
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Submit your manuscripts atwwwhindawicom
2 Evidence-Based Complementary and Alternative Medicine
(ie intracranial pressure) and the preventive measures toreduce morbidity and mortality [14] Despite the fact thatdetailed medicines contain free-radical scavengers antago-nists of N-methyl-D-aspartate and calcium channel blockers[15] the results of the controlled clinical trials of these drugsare disappointing [16] Neuroscientists and doctors tend tosearch for potential novel drugs from traditional Chinesemedicine (TCM) library to treat TBI [17]
TCM is a comprehensive medicinal system that has beenused in clinical practice for thousands of years and playsan important role in the health maintenance for people allover the world [18 19] The validated curative effects of TCMmake it a feasible alternative therapeutic agent for diseasetreatment Xuefu Zhuyu decoction (XFZYD) a representa-tive TCM formula was first recorded in Correction of Errorsin Medical Works by Qing-ren Wang XFZYD consists of11 crude herbs Persicae Semen (Tao ren) Carthami Flos(Hong hua) Radix Paeoniae Rubra (Chi shao) ChuanxiongRhizoma (Chuan xiong) Achyranthis Bidentatae Radix (Niuxi) Angelicae Sinensis Radix (Dang gui) Rehmannia glutinosaLibosch (Sheng di huang) Platycodon Grandiforus (Jie geng)Aurantii Fructus (zhi qiao) Radix Bupleuri (chai hu) andlicorice (Gan cao)Themain chemicals from XFZYD includeflavonoids organic acids terpenoids and steroidal saponins[20ndash22] The formula has been proven reliable and effectivefor curing various diseases including unstable angina pectoris[23 24] coronary artery disease [25] thromboembolic stroke[26] ischemic stroke [27] and TBI The therapeutic agent ofXFZYD is to promote blood circulation and remove bloodstasis according to the TCM theory Several randomizedcontrolled clinical trials and animal experiments have showeddefinite therapeutic effects of XFZYD for the treatment ofTBI [28ndash31] Recent researches demonstrate that XFZYDprovides neuroprotection via anti-inflammatory pathway andcognitive improvement through synaptic regulation [32 33]However merely these evidences to explain the multipletherapeutic mechanisms of TCM for TBI treatment areunavailable Because the effects of TCM are always contro-versial in terms of their abstract theory unclear basis com-plex interactions between various ingredients and complexinteractive biological systems [25] it is essential to developan advanced technique to deeply uncover the synthesizedpharmacological effects of XFZYD in the treatment of TBI
With the development of TCM modernization networkpharmacology has become a novel method to elucidate themulti-druggable targets effects of TCM [34] TCM networkpharmacology first proposed by Shao Li [35] makes itfeasible to understand the effective constituents and targetsof the herbs from TCM formula This analytical methodintegrates bioinformatics systems biology and polypharma-cology and further utilizes network analysis to imply themultiple actions of drugs across multiple scales ranging frommolecularcellular to tissueorganism levels [36 37] Coin-ciding with the holistic and systemic characteristics of TCMnetwork pharmacology is expected to bridge the gap betweenTCM and modern medicine [25] Previous researches haveclarified the scientific basis and systematic features of herbalmedicine to treat diseases through network pharmacologysuch asQing-Luo-Yin andMa-HuangDecoction etc [38 39]
In the present work we explored the pharmacologicalmechanisms of XFZYD acting on TBI via a network pharma-cology approach Network analyses and molecular dockingmethod were used to reveal candidate drug targets relatedto TBI Target analysis suggested that XFZYD regulatedseveral key biological processes of TBI development suchas apoptosis inflammation blood coagulation and axongenesis These processes contributed to the clarifying of themolecular mechanisms of XFZYD for TBI treatment Thiswill help to improve the effectiveness and specificity of TCMclinical usage (Figure 1 depicts a flowchart of the entireresearch procedure)
2 Methods
21 Database Construction The chemical ingredients of11 herbs in XFZYD were screened from Traditional Chi-nese Medicine Systems Pharmacology database (TCMSPhttplspnwueducntcmspphp) [40] As a chemically ori-ented herbal encyclopedia TCMSP can provide comprehen-sive information about herb ingredients including chemicalstructural data oral bioavailability drug targets and theirrelationships with diseases as well as the biological or physio-logical consequences of drug actions involving drug-likenessintestinal epithelial permeability and aqueous solubility [40]The structures of these compoundswere saved asmol2 formatfor further analysis Discovery studio 25 was employed tooptimize these molecules with a Merck molecular force field(MMFF) All detailed information about these ingredients isprovided in Table S1
22 Pharmacokinetic Prediction Due to the disadvantagesof biological experiments as being time-consuming and ofhigh cost identification of ADME (absorption distributionmetabolism and excretion) properties by in silico toolshas now become a necessary paradigm in pharmaceuticalresearch In this study 2 ADME-related models includingthe evaluation of oral bioavailability (OB) and drug-likeness(DL) were employed to identify the potential bioactivecompounds of XFZYD
Oral bioavailability (OB) one of the most importantpharmacokinetic parameters represents the speed of a drugof becoming available to the body and the eventuallyabsorbed extent of the oral dose [41] which is particularly sig-nificant in drug discovery of TCM formost oral Chinese herbformulas Poor OB is indeed the main reason responsible forthe unsuccessful development of compounds into therapeuticdrugs in drug screening cascades Here a reliable in silicomodel OBioavail 11 [42] which integrates the metabolism(P450 3A4) and transport (P-glycoprotein) information wasemployed to calculate the OB values of herbal ingredientsIn this study OBge30 (a suggested criterion by TCMSPdatabase) was regarded as one threshold for screening pos-sible candidate drugs presently while 2 compounds withOB le 30 were also taken into consideration due to theirtherapeutic effects according to literatures such as amygdalinand hydroxysafflor yellow A [43 44]
Drug-likeness (DL) is a qualitative profile used in drugdesign to evaluate whether a compound is chemically suitable
Evidence-Based Complementary and Alternative Medicine 3
TCMSP database Molecule information ofcompounds in XFZYD
Candidate compounds profile
Candidate targets profileCandidate targets overlap analysisamong three group of herbs
Candidate compounds overlapanalysis among three group of herbs
HB-cC-cT network
Known therapeuticproteins for TBI
TBI specific proteins
OB DL
STRING
Overlap analysis of targets betweenXFZYD and TBI specific proteins
Cytoscape 340
TTD amp OMIM
HB-pC-pT network TBI specific PPInetwork
Molecule dockingGO and KEGG
pathway analysis
Binding mode between potentialtargets and compounds in XFZYD
Biological significance of potentialtargets of XFZYD on TBI
Synergistic effects ofTCM formula
HPRDTarget prediction
Figure 1 A schematic diagram of the network pharmacology-based strategies for determining the pharmacological mechanisms of the herbalformula XFZYD on TBI
for drug and how drug-like a molecule is with respect toparameters affecting its pharmacodynamic and pharmacoki-netic profiles which ultimately impacts its ADME properties[45] In this study the drug-likeness (DL) index (see (1))using the Tanimoto coefficient [46] was computed for eachcompound in XFZYD
119879 (119883119884) =119883 sdot 119884
1198832 + 1198842 minus 119883 sdot 119884(1)
where X represents the molecular descriptors of herb com-pounds and Y is the average molecular properties of all com-pounds in Drugbank database (httpwwwdrugbankca)Compounds with DL ge018 (average value for Drugbank)were selected as bioactive compounds in XFZYD
In summary compounds with OB ge30 and DLge018were selected for subsequent research and others wereexcluded The criteria used here were mainly for (1) extract-ing information from the herbs as much as possible with theleast number of components and (2) explaining the obtainedmodel by the reported pharmacological data
23 Target Prediction To obtain the molecular targets ofthese active ingredients an in-house developedmodel SysDTbased on Random Forest (RF) and Support Vector Machine(SVM) methods [47] was proposed which efficiently inte-grated large-scale information on chemistry genomics andpharmacology This approach shows impressive performanceof prediction for drug-target interactions with a concordanceof 8283 a sensitivity of 8133 and a specificity of 9362
respectively [40] UniProtKB (httpwwwuniprotorg) wasemployed to obtain the standard name of the predicted targetproteins
24 TBI-Specific Protein Collecting The known therapeutictarget proteins of TBI were screened fromTherapeutic TargetDatabase (TTD available httpbiddnusedusggroupcjttd)TTD is a publicly accessible database which provides com-prehensive information about the known therapeutic proteinand nucleic acid targets described in the literature thetargeted disease conditions the pathway information and thecorresponding drugsligands directed at each of these targets[48] We also searched for Online Mendelian Inheritancein Man database (OMIM available httpomimorg) toget proteins related to TBI OMIM catalogues all knowndiseases with a genetic component and then possibly linksthem to the relevant genes in the human genome andprovides references for further research and tools for genomicanalysis of a catalogued gene [49] Then the proteinsacquired from both databases were used as hub proteinsand submitted to Human protein Reference Database [50](HPRD available httpwwwhprdorg) and STRING [51](httpsstring-dborg) to generate the proteins interactingwith these hub proteins HPRD is a database containingcurated proteomic information pertaining to human pro-teins The human protein-protein interaction (PPI) data onHPRD (Release 9) consists of 39240 interactions among 9617genesThe STRINGdatabase provides both experimental andpredicted interaction information providing a probabilisticassociation confidence score
4 Evidence-Based Complementary and Alternative Medicine
25 Molecule Docking The LibDock algorithm based on theCHARMm Force Field in Discovery Studio (DS) 25 wasused in this study to evaluate the potential molecular bindingmode between bioactive compounds and putative targetsThecrystal structure of the target proteins of XFZYD for treatingTBI was downloaded from the RCSB Protein Data Bank(wwwrcsborg) The 3D chemical structures of bioactivecompounds were downloaded from PubMed Compounddatabase or TCMSP database and subjected to minimize theenergy by using molecular mechanics-2 (MM2) force fieldThe protein preparation protocol was used before dockingsuch as inserting missing atoms in incomplete residuesremoving water and protonating titratable residues The lig-and preparation protocol was employed before docking suchas removing duplicates enumerating isomers and generating3D conformations The protein-ligand docking active sitewas defined by the location of the original ligand All otherdocking and consequent scoring parameters were kept attheir default settings The compound was considered to be apotentially active ingredient if the LibDock score was higherthan the original ligand
26 Network Construction and Analysis Network construc-tion was performed as follows
(1) The herbs candidate compounds and candidate tar-gets of XFZYD were used to construct an herb-candidate compound-candidate target (HB-cC-cT)network
(2) The PPI data obtained above was used to establish theTBI-specific protein interaction network
(3) Herbs potential compounds and putative targetsfrom XFZYD for treating TBI were used to builda herb-potential compounds-potential targets (HB-pC-pT) network
(4) Potential targets and the biology process they partici-pate in were used to construct the pT-F network
(5) Compounds and targets through molecule dockingvalidating were used to build a compound-target (C-T) network
All networks were generated and analyzed by Cytoscape 340[52] an open source of bioinformatic package for biologicalnetwork analysis and visualization Two topological param-eters including degree and betweenness were calculated forthe obtained networks which disclose the significance ofa node
27 Gene Ontology (GO) and Pathway Enrichment Analy-sis The functional enrichment tool DAVID [53] (DAVIDhttpsdavidncifcrfgov) ver 68) was used to calculateboth the KEGG pathway and GO biological processes (BP)enrichment
28 Statistical Analysis All data were expressed as mean plusmnstandard deviation (SD) The molecule descriptors data wereanalyzed by one-way ANOVA The criterion for statisticalsignificance was p lt 005 Statistical analyses were conductedusing the SPSS 240
3 Results
TCM an experience-based medicine has been widely usedfor thousands of years It has accumulated abundant clinicalexperience forming a comprehensive and unique medicalsystem [35] The complexity of the phytochemical compo-nents makes it extremely difficult to illustrate the actionmechanisms of XFZYD from a molecule or system levelAs a chief mean of treating diseases clinically generallyTCM doctors prescribe formula based on the principle ofldquoJun-Chen-Zuo-Shirdquo ldquoJunrdquo (monarch) treats the main causeor primary symptoms of the disease ldquoChenrdquo (minister)enhances the actions of ldquoJunrdquo or treats the accompanyingsymptoms ldquoZuordquo (adjuvant) not only reduces or eliminatesthe possible toxic effects of the Jun or Chen but also treatsthe accompanying symptoms ldquoShirdquo (guide) helps to deliveror guide the other herbs to the target organs [18] Accordingto the unique feature of TCM ourwork tried to perform ldquoJun-Chen-Zuo-Shirdquo based system study to clarify the multiplemechanisms of XFZYD in the treatment of TBI
31 Herbal Ingredient Comparison and Target Predictionof XFZYD We obtained 162 components originated fromXFZYD Of these compounds 160 chemicals that were inaccord with standard requirements were searched from theTCMSP database The other 2 components amygdalin andhydroxysafflor yellow A were taken into consideration fortheir obvious pharmacological action as well The detailedinformation of these compounds is showed in Table S1Persicae Semen (Tao ren) and Carthami Flos (Hong hua)the Jun (monarch) herbs of XFZYD contained 36 bioactivecomponents which accounted for 22 of the 162 chemicalsRadix Paeoniae Rubra (Chi shao) Chuanxiong Rhizoma(Chuan xiong) andAchyranthis Bidentatae Radix (Niu xi) theChen (minister) herbs contained 31 bioactive componentswhich accounted for 19 of the 162 chemicals AngelicaeSinensis Radix (Dang gui) Rehmannia glutinosa Libosch(Sheng di huang) Platycodon Grandiforus (Jie geng) AurantiiFructus (zhi ke) Radix Bupleuri (chai hu) and licorice (Gancao) the Zuo-Shi (adjuvant and guide) herbs of XFZYDcontained 109 bioactive components which accounted for67 of the 162 chemicals Ingredients from these herbswere compared based on the 6 important drug-associateddescriptors including molecular weight (MW) number ofhydrogen-bond donors (nHdon) number of hydrogen-bondacceptors (nHacc) partition coefficient between octanol andwater (AlogP) oral bioavailability (OB) and drug-likeness(DL)The distributions of the 6 descriptors of the ingredientsfrom the three groups are shown in Table 1 and Figure 2 Wefoundno significant differences in the values ofMW(p=016)nHdon (p=032) nHacc (p=061) and AlogP (p=082) amongthe 3 groups However the average OB value of compoundsfrom the march herbs is 5559plusmn2391 which was significantlydifferent from the Chen herbs (OB=4564plusmn1327 P=0004)and the Zuo-Shi herbs (OB=4877plusmn1505 P=0021) Thefollowing 2 groups had no significant difference in OBvalue (P=0272) As for DL the Chen herbs revealed thehighest DL index (053plusmn023) which displayed significantdifference from the Zuo-Shi herbs (044plusmn019 p=0012) while
Evidence-Based Complementary and Alternative Medicine 5
Table 1 Comparison of molecular properties among the Jun Chen and Zuo-Shi herbs
INDEX MW nHdon nHacc AlogP OB DL(mean plusmn SD) (mean plusmn SD) (mean plusmn SD) (mean plusmn SD) (mean plusmn SD) (mean plusmn SD)
Jun herbs 38028 (9423) 265 (226) 511 (321) 337 (436) 5559 (2391) 049 (018)Chen herbs 38180 (9160) 218 (195) 55 (347) 314 (315) 4564 (1327) 053 (023)Zuo-shi herbs 35745 (8822) 261 (160) 56 (249) 344 (185) 4877 (1505) 044 (019)OB oral bioavailability MW molecular weight DL drug-likeness AlogP partition coefficient between octanol and water nHacc number of hydrogen-bondacceptors nHdon number of hydrogen-bond donors
Table 2 Top 10 candidate compounds according to 2 centrality indicators
Compounds Degree Compounds Betweennessquercetin 153 quercetin 035875838kaempferol 65 naringenin 008768253luteolin 48 kaempferol 007665228wogonin 46 luteolin 0057362047-Methoxy-2-methyl isoflavone 44 baicalein 005542898beta-sitosterol 41 beta-sitosterol 0041007baicalein 40 wogonin 003968963formononetin 39 Stigmasterol 003586788naringenin 39 nobiletin 003057461isorhamnetin 38 formononetin 003051437
showing nodifferencewith the Junherbs (049plusmn018 p=0333)Figure 3(a) indicated that 5 bioactive compounds wereshared by the Jun Chen and Zuo-Shi herbs One compoundoverlapped between the Jun andChen herbs while there were2 compounds shared by the Chen and Zuo-Shi herbs Onecompound overlapped between the Jun and Zuo-Shi herbs
32 Target Prediction of XFZYD A total of 285 potentialtargets from the 162 compounds were generated using thetarget prediction model The amounts of potential targets hitby the Jun Chen and Zuo-Shi drugs were 217 218 and 261respectively The detailed data of the targets is shown in TableS2 As depicted in Figure 3(b) there was a significant targetoverlap among the 3 groups (189 candidate targets) but lessoverlap between the Jun andChenherbs (9 candidate targets)The number of targets shared by the Jun and Zuo-Shi herbswas 14 while 10 targets were overlapped between the Chenand Zuo-Shi herbs
33 HB-cC-cT Network Construction and Analysis We nextestablished aHB-cC-cT network through network analysis toilluminate the relationship among the herbs candidate com-pounds and candidate targets (Figure 3(c)) This networkconsisted of 485 nodes (11 herbs 162 candidate compoundsand 285 candidate targets) and 2585 edges A herb (triangle)and cC (square) are connected if the compound is containedin this herb and the edges between cC and cT represent theinteraction The size of nodes is proportional to the valueof degree The larger size of the node means more phar-macologically important Two centrality indicators degreeand betweenness identify the important nodes within thenetwork Different centralities reflect different importanceof nodes in a network from different angles Interestingly
both of the two types of centrality indicators uniformlyconfirmed themost important 10 candidate compounds fromXFZYD and the top 10 targets anchored by XFZYD (Tables2 and 3) Figure 3(c) demonstrated that licorice possessedthe largest degree (88) compared with other herbs originatedfrom XFZYD This implicated that it contained the mostbioactive compounds (88) including quercetin (Mol 148degree=153) kaempferol (Mol 108 degree=65) 7-Methoxy-2-methyl isoflavone (Mol 33 degree=44) formononetin (Mol63 degree=39) naringenin (Mol 133 degree=39) isorham-netin (Mol 105 degree=38) medicarpin (Mol 131 degree=35)and licochalcone a (Mol 113 degree=33) followed byPersicae Semen (degree=19) Carthami Flos (degree=18)Achyranthis Bidentatae Radix (degree=17) Radix PaeoniaeRubra (degree=14) Radix Bupleuri (degree=12) ChuanxiongRhizoma (degree=6) Aurantii Fructus (degree=4) Platy-codon Grandiforus (degree=4) Rehmannia glutinosa Libosch(degree=3) andAngelicae Sinensis Radix (degree=2) PersicaeSemen (degree=19) and Carthami Flos (degree=18) the Junherbs from XFZYD possessed 29 specific compounds and5 unique target proteins including ALB CTNNB1 MMP10LYZ andNFKB1 Twenty-three Chen-specific potential com-pounds targeted 10 specific proteins including CD14 LBPNR3C1 BBC3 TEP1 PRKCD FN1 PDE10A GSTA1 andGSTA2 The Zuo-Shi herbs possessed the largest number ofspecific compounds (101) and 48 unique proteins such asHTR3A ADRA1D PYGM OLR1 CHRM5 RXRB STAT3MAPK10 OPRD1 and MAPK3 There were 189 proteinsanchored by the Jun Chen and Zuo-Shi drugs
Among the 162 candidate compounds quercetin hadthe largest value of degree (153) implicating its critical rolein XFZYD The four herbs namely Carthami Flos (Jun)Achyranthis Bidentatae Radix (Chen) and licorice and
6 Evidence-Based Complementary and Alternative Medicine
50
40
30
20
10
0
50
40
40
30
20
20
10
0
0
10 30 50 70 90 110
60
60
60
01 02 03 04 05 06 07 08
225 275 325 375 425 475 525 575 625 minus6minus5minus4minus3minus2minus1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
minus2 minus1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
70
minus1 1 3 5 7 9 11 13 15 17
perc
entage
s (
)
perc
entage
s (
)50
40
30
20
10
0
perc
entage
s (
)
perc
entage
s (
)
40
20
0
60
perc
entage
s (
)50
40
30
20
10
0
perc
entage
s (
)
OB
MW
nHdon
DL
nHacc
Distribution
Distribution
Distribution
Distribution
Distribution
Distribution
AlogP
Jun herbsChen herbsZuo-Shi herbs
Jun herbsChen herbsZuo-Shi herbs
Jun herbsChen herbsZuo-Shi herbs
Jun herbsChen herbsZuo-Shi herbs
Jun herbsChen herbsZuo-Shi herbs
Jun herbsChen herbsZuo-Shi herbs
Figure 2 The profile distributions of six important molecular properties for all ingredients from the Jun Chen and Zuo-Shi herbs OBOral bioavailability MW molecular weight DL drug-likeness AlogP partition coefficient between octanol and water nHacc number ofhydrogen-bond acceptors nHdon number of hydrogen-bond donors
Evidence-Based Complementary and Alternative Medicine 7
Jun herbs(29)
Zuo-
Shi h
erbs
(101
)
Chen herbs
(23)
1
2
15
(a)
Jun herbs(5)
Zuo-
Shi h
erbs
(48)
Chen herbs
(10)
9
10
14189
(b)
20Mol 1200000
MMMol 152Mol 15Mol 1521Mol 15Mol 15
Mol 8888
Mol 29Mol 2Mol 2Mol 2Mol 29
Mol 3Mol 3Mol 35Mol 3535
1Mol 121Mol 121Mol 122
Mol 124Mol 124Mol 1Mol 124Mol 124Mol 124
MMol 10Mol 1Mol 10M 00MMMM
MMol 73Mol 77333222
ABATABATA
MAPK3MAPKMAPK
UGT1A1GT1UGT1A1
GOT1GOTT1
SREBF1REBFSREBF1
RXRBRXRBRXRB
CYP19A1CYP19A1YP19A
PLB1PLBPLB1
FASLGFASLGASL
BCHEBCHHE
HSD3B1HSD3
MTTPMTT
CES1CES
TIMP1TIMP
ATP5BATP5
MAPK10MAPK 0
BADBAD
CHRM5CHRM5
EPHBEPHBPHB2
HSD3B2HSD3B2SD3B
SOAT2OATSOAT2
FASNFASNFASN
MT-ND6MT-N
HTR3AHTR3HTR3APLA2G4APLA2G4AA2G
PKIAAIA
ADH1AADHAD
AKR1C1KR1AKR1C1
CREB1CREBB1
HMGCRMGHMGCR
SOAT1OAT
ABCC1ABCC
OPRD1OPRDOPRD1
VCPVCP
LDLRLDLR
ADRA1DDRAA1D
NFATC1FATCFATCATCFATCA
EGLN1GLNGLNGLNGLN
FABP5ABP5ABPABPABPABP5
CYCSCYCSCYCSCYCSYCSFOSL1OSLOSLOSLOSL
ALOX12LOXLOXOXLOX
TDRD7DRDDDRDDRDDRDAPODAPODAPOPOAPOD
NNNNOXNOXNOXNOX5NOXX
Mol 40
NFKB1NFKBFKBFKBFKBLYZLYZLYZZLYZ CTNNB1TNNBTNNNNNN MMP10MMPMPMPMMP1ALBALBALBALBALB
3Mol 433MMol 28Mol 28Mol 2Mol 28
HSPA5SPASPASPA5HSPA5AERBB2ERBB2ERBBRBBB2ERBB2
HSPB1HSPBSPBSPBHSPB1HSPB1B
NQO1NQO1NQONQO1NQONQO1TOP1TOP1TOP1TOPTOPT
PORPPORPORPORPORR ACPPACPPACPPACPPCPP
SLC2ALC2A4LC2A4C2A4LC2A4LC2A4LC
SERPINE1SERPINE1PINRPINERPINPIN
JUNJUNNJUNNN
PTGER3PTGER3TGERTGERGERTGER
COL1A1OL1AOL1AOL1AA1CO
SSLPISLPSLPISLPISLPII
CCCALCCC MMMMMMSS
MMMMP1MMPMMPMMPTNFTNFTNFFTNF
CCND1CCNDCCNDCNDCNDCCND
SMDPSMDPSMDPSMD3PSMD3PSMDNFNFNFFFNF
ELK 1ELKELK LK ELK ELK CCL2CCL2CCLCCL2CCL2CCL2
MMol 14MMol 14Mol 1447
MMol 15MMol 1Mol 115Mol 15M
Mol 122Mol 122Mol 122MMol 12Mol 122222
MMol 24Mol 2Mol 2Mol 2444
Mol 78MMol 78Mol 7Mol 7Mol 788
Mol 38MMMMol 3Mol 3338
Mol 154Mol 154MMol 15ol 1ol 155M
Mol 67MMol 6Mol 6Mol Mol 67Mol 677766
Mol 64MMol 64Mol 6Mol 6Mol 64
Mol 48Mol 48MMMol 4Mol 4448M 4
PPPPARAPARPARPAR
PCOLCEPCOLCECOLCOLCCOLC
SPP1SPP1SPP1PP1SPPP1 AKR1C3AKR1C3KR1CKR1CKR1CKR
NNNOSNN 2222 DUOXDUOXDUOXDUOX2DUOXDUOX2
MCL1MCL1MCLMCLMCLMCL
PARP1ARP1ARPARPPARP1PEEMPOMPOMPOMPOM DDDUDUDUDDU
NGASYNGASYNGAP1NGAPNGAAP1RARAARARA
BAXXXXB X ESR1
GSTM2GSTM2GSTMSTMSTMGSTMSHSAAHSAAHSA1AHSA1AHSAHSAA GABRAGABRA2GABRA2GABRAGABRAGABRAMM2MMM2
CHRM2CHRM2CHRMHRMHRMCHRM2
VDRVDRVDRRVDRVDRARGABRA1GABRA1GABRAA11GABRA1G
IL888IL88
PPPPRSPRSPRSSSSSSSPPARDPARDPARPARDPARDPARDSELESELESELESELEEE
COL3A1COL3A1OL3AOL3AOL3AA1OL3ARB11111
CYP1A2222222 NCOANCOANCOANCOANCOANCOA22222211111 RRRRRR
Quercetin
Mol 9444444
Mol 99
Mol 63
Mol 75555
Mol 37Mol 37Mol 37Mol 37MolMol 33Mol 37MolM
Mol 158
Mol 1Mol 10Mol 1Mol 10000
Mol 49Mol 4Mol 4Mol 4Mol 44Mol 494Mol 4
Mol 6Mol 6666M 66
MMol 60Mol 60Mol 6MM
Mol 101Mol 101Mol 101Mol 10011
Mol 12Mol 12Mol 12Mol 1Mol 12221
Mol 11MMol 1155Mol 11555M6060660
Mol 85Mol 85Mol 85MMol 88558554MMol 114Mol 114444M
Mol 116Mol 116Mol 116MMol 1ol 1Mol 11616M 1
Mol 9000
Mol 100Mol 1000Mol 106MMMol 106Mol 10Mol 1066Mol 322 Mol 30Mol 30Mol 30000MM
Mol 140MMol 14MMol 14Mol 1440Mol 14
PlatycodPlatycodatycodPlatycodP ononoGrandifondifoGrandGrand rususus
RadiRadixR diBupleurBupleuriBupleuriu
RehmanniRehmanniRehmanniiaaaglutinosglutinolutinoglutin aaaLiboschLiboschLiboschLLiLiLicoricececece
AngelicaAngelicaAngelican l eeSinensisSinenS nensisnensis
Radixadixdix
Mol 84
Mol 23 Mol 130Mol 13Mol 13Mol 11Mol 130Mol 13Mol 1MMol 13
Mol 11Mol 1Mol 1Mol 11Mol 11
0Mol 200000
Mol 34Mol 3Mol 3MolMol 3344Mol 3Mol 134
Mol 151
Mol 126Mol 126Mol 12666
Mol 8111AurantiiAurantAuranAurantiitFructusructusFructuructuFr
Mol 77Mol 7Mol 7Mol 77777
Mol 444
MMol 82Mol 8Mol 8222Mol 88
Mol 133
Mol 123MMol 12Mol 12ol 1Mol 121
Mol 142Mol 1422
Mol 36
0Mol 80Mol 88Mol 80M 80
Mol 277Mol 135Mol 13555M 555
Mol 141MMol 144Mol 14111
Mol 104Mol 104MMMol 10Mol 104044M
Mol 922222Mol 22 Mol 2Mol 2Mol 2Mol Mol 2Mol 22M 2
MMol 83333
Mol 110Mol 110
Mol 119MMol 11999999
Mol 88888 Mol 109Mol 10MMol 1Mol 100Mol 109M
Mol 5444
Mol 39999999
Mol 150MMol 155000M 0
Mol 611
21Mol 21MMol 221111
Mol 13
Mol 9Mol 91MMol 9911
Mol 118MMol 11Mol 1Mol 11Mol 1Mol 11ol
Mol 466
Mol 96Mol 96Mol 96Mol 996Mol 9696 Mol 53Mol 5Mol 5MolMol 5Mol 5Mol 535Mol 50Mol 50Mol 5Mol 5Mol 550Mol 5
Mol 107Mol 107MMol 10077Mol 1077
M l 149MMol 1449Mol 149Mol 149
Mol 14Mol 1444
Mol 89Mol 125MMol 1ol 1Mol 12MMMol 1MM
Mol 87
Mol 72MolMol 7Mol 7MolMol 72M 7M Mol 933Mol 112Mol 112Mol 112Mol 1122M
Mol 1Mol MMolMollMol 1Mol Mol 105
MAP2MAP2MAPMAPMAP
IL2I 2L22L22
SLC6A2LC6A2LC6A2LC6A2C6A2S
GABRA5ABRAABRGABRA5AABRCHRNA2CHRNA2HRNA2HRNHRNA2HRNA2
DRD1D1DRDDRDDRDDD1 MMAPKMAPK1MAMADRA1AADDDRADRA1ADRAADRA1A CASP3CASP3CASP3CASP3C
F2
Mol 17 Mol 7666Mol 33
Mol 86Mol 86Mol 8Mol 8Mol 866686
Mol 161Mol 161Mol 161Mol 16Mol 16161MMo
Mol 56Mol 5Mol 556666Mol 113
Mol 97Mol 162MMol 16Mol 1Mol 1ol 111M 1
Mol 131
Mol 99Mol 9MMMolMol 9MMol 9Mol 99Mol 9ol
Mol 111
Mol 128Mol 128Mol 1ol 1Mol 121MolMol 199Mol 117Mol 117777
Mol 744444Mol 77
2Mol 52Mol 52Mol 5MM
MMMol 5Mol 5MMol 5Mol 5Mol 5M
MMol 58Mol 58Mol 58M 8
Mol 138MMol 1388M
Mol 102Mol 102Mol 10Mol 102MMol 10Mol 10
Mol 42MMMol 42M
MMMol 3Mol 3
Mol 137Mol 13Mol 13ol 13Mol 13Mol 13733
Mol 59Mol 59olMol 5Mol 59oMol 5Mol 559
Mol 129lMol 12ol 12ol 12Mol 129o 2ol 12o
Mol 62Mol 62Mol 6Mol 6Mol 6Mol 62Mol 6Mol 6ol
Mol 144Mol 144Mol 14Mol 14ol 14Mol 144ol 1
AchyraAchyraAchyranthisnthisnthisBidentBidentBidentataeataeatae
RadixRadixRadix
Mol 139Mool 13ol 13ol 139Mol 139Mool 13
ChuanxioChuanxioChuanxiongngngRhizomaRhizomaRhizoma
Mol 159Mol 159MoMol 15ol 15ol 15Mol 159o 5
Mol 136Mol 136ol 13ol 13ol 1ol 13ool 13
CXCL11CXCL11XCL1XCL1XCLXCL1p53p53p53p
Mol 47Mol 47Mol 4Mol 4Mol 4ol 47Mol 4o
MMol 156ol 15ol 15ol 1ol 15ooPRKCARKCRKCPRKCACARKC
PPP3CAPP3CAP3CAP3CAP3CAPPP3CAP
CDKN1BDKNDKNDKN1CDKN1BPTENPTENPTENPTENPTENPTENBBB
RadixRadixRadixPaeoniaePaeoniaePaeoniaePae
RubraRubraRubraNR1I3NR1I3NR1INR1INR1INR1I3
HERC5ERCERC5HERC5ERC5OODC1ODCODCODC1CERBB3RBBRBBERBB3RBBRBB3
CTSDCTSDCTSDCTSDCTSDCTSD Mol 18Mol 1Mol 1ol 18Mol 18MolMol 1
PLATPLATPLAPLATPLATL
DIOOO1OO1OEGFEGFEGEGFEGDIODDIODIO
BIL1BIL1BIL1BIL1BB
MMP 3MMP 3MMP 3MMPMMP 3M
THBDTHBDTHBTHBTHBTHBD
CYP1B1YP1BYP1BYP1BYP1BB1
PPPPPARPP GGGGGGPPP
KDR LLAACTACTACTLACTBLACTBACTTT
ESR2
CHEK1CHEK1CHEK1CHEK1CHEK1CHEK111CCHEK1C
CATCATCATCATTTCATCATT
CDK2
CCNA2CCNA2CCNA2CCNA2CCNA2CCNA2CCNA2CCCNA2CC
PIM1 GSK3B
MAPKKK1414MMAPKKKMM KMMM
IL10IL10IL10IL10IL10IL10
S2HAS2HAS2HAS22HAS2ADADDHDH1CADAD HAHAHHHHA
CDKN2ACDKN2CDKNCDKNDKN2ACDKN
Mol 155Mol 155Mol 15Mol 15Mol 15ol 1ol 15ol 15Mol 15MMo
Mol 153Mol 15Mol 15ol 15Mol 153ol 15Mol 15Mol 15
CHEKCHEKCHEKHEK2HEK2HEK
MMP2MMP2MMPMMPMMPMMP2
MGAMMGAMMGAMGAAM
KCNH2222
CXCL2CXCL2CXXCLXCLXCL
GJA1GJA1GJAGJA1GJA1GJA1KK222K2
IKBKBIKBKBIKBKBIKCNH2KCNH2
IFNGIFNGIFNGIFNGIFNGIFNGFNEIF666F6IGHG1IGHG1GHGIGHG1IGGHG
SSStigSStiggmmmaS gg steeerolerol
PPTPNPTPN1PTPN1P 1SCN5ASCN5AASCN5ASCN5A BetaBetaaaa-sata istosssststerolerolllroNR3C1R3CGSTA2STA BBC3BBC3LBPLBP TTEP1TEP1 RKCDPRKCD PDE10ADE10GSTA1STA FN1FN1CD14CD14
BACE1ACEBACE1 SLC6A1LC6AACD163CD16 GSRGSRR ADIPOQDIPOQSTAT3TATOPRM1OPRM1OPRMOPRMOPRMO ADRA1BADRA1BAADRA1BBBB
SLC6A4LC6ALC6ASLC6A44SLCYCYP1A1YP1A1YP1A1CYP1AYP1A1
SLC6A3SLC6A3SLC6ASLC6A3SLC6A3
PDE3APDE3AA
CHRNA7CHRNA7HRNAHRNAHRNAA
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BIRCBIRC5BIRCBIRCBIRC55
IL6666
HK2HK2HK2HK2HK2HK2
RUNX2UNXUNXUNXRUNX2UNXHSF1HSF1HSF1HSF1HSF1HSF1
IGFBP3GFBPGFBPFBPIGFBP3
STAT1T1TAT1TATSTAT1TATT
NCF1NCFNCFNCF1NCFN
PRSS3RSSSIRT1SIRTGATMGAT OLR1OLRAPOBAPOPYGMPYGM
TGFB1GFBGFBGFBGFBTGFB1F10 MMP9MMPMMPMMPP9PNFE2L2FE2LFE2LFE2LNFE2L2FE2L2L
Mol 160Mol 44Mol 44ol 4Mol 444Mol 444ol 4ol 4ol 44
Mol 55MoMol 5Mol 5Mol 555Mol 55Mol 5Mol 5MoMol 5
MMMol 132
MMMol 57
CRPCRPCRPCRPPHH
RUNX1T1UNX1NX1NX1NX1T1R
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CXCL10CXCL10XCXCLCXCLCXCL IGG
CLDN4LDN4LDN4LDN4ABABABAAB RRAHRAHRBBBIB
RARRAF1RAF1RAF11KKKKKKaemKKKKK pffefeferereerf referololol
CASP9CCASPCASP9PMDM2MDMMMDMMDMMDM2MDMMDM
BMAOBMAOBMAOBMAOBMAOBINSRINSRINSRINSRINSRRRNCOA1NCOA11NCOA1
HTR2AHTR2HTR2HTR222A
CHRMCHRMCHRM3MM3BCL2LCL2CL2L1LCL2BCL2L1111 CHCCHCCCCH
BCL2222BCL2CASP8CASP8CASP8CASP8CASP
RELARELARELAAAR
BaicalBaicalBaBaicalcacacalicaBB calcaac ininininininninninnCHUCHUKHUKCHUKHUKUKH AAAAAA
EGFREGFRGFREGFREGFR
NFKBIAFKBIFKBFKBNFKBIAFKBI
E2F2E2F2E2F2E2F2E
APPAPAPPAPPPAPPAPPA
CASP7CASPCASPASPCASPCASPPP IL4IL444L4L44
NUF2NUFNUFNUFNUFNUFNUFNUFN
CDK4CDKCDKCDKCDKCDKCD
XIAPXIAPXIAXIAXIAXIAX MEMEMETMETMETMEETMMME
ADCY2ADCYDCYDCDCYDCD
Mol 127
NNR3C2NR3C2NR3CNR3C2N
DPP4
DCAF5DCACAFDCAF55DCAFAKR1B1AKR1BKKR1BKR1BKR1BCTRB1CTRBTRBCTRBCTRB
LTA4HLTA4LTA4TA4LTA4LTA4H4
XDHXDHXDHHHXDH
PTGS2
Mol 51Mol 51Mol 5MMolMolMol 5Mol 5MMol 5
Mol 145Mol 14Mol 14ol 1ol 1ol 1Mol 1Mo
Mol 146MMMMool 1ol 11461
Mol 143Mol 14Mol 14Mool 1ol 11Mol 1411
Mol 70MMMolMol 7Mol 77Mol 70Mol 7Mol 7
Mol 71Mol 7Mol Mol 777Mol 71Mol 7Mol
Mol 25Mol 25MMMol Mol 22Mol 2
Mol 79Mol 7Mol 7Mol Mol 7Mol 779MM 7
Mol 26Mol 26MMol 2Mol Mol 22Mol 2M 22
CA22222222ADH1BADHADHDHADH1ADH1DH KCNMA1CNMCNMCNMCNMKCNMAKCNMCGABRA6GABRAABRAABRAABRAGABRAGA A6GA RAGRIA2GRIAGRIAGRIAGRIAAAA2GRIA
RASSFRASSF1ASSFASSFASSF
Mol 31MMol 3Mol 3Mol 3311M 3Mol 98Mol 98Mol 98MMol 9Mol 999Mol 98MM
GSTP1GSTP1GSTPGSTPGSTPGSTPF33333
Mol 69Mol 6MMol 6Mol 6Mol 66Mol 69Mol 69M 66
VCAM1VCAMCAMCAMVCAM1C
MAPK8MAPK8MAPKMAPMAPKM
CYP3A4CYP3A4YP3AYP3AYP3AY
Mol 68Mol 68MMol 6Mol 6Mol 66MMol 68Mol 6MMol 16Mol 16Mol 1Mol 1Mol Mol 1Mol 16
MMMMol 6MMol 66Mol 666Mo
CCartCarta hamihamimiiiiiFlosFlosFlosossoss
Mol 9566Mol 65Mol 6Mol 6MMol MolMol 6M 666M
PersPersPerserrr icaeicaeicaesemesemesemeemememeem nnn
ICAM1CAMCAMM1CAMI
E2F1E2FE2F1E2F1E2F1ENPEPPSPEPPSPEPPSEPPSPEPPSNPEPP
IGF2IGF2IGF2IGF2F
FOSFOSFOSFOSSFOSFOSSSS
CCNB1CCNBCNBCNBCCNB1CCNB1CDK1CDK1CDKCDKCDKCC
PRKCBPRKCBRKCRKCRKCR CB
CHRM1CHRM11CHRM11
HHHHSP9HHHH 0ABABAB2B2B2B2PPPPPPPPPPPPTOP2A ACACACACACACACACAACACAACACA
PRKACA
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CAV1CCAVCAVCAV1V1CAV1
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ADRB1ADRBRBADRBADR
ADRADRADRA2A2AA2A
SOD1SSODSOD1D11
NOS33
PLAUPLAUPLAAUUPLAUPRXRA
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HHMOX11MOXMOXHMOX
GSTM1GSTM1GSTMGSTMSTMAAA
HIF1AHIF1HIF1HIF11AHIF1A
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ALOX5ALOXALOXALOXALOX5
VEGFAVEGFAVEGFAVEGFAV FFA
PON1PONPONPONN1PON
PIK3CGPIK3CGGG
SULT1E1SULT1E1ULT1ELT1LT1GAGAABRAGABRA3AGG
AKT1AKTT1AKT11A
CHRMCHRMCHRMCHRM4CHRM4RMM
MAOAMAOAMAOAMAOMAOCD40LGCD40LGCD40LGD40LGCD40LGCD40LBRA3RAAABRA3CHRCHHHCHR
ADRB2
(c)
Figure 3 HB-cC-cT network of XFZYD (a) and (b) The distribution of different candidate compounds and targets in the network (red theJun herbs-specific cC (a) and cT (b) aqua the Chen herbs-specific cC (a) and cT (b) periwinkle the Zuo-Shi herbs-specific cC (a) and cT(b) claybank common cC (a) and cT (b) between the Jun and Chen herbs blue common cC (a) and cT (b) between the Jun and Zuo-Shiherbs green common cC (a) and cT (b) between the Chen and Zuo-Shi herbs purple common cC (a) and cT (b) among the 3 group ofherbs (c) The triangles with circle backgrounds represent the herbs (HB) the squares and circles represent the candidate compounds (cC)and candidate targets (cT) The red triangles squares and circles represent corresponding HB cC and cT in the Jun herbs the same is toaqua representing the Chen herbs and periwinkle representing the Zuo-Shi herbs The claybank squares and circles represent correspondingcC and cT overlap between the Jun and Chen herbs the same is to blue representing the overlap between the Jun and Zuo-Shi herbs and thegreen representing the overlap between the Chen and Zuo-Shi herbs The purple squares and circles represent the corresponding cC and cTshared by the 3 group of herbs The size of the node is proportional to the value of degree
Radix Bupleuri (Zuo-Shi) contained quercetin It targeted149 bioactive proteins PTGS2 (degree=126) HSP90AB2P(degree=85) AR (degree=81) NCOA2 (degree=75) PRSS1(degree=68) PTGS1 (degree=67) PPARG (degree=66)and F10 (degree=60) were predicted as the major candidatetargets of quercetin followed by kaempferol (degree=65)which was contained by Carthami Flos (Jun) AchyranthisBidentatae Radix (Chen) and Radix Bupleuri licorice(Zuo-Shi) It targeted 61 bioactive proteins includingPTGS2 (degree=126) HSP90AB2P (degree=85) CALM(degree=81) AR (degree=81) NOS2 (degree=76) andNCOA2 (degree=75) Quercetin stigmasterol kaempferol
baicalin and beta-sitosterol existed in 3 the Jun Chenand Zuo-Shi drugs demonstrating crucial roles of thesecomponents The 5 ingredients in the Jun Chen and Zuo-Shiherbs targeted 186 bioactive proteins which accounted for65 targets of XFZYD Baicalein existed in the Jun andChen herbs Luteolin existed in the Jun and Zuo-Shi herbsSpinasterol and sitosterol existed in the Chen and Zuo-Shiherbs 126 bioactive compounds targeted PTGS2 followedby ESR1 (88) HSP90AB2P (85) AR (81) CALM (81) NOS2(76) NCOA2 (75) PRSS1 (68) PTGS1 (67) and PPARG(66) As depicted in Figure 3(c) these target proteins wereanchored by ingredients in the 3 group drugs
8 Evidence-Based Complementary and Alternative Medicine
Table 3 Top 10 target proteins of XFZYD according to 2 centrality indicators
Proteins Degree Proteins BetweennessPTGS2 126 PTGS2 0128936ESR1 88 NCOA2 0060806HSP90AB2P 85 HSP90AB2P 0049647AR 81 PRKACA 0046484CALM 81 PTGS1 004365NOS2 76 AR 0030252NCOA2 75 PRSS1 0025575PRSS1 68 ESR1 0022212PTGS1 67 PPARG 0021403PPARG 66 PGR 0020685Note the centrality indicators identify the important nodes within the network Higher degree centrality and betweenness centrality indicate greaterimportance
The analysis of the network revealed that quercetin (Mol148) kaempferol (Mol 108) luteolin (Mol 127) wogonin (Mol160) 7-Methoxy-2-methyl (Mol 33) and beta-sitosterol (Mol41) were predicted as the major active compounds of XFZYDTheproteins including F2 NOS2 PTGS1 PTGS2 and CALMwere predicted as essential pharmacological proteins for thetherapeutic effects of XFZYD
34 Analyses on TBI Based Specific Protein Interaction Net-work Network biology integrated with different kinds ofdata including physical or functional networks and diseasegene sets is used to interpret humandiseases Protein-proteininteraction networks (PPI) are fundamental to understandthe cellular organizations biological processes and proteinfunctions [54] From the systematic perspective the analysisof TBI-related PPI will improve the understanding of thecomplicated molecular pathways and the dynamic processesunderlying TBI We screened the TBI-specific genes andprotein targets using Online Mendelian Inheritance in Mandatabase (OMIM) and Therapeutic Target Database (TTD)Figure 4 indicated that 21 TBI-specific genesproteins wereacquired Further these hub-proteins were submitted toHPRD and STRING to establish the TBI-specific proteininteraction network The detailed information of the TBI-specific proteins is shown in Table S3 The results suggestedthat the network consisted of 489 nodes and 5738 edges(Figure 4(a)) We obtained top 10 TBI-related proteinsaccording to 2 centrality indicators generated and summa-rized in Table 4 Interestingly we found that the node withhigher betweenness tends to possess larger degree (Fig-ure 5) Network topology analysis showed that protoonco-gene tyrosine-protein kinase Src (SRC degree=164 between-ness=0059) RAC-alpha serinethreonine-protein kinase(AKT1 degree=157 betweenness=0045) Serum albumin(ALB degree=153 betweenness=0069) Epidermal growthfactor receptor (EGFR degree=139 betweenness=0053)and Amyloid beta A4 protein (APP degree=134 between-ness=0072) contributed to the essential role in the patho-physiology of TBI All of these indicated that the top mutualtarget proteins performed various beneficial functions to treatTBI at the molecular level For example SRC is activatedfollowing engagement of many different classes of cellular
receptors It participates in signal pathways that controla diverse spectrum of biological activities including genetranscription immune response cell adhesion cell cycleprogression apoptosis migration and transformation [55]SRC can result in blood-brain barrier (BBB) disruptionand brain edema at the acute stage the inhibition of SRCfamily kinases can protect hippocampal neurons and improvecognitive function after TBI [56] AKT1 regulates manyprocesses including metabolism proliferation cell survivalgrowth and angiogenesis [57] The PI3KAKTPTEN path-way has been shown to play a pivotal role in neuroprotectionenhancing cell survival by stimulating cell proliferation andinhibiting apoptosis after TBI [58] ALB is the main proteinof plasma and can be a biomarker to predict outcome ofTBI [59] Its main function is the regulation of the colloidalosmotic pressure of blood We found that it may participatein pathological process of TBI
KEGG pathway analysis was also used to determine thefunctions of proteins Table 5 describes the top 10 significantlyenriched KEGG pathways These pathways play crucial rolesin pathophysiology process which have also been widelydiscussed in existing literature For instanceMAPK signalingpathway can promote pathological axonal death throughtriggering a local energy deficit [60] Neurotrophin signalingthrough Trk receptors regulates cell survival proliferationthe fate of neural precursors axon and dendrite growth andpatterning and the expression and activity of functionallyimportant proteins such as ion channels and neurotransmit-ter receptors [61] Engagement of cells with the extracellularmatrix (ECM) proteins is crucial for various biological pro-cesses including cell adhesion differentiation and apoptosiscontributing to maintenance of tissue integrity and woundhealing [62]The 47 TBI-specific proteins targeted byXFZYD(yellow and green) were further discussed below
35 HB-pC-pT Network pT-F Network Construction andMolecule Docking Analyses of XFZYD for the Treatment ofTBI To investigate the therapeutic mechanisms of XFZYDfor the treatment of TBI a HB-pC-pT network of XFZYDfor treating TBI was built (Figure 6) 47 TBI-specific targetproteins (circles) were targeted by 119 potential compounds(squares) from XFZYD (Figure 6) Detailed information
Evidence-Based Complementary and Alternative Medicine 9
BLMH
GPRASP2
GJC2
CAMK2N1
OBSL1
ZNF292
RLFAMPD3
NANOS1CAMTA1HYPKCCS
DLGAP4PF4V1
TRIM2
CHKB
F8A1
TIAM1
BMX
RAPGEF1
APBB1
KCNMA1
XRCC6
TTR
TGM2MAP2
PRNP
RASGRF1 PPP3CADMD SPTAN1
ACE
TGFB2
AP2M1GRAP2
SCN5A
MYH9
PKN1
ACHE
SLC25A13
PHKG1
SPAST
UMOD
PRPF40B
BBC3
TUBA4A TTPALRYR2
STUB1KRT6A
AKAP8L
GFI1BTHRAP3
ZNF232
SOD1
CRKL
CDH2
MBP MAP2K2
DCN
MAP3K5NGFR
PIK3CA
ITGA2
DNM1
RAP1A
GRIN2B
PTPN11
PRKCA
LDLRAP1
DNAJA3INA
SP3
CACNA2D3
CSF1
CNTF
TNFRSF1B
BCL10
KLC1
DCTN1
IDE
SERPINA3
HSPA1A
HSPG2
FN1
LRP2
TUBB
GRIN2A
YWHAB
CAMK2D
HTRA2
PRKCE
CRYAB
APC
GIT1
PIAS4
HPX
TRIP10
PDE4B
MARK4
PLAT
UBE2K
APOC2
LIN7B BGN
MAP3K10MAPK8IP2
CBS
SH3GL3
STAU1
CASP6
PPP5C
KRT16
DAB1
CASP1SHC2
CTSD
UCHL1
HP
SNCACAMK2G
TGFB1
GNB1
PTPN1PIN1
TJP1
CASP8PLCG1
NPHS1
PPP2R2A
DNAJB1
ADRBK1
INADL
NCSTN
MATK
NEDD4L
KAT5IFNGTBP
FRS2
CSNK1A1
CALRKNG1
SGOL2
KRT6B
TRAF3 DLG3
SACS
BDKRB2PRKCB
CAV1
MAPK3F2 SMAD3
APOEAKAP9 RANBP2
CLTC
TSC22D1GAB2PSEN2
MLF1CROT
KRT9
ADRA1B
EPB41
COL4A1
SHC3
ARHGAP32
ATM
PPP2R5A
CAMK2A
NTRK1
KRT18
TNFAPOA2
LAT
GRIN2D
COL4A6
KLK3
VLDLR
COL4A5
COL4A2
NEFH
OR8D2
MTSS1
PDZRN4
PTPN4
OR3A2
OR2T6
DGKG
CASP3
IGF1R
CASK
DRD2
TRAF2
EPHB2
PPM1A
PRKCG
CACNB3
HNRNPUSIN3A
DLG1
CLU
RAP2A
ERBB4CANX
BACE1 GABBR1
KARS
CNOT1
UNG
AGA CDCP1
UTP14A PLAG1
EXOC6
HRAS
CTNNB1
SP1
NOS3 SUMO1
PSEN1
HSPA8
HSP90AA1
BCL2
GNAO1
RPS6KB1
PARK2
DLG4
PTK2B
GSN
NAE1 TLN2LRFN2
PLTP
NUMBLSTX1A
DERL1
NEFM
HMOX2
AATF
PPIAMTRNR2L2AP1M1
OGT
DDB1
AP4M1
MED31
RGS3
TDP2
KRT5
ADAM9
REST
PACSIN1
SHB
CTSL1
LAMA1
SH2B1
PLA2G4F
SMURF2
SLC9A8
KRT14
CIT
TGFB1I1
PPP2R1APIK3R1
NUMB
ADAM17
DLG2
LRP1
APOA4
LTA
IRS1
GSK3B
EGFR
CTSB
TRAF6
SHC1
PPP2CB
NGF
HTT
PPIDLDB3
APOC1HIP1
ZDHHC17COL25A1MAGI3
ITGB5
ACTB
GNA11
ACTN2CAMK2B
CDK1
LIN7C
S100B
PDLIM5
DLGAP1
LIN7A
TPR
SYNGAP1
CFD
GRIN3A
F12
SLC1A3
GRK5
CACNA2D2
TANC1
DUSP4
EXOC4
AHSG
GRIN3B
UCP2
TTN
GPC1
NSF
FGA
DYNLL1
MAPK8IP1
PICALM
PPBP
ITM2B
NID1
STXBP1
CTBP1
SGK1
TRAF1
SQSTM1
TNFRSF1A
ACTN1
OPTN
UBE2D2
AP2A2
FUS
APOA1
DICER1
CACNA1B
CST3
NCOA3
SCARB1
APOC3
SLC6A3
KIAA1551 KRT13
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ENSG00000258818
SPATA31A7
CTAGE5
CEP44
SH2B2
EPHB4
CAPN1
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APBA2
COL4A3
LDLR
GIPC1
GFAP
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SRC
MAPK12APP
GAPDH
CDK5
CREBBP
NOS1
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YWHAQGRIN1
RPS6KA3
COL1A2
TCERG1
A2M
CASP7
LRP8
PDK2
AGTR1
FYN
ALBGRB2
MAPK1AKT1
STAT1
MAPT
GSK3A
UBB
PPP2CA
SMAD4
PRKCD
RGS12
SPTB
SH3BP5
CACNA1I
IL16
SLA2
PFN2
NLRC4
TNFRSF14
PRTN3
SNCB
FBLN1
BACE2
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FANCD2
SAP30
RANBP3
PALB2
SLC1A5RASA1
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ADRBK2
CDC45
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DRD1
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GRK6
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CHD3
PRSS3
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CHRNA7
KRT10
RIMS1
CRMP1
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SORBS3
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GCN1L1
RNF19A
HOMER3
GRK4
JARID2
MYLK3
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NCOR1
CASP4
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AP4E1
CLCA2
IGDCC4
NECAB3
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SH3GLB1
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PLCG2
(a)
STAT1
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FN1
PLAT
SOD1
PRKCA CDK1
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ALB APP
PPP3CA
IFNG
BBC3
SCN5A
KCNMA1
MAP2
ACHE
MAPK3F2
PTPN1
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PRKCB
TGFB1CASP8
TNF
AKT1
F7
MAPK1LDLR
PRKCD
SLC6A3
DRD1
CASP3
CHRNA7
PRSS3
TP53BP2
CASP7
EPHB2
SYNGAP1
CALM1
CTSD
BACE1
ADRA1B
(b)
Figure 4 TBI-related protein interaction network (a) 21 hub proteins (red and green) were identified through the analysis of TherapeuticTargetDatabase (TTD) aswell asOnlineMendelian Inheritance inMan (OMIM) 4 overlapped protein targets (green)were obtained between21 hub proteins in TBI and candidate targets of XFZYD Periwinkle proteins fromHPRD and STRING analyses were not targeted by XFZYD(b) 47 candidate protein targets of XFZYD were screened for treating TBI Yellow candidate protein targets of XFZYD The size of nodes isproportional to the value of degree
10 Evidence-Based Complementary and Alternative Medicine
Table 4 Top 10 proteins of TBI specific proteins according to 2 centrality indicators
Proteins Degree Proteins BetweennessSRC 164 APP 0071814AKT1 157 ALB 0069343ALB 153 SRC 0058568EGFR 139 EGFR 0052938APP 134 AKT1 0045466GAPDH 131 HTT 0037164HSP90AA1 108 GAPDH 0034549BCL2 106 HSP90AA1 0027596MAPK1 105 CTNNB1 0021759HRAS 105 GNB1 0019492Note the centrality indicators identify the important nodes within the network Higher degree centrality and betweenness centrality indicate greaterimportance
Table 5 Top 10 significantly enriched KEGG pathways in TBI-specific proteins
Pathway ID Pathway description Gene count FDR4010 MAPK signaling pathway 44 299E-235200 Pathways in cancer 43 113E-184722 Neurotrophin signaling pathway 41 101E-344151 PI3K-Akt signaling pathway 40 111E-154510 Focal adhesion 39 292E-225205 Proteoglycans in cancer 39 410E-215010 Alzheimer s disease 34 124E-204015 Rap1 signaling pathway 33 769E-174014 Ras signaling pathway 33 646E-164020 Calcium signaling pathway 31 505E-17
0
001
002
003
004
005
006
007
008
0 20 40 60 80 100 120 140 160 180
Betw
eenn
ess
Degree
APP
ALB SRCEGFR
AKT1HTT
Figure 5 Relationship between degree and betweenness centralityin the TBI-specific protein interaction network
for the 119 potential compounds is shown in Table S4Similarly 5 pharmacologically active ingredients in the JunChen and Zuo-Shi groups including quercetin stigmasterolkaempferol baicalin and beta-sitosterol anchored 33 TBI-specific proteins such as CALM SCN5A F2 ACHE F7ADRA1B NOS3 BCL2 CASP3 and AKT1 11 Jun-specificcompounds targeted 2 specific targets (ALB CTNNB1) while12 Chen-specific compounds anchored 3 unique proteins(PRKCD FN1 and BC3) The Zuo-Shi herbs possessed thelargest number of active compounds (89) and targeted 5
unique proteins including EPHB2 BACE1 LDLR MAPK3and PRSS3 GSK3Bwas the common target between theChenandZuo-Shi drugs KCNMA1 CASP7 andAPPwere targetedby the Jun and Zuo-Shi drugs
The top 10 candidate compounds and targets to treat TBIwere showed in Tables 6 and 7 Formost of active compoundsfrom XFZYD each component hit more than one targetTable 6 demonstrated that quercetin had the highest numberof targets (degree =76) followed by kaempferol (degree=43) beta-sitosterol (degree =35) stigmasterol (degree =19)luteolin (degree=18) baicalein (degree=15) 7-Methoxy-2-methyl isoflavone (degree=12) wogonin (degree=12)nobiletin (degree=11) and naringenin (degree=9) Forinstance quercetin (3310158404101584057-pentahydroxyflavone) is anaturally occurring flavonoid commonly found in fruits andvegetables It regulates multiple biological pathways elicitinginduction of apoptosis as well as inhibiting angiogenesisand proliferation [63 64] Quercetin can attenuate neuronalautophagy and apoptosis in rat traumatic brain injury modelvia activation of PI3KAkt signaling pathway [65] It has alsobeen reported to have a protective ability against oxidativestress and mutagenesis in normal cells [66 67] Kaempferol(3 41015840 5 7 tetrahydroxy flavone) is a yellow-colored flavonoidthat is widely distributed in many botanical families[68] It has been shown to possess a variety of biologicalcharacteristics including effects of anti-inflammatory[69] antioxidative [70] tumor growth inhibition [71] and
Evidence-Based Complementary and Alternative Medicine 11
Mol 22Mol 33
Mol 29Mol 32 Mol 23Mol 20
Mol 97Mol 106
Mol 104Mol 100Mol 94
Mol 63Mol 90
Mol 87
Mol 91Mol 61
Mol 19
Mol 109
Mol 161
Mol 118
Mol 111
Mol 158
Mol 114
Mol 149
Mol 141
Mol 152
Mol 14
CASP3
BCL2
NOS3
SCN5A
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ADRA1B
ACHE
F2
Mol 4
Mol 49
Mol 127
APP CASP7
Mol 92
Mol 54
Mol 6
Mol 36
KCNMA1
Mol 27
Mol 13Mol 105Mol 140
Mol 17
Mol 39
Mol 2
Mol 21
Mol 12
Mol 142
Mol 126
Mol 135
Mol 121
Mol 119
Mol 124
Mol 120
Mol 117
Mol 133
Mol 131
Mol 134
PlatycodonGrandiforus
Aurantii Fructus
Licorice
Radix Bupleuri
RehmanniaglutinosaLibosch
AngelicaeSinensis Radix
Mol 8
Mol 73
Mol 75
Mol 77
Mol 80
Mol 7
Mol 82
Mol 76
Mol 74
Mol 60
Mol 46
Mol 150
Mol 151
Mol 112
Mol 115
Mol 11
Mol 116
Mol 113
Mol 110
Mol 1
Mol 9Mol 101Mol 10Mol 93
Mol 103Mol 89 Mol 107
Mol 96PRSS3 LDLRMAPK3 BACE1 EPHB2
Mol 35Mol 81
Mol 86
Mol 85
Mol 84
Mol 88
Mol 83Mol 30
Mol 137
ChuanxiongRhizoma
Mol 3
Mol 57
Mol 139
Achyranthis
Bidentatae
RadixMol 102
CASP8
IGHG1AKT1p53
CHRNA7
Mol 40
SLC6A3 PTPN1
SOD1MAPK1
Mol 38
Mol 95
Carthami Flos
Mol 24
Mol 78 Persicae Semen
Mol 122
Mol 25
Mol 98
TGFB1
GSK3B
PRKCA
Radix Paeoniae Rubra
Mol 52
Mol 42
Mol 62
Mol 138
EGFR
STAT1 PPP3CAPLAT
Stigmasterol
MAP2
Beta-sistosterol Quercetin
Kaempferol
SYNGAP1
CALM
Baicalin
PRKCB
CAV1
CTSD
TNF
DRD1
CDK1
IFNG
FN1BBC3 PRKCD
Mol 132
Mol 160
Mol 58
Mol 67
Mol 69 Mol 43ALB
Mol 64CTNNB1
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RehmanniaRe mRe m nnRehmanniamReRehmhmannnniamanniaRehmehmanniRR nosasasasaosaosglutilutglutgluulutiglgl titinos
LiboschLibLLibo hschib chLiboschboscLLib chschLibos
icaecaecacaeicaeicAngelAnAngeAngelAnAng lAAngeelielicdix dix dixdixdSinensinensSS nennensSinSSiSinensSinensSine sissis Rad
FloslololFlhamaCartharthhaamami Fl
menmennnnmicae Scae SSem
Figure 6 HB-pC-pT network of XFZYD for treating TBI The triangles with circle backgrounds represent the herbs (HB) the squares andcircles represent the potential compounds (pC) and targets (pT)The red triangles squares and circles represent corresponding HB pC andpT in the Junherbs the same is to aqua representing theChen herbs and periwinkle representing the Zuo-Shi herbsThe claybank squares andcircles represent corresponding pC and pT shared by the Chen and Zuo-Shi herbs the same is to blue representing the overlap between theJun and Zuo-Shi herbs and the green representing the overlap between the Chen and Zuo-Shi herbsThe purple squares and circles representthe corresponding pC and pT shared by the 3 groups of herbs
alleviating insulin resistance in type 2 diabetic rats [72]Beta-sitosterol (BS) is a vegetable-derived compound foundin various plants and is suggested to modulate the immunefunction inflammation and pain levels by controlling theproduction of inflammatory cytokines [73]
For targets analysis CALM possesses the largest degree(degree =84) followed by GSK3B (degree =61) SCN5A(degree = 59) F2 (degree = 43) ACHE (degree=28)F7 (degree=28) ADRA1B (degree=28) NOS3 (degree=20)BCL2 (degree=18) and CASP3 (degree=18) which demon-strated their crucial therapeutic effects for treating TBI Forinstance CALM possess an essential position in calciumsignaling pathway and is related to morphological changesmigration proliferation and secretion of cytokines andreactive oxygen species ofMicroglial cells [74]The activationof CaMKII120572 major isoform of Ca2+calmodulin-dependentprotein kinase (CaMK) in brain is directly associated withthe production of proinflammatory cytokines such as TNF-120572and IL-1120573 [75] GSK-3120573 is a serinethreonine-protein kinasewhich is abundant in the central nervous system (CNS)particularly in neurons [76] It can control gene transcrip-tion axonal transport and cytoskeletal dynamics in growthcones [77] The inhibition of GSK-3120573 attenuates apoptotic
signals and prevents neuronal death [78] There is increasingevidence that prothrombin (F2) and its active derivativethrombin are expressed locally in the central nervous systemBeside the central role in the coagulation cascade the gener-ation of thrombin leads to receptor mediated inflammatoryresponses cell proliferationmodulation cell protection andapoptosis [79 80] The role in brain injury depends upon itsconcentration as higher amounts cause neuroinflammationand apoptosis while lower concentrations might even becytoprotective [81]
Direct tissue damage aswell as hypoxic-ischemic increas-ing anaerobic glycolysis of the brain tissue results in theATP-stores depletion and failure of energy-dependent mem-brane ion pumps especially for voltage-dependent Ca2+ andNa+-channels Accumulated Ca2+ activates lipid peroxidasesproteases and phospholipases and caspases at the sametime increasing the intracellular concentration of free fattyacids and free radicals leading to necrosis or apoptosis ofneurocyte [82] At the same time the activation of residentglial cells microglia and astrocytes and the infiltration ofblood leukocytes secrete various immune mediators elicitedinflammatory responses which subsequently intersect withadjacent pathological cascades including oxidative stress
12 Evidence-Based Complementary and Alternative Medicine
Table 6 Top 10 potential candidate compounds of XFZYD for treating TBI according to 2 centrality indicators
Compound Degree Compound Betweennessquercetin 76 quercetin 01682179kaempferol 43 kaempferol 005501511beta-sitosterol 35 wogonin 005141376Stigmasterol 19 beta-sitosterol 004451294luteolin 18 naringenin 003251738baicalein 15 beta-carotene 002977217-Methoxy-2-methyl isoflavone 12 nobiletin 002787992wogonin 12 7-Methoxy-2-methyl isoflavone 002096439nobiletin 11 luteolin 002090223naringenin 9 baicalein 001453488
Table 7 Top 10 potential targets of XFZYD for treating TBI according to 2 centrality indicators
Targets Degree Targets BetweennessCALM 84 CALM 021452046GSK3B 61 SCN5A 011441591SCN5A 59 GSK3B 009553896F2 43 F2 00689171ACHE 28 BCL2 002651903F7 28 CASP3 002372836ADRA1B 28 F7 002032647NOS3 20 ACHE 001845996BCL2 18 ADRA1B 001704505CASP3 18 NOS3 00166699
excitotoxicity or reparative events including angiogenesisscarring and neurogenesis [83] Function analysis of the 47target proteins regulated by XFZYD was mainly associatedwith core pathophysiology process of TBI (Figure 7) 47 targetproteins were connected with 16 key process related to TBIMost of the targets have one or more links to other biolog-ical process such as apoptosis cell proliferation superoxideanion generation nitric oxide biosynthetic process responseto calcium ion I-kappaB kinaseNF-kappaB signaling andregulation of inflammation The above analysis implied themultifunction character of these target proteins regulated byXFZYD Of these target proteins 25 proteins (53 of the 47)such as TGFB1 EGFR CAV1 MAPK1 PRKCB and AKT1were responsible for regulating the apoptosis process 17 forblood coagulation 14 for cell proliferation and axon genesisand 12 for hypoxia andMAPK cascade We found that TGFB1was the crucial protein because it participated in 9 biologicalprocesses related to TBI such as apoptosis blood coagulationcell proliferation and MAPK cascade followed by EGFR (8)CAV1 (7) MAPK1 (6) PRKCB (6) and AKT1 (6)
Overall these observations strongly support the evidencethat the generated HB-pCminuspT network and pT-F networkhave important roles in treating TBI further validating thedrug targeting approach
Molecule docking was used to further validate the bind-ing mode between candidate compounds and their targetproteins We found that 18 TBI-specific target proteins inter-acted with 91 candidate compounds from XFZYD (Figure 8)
Other 29 target proteins were not discussed for the lackof proper protein crystal structure The detailed moleculedocking results are shown in Table S5The 6 essential proteinsincluding GSK3B AKT1 CDK1 F2 NOS3 and ACHEwere used to elucidate the exact binding mode (Figure 9)Quercetin was located within the binding cavity of AKT1 andCDK1 (Figures 9(a) and 9(b) and S1A B) Four conventionalhydrogen bonds were formed between quercetin and AKT1by interacting with the key amino acids including ILE-290 THR-211 and SER 205 Additionally 120587-120587 interactionsbetween quercetin and TRP-90 were found in the activesite which helped the stabilization of the compound at thebinding site (Figure 9(a) S1A) Figure 9(b) and S1B suggestedthat five conventional hydrogen bonds (LEU-83 ASP-146and LYS-33) and 120587-120587 interaction (PHE-80) were formedbetween quercetin and CDK1 The GSK3B-FA complexes(Figure 9(c) S1C) were stabilized by 6 hydrogen-bondinginteractions between FA and LYS-85 GLU-97 TYR-134 andARG-141 Glyasperin Bmainly bonds to F2 through hydrogenbonds by interacting with the key amino acids includingGLY-193 SER-195 and GLY-219 (Figure 9(d) S1D) andan edge-to-face 120587 minus 120587 interaction was also observed withTYR-228 The GLN-247 GLU-351 and GLY-355 from theactive site pocket of NOS3 participated in the hydrogen-bondformationwith 1-Methoxyphaseollidin (Figure 9(e) S1E)The(-)-Medicocarpin formed a total of 6 hydrogen bonds withSER-293 PHE-295 ARG-296 and TRY-341 in the active siteof ACHE (Figure 9(f) S1F) Besides an edge-to-face 120587 minus 120587
Evidence-Based Complementary and Alternative Medicine 13
KCNMA1
P53
IFNG
PPP3CA
CASP8
SCN5A
I-kappaB kinase NF-kappaB signaling
Regulation of inflammation
Response to calcium ion
TGFB1
TNF
PRKCA
CHRNA7
CAV1
STAT1
MAPK cascadeAKT1
BCL2
APP
EGFR
MAPK1
Angiogenesis
SYNGAP1
MAP2
PTPN1
Axonogenesis
GSK3B
EPHB2
CDK1
CASP7FN1
MAPK3
CTNNB1
F2
Autophagy
Cell proliferation
BACE1
SLC6A3
DRD1
ADRA1BACHE
CALMCASP3
PLAT
Apoptotic process
NOS3
PRKCB
Blood coagulation
Response to hypoxia
Superoxide anion generation
Nitric oxide biosynthetic process
Astrocyte activation
Amyloid-beta regulation
Microglial cell activation
LDLR
PRSS3
F7
PRKCD
SOD1ALB
BBC3
Figure 7 pT-F network of XFZYD for treating TBI 16 biological processes (red square) the 47 target proteins (periwinkle circle) of XFZYDparticipate in for treating TBI
interactionwas also observedwith TYR-337 From the resultshydrogen-bonding and edge-to-face 120587 minus 120587 interactions playkey roles in the proteinminusligand recognition and stabilitywhich may be helpful in determining the inhibitor activitiesAnd the C-T network confirmed the potential therapeuticeffects of the candidate compounds from XFZYD to treatTBI through interacting with the relevant proteins The com-putational analysis further elucidated the accurate moleculemechanisms between active compounds and targets
4 Discussion
Traumatic brain injury (TBI) is a growing public healthproblem worldwide and is a leading cause of death anddisability [84] Although major progress has been made in
understanding the pathophysiology of this injury this hasnot yet led to substantial improvements in outcome by alack of treatments which have proven successful during phaseIII trials for modern medicine [85 86] TCM rooted inthousands of years of history may offer an alternative or acomplementary strategy for the treatment of TBI XFZYDa representative formula in TCM has been used for yearsto treat TBI in China and has been demonstrated to beeffective in clinical practice However its ldquomulticomponentsrdquoand ldquomultitargetsrdquo features make it much difficult to decipherthemolecular mechanisms of XFZYD in the treatment of TBIfrom a systematic perspective if employing routine methods
In the present study a network pharmacology-basedmethod was employed to elucidate the pharmacologicalmechanisms of XFZYD to treat TBI according to the drug
14 Evidence-Based Complementary and Alternative Medicine
Figure 8 C-T network through molecule docking validating 119 potential compounds (green triangles) interacting with 18 potential targets(yellow circles) of XFZYDThe size of the nodes is proportional to the value of degree
combination principle of TCM We first proposed a newmodeling system combining OB and DL screening multipledrug targets prediction and validation network constructionand molecule docking to probe the efficiency of a typicalTCM formula XFZYD for the treatment of TBI The 11herbs from XFZYD possessed 162 bioactive compounds andtargeted 285 proteins There were 5 compounds and 189
target proteins overlapped among the Jun Chen and Zuo-Shigroup Furthermore 47 TBI-specific proteins were targetedby 119 (73) bioactive compounds from XFZYD Similarly 5common compounds and 33 (70) common target proteinsamong the 3 groups of drugs were observed Most of thebioactive ingredients targeted more than one protein The 47target proteins regulated several essential pathophysiological
Evidence-Based Complementary and Alternative Medicine 15
(a) (b) (c)
(d) (e) (f)
Figure 9 Hydrogen-bonding networks within the binding site of the compoundminustarget complexes obtained from molecule docking(a) AKT1-quercetin (b) CDK1-quercetin (c) GSK3B-FA (d) F2-glyasperin B (e) NOS3-1-Methoxyphaseollidin and (f) ACHE-(-)-Medicocarpin The molecules are presented as ball and stick models Active site amino acid residues are represented as lines Dotted bluelines in these pictures represent hydrogen bonds with distance unit of A Other O and N atoms are colored as red and blue respectively
processes of TBI that referred to apoptosis inflammationcell proliferation superoxide anion generation nitric oxidebiosynthetic process response to calcium ion etc The aboveanalysis reveals that the synergistic action mechanisms ofXFZYDmay be (1) bioactive compounds overlapping amongthe different group of herbs (2) specific bioactive com-pounds from different groups of herbs targeting the sameproteins (3) specific bioactive compounds from differentgroups of herbs targeting different proteins which participatein the same pathophysiological process of the disease Toa certain degree the 5 compounds including quercetinstigmasterol kaempferol baicalin and beta-sitosterol playedessential role in XFZYD for TBI treatment MAPK3MAPK1AKT1 PRKCA TNF PRKCB EGFR BCL2 GSK3B CASP3PPP3CA and NOS3 were the main target proteins regulatedby XFZYD in the treatment of TBI
Interestingly Beta-carotene (Mol 43) from Carthami Flos(the Jun herb) specifically regulated 120573-Catenin (CTNNB1)and played critical role for curing TBI The 120573-Catenin is acritical downstream component of the Wnt pathway whichplays essential role in the regulation of mammalian neuraldevelopment [87] In vitro and in vivo studies demonstrate
that the Wnt120573-catenin pathway regulates the proliferationand differentiation of neural progenitor cells [88] Neuronaldifferentiation is induced by overexpression of 120573-cateninor the pharmacological inhibition of GSK3120573 (the phospho-rylating enzyme of 120573-catenin) [89 90] This pathway alsopromotes blood vessel formation during vascular develop-ment as well as the vascular repair process after TBI [91]In addition wogonin (Mol 160) from Achyranthis BidentataeRadix (the Chen herb) specifically targeted fibronectin (FN1)Bcl-2-binding component 3 (BBC3) and Protein KinaseC delta type (PRKCD) FN1 an important component ofthe extracellular matrix (ECM) environment promotes cellmigration neurite outgrowth and synapse formation dur-ing neural development [92] It aggregates in the injuredbrain and plays a neuroprotection role through antiapoptosisand anti-inflammation ways following TBI [93 94] BBC3namely p53 upregulated modulator of apoptosis (PUMA) iscritical for the p53-dependent apoptosis pathway which playsan important role in hippocampal neuronal loss and associ-ated cognitive deficits [95] PRKCD one of PKC isoformsactivates signal transduction pathways involved in neuronalregeneration [96] synaptic transmissionplasticity [97] and
16 Evidence-Based Complementary and Alternative Medicine
activation of apoptosis processes [98] as well as higher brainfunctions such as learning andmemory [99]The activators ofPKCare effective for the treatment of TBI [100] FurthermoreMitogen-activated protein kinase 3 (MAPK3) Beta-secretase1 (BACE1) Ephrin type-B receptor 2 (EphB) and low-densitylipoprotein receptor (LDLR) were specifically targeted bythe ingredients in the Zuo-Shi herbs Naringenin (Mol133) targeted LDLR MAPK3 while euchrenone (Mol 60)anchored BACE1 and nobiletin (Mol 134) targeted EphB2MAPK3 is an essential component of the MAP kinase signaltransduction pathway It has been appreciated recently thatthe ERK12 cascade plays a fundamental role in synapticplasticity and memory [101] BACE1 is responsible for pro-duction of A120573 from amyloid precursor protein (APP) A120573can cause cell death activate inflammatory pathways [102]and prime proapoptotic pathways for activation by otherinsults [103] The blocking of BACE1 can ameliorate motorand cognitive deficits and reduce cell loss after experimentalTBI in mice [104] EphB is localized to synaptic sites inhippocampal neurons [105] The interaction between EphBand NMDA receptors regulates excitatory synapse formation[106] LDLR acts as an important receptor that facilitatesbrain A120573 clearance and inhibits amyloid deposition [107]and then ameliorates Alzheimerrsquos disease neuropathologyafter TBI [108] The analysis above indicates the ldquoJunrdquoldquoChenrdquo and ldquoZuo-Shirdquo herbs from XFZYD trigger theirspecific targets regulation respectively for the therapeuticeffects
XFZYD is a very famous traditional Chinese formula inpromoting qi circulation and removing blood stasis accord-ing to TCM theory However several limited researches havedemonstrated its efficacy for treating TBI such as anti-inflammatory and synaptic regulation [30 31] which arein accord with our study However previous studies merelypartially deciphered the molecule mechanism of XFZYD fortreating TBI This study reports 119 bioactive compounds inXFZYD that target 47 TBI-specific proteins such as MAPK3MAPK1 AKT1 PRKCA TNF PRKCB and EGFR Theseproteins regulate several crucial pathophysiological processesof TBI such as apoptosis inflammation blood coagulationand axon genesis Our study demonstrates that the therapeu-tic actions of XFZYD refer to ldquomulticompoundsrdquo ldquomultitar-getsrdquo features rather than only the improvement of bloodcirculation With the help of molecule docking methodwe further validate the interactions between bioactive com-pounds and potential targets of XFZYD The hydrogen-bonding and edge-to-face 120587 minus120587 interactions play key roles inthe proteinminusligand recognition and stability This provides avaluable reference for further experimental investigations ofbioactive ingredients and therapeutic targets of XFZYD fortreating TBI
5 Conclusion
Our work successfully illuminates the efficiency of XFZYDfor the treatment of TBI as well as herb combination ruleof TCM formula Network pharmacology with moleculedocking method confirms the ldquomulticompounds multitar-getsrdquo therapeutic actions of XFZYD in the treatment of TBI
The present work may provide valuable evidence for furtherclinical application of XFZYD for treating TBI
Abbreviations
TBI Traumatic brain injuryXFZYD Xuefu Zhuyu decoctionTCM Traditional Chines medicineDL Drug-likenessOB Oral bioavailabilityHB-cC-cT Herb-candidate compound-candidate
targetHB-pC-pT Herb-potential compound-potential targetCALM CalmodulinGSK3B Glycogen synthase kinase-3 betaSCN5A Sodium channel protein type 5 subunit
alphaF2 ProthrombinACHE AcetylcholinesteraseGO Gene OntologyKEGG Kyoto Encyclopedia of Genes and
Genomes
Data Availability
The data used to support the findings of this study areavailable from the corresponding author upon request
Conflicts of Interest
The authors declare that they have no conflicts of interest
Authorsrsquo Contributions
YangWang and Zebing Huang participated in the conceptionand design of the study YuanyuanZhong YangWang ZebingHuang Jiekun Luo Tao Tang and Pengfei Li acquired andanalyzed the data Yuanyuan Zhong Tao Liu and HanjinCui drafted and revised the manuscript The correspondingauthor and all of the authors have read and approved the finalsubmitted manuscript
Acknowledgments
This work was supported by the National Natural ScienceFoundation of China (Nos 81673719 81874409 81603670and 81803948) Project funded byChina Postdoctoral ScienceFoundation (Nos 2016M600639 and 2017T100614)
Supplementary Materials
Table S1 162 bioactive compounds in XFZYD Table S2 targetproteins of XFZYD Table S3 TBI-specific proteins Table S4119 potential compounds of XFZYD for treating TBI TableS5 docking result of 18 target proteins with 91 potential com-pounds Fig S1 the exact bindingmode between active ingre-dients and protein targets obtained from molecule docking(A) AKT1-quercetin (B) CDK1-quercetin (C) GSK3B-FA
Evidence-Based Complementary and Alternative Medicine 17
(D) F2-glyasperin B (E) NOS3-1-Methoxyphaseollidin and(F)ACHE-(-)-Medicocarpin (Supplementary Materials)
References
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[2] J A Langlois W Rutland-Brown and M M Wald ldquoTheepidemiology and impact of traumatic brain injury a briefoverviewrdquo The Journal of Head Trauma Rehabilitation vol 21no 5 pp 375ndash378 2006
[3] H A Alsulaim B J Smart A O Asemota et al ldquoConsciousstatus predictsmortality among patientswith isolated traumaticbrain injury in administrative datardquo The American Journal ofSurgery vol 214 no 2 pp 207ndash210 2017
[4] M Majdan D Plancikova A Maas et al ldquoYears of life lost dueto traumatic brain injury in Europe A cross-sectional analysisof 16 countriesrdquo PLoS Medicine vol 14 no 7 p e1002331 2017
[5] P Cheng P Yin P Ning et al ldquoTrends in traumatic braininjury mortality in China 2006ndash2013 A population-basedlongitudinal studyrdquo PLoS Medicine vol 14 no 7 Article IDe1002332 2017
[6] M V Russo and D B McGavern ldquoInflammatory neuroprotec-tion following traumatic brain injuryrdquo Science vol 353 no 6301pp 783ndash785 2016
[7] WWang H Li J Yu et al ldquoProtective Effects of ChineseHerbalMedicine Rhizoma drynariae in Rats After Traumatic BrainInjury and Identification of Active Compoundrdquo MolecularNeurobiology vol 53 no 7 pp 4809ndash4820 2016
[8] M Das S Mohapatra and S S Mohapatra ldquoNew perspectiveson central and peripheral immune responses to acute traumaticbrain injuryrdquo Journal of Neuroinflammation vol 9 p 236 2012
[9] J W Finnie ldquoNeuroinflammation Beneficial and detrimentaleffects after traumatic brain injuryrdquo Inflammopharmacologyvol 21 no 4 pp 309ndash320 2013
[10] T Cheng W Wang Q Li et al ldquoCerebroprotection of flavanol(minus)-epicatechin after traumatic brain injury viaNrf2-dependentand -independent pathwaysrdquo Free Radical Biology amp Medicinevol 92 pp 15ndash28 2016
[11] W Young ldquoRole of calcium in central nervous system injuriesrdquoJournal of Neurotrauma vol 9 Suppl 1 pp S9ndashS25 1992
[12] R Bullock A Zauner J S Myseros et al ldquoEvidence forProlonged Release of Excitatory Amino Acids in Severe HumanHead Trauma Relationship to Clinical Eventsrdquo Annals of theNew York Academy of Sciences vol 765 no 1 pp 290ndash297 1995
[13] A T Mazzeo A Beat A Singh and M R Bullock ldquoTherole of mitochondrial transition pore and its modulation intraumatic brain injury and delayed neurodegeneration afterTBIrdquo Experimental Neurology vol 218 no 2 pp 363ndash370 2009
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[15] J Ghajar ldquoTraumatic brain injuryrdquo The Lancet vol 356 no9233 pp 923ndash929 2000
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[17] Y Wang X Fan T Tang et al ldquoRhein and rhubarb simi-larly protect the blood-brain barrier after experimental trau-matic brain injury via gp91phox subunit of NADPH oxidaseROSERKMMP-9 signaling pathwayrdquo Scientific Reports vol 6p 37098 2016
[18] D-X Kong X-J Li and H-Y Zhang ldquoWhere is the hopefor drug discovery Let history tell the futurerdquo Drug DiscoveryTherapy vol 14 no 3-4 pp 115ndash119 2009
[19] F Cheung ldquoTCM made in Chinardquo Nature vol 480 no 7378pp S82ndashS83 2011
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[21] H-J Zhang and Y-Y Cheng ldquoAn HPLCMS method for iden-tifying major constituents in the hypocholesterolemic extractsof Chinese medicine formula lsquoXue-Fu-Zhu-Yu decoctionrsquordquoBiomedical Chromatography vol 20 no 8 pp 821ndash826 2006
[22] L Zhang L Zhu YWang et al ldquoCharacterization and quantifi-cation of major constituents of Xue Fu Zhu Yu by UPLC-DAD-MSMSrdquo Journal of Pharmaceutical and Biomedical Analysisvol 62 pp 203ndash209 2012
[23] X Yang X Xiong G Yang and J Wang ldquoChinese patentmedicine Xuefu Zhuyu capsule for the treatment of unstableangina pectoris a systematic review of randomized controlledtrialsrdquo Complementary Therapies in Medicine vol 22 no 2 pp391ndash399 2014
[24] J Wang X Yang F Chu et al ldquoThe effects of Xuefu Zhuyuand Shengmai on the evolution of syndromes and inflammatorymarkers in patients with unstable angina pectoris after percuta-neous coronary intervention a randomised controlled clinicaltrialrdquoEvidence-BasedComplementary andAlternativeMedicinevol 2013 Article ID 896467 9 pages 2013
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18 Evidence-Based Complementary and Alternative Medicine
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[39] Bo Zhang Xu Wang and Shao Li ldquoAn Integrative Platform ofTCM Network Pharmacology and Its Application on a HerbalFormula Qing-Luo-Yinrdquo Evidence-Based Complementary andAlternativeMedicine vol 2013 Article ID 456747 12 pages 2013
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[50] T S Keshava Prasad RGoel K Kandasamy et al ldquoHuman pro-tein reference databasemdash2009 updaterdquo Nucleic Acids Researchvol 37 no 1 pp D767ndashD772 2009
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[54] L Yang X Zhao and X Tang ldquoPredicting disease-related pro-teins based on clique backbone in protein-protein interactionnetworkrdquo International Journal of Biological Sciences vol 10 no7 pp 677ndash688 2014
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[58] Y Kitagishi and S Matsuda ldquoDiets involved in PPAR andPI3KAKTPTEN pathway may contribute to neuroprotectionin a traumatic brain injuryrdquoAlzheimerrsquos ResearchampTherapy vol5 no 5 p 42 2013
[59] D Chen L Bao S-Q Lu and F Xu ldquoSerum albumin andprealbuminpredict the poor outcomeof traumatic brain injuryrdquoPLoS ONE vol 9 no 3 Article ID e93167 2014
[60] J Yang Z Wu N Renier et al ldquoPathological axonal deaththrough aMapk cascade that triggers a local energy deficitrdquoCellvol 160 no 1-2 pp 161ndash176 2015
[61] E J Huang and L F Reichardt ldquoTrk receptors roles in neuronalsignal transductionrdquoAnnual Review of Biochemistry vol 72 pp609ndash642 2003
[62] J W Lee and R Juliano ldquoMitogenic signal transductionby integrin- and growth factor receptor-mediated pathwaysrdquoMolecules and Cells vol 17 no 2 pp 188ndash202 2004
[63] C S Yang J M Landau M T Huang and H L NewmarkldquoInhibition of carcinogenesis by dietary polyphenolic com-poundsrdquo Annual Review of Nutrition vol 21 pp 381ndash406 2001
[64] A Murakami H Ashida and J Terao ldquoMultitargeted cancerprevention by quercetinrdquoCancer Letters vol 269 no 2 pp 315ndash325 2008
[65] G Du Z Zhao Y Chen et al ldquoQuercetin attenuates neuronalautophagy and apoptosis in rat traumatic brain injury modelvia activation of PI3KAkt signaling pathwayrdquo NeurologicalResearch vol 38 no 11 pp 1ndash8 2016
Evidence-Based Complementary and Alternative Medicine 19
[66] Q Cai R O Rahn and R Zhang ldquoDietary flavonoidsquercetin luteolin andgenistein reduce oxidativeDNAdamageand lipid peroxidation and quench free radicalsrdquoCancer Lettersvol 119 no 1 pp 99ndash107 1997
[67] G A Bongiovanni E A Soria and A R Eynard ldquoEffectsof the plant flavonoids silymarin and quercetin on arsenite-induced oxidative stress in CHO-K1 cellsrdquo Food and ChemicalToxicology vol 45 no 6 pp 971ndash976 2007
[68] H Li L Yang Y Zhang et al ldquoKaempferol inhibits fibroblastcollagen synthesis proliferation and activation in hypertrophicscar via targeting TGF-beta receptor type Irdquo Biomedicine ampPharmacotherapy = Biomedecine amp Pharmacotherapie vol 83pp 967ndash974 2016
[69] K P Devi D S Malar S F Nabavi et al ldquoKaempferol andinflammation From chemistry to medicinerdquo PharmacologicalResearch vol 99 pp 1ndash10 2015
[70] M Zhou H Ren J Han W Wang Q Zheng and DWang ldquoProtective effects of kaempferol against myocardialischemiareperfusion injury in isolated rat heart via antioxidantactivity and inhibition of glycogen synthase kinase-3120573rdquo Oxida-tive Medicine and Cellular Longevity vol 2015 p 481405 2015
[71] C-F Lee J-S Yang F-J Tsai et al ldquoKaempferol inducesATMp53-mediated death receptor and mitochondrial apop-tosis in human umbilical vein endothelial cellsrdquo InternationalJournal of Oncology vol 48 no 5 pp 2007ndash2014 2016
[72] C Luo H Yang C Tang et al ldquoKaempferol alleviates insulinresistance via hepatic IKKNF-120581B signal in type 2 diabetic ratsrdquoInternational Immunopharmacology vol 28 no 1 pp 744ndash7502015
[73] A B Awad and C S Fink ldquoPhytosterols as anticancer dietarycomponents evidence and mechanism of actionrdquo Journal ofNutrition vol 130 no 9 pp 2127ndash2130 2000
[74] K Farber and H Kettenmann ldquoFunctional role of calciumsignals for microglial functionrdquoGlia vol 54 no 7 pp 656ndash6652006
[75] Y Lu Y Gu X Ding et al ldquoIntracellular Ca2+ homeostasisand JAK1STAT3 pathway are involved in the protective effectof propofol on BV2 microglia against hypoxia-induced inflam-mation and apoptosisrdquo PLoS ONE vol 12 no 5 p e01780982017
[76] K Leroy and J-P Brion ldquoDevelopmental expression and local-ization of glycogen synthase kinase-3120573 in rat brainrdquo Journal ofChemical Neuroanatomy vol 16 no 4 pp 279ndash293 1999
[77] C-M Liu E-M Hur and F-Q Zhou ldquoCoordinating geneexpression and axon assembly to control axon growth Potentialrole ofGSK3 signalingrdquo Frontiers inMolecularNeuroscience vol3 p 3 2012
[78] D A E Cross A A Culbert K A Chalmers L Facci S DSkaper and A D Reith ldquoSelective small-molecule inhibitors ofglycogen synthase kinase-3 activity protect primary neuronesfrom deathrdquo Journal of Neurochemistry vol 77 no 1 pp 94ndash102 2001
[79] V S Ossovskaya and NW Bunnett ldquoProtease-activated recep-tors contribution to physiology and diseaserdquo PhysiologicalReviews vol 84 no 2 pp 579ndash621 2004
[80] M Steinhoff J Buddenkotte V Shpacovitch et al ldquoProteinase-activated receptors transducers of proteinase-mediated signal-ing in inflammation and immune responserdquoEndocrine Reviewsvol 26 no 1 pp 1ndash43 2005
[81] H Krenzlin V Lorenz S Danckwardt O Kempski and BAlessandri ldquoThe importance of thrombin in cerebral injury and
diseaserdquo International Journal of Molecular Sciences vol 17 no1 2016
[82] M J McGinn and J T Povlishock ldquoPathophysiology of Trau-matic Brain InjuryrdquoNeurosurgery Clinics of North America vol27 no 4 pp 397ndash407 2016
[83] T Woodcock and M C Morganti-Kossmann ldquoThe role ofmarkers of inflammation in traumatic brain injuryrdquo Frontiersin Neurology vol 4 article 18 2013
[84] F Tanriverdi H J Schneider G Aimaretti B E Masel F FCasanueva and F Kelestimur ldquoPituitary dysfunction after trau-matic brain injury A clinical and pathophysiological approachrdquoEndocrine Reviews vol 36 no 3 pp 305ndash342 2015
[85] J V Rosenfeld A I Maas P Bragge M C Morganti-Kossmann G T Manley and R L Gruen ldquoEarly managementof severe traumatic brain injuryrdquoThe Lancet vol 380 no 9847pp 1088ndash1098 2012
[86] B M Aertker S Bedi and C S Cox ldquoStrategies for CNS repairfollowing TBIrdquo Experimental Neurology vol 275 no 3 pp 411ndash426 2016
[87] K Adachi Z Mirzadeh M Sakaguchi et al ldquo120573-catenin signal-ing promotes proliferation of progenitor cells in the adultmousesubventricular zonerdquo Stem Cells vol 25 no 11 pp 2827ndash28362007
[88] Y Hirabayashi and Y Gotoh ldquoStage-dependent fate determina-tion of neural precursor cells in mouse forebrainrdquo NeuroscienceResearch vol 51 no 4 pp 331ndash336 2005
[89] S Ding T Y H Wu A Brinker et al ldquoSynthetic smallmolecules that control stem cell faterdquo Proceedings of theNationalAcadamy of Sciences of the United States of America vol 100 no13 pp 7632ndash7637 2003
[90] N Israsena M Hu W Fu L Kan and J A Kessler ldquoThepresence of FGF2 signaling determines whether 120573-cateninexerts effects on proliferation or neuronal differentiation ofneural stem cellsrdquo Developmental Biology vol 268 no 1 pp220ndash231 2004
[91] A Salehi A Jullienne M Baghchechi et al ldquoUp-regulation ofWnt120573-catenin expression is accompanied with vascular repairafter traumatic brain injuryrdquo Journal of Cerebral Blood Flow ampMetabolism vol 38 no 2 pp 274ndash289 2017
[92] L F Reichardt and K J Tomaselli ldquoExtracellular matrixmolecules and their receptors Functions in neural develop-mentrdquoAnnual Review of Neuroscience vol 14 pp 531ndash570 1991
[93] R M Gibson S E Craig L Heenan C Tournier and M JHumphries ldquoActivation of integrin 12057251205731 delays apoptosis ofNtera2 neuronal cellsrdquoMolecularandCellularNeuroscience vol28 no 3 pp 588ndash598 2005
[94] C C Tate A J Garcıa andMC LaPlaca ldquoPlasma fibronectin isneuroprotective following traumatic brain injuryrdquoExperimentalNeurology vol 207 no 1 pp 13ndash22 2007
[95] C Culmsee and M P Mattson ldquop53 in neuronal apoptosisrdquoBiochemical and Biophysical Research Communications vol 331no 3 pp 761ndash777 2005
[96] M S Geddis and V Rehder ldquoThe phosphorylation state ofneuronal processes determines growth cone formation afterneuronal injuryrdquo Journal of Neuroscience Research vol 74 no2 pp 210ndash220 2003
[97] M L Craske M Fivaz N N Batada and T Meyer ldquoSpines andneurite branches function as geometric attractors that enhanceprotein kinase C actionrdquoThe Journal of Cell Biology vol 170 no7 pp 1147ndash1158 2005
20 Evidence-Based Complementary and Alternative Medicine
[98] A Muscella C Vetrugno L G Cossa et al ldquoApoptosis by[Pt(OOrsquo-acac)(gamma-acac)(DMS)] requires PKC-deltamedi-ated p53 activation in malignant pleural mesotheliomardquo PloSone vol 12 no 7 Article ID e0181114 2017
[99] J S Bonini W C Da Silva L R M Bevilaqua J H MedinaI Izquierdo and M Cammarota ldquoOn the participation ofhippocampal PKC in acquisition consolidation and reconsol-idation of spatial memoryrdquo Neuroscience vol 147 no 1 pp 37ndash45 2007
[100] O Zohar R Lavy X Zi et al ldquoPKC activator therapeutic formild traumatic brain injury in micerdquo Neurobiology of Diseasevol 41 no 2 pp 329ndash337 2011
[101] J David Sweatt ldquoTheneuronalMAPkinase cascade a biochem-ical signal integration system subserving synaptic plasticity andmemoryrdquo Journal ofNeurochemistry vol 76 no 1 pp 1ndash10 2001
[102] Y Matsuoka M Picciano B Maleste et al ldquoInflammatoryresponses to amyloidosis in a transgenic mouse model ofAlzheimerrsquos diseaserdquo The American Journal of Pathology vol158 no 4 pp 1345ndash1354 2001
[103] L Esposito L Gan G-Q Yu C Essrich and L MuckeldquoIntracellularly generated amyloid-120573 peptide counteracts theantiapoptotic function of its precursor protein and primesproapoptotic pathways for activation by other insults in neu-roblastoma cellsrdquo Journal of Neurochemistry vol 91 no 6 pp1260ndash1274 2004
[104] D J Loane A Pocivavsek C E-H Moussa et al ldquoAmyloidprecursor protein secretases as therapeutic targets for traumaticbrain injuryrdquo Nature Medicine vol 15 no 4 pp 377ndash379 2009
[105] R Torres B L Firestein H Dong et al ldquoPDZ proteins bindcluster and synaptically colocalize with Eph receptors and theirephrin ligandsrdquo Neuron vol 21 no 6 pp 1453ndash1463 1998
[106] M B Dalva M A Takasu M Z Lin et al ldquoEphB receptorsinteract with NMDA receptors and regulate excitatory synapseformationrdquo Cell vol 103 no 6 pp 945ndash956 2000
[107] J M Basak P B Verghese H Yoon J Kim and D MHoltzman ldquoLow-density lipoprotein receptor represents anapolipoprotein E-independent pathway of A120573 uptake anddegradation by astrocytesrdquoThe Journal of Biological Chemistryvol 287 no 17 pp 13959ndash13971 2012
[108] L Yao X Gu Q Song et al ldquoNanoformulated alpha-mangostinameliorates Alzheimerrsquos disease neuropathology by elevatingLDLR expression and accelerating amyloid-beta clearancerdquoJournal of Controlled Release vol 226 pp 1ndash14 2016
Stem Cells International
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Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 3
TCMSP database Molecule information ofcompounds in XFZYD
Candidate compounds profile
Candidate targets profileCandidate targets overlap analysisamong three group of herbs
Candidate compounds overlapanalysis among three group of herbs
HB-cC-cT network
Known therapeuticproteins for TBI
TBI specific proteins
OB DL
STRING
Overlap analysis of targets betweenXFZYD and TBI specific proteins
Cytoscape 340
TTD amp OMIM
HB-pC-pT network TBI specific PPInetwork
Molecule dockingGO and KEGG
pathway analysis
Binding mode between potentialtargets and compounds in XFZYD
Biological significance of potentialtargets of XFZYD on TBI
Synergistic effects ofTCM formula
HPRDTarget prediction
Figure 1 A schematic diagram of the network pharmacology-based strategies for determining the pharmacological mechanisms of the herbalformula XFZYD on TBI
for drug and how drug-like a molecule is with respect toparameters affecting its pharmacodynamic and pharmacoki-netic profiles which ultimately impacts its ADME properties[45] In this study the drug-likeness (DL) index (see (1))using the Tanimoto coefficient [46] was computed for eachcompound in XFZYD
119879 (119883119884) =119883 sdot 119884
1198832 + 1198842 minus 119883 sdot 119884(1)
where X represents the molecular descriptors of herb com-pounds and Y is the average molecular properties of all com-pounds in Drugbank database (httpwwwdrugbankca)Compounds with DL ge018 (average value for Drugbank)were selected as bioactive compounds in XFZYD
In summary compounds with OB ge30 and DLge018were selected for subsequent research and others wereexcluded The criteria used here were mainly for (1) extract-ing information from the herbs as much as possible with theleast number of components and (2) explaining the obtainedmodel by the reported pharmacological data
23 Target Prediction To obtain the molecular targets ofthese active ingredients an in-house developedmodel SysDTbased on Random Forest (RF) and Support Vector Machine(SVM) methods [47] was proposed which efficiently inte-grated large-scale information on chemistry genomics andpharmacology This approach shows impressive performanceof prediction for drug-target interactions with a concordanceof 8283 a sensitivity of 8133 and a specificity of 9362
respectively [40] UniProtKB (httpwwwuniprotorg) wasemployed to obtain the standard name of the predicted targetproteins
24 TBI-Specific Protein Collecting The known therapeutictarget proteins of TBI were screened fromTherapeutic TargetDatabase (TTD available httpbiddnusedusggroupcjttd)TTD is a publicly accessible database which provides com-prehensive information about the known therapeutic proteinand nucleic acid targets described in the literature thetargeted disease conditions the pathway information and thecorresponding drugsligands directed at each of these targets[48] We also searched for Online Mendelian Inheritancein Man database (OMIM available httpomimorg) toget proteins related to TBI OMIM catalogues all knowndiseases with a genetic component and then possibly linksthem to the relevant genes in the human genome andprovides references for further research and tools for genomicanalysis of a catalogued gene [49] Then the proteinsacquired from both databases were used as hub proteinsand submitted to Human protein Reference Database [50](HPRD available httpwwwhprdorg) and STRING [51](httpsstring-dborg) to generate the proteins interactingwith these hub proteins HPRD is a database containingcurated proteomic information pertaining to human pro-teins The human protein-protein interaction (PPI) data onHPRD (Release 9) consists of 39240 interactions among 9617genesThe STRINGdatabase provides both experimental andpredicted interaction information providing a probabilisticassociation confidence score
4 Evidence-Based Complementary and Alternative Medicine
25 Molecule Docking The LibDock algorithm based on theCHARMm Force Field in Discovery Studio (DS) 25 wasused in this study to evaluate the potential molecular bindingmode between bioactive compounds and putative targetsThecrystal structure of the target proteins of XFZYD for treatingTBI was downloaded from the RCSB Protein Data Bank(wwwrcsborg) The 3D chemical structures of bioactivecompounds were downloaded from PubMed Compounddatabase or TCMSP database and subjected to minimize theenergy by using molecular mechanics-2 (MM2) force fieldThe protein preparation protocol was used before dockingsuch as inserting missing atoms in incomplete residuesremoving water and protonating titratable residues The lig-and preparation protocol was employed before docking suchas removing duplicates enumerating isomers and generating3D conformations The protein-ligand docking active sitewas defined by the location of the original ligand All otherdocking and consequent scoring parameters were kept attheir default settings The compound was considered to be apotentially active ingredient if the LibDock score was higherthan the original ligand
26 Network Construction and Analysis Network construc-tion was performed as follows
(1) The herbs candidate compounds and candidate tar-gets of XFZYD were used to construct an herb-candidate compound-candidate target (HB-cC-cT)network
(2) The PPI data obtained above was used to establish theTBI-specific protein interaction network
(3) Herbs potential compounds and putative targetsfrom XFZYD for treating TBI were used to builda herb-potential compounds-potential targets (HB-pC-pT) network
(4) Potential targets and the biology process they partici-pate in were used to construct the pT-F network
(5) Compounds and targets through molecule dockingvalidating were used to build a compound-target (C-T) network
All networks were generated and analyzed by Cytoscape 340[52] an open source of bioinformatic package for biologicalnetwork analysis and visualization Two topological param-eters including degree and betweenness were calculated forthe obtained networks which disclose the significance ofa node
27 Gene Ontology (GO) and Pathway Enrichment Analy-sis The functional enrichment tool DAVID [53] (DAVIDhttpsdavidncifcrfgov) ver 68) was used to calculateboth the KEGG pathway and GO biological processes (BP)enrichment
28 Statistical Analysis All data were expressed as mean plusmnstandard deviation (SD) The molecule descriptors data wereanalyzed by one-way ANOVA The criterion for statisticalsignificance was p lt 005 Statistical analyses were conductedusing the SPSS 240
3 Results
TCM an experience-based medicine has been widely usedfor thousands of years It has accumulated abundant clinicalexperience forming a comprehensive and unique medicalsystem [35] The complexity of the phytochemical compo-nents makes it extremely difficult to illustrate the actionmechanisms of XFZYD from a molecule or system levelAs a chief mean of treating diseases clinically generallyTCM doctors prescribe formula based on the principle ofldquoJun-Chen-Zuo-Shirdquo ldquoJunrdquo (monarch) treats the main causeor primary symptoms of the disease ldquoChenrdquo (minister)enhances the actions of ldquoJunrdquo or treats the accompanyingsymptoms ldquoZuordquo (adjuvant) not only reduces or eliminatesthe possible toxic effects of the Jun or Chen but also treatsthe accompanying symptoms ldquoShirdquo (guide) helps to deliveror guide the other herbs to the target organs [18] Accordingto the unique feature of TCM ourwork tried to perform ldquoJun-Chen-Zuo-Shirdquo based system study to clarify the multiplemechanisms of XFZYD in the treatment of TBI
31 Herbal Ingredient Comparison and Target Predictionof XFZYD We obtained 162 components originated fromXFZYD Of these compounds 160 chemicals that were inaccord with standard requirements were searched from theTCMSP database The other 2 components amygdalin andhydroxysafflor yellow A were taken into consideration fortheir obvious pharmacological action as well The detailedinformation of these compounds is showed in Table S1Persicae Semen (Tao ren) and Carthami Flos (Hong hua)the Jun (monarch) herbs of XFZYD contained 36 bioactivecomponents which accounted for 22 of the 162 chemicalsRadix Paeoniae Rubra (Chi shao) Chuanxiong Rhizoma(Chuan xiong) andAchyranthis Bidentatae Radix (Niu xi) theChen (minister) herbs contained 31 bioactive componentswhich accounted for 19 of the 162 chemicals AngelicaeSinensis Radix (Dang gui) Rehmannia glutinosa Libosch(Sheng di huang) Platycodon Grandiforus (Jie geng) AurantiiFructus (zhi ke) Radix Bupleuri (chai hu) and licorice (Gancao) the Zuo-Shi (adjuvant and guide) herbs of XFZYDcontained 109 bioactive components which accounted for67 of the 162 chemicals Ingredients from these herbswere compared based on the 6 important drug-associateddescriptors including molecular weight (MW) number ofhydrogen-bond donors (nHdon) number of hydrogen-bondacceptors (nHacc) partition coefficient between octanol andwater (AlogP) oral bioavailability (OB) and drug-likeness(DL)The distributions of the 6 descriptors of the ingredientsfrom the three groups are shown in Table 1 and Figure 2 Wefoundno significant differences in the values ofMW(p=016)nHdon (p=032) nHacc (p=061) and AlogP (p=082) amongthe 3 groups However the average OB value of compoundsfrom the march herbs is 5559plusmn2391 which was significantlydifferent from the Chen herbs (OB=4564plusmn1327 P=0004)and the Zuo-Shi herbs (OB=4877plusmn1505 P=0021) Thefollowing 2 groups had no significant difference in OBvalue (P=0272) As for DL the Chen herbs revealed thehighest DL index (053plusmn023) which displayed significantdifference from the Zuo-Shi herbs (044plusmn019 p=0012) while
Evidence-Based Complementary and Alternative Medicine 5
Table 1 Comparison of molecular properties among the Jun Chen and Zuo-Shi herbs
INDEX MW nHdon nHacc AlogP OB DL(mean plusmn SD) (mean plusmn SD) (mean plusmn SD) (mean plusmn SD) (mean plusmn SD) (mean plusmn SD)
Jun herbs 38028 (9423) 265 (226) 511 (321) 337 (436) 5559 (2391) 049 (018)Chen herbs 38180 (9160) 218 (195) 55 (347) 314 (315) 4564 (1327) 053 (023)Zuo-shi herbs 35745 (8822) 261 (160) 56 (249) 344 (185) 4877 (1505) 044 (019)OB oral bioavailability MW molecular weight DL drug-likeness AlogP partition coefficient between octanol and water nHacc number of hydrogen-bondacceptors nHdon number of hydrogen-bond donors
Table 2 Top 10 candidate compounds according to 2 centrality indicators
Compounds Degree Compounds Betweennessquercetin 153 quercetin 035875838kaempferol 65 naringenin 008768253luteolin 48 kaempferol 007665228wogonin 46 luteolin 0057362047-Methoxy-2-methyl isoflavone 44 baicalein 005542898beta-sitosterol 41 beta-sitosterol 0041007baicalein 40 wogonin 003968963formononetin 39 Stigmasterol 003586788naringenin 39 nobiletin 003057461isorhamnetin 38 formononetin 003051437
showing nodifferencewith the Junherbs (049plusmn018 p=0333)Figure 3(a) indicated that 5 bioactive compounds wereshared by the Jun Chen and Zuo-Shi herbs One compoundoverlapped between the Jun andChen herbs while there were2 compounds shared by the Chen and Zuo-Shi herbs Onecompound overlapped between the Jun and Zuo-Shi herbs
32 Target Prediction of XFZYD A total of 285 potentialtargets from the 162 compounds were generated using thetarget prediction model The amounts of potential targets hitby the Jun Chen and Zuo-Shi drugs were 217 218 and 261respectively The detailed data of the targets is shown in TableS2 As depicted in Figure 3(b) there was a significant targetoverlap among the 3 groups (189 candidate targets) but lessoverlap between the Jun andChenherbs (9 candidate targets)The number of targets shared by the Jun and Zuo-Shi herbswas 14 while 10 targets were overlapped between the Chenand Zuo-Shi herbs
33 HB-cC-cT Network Construction and Analysis We nextestablished aHB-cC-cT network through network analysis toilluminate the relationship among the herbs candidate com-pounds and candidate targets (Figure 3(c)) This networkconsisted of 485 nodes (11 herbs 162 candidate compoundsand 285 candidate targets) and 2585 edges A herb (triangle)and cC (square) are connected if the compound is containedin this herb and the edges between cC and cT represent theinteraction The size of nodes is proportional to the valueof degree The larger size of the node means more phar-macologically important Two centrality indicators degreeand betweenness identify the important nodes within thenetwork Different centralities reflect different importanceof nodes in a network from different angles Interestingly
both of the two types of centrality indicators uniformlyconfirmed themost important 10 candidate compounds fromXFZYD and the top 10 targets anchored by XFZYD (Tables2 and 3) Figure 3(c) demonstrated that licorice possessedthe largest degree (88) compared with other herbs originatedfrom XFZYD This implicated that it contained the mostbioactive compounds (88) including quercetin (Mol 148degree=153) kaempferol (Mol 108 degree=65) 7-Methoxy-2-methyl isoflavone (Mol 33 degree=44) formononetin (Mol63 degree=39) naringenin (Mol 133 degree=39) isorham-netin (Mol 105 degree=38) medicarpin (Mol 131 degree=35)and licochalcone a (Mol 113 degree=33) followed byPersicae Semen (degree=19) Carthami Flos (degree=18)Achyranthis Bidentatae Radix (degree=17) Radix PaeoniaeRubra (degree=14) Radix Bupleuri (degree=12) ChuanxiongRhizoma (degree=6) Aurantii Fructus (degree=4) Platy-codon Grandiforus (degree=4) Rehmannia glutinosa Libosch(degree=3) andAngelicae Sinensis Radix (degree=2) PersicaeSemen (degree=19) and Carthami Flos (degree=18) the Junherbs from XFZYD possessed 29 specific compounds and5 unique target proteins including ALB CTNNB1 MMP10LYZ andNFKB1 Twenty-three Chen-specific potential com-pounds targeted 10 specific proteins including CD14 LBPNR3C1 BBC3 TEP1 PRKCD FN1 PDE10A GSTA1 andGSTA2 The Zuo-Shi herbs possessed the largest number ofspecific compounds (101) and 48 unique proteins such asHTR3A ADRA1D PYGM OLR1 CHRM5 RXRB STAT3MAPK10 OPRD1 and MAPK3 There were 189 proteinsanchored by the Jun Chen and Zuo-Shi drugs
Among the 162 candidate compounds quercetin hadthe largest value of degree (153) implicating its critical rolein XFZYD The four herbs namely Carthami Flos (Jun)Achyranthis Bidentatae Radix (Chen) and licorice and
6 Evidence-Based Complementary and Alternative Medicine
50
40
30
20
10
0
50
40
40
30
20
20
10
0
0
10 30 50 70 90 110
60
60
60
01 02 03 04 05 06 07 08
225 275 325 375 425 475 525 575 625 minus6minus5minus4minus3minus2minus1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
minus2 minus1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
70
minus1 1 3 5 7 9 11 13 15 17
perc
entage
s (
)
perc
entage
s (
)50
40
30
20
10
0
perc
entage
s (
)
perc
entage
s (
)
40
20
0
60
perc
entage
s (
)50
40
30
20
10
0
perc
entage
s (
)
OB
MW
nHdon
DL
nHacc
Distribution
Distribution
Distribution
Distribution
Distribution
Distribution
AlogP
Jun herbsChen herbsZuo-Shi herbs
Jun herbsChen herbsZuo-Shi herbs
Jun herbsChen herbsZuo-Shi herbs
Jun herbsChen herbsZuo-Shi herbs
Jun herbsChen herbsZuo-Shi herbs
Jun herbsChen herbsZuo-Shi herbs
Figure 2 The profile distributions of six important molecular properties for all ingredients from the Jun Chen and Zuo-Shi herbs OBOral bioavailability MW molecular weight DL drug-likeness AlogP partition coefficient between octanol and water nHacc number ofhydrogen-bond acceptors nHdon number of hydrogen-bond donors
Evidence-Based Complementary and Alternative Medicine 7
Jun herbs(29)
Zuo-
Shi h
erbs
(101
)
Chen herbs
(23)
1
2
15
(a)
Jun herbs(5)
Zuo-
Shi h
erbs
(48)
Chen herbs
(10)
9
10
14189
(b)
20Mol 1200000
MMMol 152Mol 15Mol 1521Mol 15Mol 15
Mol 8888
Mol 29Mol 2Mol 2Mol 2Mol 29
Mol 3Mol 3Mol 35Mol 3535
1Mol 121Mol 121Mol 122
Mol 124Mol 124Mol 1Mol 124Mol 124Mol 124
MMol 10Mol 1Mol 10M 00MMMM
MMol 73Mol 77333222
ABATABATA
MAPK3MAPKMAPK
UGT1A1GT1UGT1A1
GOT1GOTT1
SREBF1REBFSREBF1
RXRBRXRBRXRB
CYP19A1CYP19A1YP19A
PLB1PLBPLB1
FASLGFASLGASL
BCHEBCHHE
HSD3B1HSD3
MTTPMTT
CES1CES
TIMP1TIMP
ATP5BATP5
MAPK10MAPK 0
BADBAD
CHRM5CHRM5
EPHBEPHBPHB2
HSD3B2HSD3B2SD3B
SOAT2OATSOAT2
FASNFASNFASN
MT-ND6MT-N
HTR3AHTR3HTR3APLA2G4APLA2G4AA2G
PKIAAIA
ADH1AADHAD
AKR1C1KR1AKR1C1
CREB1CREBB1
HMGCRMGHMGCR
SOAT1OAT
ABCC1ABCC
OPRD1OPRDOPRD1
VCPVCP
LDLRLDLR
ADRA1DDRAA1D
NFATC1FATCFATCATCFATCA
EGLN1GLNGLNGLNGLN
FABP5ABP5ABPABPABPABP5
CYCSCYCSCYCSCYCSYCSFOSL1OSLOSLOSLOSL
ALOX12LOXLOXOXLOX
TDRD7DRDDDRDDRDDRDAPODAPODAPOPOAPOD
NNNNOXNOXNOXNOX5NOXX
Mol 40
NFKB1NFKBFKBFKBFKBLYZLYZLYZZLYZ CTNNB1TNNBTNNNNNN MMP10MMPMPMPMMP1ALBALBALBALBALB
3Mol 433MMol 28Mol 28Mol 2Mol 28
HSPA5SPASPASPA5HSPA5AERBB2ERBB2ERBBRBBB2ERBB2
HSPB1HSPBSPBSPBHSPB1HSPB1B
NQO1NQO1NQONQO1NQONQO1TOP1TOP1TOP1TOPTOPT
PORPPORPORPORPORR ACPPACPPACPPACPPCPP
SLC2ALC2A4LC2A4C2A4LC2A4LC2A4LC
SERPINE1SERPINE1PINRPINERPINPIN
JUNJUNNJUNNN
PTGER3PTGER3TGERTGERGERTGER
COL1A1OL1AOL1AOL1AA1CO
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CCCALCCC MMMMMMSS
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CCND1CCNDCCNDCNDCNDCCND
SMDPSMDPSMDPSMD3PSMD3PSMDNFNFNFFFNF
ELK 1ELKELK LK ELK ELK CCL2CCL2CCLCCL2CCL2CCL2
MMol 14MMol 14Mol 1447
MMol 15MMol 1Mol 115Mol 15M
Mol 122Mol 122Mol 122MMol 12Mol 122222
MMol 24Mol 2Mol 2Mol 2444
Mol 78MMol 78Mol 7Mol 7Mol 788
Mol 38MMMMol 3Mol 3338
Mol 154Mol 154MMol 15ol 1ol 155M
Mol 67MMol 6Mol 6Mol Mol 67Mol 677766
Mol 64MMol 64Mol 6Mol 6Mol 64
Mol 48Mol 48MMMol 4Mol 4448M 4
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PPPPRSPRSPRSSSSSSSPPARDPARDPARPARDPARDPARDSELESELESELESELEEE
COL3A1COL3A1OL3AOL3AOL3AA1OL3ARB11111
CYP1A2222222 NCOANCOANCOANCOANCOANCOA22222211111 RRRRRR
Quercetin
Mol 9444444
Mol 99
Mol 63
Mol 75555
Mol 37Mol 37Mol 37Mol 37MolMol 33Mol 37MolM
Mol 158
Mol 1Mol 10Mol 1Mol 10000
Mol 49Mol 4Mol 4Mol 4Mol 44Mol 494Mol 4
Mol 6Mol 6666M 66
MMol 60Mol 60Mol 6MM
Mol 101Mol 101Mol 101Mol 10011
Mol 12Mol 12Mol 12Mol 1Mol 12221
Mol 11MMol 1155Mol 11555M6060660
Mol 85Mol 85Mol 85MMol 88558554MMol 114Mol 114444M
Mol 116Mol 116Mol 116MMol 1ol 1Mol 11616M 1
Mol 9000
Mol 100Mol 1000Mol 106MMMol 106Mol 10Mol 1066Mol 322 Mol 30Mol 30Mol 30000MM
Mol 140MMol 14MMol 14Mol 1440Mol 14
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Radixadixdix
Mol 84
Mol 23 Mol 130Mol 13Mol 13Mol 11Mol 130Mol 13Mol 1MMol 13
Mol 11Mol 1Mol 1Mol 11Mol 11
0Mol 200000
Mol 34Mol 3Mol 3MolMol 3344Mol 3Mol 134
Mol 151
Mol 126Mol 126Mol 12666
Mol 8111AurantiiAurantAuranAurantiitFructusructusFructuructuFr
Mol 77Mol 7Mol 7Mol 77777
Mol 444
MMol 82Mol 8Mol 8222Mol 88
Mol 133
Mol 123MMol 12Mol 12ol 1Mol 121
Mol 142Mol 1422
Mol 36
0Mol 80Mol 88Mol 80M 80
Mol 277Mol 135Mol 13555M 555
Mol 141MMol 144Mol 14111
Mol 104Mol 104MMMol 10Mol 104044M
Mol 922222Mol 22 Mol 2Mol 2Mol 2Mol Mol 2Mol 22M 2
MMol 83333
Mol 110Mol 110
Mol 119MMol 11999999
Mol 88888 Mol 109Mol 10MMol 1Mol 100Mol 109M
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Mol 39999999
Mol 150MMol 155000M 0
Mol 611
21Mol 21MMol 221111
Mol 13
Mol 9Mol 91MMol 9911
Mol 118MMol 11Mol 1Mol 11Mol 1Mol 11ol
Mol 466
Mol 96Mol 96Mol 96Mol 996Mol 9696 Mol 53Mol 5Mol 5MolMol 5Mol 5Mol 535Mol 50Mol 50Mol 5Mol 5Mol 550Mol 5
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M l 149MMol 1449Mol 149Mol 149
Mol 14Mol 1444
Mol 89Mol 125MMol 1ol 1Mol 12MMMol 1MM
Mol 87
Mol 72MolMol 7Mol 7MolMol 72M 7M Mol 933Mol 112Mol 112Mol 112Mol 1122M
Mol 1Mol MMolMollMol 1Mol Mol 105
MAP2MAP2MAPMAPMAP
IL2I 2L22L22
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Mol 17 Mol 7666Mol 33
Mol 86Mol 86Mol 8Mol 8Mol 866686
Mol 161Mol 161Mol 161Mol 16Mol 16161MMo
Mol 56Mol 5Mol 556666Mol 113
Mol 97Mol 162MMol 16Mol 1Mol 1ol 111M 1
Mol 131
Mol 99Mol 9MMMolMol 9MMol 9Mol 99Mol 9ol
Mol 111
Mol 128Mol 128Mol 1ol 1Mol 121MolMol 199Mol 117Mol 117777
Mol 744444Mol 77
2Mol 52Mol 52Mol 5MM
MMMol 5Mol 5MMol 5Mol 5Mol 5M
MMol 58Mol 58Mol 58M 8
Mol 138MMol 1388M
Mol 102Mol 102Mol 10Mol 102MMol 10Mol 10
Mol 42MMMol 42M
MMMol 3Mol 3
Mol 137Mol 13Mol 13ol 13Mol 13Mol 13733
Mol 59Mol 59olMol 5Mol 59oMol 5Mol 559
Mol 129lMol 12ol 12ol 12Mol 129o 2ol 12o
Mol 62Mol 62Mol 6Mol 6Mol 6Mol 62Mol 6Mol 6ol
Mol 144Mol 144Mol 14Mol 14ol 14Mol 144ol 1
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Mol 159Mol 159MoMol 15ol 15ol 15Mol 159o 5
Mol 136Mol 136ol 13ol 13ol 1ol 13ool 13
CXCL11CXCL11XCL1XCL1XCLXCL1p53p53p53p
Mol 47Mol 47Mol 4Mol 4Mol 4ol 47Mol 4o
MMol 156ol 15ol 15ol 1ol 15ooPRKCARKCRKCPRKCACARKC
PPP3CAPP3CAP3CAP3CAP3CAPPP3CAP
CDKN1BDKNDKNDKN1CDKN1BPTENPTENPTENPTENPTENPTENBBB
RadixRadixRadixPaeoniaePaeoniaePaeoniaePae
RubraRubraRubraNR1I3NR1I3NR1INR1INR1INR1I3
HERC5ERCERC5HERC5ERC5OODC1ODCODCODC1CERBB3RBBRBBERBB3RBBRBB3
CTSDCTSDCTSDCTSDCTSDCTSD Mol 18Mol 1Mol 1ol 18Mol 18MolMol 1
PLATPLATPLAPLATPLATL
DIOOO1OO1OEGFEGFEGEGFEGDIODDIODIO
BIL1BIL1BIL1BIL1BB
MMP 3MMP 3MMP 3MMPMMP 3M
THBDTHBDTHBTHBTHBTHBD
CYP1B1YP1BYP1BYP1BYP1BB1
PPPPPARPP GGGGGGPPP
KDR LLAACTACTACTLACTBLACTBACTTT
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CDK2
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IL10IL10IL10IL10IL10IL10
S2HAS2HAS2HAS22HAS2ADADDHDH1CADAD HAHAHHHHA
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Mol 155Mol 155Mol 15Mol 15Mol 15ol 1ol 15ol 15Mol 15MMo
Mol 153Mol 15Mol 15ol 15Mol 153ol 15Mol 15Mol 15
CHEKCHEKCHEKHEK2HEK2HEK
MMP2MMP2MMPMMPMMPMMP2
MGAMMGAMMGAMGAAM
KCNH2222
CXCL2CXCL2CXXCLXCLXCL
GJA1GJA1GJAGJA1GJA1GJA1KK222K2
IKBKBIKBKBIKBKBIKCNH2KCNH2
IFNGIFNGIFNGIFNGIFNGIFNGFNEIF666F6IGHG1IGHG1GHGIGHG1IGGHG
SSStigSStiggmmmaS gg steeerolerol
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BACE1ACEBACE1 SLC6A1LC6AACD163CD16 GSRGSRR ADIPOQDIPOQSTAT3TATOPRM1OPRM1OPRMOPRMOPRMO ADRA1BADRA1BAADRA1BBBB
SLC6A4LC6ALC6ASLC6A44SLCYCYP1A1YP1A1YP1A1CYP1AYP1A1
SLC6A3SLC6A3SLC6ASLC6A3SLC6A3
PDE3APDE3AA
CHRNA7CHRNA7HRNAHRNAHRNAA
PPPTGPP S1S1S1S1SS111NKX3-1NKX3-1NKX3KX3NKX3NKX3-
BIRCBIRC5BIRCBIRCBIRC55
IL6666
HK2HK2HK2HK2HK2HK2
RUNX2UNXUNXUNXRUNX2UNXHSF1HSF1HSF1HSF1HSF1HSF1
IGFBP3GFBPGFBPFBPIGFBP3
STAT1T1TAT1TATSTAT1TATT
NCF1NCFNCFNCF1NCFN
PRSS3RSSSIRT1SIRTGATMGAT OLR1OLRAPOBAPOPYGMPYGM
TGFB1GFBGFBGFBGFBTGFB1F10 MMP9MMPMMPMMPP9PNFE2L2FE2LFE2LFE2LNFE2L2FE2L2L
Mol 160Mol 44Mol 44ol 4Mol 444Mol 444ol 4ol 4ol 44
Mol 55MoMol 5Mol 5Mol 555Mol 55Mol 5Mol 5MoMol 5
MMMol 132
MMMol 57
CRPCRPCRPCRPPHH
RUNX1T1UNX1NX1NX1NX1T1R
BCGABCGABCG2BCGA
CXCL10CXCL10XCXCLCXCLCXCL IGG
CLDN4LDN4LDN4LDN4ABABABAAB RRAHRAHRBBBIB
RARRAF1RAF1RAF11KKKKKKaemKKKKK pffefeferereerf referololol
CASP9CCASPCASP9PMDM2MDMMMDMMDMMDM2MDMMDM
BMAOBMAOBMAOBMAOBMAOBINSRINSRINSRINSRINSRRRNCOA1NCOA11NCOA1
HTR2AHTR2HTR2HTR222A
CHRMCHRMCHRM3MM3BCL2LCL2CL2L1LCL2BCL2L1111 CHCCHCCCCH
BCL2222BCL2CASP8CASP8CASP8CASP8CASP
RELARELARELAAAR
BaicalBaicalBaBaicalcacacalicaBB calcaac ininininininninninnCHUCHUKHUKCHUKHUKUKH AAAAAA
EGFREGFRGFREGFREGFR
NFKBIAFKBIFKBFKBNFKBIAFKBI
E2F2E2F2E2F2E2F2E
APPAPAPPAPPPAPPAPPA
CASP7CASPCASPASPCASPCASPPP IL4IL444L4L44
NUF2NUFNUFNUFNUFNUFNUFNUFN
CDK4CDKCDKCDKCDKCDKCD
XIAPXIAPXIAXIAXIAXIAX MEMEMETMETMETMEETMMME
ADCY2ADCYDCYDCDCYDCD
Mol 127
NNR3C2NR3C2NR3CNR3C2N
DPP4
DCAF5DCACAFDCAF55DCAFAKR1B1AKR1BKKR1BKR1BKR1BCTRB1CTRBTRBCTRBCTRB
LTA4HLTA4LTA4TA4LTA4LTA4H4
XDHXDHXDHHHXDH
PTGS2
Mol 51Mol 51Mol 5MMolMolMol 5Mol 5MMol 5
Mol 145Mol 14Mol 14ol 1ol 1ol 1Mol 1Mo
Mol 146MMMMool 1ol 11461
Mol 143Mol 14Mol 14Mool 1ol 11Mol 1411
Mol 70MMMolMol 7Mol 77Mol 70Mol 7Mol 7
Mol 71Mol 7Mol Mol 777Mol 71Mol 7Mol
Mol 25Mol 25MMMol Mol 22Mol 2
Mol 79Mol 7Mol 7Mol Mol 7Mol 779MM 7
Mol 26Mol 26MMol 2Mol Mol 22Mol 2M 22
CA22222222ADH1BADHADHDHADH1ADH1DH KCNMA1CNMCNMCNMCNMKCNMAKCNMCGABRA6GABRAABRAABRAABRAGABRAGA A6GA RAGRIA2GRIAGRIAGRIAGRIAAAA2GRIA
RASSFRASSF1ASSFASSFASSF
Mol 31MMol 3Mol 3Mol 3311M 3Mol 98Mol 98Mol 98MMol 9Mol 999Mol 98MM
GSTP1GSTP1GSTPGSTPGSTPGSTPF33333
Mol 69Mol 6MMol 6Mol 6Mol 66Mol 69Mol 69M 66
VCAM1VCAMCAMCAMVCAM1C
MAPK8MAPK8MAPKMAPMAPKM
CYP3A4CYP3A4YP3AYP3AYP3AY
Mol 68Mol 68MMol 6Mol 6Mol 66MMol 68Mol 6MMol 16Mol 16Mol 1Mol 1Mol Mol 1Mol 16
MMMMol 6MMol 66Mol 666Mo
CCartCarta hamihamimiiiiiFlosFlosFlosossoss
Mol 9566Mol 65Mol 6Mol 6MMol MolMol 6M 666M
PersPersPerserrr icaeicaeicaesemesemesemeemememeem nnn
ICAM1CAMCAMM1CAMI
E2F1E2FE2F1E2F1E2F1ENPEPPSPEPPSPEPPSEPPSPEPPSNPEPP
IGF2IGF2IGF2IGF2F
FOSFOSFOSFOSSFOSFOSSSS
CCNB1CCNBCNBCNBCCNB1CCNB1CDK1CDK1CDKCDKCDKCC
PRKCBPRKCBRKCRKCRKCR CB
CHRM1CHRM11CHRM11
HHHHSP9HHHH 0ABABAB2B2B2B2PPPPPPPPPPPPTOP2A ACACACACACACACACAACACAACACA
PRKACA
TOP2TOP2
My c FF7FFIRF1IRF1IRF1IRF1IRF1IR
CAV1CCAVCAVCAV1V1CAV1
ACHE
ADRB1ADRBRBADRBADR
ADRADRADRA2A2AA2A
SOD1SSODSOD1D11
NOS33
PLAUPLAUPLAAUUPLAUPRXRA
PGR
HHMOX11MOXMOXHMOX
GSTM1GSTM1GSTMGSTMSTMAAA
HIF1AHIF1HIF1HIF11AHIF1A
FOSL2OSLOSLOSL22FO
ALOX5ALOXALOXALOXALOX5
VEGFAVEGFAVEGFAVEGFAV FFA
PON1PONPONPONN1PON
PIK3CGPIK3CGGG
SULT1E1SULT1E1ULT1ELT1LT1GAGAABRAGABRA3AGG
AKT1AKTT1AKT11A
CHRMCHRMCHRMCHRM4CHRM4RMM
MAOAMAOAMAOAMAOMAOCD40LGCD40LGCD40LGD40LGCD40LGCD40LBRA3RAAABRA3CHRCHHHCHR
ADRB2
(c)
Figure 3 HB-cC-cT network of XFZYD (a) and (b) The distribution of different candidate compounds and targets in the network (red theJun herbs-specific cC (a) and cT (b) aqua the Chen herbs-specific cC (a) and cT (b) periwinkle the Zuo-Shi herbs-specific cC (a) and cT(b) claybank common cC (a) and cT (b) between the Jun and Chen herbs blue common cC (a) and cT (b) between the Jun and Zuo-Shiherbs green common cC (a) and cT (b) between the Chen and Zuo-Shi herbs purple common cC (a) and cT (b) among the 3 group ofherbs (c) The triangles with circle backgrounds represent the herbs (HB) the squares and circles represent the candidate compounds (cC)and candidate targets (cT) The red triangles squares and circles represent corresponding HB cC and cT in the Jun herbs the same is toaqua representing the Chen herbs and periwinkle representing the Zuo-Shi herbs The claybank squares and circles represent correspondingcC and cT overlap between the Jun and Chen herbs the same is to blue representing the overlap between the Jun and Zuo-Shi herbs and thegreen representing the overlap between the Chen and Zuo-Shi herbs The purple squares and circles represent the corresponding cC and cTshared by the 3 group of herbs The size of the node is proportional to the value of degree
Radix Bupleuri (Zuo-Shi) contained quercetin It targeted149 bioactive proteins PTGS2 (degree=126) HSP90AB2P(degree=85) AR (degree=81) NCOA2 (degree=75) PRSS1(degree=68) PTGS1 (degree=67) PPARG (degree=66)and F10 (degree=60) were predicted as the major candidatetargets of quercetin followed by kaempferol (degree=65)which was contained by Carthami Flos (Jun) AchyranthisBidentatae Radix (Chen) and Radix Bupleuri licorice(Zuo-Shi) It targeted 61 bioactive proteins includingPTGS2 (degree=126) HSP90AB2P (degree=85) CALM(degree=81) AR (degree=81) NOS2 (degree=76) andNCOA2 (degree=75) Quercetin stigmasterol kaempferol
baicalin and beta-sitosterol existed in 3 the Jun Chenand Zuo-Shi drugs demonstrating crucial roles of thesecomponents The 5 ingredients in the Jun Chen and Zuo-Shiherbs targeted 186 bioactive proteins which accounted for65 targets of XFZYD Baicalein existed in the Jun andChen herbs Luteolin existed in the Jun and Zuo-Shi herbsSpinasterol and sitosterol existed in the Chen and Zuo-Shiherbs 126 bioactive compounds targeted PTGS2 followedby ESR1 (88) HSP90AB2P (85) AR (81) CALM (81) NOS2(76) NCOA2 (75) PRSS1 (68) PTGS1 (67) and PPARG(66) As depicted in Figure 3(c) these target proteins wereanchored by ingredients in the 3 group drugs
8 Evidence-Based Complementary and Alternative Medicine
Table 3 Top 10 target proteins of XFZYD according to 2 centrality indicators
Proteins Degree Proteins BetweennessPTGS2 126 PTGS2 0128936ESR1 88 NCOA2 0060806HSP90AB2P 85 HSP90AB2P 0049647AR 81 PRKACA 0046484CALM 81 PTGS1 004365NOS2 76 AR 0030252NCOA2 75 PRSS1 0025575PRSS1 68 ESR1 0022212PTGS1 67 PPARG 0021403PPARG 66 PGR 0020685Note the centrality indicators identify the important nodes within the network Higher degree centrality and betweenness centrality indicate greaterimportance
The analysis of the network revealed that quercetin (Mol148) kaempferol (Mol 108) luteolin (Mol 127) wogonin (Mol160) 7-Methoxy-2-methyl (Mol 33) and beta-sitosterol (Mol41) were predicted as the major active compounds of XFZYDTheproteins including F2 NOS2 PTGS1 PTGS2 and CALMwere predicted as essential pharmacological proteins for thetherapeutic effects of XFZYD
34 Analyses on TBI Based Specific Protein Interaction Net-work Network biology integrated with different kinds ofdata including physical or functional networks and diseasegene sets is used to interpret humandiseases Protein-proteininteraction networks (PPI) are fundamental to understandthe cellular organizations biological processes and proteinfunctions [54] From the systematic perspective the analysisof TBI-related PPI will improve the understanding of thecomplicated molecular pathways and the dynamic processesunderlying TBI We screened the TBI-specific genes andprotein targets using Online Mendelian Inheritance in Mandatabase (OMIM) and Therapeutic Target Database (TTD)Figure 4 indicated that 21 TBI-specific genesproteins wereacquired Further these hub-proteins were submitted toHPRD and STRING to establish the TBI-specific proteininteraction network The detailed information of the TBI-specific proteins is shown in Table S3 The results suggestedthat the network consisted of 489 nodes and 5738 edges(Figure 4(a)) We obtained top 10 TBI-related proteinsaccording to 2 centrality indicators generated and summa-rized in Table 4 Interestingly we found that the node withhigher betweenness tends to possess larger degree (Fig-ure 5) Network topology analysis showed that protoonco-gene tyrosine-protein kinase Src (SRC degree=164 between-ness=0059) RAC-alpha serinethreonine-protein kinase(AKT1 degree=157 betweenness=0045) Serum albumin(ALB degree=153 betweenness=0069) Epidermal growthfactor receptor (EGFR degree=139 betweenness=0053)and Amyloid beta A4 protein (APP degree=134 between-ness=0072) contributed to the essential role in the patho-physiology of TBI All of these indicated that the top mutualtarget proteins performed various beneficial functions to treatTBI at the molecular level For example SRC is activatedfollowing engagement of many different classes of cellular
receptors It participates in signal pathways that controla diverse spectrum of biological activities including genetranscription immune response cell adhesion cell cycleprogression apoptosis migration and transformation [55]SRC can result in blood-brain barrier (BBB) disruptionand brain edema at the acute stage the inhibition of SRCfamily kinases can protect hippocampal neurons and improvecognitive function after TBI [56] AKT1 regulates manyprocesses including metabolism proliferation cell survivalgrowth and angiogenesis [57] The PI3KAKTPTEN path-way has been shown to play a pivotal role in neuroprotectionenhancing cell survival by stimulating cell proliferation andinhibiting apoptosis after TBI [58] ALB is the main proteinof plasma and can be a biomarker to predict outcome ofTBI [59] Its main function is the regulation of the colloidalosmotic pressure of blood We found that it may participatein pathological process of TBI
KEGG pathway analysis was also used to determine thefunctions of proteins Table 5 describes the top 10 significantlyenriched KEGG pathways These pathways play crucial rolesin pathophysiology process which have also been widelydiscussed in existing literature For instanceMAPK signalingpathway can promote pathological axonal death throughtriggering a local energy deficit [60] Neurotrophin signalingthrough Trk receptors regulates cell survival proliferationthe fate of neural precursors axon and dendrite growth andpatterning and the expression and activity of functionallyimportant proteins such as ion channels and neurotransmit-ter receptors [61] Engagement of cells with the extracellularmatrix (ECM) proteins is crucial for various biological pro-cesses including cell adhesion differentiation and apoptosiscontributing to maintenance of tissue integrity and woundhealing [62]The 47 TBI-specific proteins targeted byXFZYD(yellow and green) were further discussed below
35 HB-pC-pT Network pT-F Network Construction andMolecule Docking Analyses of XFZYD for the Treatment ofTBI To investigate the therapeutic mechanisms of XFZYDfor the treatment of TBI a HB-pC-pT network of XFZYDfor treating TBI was built (Figure 6) 47 TBI-specific targetproteins (circles) were targeted by 119 potential compounds(squares) from XFZYD (Figure 6) Detailed information
Evidence-Based Complementary and Alternative Medicine 9
BLMH
GPRASP2
GJC2
CAMK2N1
OBSL1
ZNF292
RLFAMPD3
NANOS1CAMTA1HYPKCCS
DLGAP4PF4V1
TRIM2
CHKB
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BMX
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APBB1
KCNMA1
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TTR
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PIK3CA
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CACNA2D3
CSF1
CNTF
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BCL10
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RGS3
TDP2
KRT5
ADAM9
REST
PACSIN1
SHB
CTSL1
LAMA1
SH2B1
PLA2G4F
SMURF2
SLC9A8
KRT14
CIT
TGFB1I1
PPP2R1APIK3R1
NUMB
ADAM17
DLG2
LRP1
APOA4
LTA
IRS1
GSK3B
EGFR
CTSB
TRAF6
SHC1
PPP2CB
NGF
HTT
PPIDLDB3
APOC1HIP1
ZDHHC17COL25A1MAGI3
ITGB5
ACTB
GNA11
ACTN2CAMK2B
CDK1
LIN7C
S100B
PDLIM5
DLGAP1
LIN7A
TPR
SYNGAP1
CFD
GRIN3A
F12
SLC1A3
GRK5
CACNA2D2
TANC1
DUSP4
EXOC4
AHSG
GRIN3B
UCP2
TTN
GPC1
NSF
FGA
DYNLL1
MAPK8IP1
PICALM
PPBP
ITM2B
NID1
STXBP1
CTBP1
SGK1
TRAF1
SQSTM1
TNFRSF1A
ACTN1
OPTN
UBE2D2
AP2A2
FUS
APOA1
DICER1
CACNA1B
CST3
NCOA3
SCARB1
APOC3
SLC6A3
KIAA1551 KRT13
DCD
KIAA0232
ENSG00000258818
SPATA31A7
CTAGE5
CEP44
SH2B2
EPHB4
CAPN1
F7
APBA2
COL4A3
LDLR
GIPC1
GFAP
SERPING1
SRC
MAPK12APP
GAPDH
CDK5
CREBBP
NOS1
YWHAZCALM1
YWHAG
YWHAQGRIN1
RPS6KA3
COL1A2
TCERG1
A2M
CASP7
LRP8
PDK2
AGTR1
FYN
ALBGRB2
MAPK1AKT1
STAT1
MAPT
GSK3A
UBB
PPP2CA
SMAD4
PRKCD
RGS12
SPTB
SH3BP5
CACNA1I
IL16
SLA2
PFN2
NLRC4
TNFRSF14
PRTN3
SNCB
FBLN1
BACE2
SETX
FANCD2
SAP30
RANBP3
PALB2
SLC1A5RASA1
HAP1
LRRC7
CFB
ADRBK2
CDC45
CLSTN3
PCED1B
SCAF1KIAA1377
FAM71E2
FEZ1 ACSBG2
PCDH1
TRAPPC11QTRTD1
KRT1
LRP1B
HADHB
TP53BP2
DRD1
ST13
PRPF40A
TRAIP
KIDINS220
GRK6
MMP17
LTBR
EIF6
PGAM1
CHD3
PRSS3
APBB3
CHRNA7
KRT10
RIMS1
CRMP1
PDIK1L
SORBS3
SYMPK
GCN1L1
RNF19A
HOMER3
GRK4
JARID2
MYLK3
EFS
CRB1
HSD17B10
TST
HOMER2
PHC3
TM2D1 FICD
PEG3
CABLES1
LGALS2
ITIH1
HOXB2
TAF4FLOT1
BABAM1
CFH
HIP1R
LTBMYL4
NCOR1
CASP4
NF1
AP4E1
CLCA2
IGDCC4
NECAB3
CXorf27
SH3GLB1
CTCF
PLCG2
(a)
STAT1
EGFR
FN1
PLAT
SOD1
PRKCA CDK1
GSK3B
ALB APP
PPP3CA
IFNG
BBC3
SCN5A
KCNMA1
MAP2
ACHE
MAPK3F2
PTPN1
NOS3 BCL2
CAV1
CTNNB1
PRKCB
TGFB1CASP8
TNF
AKT1
F7
MAPK1LDLR
PRKCD
SLC6A3
DRD1
CASP3
CHRNA7
PRSS3
TP53BP2
CASP7
EPHB2
SYNGAP1
CALM1
CTSD
BACE1
ADRA1B
(b)
Figure 4 TBI-related protein interaction network (a) 21 hub proteins (red and green) were identified through the analysis of TherapeuticTargetDatabase (TTD) aswell asOnlineMendelian Inheritance inMan (OMIM) 4 overlapped protein targets (green)were obtained between21 hub proteins in TBI and candidate targets of XFZYD Periwinkle proteins fromHPRD and STRING analyses were not targeted by XFZYD(b) 47 candidate protein targets of XFZYD were screened for treating TBI Yellow candidate protein targets of XFZYD The size of nodes isproportional to the value of degree
10 Evidence-Based Complementary and Alternative Medicine
Table 4 Top 10 proteins of TBI specific proteins according to 2 centrality indicators
Proteins Degree Proteins BetweennessSRC 164 APP 0071814AKT1 157 ALB 0069343ALB 153 SRC 0058568EGFR 139 EGFR 0052938APP 134 AKT1 0045466GAPDH 131 HTT 0037164HSP90AA1 108 GAPDH 0034549BCL2 106 HSP90AA1 0027596MAPK1 105 CTNNB1 0021759HRAS 105 GNB1 0019492Note the centrality indicators identify the important nodes within the network Higher degree centrality and betweenness centrality indicate greaterimportance
Table 5 Top 10 significantly enriched KEGG pathways in TBI-specific proteins
Pathway ID Pathway description Gene count FDR4010 MAPK signaling pathway 44 299E-235200 Pathways in cancer 43 113E-184722 Neurotrophin signaling pathway 41 101E-344151 PI3K-Akt signaling pathway 40 111E-154510 Focal adhesion 39 292E-225205 Proteoglycans in cancer 39 410E-215010 Alzheimer s disease 34 124E-204015 Rap1 signaling pathway 33 769E-174014 Ras signaling pathway 33 646E-164020 Calcium signaling pathway 31 505E-17
0
001
002
003
004
005
006
007
008
0 20 40 60 80 100 120 140 160 180
Betw
eenn
ess
Degree
APP
ALB SRCEGFR
AKT1HTT
Figure 5 Relationship between degree and betweenness centralityin the TBI-specific protein interaction network
for the 119 potential compounds is shown in Table S4Similarly 5 pharmacologically active ingredients in the JunChen and Zuo-Shi groups including quercetin stigmasterolkaempferol baicalin and beta-sitosterol anchored 33 TBI-specific proteins such as CALM SCN5A F2 ACHE F7ADRA1B NOS3 BCL2 CASP3 and AKT1 11 Jun-specificcompounds targeted 2 specific targets (ALB CTNNB1) while12 Chen-specific compounds anchored 3 unique proteins(PRKCD FN1 and BC3) The Zuo-Shi herbs possessed thelargest number of active compounds (89) and targeted 5
unique proteins including EPHB2 BACE1 LDLR MAPK3and PRSS3 GSK3Bwas the common target between theChenandZuo-Shi drugs KCNMA1 CASP7 andAPPwere targetedby the Jun and Zuo-Shi drugs
The top 10 candidate compounds and targets to treat TBIwere showed in Tables 6 and 7 Formost of active compoundsfrom XFZYD each component hit more than one targetTable 6 demonstrated that quercetin had the highest numberof targets (degree =76) followed by kaempferol (degree=43) beta-sitosterol (degree =35) stigmasterol (degree =19)luteolin (degree=18) baicalein (degree=15) 7-Methoxy-2-methyl isoflavone (degree=12) wogonin (degree=12)nobiletin (degree=11) and naringenin (degree=9) Forinstance quercetin (3310158404101584057-pentahydroxyflavone) is anaturally occurring flavonoid commonly found in fruits andvegetables It regulates multiple biological pathways elicitinginduction of apoptosis as well as inhibiting angiogenesisand proliferation [63 64] Quercetin can attenuate neuronalautophagy and apoptosis in rat traumatic brain injury modelvia activation of PI3KAkt signaling pathway [65] It has alsobeen reported to have a protective ability against oxidativestress and mutagenesis in normal cells [66 67] Kaempferol(3 41015840 5 7 tetrahydroxy flavone) is a yellow-colored flavonoidthat is widely distributed in many botanical families[68] It has been shown to possess a variety of biologicalcharacteristics including effects of anti-inflammatory[69] antioxidative [70] tumor growth inhibition [71] and
Evidence-Based Complementary and Alternative Medicine 11
Mol 22Mol 33
Mol 29Mol 32 Mol 23Mol 20
Mol 97Mol 106
Mol 104Mol 100Mol 94
Mol 63Mol 90
Mol 87
Mol 91Mol 61
Mol 19
Mol 109
Mol 161
Mol 118
Mol 111
Mol 158
Mol 114
Mol 149
Mol 141
Mol 152
Mol 14
CASP3
BCL2
NOS3
SCN5A
F7
ADRA1B
ACHE
F2
Mol 4
Mol 49
Mol 127
APP CASP7
Mol 92
Mol 54
Mol 6
Mol 36
KCNMA1
Mol 27
Mol 13Mol 105Mol 140
Mol 17
Mol 39
Mol 2
Mol 21
Mol 12
Mol 142
Mol 126
Mol 135
Mol 121
Mol 119
Mol 124
Mol 120
Mol 117
Mol 133
Mol 131
Mol 134
PlatycodonGrandiforus
Aurantii Fructus
Licorice
Radix Bupleuri
RehmanniaglutinosaLibosch
AngelicaeSinensis Radix
Mol 8
Mol 73
Mol 75
Mol 77
Mol 80
Mol 7
Mol 82
Mol 76
Mol 74
Mol 60
Mol 46
Mol 150
Mol 151
Mol 112
Mol 115
Mol 11
Mol 116
Mol 113
Mol 110
Mol 1
Mol 9Mol 101Mol 10Mol 93
Mol 103Mol 89 Mol 107
Mol 96PRSS3 LDLRMAPK3 BACE1 EPHB2
Mol 35Mol 81
Mol 86
Mol 85
Mol 84
Mol 88
Mol 83Mol 30
Mol 137
ChuanxiongRhizoma
Mol 3
Mol 57
Mol 139
Achyranthis
Bidentatae
RadixMol 102
CASP8
IGHG1AKT1p53
CHRNA7
Mol 40
SLC6A3 PTPN1
SOD1MAPK1
Mol 38
Mol 95
Carthami Flos
Mol 24
Mol 78 Persicae Semen
Mol 122
Mol 25
Mol 98
TGFB1
GSK3B
PRKCA
Radix Paeoniae Rubra
Mol 52
Mol 42
Mol 62
Mol 138
EGFR
STAT1 PPP3CAPLAT
Stigmasterol
MAP2
Beta-sistosterol Quercetin
Kaempferol
SYNGAP1
CALM
Baicalin
PRKCB
CAV1
CTSD
TNF
DRD1
CDK1
IFNG
FN1BBC3 PRKCD
Mol 132
Mol 160
Mol 58
Mol 67
Mol 69 Mol 43ALB
Mol 64CTNNB1
xiongixioChuanxC nxnxioaamaRhihihizoma
Achyranthisiistntnt
aeeaeBidenBidennntatae
RadixadixR diR di
oniae nianiaiaonRadix PaeoPaePRadix P eeoeonRRRRubra dondonddoodPlatycolatycPlatyycPl ycoyccoPlatPlatycoPlatyccocod
rususrusususruGranG anGranGranGranGrGGranndndiforu
ructusuctuc sructusuctusructusruAurantiirA ntaA ntiitiirantAurantintiurantAur iiii Fru
cecececececececeecLiLLiLLiLiLLLLLLLLiLiLiLLiLLLiLicoric
leuririeuuurieurileurleurieurieurieurieurileRadix BBRadRad Bdix Bdixx R i Bdi BBBadix Radix BBuple
RehmanniaRe mRe m nnRehmanniamReRehmhmannnniamanniaRehmehmanniRR nosasasasaosaosglutilutglutgluulutiglgl titinos
LiboschLibLLibo hschib chLiboschboscLLib chschLibos
icaecaecacaeicaeicAngelAnAngeAngelAnAng lAAngeelielicdix dix dixdixdSinensinensSS nennensSinSSiSinensSinensSine sissis Rad
FloslololFlhamaCartharthhaamami Fl
menmennnnmicae Scae SSem
Figure 6 HB-pC-pT network of XFZYD for treating TBI The triangles with circle backgrounds represent the herbs (HB) the squares andcircles represent the potential compounds (pC) and targets (pT)The red triangles squares and circles represent corresponding HB pC andpT in the Junherbs the same is to aqua representing theChen herbs and periwinkle representing the Zuo-Shi herbsThe claybank squares andcircles represent corresponding pC and pT shared by the Chen and Zuo-Shi herbs the same is to blue representing the overlap between theJun and Zuo-Shi herbs and the green representing the overlap between the Chen and Zuo-Shi herbsThe purple squares and circles representthe corresponding pC and pT shared by the 3 groups of herbs
alleviating insulin resistance in type 2 diabetic rats [72]Beta-sitosterol (BS) is a vegetable-derived compound foundin various plants and is suggested to modulate the immunefunction inflammation and pain levels by controlling theproduction of inflammatory cytokines [73]
For targets analysis CALM possesses the largest degree(degree =84) followed by GSK3B (degree =61) SCN5A(degree = 59) F2 (degree = 43) ACHE (degree=28)F7 (degree=28) ADRA1B (degree=28) NOS3 (degree=20)BCL2 (degree=18) and CASP3 (degree=18) which demon-strated their crucial therapeutic effects for treating TBI Forinstance CALM possess an essential position in calciumsignaling pathway and is related to morphological changesmigration proliferation and secretion of cytokines andreactive oxygen species ofMicroglial cells [74]The activationof CaMKII120572 major isoform of Ca2+calmodulin-dependentprotein kinase (CaMK) in brain is directly associated withthe production of proinflammatory cytokines such as TNF-120572and IL-1120573 [75] GSK-3120573 is a serinethreonine-protein kinasewhich is abundant in the central nervous system (CNS)particularly in neurons [76] It can control gene transcrip-tion axonal transport and cytoskeletal dynamics in growthcones [77] The inhibition of GSK-3120573 attenuates apoptotic
signals and prevents neuronal death [78] There is increasingevidence that prothrombin (F2) and its active derivativethrombin are expressed locally in the central nervous systemBeside the central role in the coagulation cascade the gener-ation of thrombin leads to receptor mediated inflammatoryresponses cell proliferationmodulation cell protection andapoptosis [79 80] The role in brain injury depends upon itsconcentration as higher amounts cause neuroinflammationand apoptosis while lower concentrations might even becytoprotective [81]
Direct tissue damage aswell as hypoxic-ischemic increas-ing anaerobic glycolysis of the brain tissue results in theATP-stores depletion and failure of energy-dependent mem-brane ion pumps especially for voltage-dependent Ca2+ andNa+-channels Accumulated Ca2+ activates lipid peroxidasesproteases and phospholipases and caspases at the sametime increasing the intracellular concentration of free fattyacids and free radicals leading to necrosis or apoptosis ofneurocyte [82] At the same time the activation of residentglial cells microglia and astrocytes and the infiltration ofblood leukocytes secrete various immune mediators elicitedinflammatory responses which subsequently intersect withadjacent pathological cascades including oxidative stress
12 Evidence-Based Complementary and Alternative Medicine
Table 6 Top 10 potential candidate compounds of XFZYD for treating TBI according to 2 centrality indicators
Compound Degree Compound Betweennessquercetin 76 quercetin 01682179kaempferol 43 kaempferol 005501511beta-sitosterol 35 wogonin 005141376Stigmasterol 19 beta-sitosterol 004451294luteolin 18 naringenin 003251738baicalein 15 beta-carotene 002977217-Methoxy-2-methyl isoflavone 12 nobiletin 002787992wogonin 12 7-Methoxy-2-methyl isoflavone 002096439nobiletin 11 luteolin 002090223naringenin 9 baicalein 001453488
Table 7 Top 10 potential targets of XFZYD for treating TBI according to 2 centrality indicators
Targets Degree Targets BetweennessCALM 84 CALM 021452046GSK3B 61 SCN5A 011441591SCN5A 59 GSK3B 009553896F2 43 F2 00689171ACHE 28 BCL2 002651903F7 28 CASP3 002372836ADRA1B 28 F7 002032647NOS3 20 ACHE 001845996BCL2 18 ADRA1B 001704505CASP3 18 NOS3 00166699
excitotoxicity or reparative events including angiogenesisscarring and neurogenesis [83] Function analysis of the 47target proteins regulated by XFZYD was mainly associatedwith core pathophysiology process of TBI (Figure 7) 47 targetproteins were connected with 16 key process related to TBIMost of the targets have one or more links to other biolog-ical process such as apoptosis cell proliferation superoxideanion generation nitric oxide biosynthetic process responseto calcium ion I-kappaB kinaseNF-kappaB signaling andregulation of inflammation The above analysis implied themultifunction character of these target proteins regulated byXFZYD Of these target proteins 25 proteins (53 of the 47)such as TGFB1 EGFR CAV1 MAPK1 PRKCB and AKT1were responsible for regulating the apoptosis process 17 forblood coagulation 14 for cell proliferation and axon genesisand 12 for hypoxia andMAPK cascade We found that TGFB1was the crucial protein because it participated in 9 biologicalprocesses related to TBI such as apoptosis blood coagulationcell proliferation and MAPK cascade followed by EGFR (8)CAV1 (7) MAPK1 (6) PRKCB (6) and AKT1 (6)
Overall these observations strongly support the evidencethat the generated HB-pCminuspT network and pT-F networkhave important roles in treating TBI further validating thedrug targeting approach
Molecule docking was used to further validate the bind-ing mode between candidate compounds and their targetproteins We found that 18 TBI-specific target proteins inter-acted with 91 candidate compounds from XFZYD (Figure 8)
Other 29 target proteins were not discussed for the lackof proper protein crystal structure The detailed moleculedocking results are shown in Table S5The 6 essential proteinsincluding GSK3B AKT1 CDK1 F2 NOS3 and ACHEwere used to elucidate the exact binding mode (Figure 9)Quercetin was located within the binding cavity of AKT1 andCDK1 (Figures 9(a) and 9(b) and S1A B) Four conventionalhydrogen bonds were formed between quercetin and AKT1by interacting with the key amino acids including ILE-290 THR-211 and SER 205 Additionally 120587-120587 interactionsbetween quercetin and TRP-90 were found in the activesite which helped the stabilization of the compound at thebinding site (Figure 9(a) S1A) Figure 9(b) and S1B suggestedthat five conventional hydrogen bonds (LEU-83 ASP-146and LYS-33) and 120587-120587 interaction (PHE-80) were formedbetween quercetin and CDK1 The GSK3B-FA complexes(Figure 9(c) S1C) were stabilized by 6 hydrogen-bondinginteractions between FA and LYS-85 GLU-97 TYR-134 andARG-141 Glyasperin Bmainly bonds to F2 through hydrogenbonds by interacting with the key amino acids includingGLY-193 SER-195 and GLY-219 (Figure 9(d) S1D) andan edge-to-face 120587 minus 120587 interaction was also observed withTYR-228 The GLN-247 GLU-351 and GLY-355 from theactive site pocket of NOS3 participated in the hydrogen-bondformationwith 1-Methoxyphaseollidin (Figure 9(e) S1E)The(-)-Medicocarpin formed a total of 6 hydrogen bonds withSER-293 PHE-295 ARG-296 and TRY-341 in the active siteof ACHE (Figure 9(f) S1F) Besides an edge-to-face 120587 minus 120587
Evidence-Based Complementary and Alternative Medicine 13
KCNMA1
P53
IFNG
PPP3CA
CASP8
SCN5A
I-kappaB kinase NF-kappaB signaling
Regulation of inflammation
Response to calcium ion
TGFB1
TNF
PRKCA
CHRNA7
CAV1
STAT1
MAPK cascadeAKT1
BCL2
APP
EGFR
MAPK1
Angiogenesis
SYNGAP1
MAP2
PTPN1
Axonogenesis
GSK3B
EPHB2
CDK1
CASP7FN1
MAPK3
CTNNB1
F2
Autophagy
Cell proliferation
BACE1
SLC6A3
DRD1
ADRA1BACHE
CALMCASP3
PLAT
Apoptotic process
NOS3
PRKCB
Blood coagulation
Response to hypoxia
Superoxide anion generation
Nitric oxide biosynthetic process
Astrocyte activation
Amyloid-beta regulation
Microglial cell activation
LDLR
PRSS3
F7
PRKCD
SOD1ALB
BBC3
Figure 7 pT-F network of XFZYD for treating TBI 16 biological processes (red square) the 47 target proteins (periwinkle circle) of XFZYDparticipate in for treating TBI
interactionwas also observedwith TYR-337 From the resultshydrogen-bonding and edge-to-face 120587 minus 120587 interactions playkey roles in the proteinminusligand recognition and stabilitywhich may be helpful in determining the inhibitor activitiesAnd the C-T network confirmed the potential therapeuticeffects of the candidate compounds from XFZYD to treatTBI through interacting with the relevant proteins The com-putational analysis further elucidated the accurate moleculemechanisms between active compounds and targets
4 Discussion
Traumatic brain injury (TBI) is a growing public healthproblem worldwide and is a leading cause of death anddisability [84] Although major progress has been made in
understanding the pathophysiology of this injury this hasnot yet led to substantial improvements in outcome by alack of treatments which have proven successful during phaseIII trials for modern medicine [85 86] TCM rooted inthousands of years of history may offer an alternative or acomplementary strategy for the treatment of TBI XFZYDa representative formula in TCM has been used for yearsto treat TBI in China and has been demonstrated to beeffective in clinical practice However its ldquomulticomponentsrdquoand ldquomultitargetsrdquo features make it much difficult to decipherthemolecular mechanisms of XFZYD in the treatment of TBIfrom a systematic perspective if employing routine methods
In the present study a network pharmacology-basedmethod was employed to elucidate the pharmacologicalmechanisms of XFZYD to treat TBI according to the drug
14 Evidence-Based Complementary and Alternative Medicine
Figure 8 C-T network through molecule docking validating 119 potential compounds (green triangles) interacting with 18 potential targets(yellow circles) of XFZYDThe size of the nodes is proportional to the value of degree
combination principle of TCM We first proposed a newmodeling system combining OB and DL screening multipledrug targets prediction and validation network constructionand molecule docking to probe the efficiency of a typicalTCM formula XFZYD for the treatment of TBI The 11herbs from XFZYD possessed 162 bioactive compounds andtargeted 285 proteins There were 5 compounds and 189
target proteins overlapped among the Jun Chen and Zuo-Shigroup Furthermore 47 TBI-specific proteins were targetedby 119 (73) bioactive compounds from XFZYD Similarly 5common compounds and 33 (70) common target proteinsamong the 3 groups of drugs were observed Most of thebioactive ingredients targeted more than one protein The 47target proteins regulated several essential pathophysiological
Evidence-Based Complementary and Alternative Medicine 15
(a) (b) (c)
(d) (e) (f)
Figure 9 Hydrogen-bonding networks within the binding site of the compoundminustarget complexes obtained from molecule docking(a) AKT1-quercetin (b) CDK1-quercetin (c) GSK3B-FA (d) F2-glyasperin B (e) NOS3-1-Methoxyphaseollidin and (f) ACHE-(-)-Medicocarpin The molecules are presented as ball and stick models Active site amino acid residues are represented as lines Dotted bluelines in these pictures represent hydrogen bonds with distance unit of A Other O and N atoms are colored as red and blue respectively
processes of TBI that referred to apoptosis inflammationcell proliferation superoxide anion generation nitric oxidebiosynthetic process response to calcium ion etc The aboveanalysis reveals that the synergistic action mechanisms ofXFZYDmay be (1) bioactive compounds overlapping amongthe different group of herbs (2) specific bioactive com-pounds from different groups of herbs targeting the sameproteins (3) specific bioactive compounds from differentgroups of herbs targeting different proteins which participatein the same pathophysiological process of the disease Toa certain degree the 5 compounds including quercetinstigmasterol kaempferol baicalin and beta-sitosterol playedessential role in XFZYD for TBI treatment MAPK3MAPK1AKT1 PRKCA TNF PRKCB EGFR BCL2 GSK3B CASP3PPP3CA and NOS3 were the main target proteins regulatedby XFZYD in the treatment of TBI
Interestingly Beta-carotene (Mol 43) from Carthami Flos(the Jun herb) specifically regulated 120573-Catenin (CTNNB1)and played critical role for curing TBI The 120573-Catenin is acritical downstream component of the Wnt pathway whichplays essential role in the regulation of mammalian neuraldevelopment [87] In vitro and in vivo studies demonstrate
that the Wnt120573-catenin pathway regulates the proliferationand differentiation of neural progenitor cells [88] Neuronaldifferentiation is induced by overexpression of 120573-cateninor the pharmacological inhibition of GSK3120573 (the phospho-rylating enzyme of 120573-catenin) [89 90] This pathway alsopromotes blood vessel formation during vascular develop-ment as well as the vascular repair process after TBI [91]In addition wogonin (Mol 160) from Achyranthis BidentataeRadix (the Chen herb) specifically targeted fibronectin (FN1)Bcl-2-binding component 3 (BBC3) and Protein KinaseC delta type (PRKCD) FN1 an important component ofthe extracellular matrix (ECM) environment promotes cellmigration neurite outgrowth and synapse formation dur-ing neural development [92] It aggregates in the injuredbrain and plays a neuroprotection role through antiapoptosisand anti-inflammation ways following TBI [93 94] BBC3namely p53 upregulated modulator of apoptosis (PUMA) iscritical for the p53-dependent apoptosis pathway which playsan important role in hippocampal neuronal loss and associ-ated cognitive deficits [95] PRKCD one of PKC isoformsactivates signal transduction pathways involved in neuronalregeneration [96] synaptic transmissionplasticity [97] and
16 Evidence-Based Complementary and Alternative Medicine
activation of apoptosis processes [98] as well as higher brainfunctions such as learning andmemory [99]The activators ofPKCare effective for the treatment of TBI [100] FurthermoreMitogen-activated protein kinase 3 (MAPK3) Beta-secretase1 (BACE1) Ephrin type-B receptor 2 (EphB) and low-densitylipoprotein receptor (LDLR) were specifically targeted bythe ingredients in the Zuo-Shi herbs Naringenin (Mol133) targeted LDLR MAPK3 while euchrenone (Mol 60)anchored BACE1 and nobiletin (Mol 134) targeted EphB2MAPK3 is an essential component of the MAP kinase signaltransduction pathway It has been appreciated recently thatthe ERK12 cascade plays a fundamental role in synapticplasticity and memory [101] BACE1 is responsible for pro-duction of A120573 from amyloid precursor protein (APP) A120573can cause cell death activate inflammatory pathways [102]and prime proapoptotic pathways for activation by otherinsults [103] The blocking of BACE1 can ameliorate motorand cognitive deficits and reduce cell loss after experimentalTBI in mice [104] EphB is localized to synaptic sites inhippocampal neurons [105] The interaction between EphBand NMDA receptors regulates excitatory synapse formation[106] LDLR acts as an important receptor that facilitatesbrain A120573 clearance and inhibits amyloid deposition [107]and then ameliorates Alzheimerrsquos disease neuropathologyafter TBI [108] The analysis above indicates the ldquoJunrdquoldquoChenrdquo and ldquoZuo-Shirdquo herbs from XFZYD trigger theirspecific targets regulation respectively for the therapeuticeffects
XFZYD is a very famous traditional Chinese formula inpromoting qi circulation and removing blood stasis accord-ing to TCM theory However several limited researches havedemonstrated its efficacy for treating TBI such as anti-inflammatory and synaptic regulation [30 31] which arein accord with our study However previous studies merelypartially deciphered the molecule mechanism of XFZYD fortreating TBI This study reports 119 bioactive compounds inXFZYD that target 47 TBI-specific proteins such as MAPK3MAPK1 AKT1 PRKCA TNF PRKCB and EGFR Theseproteins regulate several crucial pathophysiological processesof TBI such as apoptosis inflammation blood coagulationand axon genesis Our study demonstrates that the therapeu-tic actions of XFZYD refer to ldquomulticompoundsrdquo ldquomultitar-getsrdquo features rather than only the improvement of bloodcirculation With the help of molecule docking methodwe further validate the interactions between bioactive com-pounds and potential targets of XFZYD The hydrogen-bonding and edge-to-face 120587 minus120587 interactions play key roles inthe proteinminusligand recognition and stability This provides avaluable reference for further experimental investigations ofbioactive ingredients and therapeutic targets of XFZYD fortreating TBI
5 Conclusion
Our work successfully illuminates the efficiency of XFZYDfor the treatment of TBI as well as herb combination ruleof TCM formula Network pharmacology with moleculedocking method confirms the ldquomulticompounds multitar-getsrdquo therapeutic actions of XFZYD in the treatment of TBI
The present work may provide valuable evidence for furtherclinical application of XFZYD for treating TBI
Abbreviations
TBI Traumatic brain injuryXFZYD Xuefu Zhuyu decoctionTCM Traditional Chines medicineDL Drug-likenessOB Oral bioavailabilityHB-cC-cT Herb-candidate compound-candidate
targetHB-pC-pT Herb-potential compound-potential targetCALM CalmodulinGSK3B Glycogen synthase kinase-3 betaSCN5A Sodium channel protein type 5 subunit
alphaF2 ProthrombinACHE AcetylcholinesteraseGO Gene OntologyKEGG Kyoto Encyclopedia of Genes and
Genomes
Data Availability
The data used to support the findings of this study areavailable from the corresponding author upon request
Conflicts of Interest
The authors declare that they have no conflicts of interest
Authorsrsquo Contributions
YangWang and Zebing Huang participated in the conceptionand design of the study YuanyuanZhong YangWang ZebingHuang Jiekun Luo Tao Tang and Pengfei Li acquired andanalyzed the data Yuanyuan Zhong Tao Liu and HanjinCui drafted and revised the manuscript The correspondingauthor and all of the authors have read and approved the finalsubmitted manuscript
Acknowledgments
This work was supported by the National Natural ScienceFoundation of China (Nos 81673719 81874409 81603670and 81803948) Project funded byChina Postdoctoral ScienceFoundation (Nos 2016M600639 and 2017T100614)
Supplementary Materials
Table S1 162 bioactive compounds in XFZYD Table S2 targetproteins of XFZYD Table S3 TBI-specific proteins Table S4119 potential compounds of XFZYD for treating TBI TableS5 docking result of 18 target proteins with 91 potential com-pounds Fig S1 the exact bindingmode between active ingre-dients and protein targets obtained from molecule docking(A) AKT1-quercetin (B) CDK1-quercetin (C) GSK3B-FA
Evidence-Based Complementary and Alternative Medicine 17
(D) F2-glyasperin B (E) NOS3-1-Methoxyphaseollidin and(F)ACHE-(-)-Medicocarpin (Supplementary Materials)
References
[1] L Yang Y Chu D Tweedie et al ldquoPost-trauma administrationof the pifithrin-120572 oxygen analog improves histological andfunctional outcomes after experimental traumatic brain injuryrdquoExperimental Neurology vol 269 pp 56ndash66 2015
[2] J A Langlois W Rutland-Brown and M M Wald ldquoTheepidemiology and impact of traumatic brain injury a briefoverviewrdquo The Journal of Head Trauma Rehabilitation vol 21no 5 pp 375ndash378 2006
[3] H A Alsulaim B J Smart A O Asemota et al ldquoConsciousstatus predictsmortality among patientswith isolated traumaticbrain injury in administrative datardquo The American Journal ofSurgery vol 214 no 2 pp 207ndash210 2017
[4] M Majdan D Plancikova A Maas et al ldquoYears of life lost dueto traumatic brain injury in Europe A cross-sectional analysisof 16 countriesrdquo PLoS Medicine vol 14 no 7 p e1002331 2017
[5] P Cheng P Yin P Ning et al ldquoTrends in traumatic braininjury mortality in China 2006ndash2013 A population-basedlongitudinal studyrdquo PLoS Medicine vol 14 no 7 Article IDe1002332 2017
[6] M V Russo and D B McGavern ldquoInflammatory neuroprotec-tion following traumatic brain injuryrdquo Science vol 353 no 6301pp 783ndash785 2016
[7] WWang H Li J Yu et al ldquoProtective Effects of ChineseHerbalMedicine Rhizoma drynariae in Rats After Traumatic BrainInjury and Identification of Active Compoundrdquo MolecularNeurobiology vol 53 no 7 pp 4809ndash4820 2016
[8] M Das S Mohapatra and S S Mohapatra ldquoNew perspectiveson central and peripheral immune responses to acute traumaticbrain injuryrdquo Journal of Neuroinflammation vol 9 p 236 2012
[9] J W Finnie ldquoNeuroinflammation Beneficial and detrimentaleffects after traumatic brain injuryrdquo Inflammopharmacologyvol 21 no 4 pp 309ndash320 2013
[10] T Cheng W Wang Q Li et al ldquoCerebroprotection of flavanol(minus)-epicatechin after traumatic brain injury viaNrf2-dependentand -independent pathwaysrdquo Free Radical Biology amp Medicinevol 92 pp 15ndash28 2016
[11] W Young ldquoRole of calcium in central nervous system injuriesrdquoJournal of Neurotrauma vol 9 Suppl 1 pp S9ndashS25 1992
[12] R Bullock A Zauner J S Myseros et al ldquoEvidence forProlonged Release of Excitatory Amino Acids in Severe HumanHead Trauma Relationship to Clinical Eventsrdquo Annals of theNew York Academy of Sciences vol 765 no 1 pp 290ndash297 1995
[13] A T Mazzeo A Beat A Singh and M R Bullock ldquoTherole of mitochondrial transition pore and its modulation intraumatic brain injury and delayed neurodegeneration afterTBIrdquo Experimental Neurology vol 218 no 2 pp 363ndash370 2009
[14] S Gennai A Monsel Q Hao et al ldquoCell-Based therapy fortraumatic brain injuryrdquo British Journal of Anaesthesia vol 115no 2 pp 203ndash212 2015
[15] J Ghajar ldquoTraumatic brain injuryrdquo The Lancet vol 356 no9233 pp 923ndash929 2000
[16] D J Loane and A I Faden ldquoNeuroprotection for traumaticbrain injury translational challenges and emerging therapeuticstrategiesrdquoTrends in Pharmacological Sciences vol 31 no 12 pp596ndash604 2010
[17] Y Wang X Fan T Tang et al ldquoRhein and rhubarb simi-larly protect the blood-brain barrier after experimental trau-matic brain injury via gp91phox subunit of NADPH oxidaseROSERKMMP-9 signaling pathwayrdquo Scientific Reports vol 6p 37098 2016
[18] D-X Kong X-J Li and H-Y Zhang ldquoWhere is the hopefor drug discovery Let history tell the futurerdquo Drug DiscoveryTherapy vol 14 no 3-4 pp 115ndash119 2009
[19] F Cheung ldquoTCM made in Chinardquo Nature vol 480 no 7378pp S82ndashS83 2011
[20] C Fu Z Xia Y Liu et al ldquoQualitative analysis of majorconstituents from Xue Fu Zhu Yu Decoction using ultra highperformance liquid chromatographywith hybrid ion trap time-of-flight mass spectrometryrdquo Journal of Separation Science vol39 no 17 pp 3457ndash3468 2016
[21] H-J Zhang and Y-Y Cheng ldquoAn HPLCMS method for iden-tifying major constituents in the hypocholesterolemic extractsof Chinese medicine formula lsquoXue-Fu-Zhu-Yu decoctionrsquordquoBiomedical Chromatography vol 20 no 8 pp 821ndash826 2006
[22] L Zhang L Zhu YWang et al ldquoCharacterization and quantifi-cation of major constituents of Xue Fu Zhu Yu by UPLC-DAD-MSMSrdquo Journal of Pharmaceutical and Biomedical Analysisvol 62 pp 203ndash209 2012
[23] X Yang X Xiong G Yang and J Wang ldquoChinese patentmedicine Xuefu Zhuyu capsule for the treatment of unstableangina pectoris a systematic review of randomized controlledtrialsrdquo Complementary Therapies in Medicine vol 22 no 2 pp391ndash399 2014
[24] J Wang X Yang F Chu et al ldquoThe effects of Xuefu Zhuyuand Shengmai on the evolution of syndromes and inflammatorymarkers in patients with unstable angina pectoris after percuta-neous coronary intervention a randomised controlled clinicaltrialrdquoEvidence-BasedComplementary andAlternativeMedicinevol 2013 Article ID 896467 9 pages 2013
[25] Q Zhang H Yu J Qi et al ldquoNatural formulas and the natureof formulas Exploring potential therapeutic targets based ontraditional Chinese herbal formulasrdquo PLoS ONE vol 12 no 2p e0171628 2017
[26] J LeeWHsu T Yen et al ldquoTraditional ChinesemedicineXue-Fu-Zhu-Yu decoction potentiates tissue plasminogen activatoragainst thromboembolic stroke in ratsrdquo Journal of Ethnophar-macology vol 134 no 3 pp 824ndash830 2011
[27] Y-C Shen C-K Lu K-T Liou et al ldquoCommon and uniquemechanisms of Chinese herbal remedies on ischemic strokemice revealed by transcriptome analysesrdquo Journal of Ethnophar-macology vol 173 pp 370ndash382 2015
[28] F Xue-hai G Yan and L Jian-tao ldquoTherapeutic effect of XiefuZhuyu decoction joint brain protein hydrolysat injection intraumatic brain injury and its effect on cerebrospinal fluid ofET-1rdquo Chinese Journal of Biochemical Pharmaceutics no 02 pp55ndash58 2017
[29] L Shuxiang W Jingchun C Jie et al ldquoEffect of Xuefu Zhuyudecoction on the neural functional recovery and living abilityin patients with craniocerebral injuryrdquo Modern Journal ofIntegrated Traditional Chinese andWesternMedicine no 04 pp350ndash352 2015
[30] L Zhong W Ning and W Dong-pi ldquoModel Study of theTherapy of Replenishing Qi and Activating Blood Circulationon Protecting the Brain Impairment Caused by Hypoxic -ischemic Encephalopathy in SD Ratsrdquo Journal of TraditionalChinese Medicine vol 07 pp 1552ndash1555 2011
18 Evidence-Based Complementary and Alternative Medicine
[31] M Sun X-P Zhan C-Y Jin J-Z Shan S Xu and Y-LWang ldquoclinical observation on treatment of post-craniocerebraltraumatic mental disorder by integrative medicinerdquo ChineseJournal of Integrative Medicine vol 14 no 2 pp 137ndash141 2008
[32] Z Xing Z Xia W Peng et al ldquoXuefu Zhuyu decoction atraditional Chinese medicine provides neuroprotection in arat model of traumatic brain injury via an anti-inflammatorypathwayrdquo Scientific Reports vol 6 Article ID 20040 2016
[33] J Zhou T Liu H Cui et al ldquoXuefu zhuyu decoction improvescognitive impairment in experimental traumatic brain injuryvia synaptic regulationrdquo Oncotarget vol 8 no 42 2017
[34] S Li ldquoExploring traditional chinese medicine by a novel thera-peutic concept of network targetrdquo Chinese Journal of IntegrativeMedicine vol 22 no 9 pp 647ndash652 2016
[35] S Li and B Zhang ldquoTraditional Chinese medicine networkpharmacology theory methodology and applicationrdquo ChineseJournal of Natural Medicines vol 11 no 2 pp 110ndash120 2013
[36] A L Hopkins ldquoNetwork pharmacology the next paradigm indrug discoveryrdquoNature Chemical Biology vol 4 no 11 pp 682ndash690 2008
[37] Y Li C Han J Wang et al ldquoInvestigation into the mechanismof Eucommia ulmoides Oliv based on a systems pharmacologyapproachrdquo Journal of Ethnopharmacology vol 151 no 1 pp452ndash460 2014
[38] Y Yao X Zhang Z Wang et al ldquoDeciphering the combinationprinciples of Traditional Chinese Medicine from a systemspharmacology perspective based on Ma-huang DecoctionrdquoJournal of Ethnopharmacology vol 150 no 2 pp 619ndash638 2013
[39] Bo Zhang Xu Wang and Shao Li ldquoAn Integrative Platform ofTCM Network Pharmacology and Its Application on a HerbalFormula Qing-Luo-Yinrdquo Evidence-Based Complementary andAlternativeMedicine vol 2013 Article ID 456747 12 pages 2013
[40] J Ru P Li J Wang et al ldquoTCMSP a database of systemspharmacology for drug discovery from herbal medicinesrdquoJournal of Cheminformatics vol 6 no 1 p 13 2014
[41] J V Turner D J Maddalena and S Agatonovic-KustrinldquoBioavailability Prediction Based on Molecular Structure for aDiverse Series of Drugsrdquo Pharmaceutical Research vol 21 no 1pp 68ndash82 2004
[42] X XuW Zhang CHuang et al ldquoAnovel chemometricmethodfor the prediction of human oral bioavailabilityrdquo InternationalJournal of Molecular Sciences vol 13 no 6 pp 6964ndash6982 2012
[43] A Zhang W Pan J Lv and H Wu ldquoProtective Effect ofAmygdalin on LPS-Induced Acute Lung Injury by InhibitingNF-120581B and NLRP3 Signaling Pathwaysrdquo Inflammation vol 40no 3 pp 745ndash751 2017
[44] X Wei H Liu X Sun et al ldquoHydroxysafflor yellow A protectsrat brains against ischemia-reperfusion injury by antioxidantactionrdquo Neuroscience Letters vol 386 no 1 pp 58ndash62 2005
[45] W PWalters andM A Murcko ldquoPrediction of lsquodrug-likenessrsquordquoAdvanced Drug Delivery Reviews vol 54 no 3 pp 255ndash2712002
[46] Y Yamanishi M Kotera M Kanehisa and S Goto ldquoDrug-target interactionprediction from chemical genomic and phar-macological data in an integrated frameworkrdquo Bioinformaticsvol 26 no 12 pp i246ndashi254 2010
[47] H Yu J Chen X Xu et al ldquoA systematic prediction ofmultiple drug-target interactions from chemical genomic andpharmacological datardquo PLoS ONE vol 7 no 5 Article IDe37608 2012
[48] X Chen Z L Ji and Y Z Chen ldquoTTD therapeutic targetdatabaserdquo Nucleic Acids Research vol 30 no 1 pp 412ndash4152002
[49] A Hamosh A F Scott J S Amberger C A Bocchini andV AMcKusick ldquoOnline Mendelian Inheritance in Man (OMIM) aknowledgebase of human genes and genetic disordersrdquo NucleicAcids Research vol 33 pp D514ndashD517 2005
[50] T S Keshava Prasad RGoel K Kandasamy et al ldquoHuman pro-tein reference databasemdash2009 updaterdquo Nucleic Acids Researchvol 37 no 1 pp D767ndashD772 2009
[51] A Franceschini D Szklarczyk S Frankild et al ldquoSTRING v91protein-protein interaction networks with increased coverageand integrationrdquoNucleic Acids Research vol 41 no 1 pp D808ndashD815 2013
[52] P Shannon A Markiel O Ozier et al ldquoCytoscape a softwareEnvironment for integratedmodels of biomolecular interactionnetworksrdquo Genome Research vol 13 no 11 pp 2498ndash25042003
[53] G Dennis Jr B T Sherman D A Hosack et al ldquoDAVIDDatabase for Annotation Visualization and IntegratedDiscov-eryrdquo Genome Biology vol 4 no 5 p P3 2003
[54] L Yang X Zhao and X Tang ldquoPredicting disease-related pro-teins based on clique backbone in protein-protein interactionnetworkrdquo International Journal of Biological Sciences vol 10 no7 pp 677ndash688 2014
[55] S MThomas and J S Brugge ldquoCellular functions regulated bySRC family kinasesrdquo Annual Review of Cell and DevelopmentalBiology vol 13 pp 513ndash609 1997
[56] D Z Liu F R Sharp K C Van et al ldquoInhibition of Src familykinases protects hippocampal neurons and improves cognitivefunction after traumatic brain injuryrdquo Journal of Neurotraumavol 31 no 14 pp 1268ndash1276 2014
[57] B D Manning and A Toker ldquoAKTPKB Signaling Navigatingthe Networkrdquo Cell vol 169 no 3 pp 381ndash405 2017
[58] Y Kitagishi and S Matsuda ldquoDiets involved in PPAR andPI3KAKTPTEN pathway may contribute to neuroprotectionin a traumatic brain injuryrdquoAlzheimerrsquos ResearchampTherapy vol5 no 5 p 42 2013
[59] D Chen L Bao S-Q Lu and F Xu ldquoSerum albumin andprealbuminpredict the poor outcomeof traumatic brain injuryrdquoPLoS ONE vol 9 no 3 Article ID e93167 2014
[60] J Yang Z Wu N Renier et al ldquoPathological axonal deaththrough aMapk cascade that triggers a local energy deficitrdquoCellvol 160 no 1-2 pp 161ndash176 2015
[61] E J Huang and L F Reichardt ldquoTrk receptors roles in neuronalsignal transductionrdquoAnnual Review of Biochemistry vol 72 pp609ndash642 2003
[62] J W Lee and R Juliano ldquoMitogenic signal transductionby integrin- and growth factor receptor-mediated pathwaysrdquoMolecules and Cells vol 17 no 2 pp 188ndash202 2004
[63] C S Yang J M Landau M T Huang and H L NewmarkldquoInhibition of carcinogenesis by dietary polyphenolic com-poundsrdquo Annual Review of Nutrition vol 21 pp 381ndash406 2001
[64] A Murakami H Ashida and J Terao ldquoMultitargeted cancerprevention by quercetinrdquoCancer Letters vol 269 no 2 pp 315ndash325 2008
[65] G Du Z Zhao Y Chen et al ldquoQuercetin attenuates neuronalautophagy and apoptosis in rat traumatic brain injury modelvia activation of PI3KAkt signaling pathwayrdquo NeurologicalResearch vol 38 no 11 pp 1ndash8 2016
Evidence-Based Complementary and Alternative Medicine 19
[66] Q Cai R O Rahn and R Zhang ldquoDietary flavonoidsquercetin luteolin andgenistein reduce oxidativeDNAdamageand lipid peroxidation and quench free radicalsrdquoCancer Lettersvol 119 no 1 pp 99ndash107 1997
[67] G A Bongiovanni E A Soria and A R Eynard ldquoEffectsof the plant flavonoids silymarin and quercetin on arsenite-induced oxidative stress in CHO-K1 cellsrdquo Food and ChemicalToxicology vol 45 no 6 pp 971ndash976 2007
[68] H Li L Yang Y Zhang et al ldquoKaempferol inhibits fibroblastcollagen synthesis proliferation and activation in hypertrophicscar via targeting TGF-beta receptor type Irdquo Biomedicine ampPharmacotherapy = Biomedecine amp Pharmacotherapie vol 83pp 967ndash974 2016
[69] K P Devi D S Malar S F Nabavi et al ldquoKaempferol andinflammation From chemistry to medicinerdquo PharmacologicalResearch vol 99 pp 1ndash10 2015
[70] M Zhou H Ren J Han W Wang Q Zheng and DWang ldquoProtective effects of kaempferol against myocardialischemiareperfusion injury in isolated rat heart via antioxidantactivity and inhibition of glycogen synthase kinase-3120573rdquo Oxida-tive Medicine and Cellular Longevity vol 2015 p 481405 2015
[71] C-F Lee J-S Yang F-J Tsai et al ldquoKaempferol inducesATMp53-mediated death receptor and mitochondrial apop-tosis in human umbilical vein endothelial cellsrdquo InternationalJournal of Oncology vol 48 no 5 pp 2007ndash2014 2016
[72] C Luo H Yang C Tang et al ldquoKaempferol alleviates insulinresistance via hepatic IKKNF-120581B signal in type 2 diabetic ratsrdquoInternational Immunopharmacology vol 28 no 1 pp 744ndash7502015
[73] A B Awad and C S Fink ldquoPhytosterols as anticancer dietarycomponents evidence and mechanism of actionrdquo Journal ofNutrition vol 130 no 9 pp 2127ndash2130 2000
[74] K Farber and H Kettenmann ldquoFunctional role of calciumsignals for microglial functionrdquoGlia vol 54 no 7 pp 656ndash6652006
[75] Y Lu Y Gu X Ding et al ldquoIntracellular Ca2+ homeostasisand JAK1STAT3 pathway are involved in the protective effectof propofol on BV2 microglia against hypoxia-induced inflam-mation and apoptosisrdquo PLoS ONE vol 12 no 5 p e01780982017
[76] K Leroy and J-P Brion ldquoDevelopmental expression and local-ization of glycogen synthase kinase-3120573 in rat brainrdquo Journal ofChemical Neuroanatomy vol 16 no 4 pp 279ndash293 1999
[77] C-M Liu E-M Hur and F-Q Zhou ldquoCoordinating geneexpression and axon assembly to control axon growth Potentialrole ofGSK3 signalingrdquo Frontiers inMolecularNeuroscience vol3 p 3 2012
[78] D A E Cross A A Culbert K A Chalmers L Facci S DSkaper and A D Reith ldquoSelective small-molecule inhibitors ofglycogen synthase kinase-3 activity protect primary neuronesfrom deathrdquo Journal of Neurochemistry vol 77 no 1 pp 94ndash102 2001
[79] V S Ossovskaya and NW Bunnett ldquoProtease-activated recep-tors contribution to physiology and diseaserdquo PhysiologicalReviews vol 84 no 2 pp 579ndash621 2004
[80] M Steinhoff J Buddenkotte V Shpacovitch et al ldquoProteinase-activated receptors transducers of proteinase-mediated signal-ing in inflammation and immune responserdquoEndocrine Reviewsvol 26 no 1 pp 1ndash43 2005
[81] H Krenzlin V Lorenz S Danckwardt O Kempski and BAlessandri ldquoThe importance of thrombin in cerebral injury and
diseaserdquo International Journal of Molecular Sciences vol 17 no1 2016
[82] M J McGinn and J T Povlishock ldquoPathophysiology of Trau-matic Brain InjuryrdquoNeurosurgery Clinics of North America vol27 no 4 pp 397ndash407 2016
[83] T Woodcock and M C Morganti-Kossmann ldquoThe role ofmarkers of inflammation in traumatic brain injuryrdquo Frontiersin Neurology vol 4 article 18 2013
[84] F Tanriverdi H J Schneider G Aimaretti B E Masel F FCasanueva and F Kelestimur ldquoPituitary dysfunction after trau-matic brain injury A clinical and pathophysiological approachrdquoEndocrine Reviews vol 36 no 3 pp 305ndash342 2015
[85] J V Rosenfeld A I Maas P Bragge M C Morganti-Kossmann G T Manley and R L Gruen ldquoEarly managementof severe traumatic brain injuryrdquoThe Lancet vol 380 no 9847pp 1088ndash1098 2012
[86] B M Aertker S Bedi and C S Cox ldquoStrategies for CNS repairfollowing TBIrdquo Experimental Neurology vol 275 no 3 pp 411ndash426 2016
[87] K Adachi Z Mirzadeh M Sakaguchi et al ldquo120573-catenin signal-ing promotes proliferation of progenitor cells in the adultmousesubventricular zonerdquo Stem Cells vol 25 no 11 pp 2827ndash28362007
[88] Y Hirabayashi and Y Gotoh ldquoStage-dependent fate determina-tion of neural precursor cells in mouse forebrainrdquo NeuroscienceResearch vol 51 no 4 pp 331ndash336 2005
[89] S Ding T Y H Wu A Brinker et al ldquoSynthetic smallmolecules that control stem cell faterdquo Proceedings of theNationalAcadamy of Sciences of the United States of America vol 100 no13 pp 7632ndash7637 2003
[90] N Israsena M Hu W Fu L Kan and J A Kessler ldquoThepresence of FGF2 signaling determines whether 120573-cateninexerts effects on proliferation or neuronal differentiation ofneural stem cellsrdquo Developmental Biology vol 268 no 1 pp220ndash231 2004
[91] A Salehi A Jullienne M Baghchechi et al ldquoUp-regulation ofWnt120573-catenin expression is accompanied with vascular repairafter traumatic brain injuryrdquo Journal of Cerebral Blood Flow ampMetabolism vol 38 no 2 pp 274ndash289 2017
[92] L F Reichardt and K J Tomaselli ldquoExtracellular matrixmolecules and their receptors Functions in neural develop-mentrdquoAnnual Review of Neuroscience vol 14 pp 531ndash570 1991
[93] R M Gibson S E Craig L Heenan C Tournier and M JHumphries ldquoActivation of integrin 12057251205731 delays apoptosis ofNtera2 neuronal cellsrdquoMolecularandCellularNeuroscience vol28 no 3 pp 588ndash598 2005
[94] C C Tate A J Garcıa andMC LaPlaca ldquoPlasma fibronectin isneuroprotective following traumatic brain injuryrdquoExperimentalNeurology vol 207 no 1 pp 13ndash22 2007
[95] C Culmsee and M P Mattson ldquop53 in neuronal apoptosisrdquoBiochemical and Biophysical Research Communications vol 331no 3 pp 761ndash777 2005
[96] M S Geddis and V Rehder ldquoThe phosphorylation state ofneuronal processes determines growth cone formation afterneuronal injuryrdquo Journal of Neuroscience Research vol 74 no2 pp 210ndash220 2003
[97] M L Craske M Fivaz N N Batada and T Meyer ldquoSpines andneurite branches function as geometric attractors that enhanceprotein kinase C actionrdquoThe Journal of Cell Biology vol 170 no7 pp 1147ndash1158 2005
20 Evidence-Based Complementary and Alternative Medicine
[98] A Muscella C Vetrugno L G Cossa et al ldquoApoptosis by[Pt(OOrsquo-acac)(gamma-acac)(DMS)] requires PKC-deltamedi-ated p53 activation in malignant pleural mesotheliomardquo PloSone vol 12 no 7 Article ID e0181114 2017
[99] J S Bonini W C Da Silva L R M Bevilaqua J H MedinaI Izquierdo and M Cammarota ldquoOn the participation ofhippocampal PKC in acquisition consolidation and reconsol-idation of spatial memoryrdquo Neuroscience vol 147 no 1 pp 37ndash45 2007
[100] O Zohar R Lavy X Zi et al ldquoPKC activator therapeutic formild traumatic brain injury in micerdquo Neurobiology of Diseasevol 41 no 2 pp 329ndash337 2011
[101] J David Sweatt ldquoTheneuronalMAPkinase cascade a biochem-ical signal integration system subserving synaptic plasticity andmemoryrdquo Journal ofNeurochemistry vol 76 no 1 pp 1ndash10 2001
[102] Y Matsuoka M Picciano B Maleste et al ldquoInflammatoryresponses to amyloidosis in a transgenic mouse model ofAlzheimerrsquos diseaserdquo The American Journal of Pathology vol158 no 4 pp 1345ndash1354 2001
[103] L Esposito L Gan G-Q Yu C Essrich and L MuckeldquoIntracellularly generated amyloid-120573 peptide counteracts theantiapoptotic function of its precursor protein and primesproapoptotic pathways for activation by other insults in neu-roblastoma cellsrdquo Journal of Neurochemistry vol 91 no 6 pp1260ndash1274 2004
[104] D J Loane A Pocivavsek C E-H Moussa et al ldquoAmyloidprecursor protein secretases as therapeutic targets for traumaticbrain injuryrdquo Nature Medicine vol 15 no 4 pp 377ndash379 2009
[105] R Torres B L Firestein H Dong et al ldquoPDZ proteins bindcluster and synaptically colocalize with Eph receptors and theirephrin ligandsrdquo Neuron vol 21 no 6 pp 1453ndash1463 1998
[106] M B Dalva M A Takasu M Z Lin et al ldquoEphB receptorsinteract with NMDA receptors and regulate excitatory synapseformationrdquo Cell vol 103 no 6 pp 945ndash956 2000
[107] J M Basak P B Verghese H Yoon J Kim and D MHoltzman ldquoLow-density lipoprotein receptor represents anapolipoprotein E-independent pathway of A120573 uptake anddegradation by astrocytesrdquoThe Journal of Biological Chemistryvol 287 no 17 pp 13959ndash13971 2012
[108] L Yao X Gu Q Song et al ldquoNanoformulated alpha-mangostinameliorates Alzheimerrsquos disease neuropathology by elevatingLDLR expression and accelerating amyloid-beta clearancerdquoJournal of Controlled Release vol 226 pp 1ndash14 2016
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4 Evidence-Based Complementary and Alternative Medicine
25 Molecule Docking The LibDock algorithm based on theCHARMm Force Field in Discovery Studio (DS) 25 wasused in this study to evaluate the potential molecular bindingmode between bioactive compounds and putative targetsThecrystal structure of the target proteins of XFZYD for treatingTBI was downloaded from the RCSB Protein Data Bank(wwwrcsborg) The 3D chemical structures of bioactivecompounds were downloaded from PubMed Compounddatabase or TCMSP database and subjected to minimize theenergy by using molecular mechanics-2 (MM2) force fieldThe protein preparation protocol was used before dockingsuch as inserting missing atoms in incomplete residuesremoving water and protonating titratable residues The lig-and preparation protocol was employed before docking suchas removing duplicates enumerating isomers and generating3D conformations The protein-ligand docking active sitewas defined by the location of the original ligand All otherdocking and consequent scoring parameters were kept attheir default settings The compound was considered to be apotentially active ingredient if the LibDock score was higherthan the original ligand
26 Network Construction and Analysis Network construc-tion was performed as follows
(1) The herbs candidate compounds and candidate tar-gets of XFZYD were used to construct an herb-candidate compound-candidate target (HB-cC-cT)network
(2) The PPI data obtained above was used to establish theTBI-specific protein interaction network
(3) Herbs potential compounds and putative targetsfrom XFZYD for treating TBI were used to builda herb-potential compounds-potential targets (HB-pC-pT) network
(4) Potential targets and the biology process they partici-pate in were used to construct the pT-F network
(5) Compounds and targets through molecule dockingvalidating were used to build a compound-target (C-T) network
All networks were generated and analyzed by Cytoscape 340[52] an open source of bioinformatic package for biologicalnetwork analysis and visualization Two topological param-eters including degree and betweenness were calculated forthe obtained networks which disclose the significance ofa node
27 Gene Ontology (GO) and Pathway Enrichment Analy-sis The functional enrichment tool DAVID [53] (DAVIDhttpsdavidncifcrfgov) ver 68) was used to calculateboth the KEGG pathway and GO biological processes (BP)enrichment
28 Statistical Analysis All data were expressed as mean plusmnstandard deviation (SD) The molecule descriptors data wereanalyzed by one-way ANOVA The criterion for statisticalsignificance was p lt 005 Statistical analyses were conductedusing the SPSS 240
3 Results
TCM an experience-based medicine has been widely usedfor thousands of years It has accumulated abundant clinicalexperience forming a comprehensive and unique medicalsystem [35] The complexity of the phytochemical compo-nents makes it extremely difficult to illustrate the actionmechanisms of XFZYD from a molecule or system levelAs a chief mean of treating diseases clinically generallyTCM doctors prescribe formula based on the principle ofldquoJun-Chen-Zuo-Shirdquo ldquoJunrdquo (monarch) treats the main causeor primary symptoms of the disease ldquoChenrdquo (minister)enhances the actions of ldquoJunrdquo or treats the accompanyingsymptoms ldquoZuordquo (adjuvant) not only reduces or eliminatesthe possible toxic effects of the Jun or Chen but also treatsthe accompanying symptoms ldquoShirdquo (guide) helps to deliveror guide the other herbs to the target organs [18] Accordingto the unique feature of TCM ourwork tried to perform ldquoJun-Chen-Zuo-Shirdquo based system study to clarify the multiplemechanisms of XFZYD in the treatment of TBI
31 Herbal Ingredient Comparison and Target Predictionof XFZYD We obtained 162 components originated fromXFZYD Of these compounds 160 chemicals that were inaccord with standard requirements were searched from theTCMSP database The other 2 components amygdalin andhydroxysafflor yellow A were taken into consideration fortheir obvious pharmacological action as well The detailedinformation of these compounds is showed in Table S1Persicae Semen (Tao ren) and Carthami Flos (Hong hua)the Jun (monarch) herbs of XFZYD contained 36 bioactivecomponents which accounted for 22 of the 162 chemicalsRadix Paeoniae Rubra (Chi shao) Chuanxiong Rhizoma(Chuan xiong) andAchyranthis Bidentatae Radix (Niu xi) theChen (minister) herbs contained 31 bioactive componentswhich accounted for 19 of the 162 chemicals AngelicaeSinensis Radix (Dang gui) Rehmannia glutinosa Libosch(Sheng di huang) Platycodon Grandiforus (Jie geng) AurantiiFructus (zhi ke) Radix Bupleuri (chai hu) and licorice (Gancao) the Zuo-Shi (adjuvant and guide) herbs of XFZYDcontained 109 bioactive components which accounted for67 of the 162 chemicals Ingredients from these herbswere compared based on the 6 important drug-associateddescriptors including molecular weight (MW) number ofhydrogen-bond donors (nHdon) number of hydrogen-bondacceptors (nHacc) partition coefficient between octanol andwater (AlogP) oral bioavailability (OB) and drug-likeness(DL)The distributions of the 6 descriptors of the ingredientsfrom the three groups are shown in Table 1 and Figure 2 Wefoundno significant differences in the values ofMW(p=016)nHdon (p=032) nHacc (p=061) and AlogP (p=082) amongthe 3 groups However the average OB value of compoundsfrom the march herbs is 5559plusmn2391 which was significantlydifferent from the Chen herbs (OB=4564plusmn1327 P=0004)and the Zuo-Shi herbs (OB=4877plusmn1505 P=0021) Thefollowing 2 groups had no significant difference in OBvalue (P=0272) As for DL the Chen herbs revealed thehighest DL index (053plusmn023) which displayed significantdifference from the Zuo-Shi herbs (044plusmn019 p=0012) while
Evidence-Based Complementary and Alternative Medicine 5
Table 1 Comparison of molecular properties among the Jun Chen and Zuo-Shi herbs
INDEX MW nHdon nHacc AlogP OB DL(mean plusmn SD) (mean plusmn SD) (mean plusmn SD) (mean plusmn SD) (mean plusmn SD) (mean plusmn SD)
Jun herbs 38028 (9423) 265 (226) 511 (321) 337 (436) 5559 (2391) 049 (018)Chen herbs 38180 (9160) 218 (195) 55 (347) 314 (315) 4564 (1327) 053 (023)Zuo-shi herbs 35745 (8822) 261 (160) 56 (249) 344 (185) 4877 (1505) 044 (019)OB oral bioavailability MW molecular weight DL drug-likeness AlogP partition coefficient between octanol and water nHacc number of hydrogen-bondacceptors nHdon number of hydrogen-bond donors
Table 2 Top 10 candidate compounds according to 2 centrality indicators
Compounds Degree Compounds Betweennessquercetin 153 quercetin 035875838kaempferol 65 naringenin 008768253luteolin 48 kaempferol 007665228wogonin 46 luteolin 0057362047-Methoxy-2-methyl isoflavone 44 baicalein 005542898beta-sitosterol 41 beta-sitosterol 0041007baicalein 40 wogonin 003968963formononetin 39 Stigmasterol 003586788naringenin 39 nobiletin 003057461isorhamnetin 38 formononetin 003051437
showing nodifferencewith the Junherbs (049plusmn018 p=0333)Figure 3(a) indicated that 5 bioactive compounds wereshared by the Jun Chen and Zuo-Shi herbs One compoundoverlapped between the Jun andChen herbs while there were2 compounds shared by the Chen and Zuo-Shi herbs Onecompound overlapped between the Jun and Zuo-Shi herbs
32 Target Prediction of XFZYD A total of 285 potentialtargets from the 162 compounds were generated using thetarget prediction model The amounts of potential targets hitby the Jun Chen and Zuo-Shi drugs were 217 218 and 261respectively The detailed data of the targets is shown in TableS2 As depicted in Figure 3(b) there was a significant targetoverlap among the 3 groups (189 candidate targets) but lessoverlap between the Jun andChenherbs (9 candidate targets)The number of targets shared by the Jun and Zuo-Shi herbswas 14 while 10 targets were overlapped between the Chenand Zuo-Shi herbs
33 HB-cC-cT Network Construction and Analysis We nextestablished aHB-cC-cT network through network analysis toilluminate the relationship among the herbs candidate com-pounds and candidate targets (Figure 3(c)) This networkconsisted of 485 nodes (11 herbs 162 candidate compoundsand 285 candidate targets) and 2585 edges A herb (triangle)and cC (square) are connected if the compound is containedin this herb and the edges between cC and cT represent theinteraction The size of nodes is proportional to the valueof degree The larger size of the node means more phar-macologically important Two centrality indicators degreeand betweenness identify the important nodes within thenetwork Different centralities reflect different importanceof nodes in a network from different angles Interestingly
both of the two types of centrality indicators uniformlyconfirmed themost important 10 candidate compounds fromXFZYD and the top 10 targets anchored by XFZYD (Tables2 and 3) Figure 3(c) demonstrated that licorice possessedthe largest degree (88) compared with other herbs originatedfrom XFZYD This implicated that it contained the mostbioactive compounds (88) including quercetin (Mol 148degree=153) kaempferol (Mol 108 degree=65) 7-Methoxy-2-methyl isoflavone (Mol 33 degree=44) formononetin (Mol63 degree=39) naringenin (Mol 133 degree=39) isorham-netin (Mol 105 degree=38) medicarpin (Mol 131 degree=35)and licochalcone a (Mol 113 degree=33) followed byPersicae Semen (degree=19) Carthami Flos (degree=18)Achyranthis Bidentatae Radix (degree=17) Radix PaeoniaeRubra (degree=14) Radix Bupleuri (degree=12) ChuanxiongRhizoma (degree=6) Aurantii Fructus (degree=4) Platy-codon Grandiforus (degree=4) Rehmannia glutinosa Libosch(degree=3) andAngelicae Sinensis Radix (degree=2) PersicaeSemen (degree=19) and Carthami Flos (degree=18) the Junherbs from XFZYD possessed 29 specific compounds and5 unique target proteins including ALB CTNNB1 MMP10LYZ andNFKB1 Twenty-three Chen-specific potential com-pounds targeted 10 specific proteins including CD14 LBPNR3C1 BBC3 TEP1 PRKCD FN1 PDE10A GSTA1 andGSTA2 The Zuo-Shi herbs possessed the largest number ofspecific compounds (101) and 48 unique proteins such asHTR3A ADRA1D PYGM OLR1 CHRM5 RXRB STAT3MAPK10 OPRD1 and MAPK3 There were 189 proteinsanchored by the Jun Chen and Zuo-Shi drugs
Among the 162 candidate compounds quercetin hadthe largest value of degree (153) implicating its critical rolein XFZYD The four herbs namely Carthami Flos (Jun)Achyranthis Bidentatae Radix (Chen) and licorice and
6 Evidence-Based Complementary and Alternative Medicine
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Jun herbsChen herbsZuo-Shi herbs
Jun herbsChen herbsZuo-Shi herbs
Jun herbsChen herbsZuo-Shi herbs
Jun herbsChen herbsZuo-Shi herbs
Jun herbsChen herbsZuo-Shi herbs
Figure 2 The profile distributions of six important molecular properties for all ingredients from the Jun Chen and Zuo-Shi herbs OBOral bioavailability MW molecular weight DL drug-likeness AlogP partition coefficient between octanol and water nHacc number ofhydrogen-bond acceptors nHdon number of hydrogen-bond donors
Evidence-Based Complementary and Alternative Medicine 7
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Mol 9444444
Mol 99
Mol 63
Mol 75555
Mol 37Mol 37Mol 37Mol 37MolMol 33Mol 37MolM
Mol 158
Mol 1Mol 10Mol 1Mol 10000
Mol 49Mol 4Mol 4Mol 4Mol 44Mol 494Mol 4
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MMol 60Mol 60Mol 6MM
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Mol 12Mol 12Mol 12Mol 1Mol 12221
Mol 11MMol 1155Mol 11555M6060660
Mol 85Mol 85Mol 85MMol 88558554MMol 114Mol 114444M
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Mol 69Mol 6MMol 6Mol 6Mol 66Mol 69Mol 69M 66
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(c)
Figure 3 HB-cC-cT network of XFZYD (a) and (b) The distribution of different candidate compounds and targets in the network (red theJun herbs-specific cC (a) and cT (b) aqua the Chen herbs-specific cC (a) and cT (b) periwinkle the Zuo-Shi herbs-specific cC (a) and cT(b) claybank common cC (a) and cT (b) between the Jun and Chen herbs blue common cC (a) and cT (b) between the Jun and Zuo-Shiherbs green common cC (a) and cT (b) between the Chen and Zuo-Shi herbs purple common cC (a) and cT (b) among the 3 group ofherbs (c) The triangles with circle backgrounds represent the herbs (HB) the squares and circles represent the candidate compounds (cC)and candidate targets (cT) The red triangles squares and circles represent corresponding HB cC and cT in the Jun herbs the same is toaqua representing the Chen herbs and periwinkle representing the Zuo-Shi herbs The claybank squares and circles represent correspondingcC and cT overlap between the Jun and Chen herbs the same is to blue representing the overlap between the Jun and Zuo-Shi herbs and thegreen representing the overlap between the Chen and Zuo-Shi herbs The purple squares and circles represent the corresponding cC and cTshared by the 3 group of herbs The size of the node is proportional to the value of degree
Radix Bupleuri (Zuo-Shi) contained quercetin It targeted149 bioactive proteins PTGS2 (degree=126) HSP90AB2P(degree=85) AR (degree=81) NCOA2 (degree=75) PRSS1(degree=68) PTGS1 (degree=67) PPARG (degree=66)and F10 (degree=60) were predicted as the major candidatetargets of quercetin followed by kaempferol (degree=65)which was contained by Carthami Flos (Jun) AchyranthisBidentatae Radix (Chen) and Radix Bupleuri licorice(Zuo-Shi) It targeted 61 bioactive proteins includingPTGS2 (degree=126) HSP90AB2P (degree=85) CALM(degree=81) AR (degree=81) NOS2 (degree=76) andNCOA2 (degree=75) Quercetin stigmasterol kaempferol
baicalin and beta-sitosterol existed in 3 the Jun Chenand Zuo-Shi drugs demonstrating crucial roles of thesecomponents The 5 ingredients in the Jun Chen and Zuo-Shiherbs targeted 186 bioactive proteins which accounted for65 targets of XFZYD Baicalein existed in the Jun andChen herbs Luteolin existed in the Jun and Zuo-Shi herbsSpinasterol and sitosterol existed in the Chen and Zuo-Shiherbs 126 bioactive compounds targeted PTGS2 followedby ESR1 (88) HSP90AB2P (85) AR (81) CALM (81) NOS2(76) NCOA2 (75) PRSS1 (68) PTGS1 (67) and PPARG(66) As depicted in Figure 3(c) these target proteins wereanchored by ingredients in the 3 group drugs
8 Evidence-Based Complementary and Alternative Medicine
Table 3 Top 10 target proteins of XFZYD according to 2 centrality indicators
Proteins Degree Proteins BetweennessPTGS2 126 PTGS2 0128936ESR1 88 NCOA2 0060806HSP90AB2P 85 HSP90AB2P 0049647AR 81 PRKACA 0046484CALM 81 PTGS1 004365NOS2 76 AR 0030252NCOA2 75 PRSS1 0025575PRSS1 68 ESR1 0022212PTGS1 67 PPARG 0021403PPARG 66 PGR 0020685Note the centrality indicators identify the important nodes within the network Higher degree centrality and betweenness centrality indicate greaterimportance
The analysis of the network revealed that quercetin (Mol148) kaempferol (Mol 108) luteolin (Mol 127) wogonin (Mol160) 7-Methoxy-2-methyl (Mol 33) and beta-sitosterol (Mol41) were predicted as the major active compounds of XFZYDTheproteins including F2 NOS2 PTGS1 PTGS2 and CALMwere predicted as essential pharmacological proteins for thetherapeutic effects of XFZYD
34 Analyses on TBI Based Specific Protein Interaction Net-work Network biology integrated with different kinds ofdata including physical or functional networks and diseasegene sets is used to interpret humandiseases Protein-proteininteraction networks (PPI) are fundamental to understandthe cellular organizations biological processes and proteinfunctions [54] From the systematic perspective the analysisof TBI-related PPI will improve the understanding of thecomplicated molecular pathways and the dynamic processesunderlying TBI We screened the TBI-specific genes andprotein targets using Online Mendelian Inheritance in Mandatabase (OMIM) and Therapeutic Target Database (TTD)Figure 4 indicated that 21 TBI-specific genesproteins wereacquired Further these hub-proteins were submitted toHPRD and STRING to establish the TBI-specific proteininteraction network The detailed information of the TBI-specific proteins is shown in Table S3 The results suggestedthat the network consisted of 489 nodes and 5738 edges(Figure 4(a)) We obtained top 10 TBI-related proteinsaccording to 2 centrality indicators generated and summa-rized in Table 4 Interestingly we found that the node withhigher betweenness tends to possess larger degree (Fig-ure 5) Network topology analysis showed that protoonco-gene tyrosine-protein kinase Src (SRC degree=164 between-ness=0059) RAC-alpha serinethreonine-protein kinase(AKT1 degree=157 betweenness=0045) Serum albumin(ALB degree=153 betweenness=0069) Epidermal growthfactor receptor (EGFR degree=139 betweenness=0053)and Amyloid beta A4 protein (APP degree=134 between-ness=0072) contributed to the essential role in the patho-physiology of TBI All of these indicated that the top mutualtarget proteins performed various beneficial functions to treatTBI at the molecular level For example SRC is activatedfollowing engagement of many different classes of cellular
receptors It participates in signal pathways that controla diverse spectrum of biological activities including genetranscription immune response cell adhesion cell cycleprogression apoptosis migration and transformation [55]SRC can result in blood-brain barrier (BBB) disruptionand brain edema at the acute stage the inhibition of SRCfamily kinases can protect hippocampal neurons and improvecognitive function after TBI [56] AKT1 regulates manyprocesses including metabolism proliferation cell survivalgrowth and angiogenesis [57] The PI3KAKTPTEN path-way has been shown to play a pivotal role in neuroprotectionenhancing cell survival by stimulating cell proliferation andinhibiting apoptosis after TBI [58] ALB is the main proteinof plasma and can be a biomarker to predict outcome ofTBI [59] Its main function is the regulation of the colloidalosmotic pressure of blood We found that it may participatein pathological process of TBI
KEGG pathway analysis was also used to determine thefunctions of proteins Table 5 describes the top 10 significantlyenriched KEGG pathways These pathways play crucial rolesin pathophysiology process which have also been widelydiscussed in existing literature For instanceMAPK signalingpathway can promote pathological axonal death throughtriggering a local energy deficit [60] Neurotrophin signalingthrough Trk receptors regulates cell survival proliferationthe fate of neural precursors axon and dendrite growth andpatterning and the expression and activity of functionallyimportant proteins such as ion channels and neurotransmit-ter receptors [61] Engagement of cells with the extracellularmatrix (ECM) proteins is crucial for various biological pro-cesses including cell adhesion differentiation and apoptosiscontributing to maintenance of tissue integrity and woundhealing [62]The 47 TBI-specific proteins targeted byXFZYD(yellow and green) were further discussed below
35 HB-pC-pT Network pT-F Network Construction andMolecule Docking Analyses of XFZYD for the Treatment ofTBI To investigate the therapeutic mechanisms of XFZYDfor the treatment of TBI a HB-pC-pT network of XFZYDfor treating TBI was built (Figure 6) 47 TBI-specific targetproteins (circles) were targeted by 119 potential compounds(squares) from XFZYD (Figure 6) Detailed information
Evidence-Based Complementary and Alternative Medicine 9
BLMH
GPRASP2
GJC2
CAMK2N1
OBSL1
ZNF292
RLFAMPD3
NANOS1CAMTA1HYPKCCS
DLGAP4PF4V1
TRIM2
CHKB
F8A1
TIAM1
BMX
RAPGEF1
APBB1
KCNMA1
XRCC6
TTR
TGM2MAP2
PRNP
RASGRF1 PPP3CADMD SPTAN1
ACE
TGFB2
AP2M1GRAP2
SCN5A
MYH9
PKN1
ACHE
SLC25A13
PHKG1
SPAST
UMOD
PRPF40B
BBC3
TUBA4A TTPALRYR2
STUB1KRT6A
AKAP8L
GFI1BTHRAP3
ZNF232
SOD1
CRKL
CDH2
MBP MAP2K2
DCN
MAP3K5NGFR
PIK3CA
ITGA2
DNM1
RAP1A
GRIN2B
PTPN11
PRKCA
LDLRAP1
DNAJA3INA
SP3
CACNA2D3
CSF1
CNTF
TNFRSF1B
BCL10
KLC1
DCTN1
IDE
SERPINA3
HSPA1A
HSPG2
FN1
LRP2
TUBB
GRIN2A
YWHAB
CAMK2D
HTRA2
PRKCE
CRYAB
APC
GIT1
PIAS4
HPX
TRIP10
PDE4B
MARK4
PLAT
UBE2K
APOC2
LIN7B BGN
MAP3K10MAPK8IP2
CBS
SH3GL3
STAU1
CASP6
PPP5C
KRT16
DAB1
CASP1SHC2
CTSD
UCHL1
HP
SNCACAMK2G
TGFB1
GNB1
PTPN1PIN1
TJP1
CASP8PLCG1
NPHS1
PPP2R2A
DNAJB1
ADRBK1
INADL
NCSTN
MATK
NEDD4L
KAT5IFNGTBP
FRS2
CSNK1A1
CALRKNG1
SGOL2
KRT6B
TRAF3 DLG3
SACS
BDKRB2PRKCB
CAV1
MAPK3F2 SMAD3
APOEAKAP9 RANBP2
CLTC
TSC22D1GAB2PSEN2
MLF1CROT
KRT9
ADRA1B
EPB41
COL4A1
SHC3
ARHGAP32
ATM
PPP2R5A
CAMK2A
NTRK1
KRT18
TNFAPOA2
LAT
GRIN2D
COL4A6
KLK3
VLDLR
COL4A5
COL4A2
NEFH
OR8D2
MTSS1
PDZRN4
PTPN4
OR3A2
OR2T6
DGKG
CASP3
IGF1R
CASK
DRD2
TRAF2
EPHB2
PPM1A
PRKCG
CACNB3
HNRNPUSIN3A
DLG1
CLU
RAP2A
ERBB4CANX
BACE1 GABBR1
KARS
CNOT1
UNG
AGA CDCP1
UTP14A PLAG1
EXOC6
HRAS
CTNNB1
SP1
NOS3 SUMO1
PSEN1
HSPA8
HSP90AA1
BCL2
GNAO1
RPS6KB1
PARK2
DLG4
PTK2B
GSN
NAE1 TLN2LRFN2
PLTP
NUMBLSTX1A
DERL1
NEFM
HMOX2
AATF
PPIAMTRNR2L2AP1M1
OGT
DDB1
AP4M1
MED31
RGS3
TDP2
KRT5
ADAM9
REST
PACSIN1
SHB
CTSL1
LAMA1
SH2B1
PLA2G4F
SMURF2
SLC9A8
KRT14
CIT
TGFB1I1
PPP2R1APIK3R1
NUMB
ADAM17
DLG2
LRP1
APOA4
LTA
IRS1
GSK3B
EGFR
CTSB
TRAF6
SHC1
PPP2CB
NGF
HTT
PPIDLDB3
APOC1HIP1
ZDHHC17COL25A1MAGI3
ITGB5
ACTB
GNA11
ACTN2CAMK2B
CDK1
LIN7C
S100B
PDLIM5
DLGAP1
LIN7A
TPR
SYNGAP1
CFD
GRIN3A
F12
SLC1A3
GRK5
CACNA2D2
TANC1
DUSP4
EXOC4
AHSG
GRIN3B
UCP2
TTN
GPC1
NSF
FGA
DYNLL1
MAPK8IP1
PICALM
PPBP
ITM2B
NID1
STXBP1
CTBP1
SGK1
TRAF1
SQSTM1
TNFRSF1A
ACTN1
OPTN
UBE2D2
AP2A2
FUS
APOA1
DICER1
CACNA1B
CST3
NCOA3
SCARB1
APOC3
SLC6A3
KIAA1551 KRT13
DCD
KIAA0232
ENSG00000258818
SPATA31A7
CTAGE5
CEP44
SH2B2
EPHB4
CAPN1
F7
APBA2
COL4A3
LDLR
GIPC1
GFAP
SERPING1
SRC
MAPK12APP
GAPDH
CDK5
CREBBP
NOS1
YWHAZCALM1
YWHAG
YWHAQGRIN1
RPS6KA3
COL1A2
TCERG1
A2M
CASP7
LRP8
PDK2
AGTR1
FYN
ALBGRB2
MAPK1AKT1
STAT1
MAPT
GSK3A
UBB
PPP2CA
SMAD4
PRKCD
RGS12
SPTB
SH3BP5
CACNA1I
IL16
SLA2
PFN2
NLRC4
TNFRSF14
PRTN3
SNCB
FBLN1
BACE2
SETX
FANCD2
SAP30
RANBP3
PALB2
SLC1A5RASA1
HAP1
LRRC7
CFB
ADRBK2
CDC45
CLSTN3
PCED1B
SCAF1KIAA1377
FAM71E2
FEZ1 ACSBG2
PCDH1
TRAPPC11QTRTD1
KRT1
LRP1B
HADHB
TP53BP2
DRD1
ST13
PRPF40A
TRAIP
KIDINS220
GRK6
MMP17
LTBR
EIF6
PGAM1
CHD3
PRSS3
APBB3
CHRNA7
KRT10
RIMS1
CRMP1
PDIK1L
SORBS3
SYMPK
GCN1L1
RNF19A
HOMER3
GRK4
JARID2
MYLK3
EFS
CRB1
HSD17B10
TST
HOMER2
PHC3
TM2D1 FICD
PEG3
CABLES1
LGALS2
ITIH1
HOXB2
TAF4FLOT1
BABAM1
CFH
HIP1R
LTBMYL4
NCOR1
CASP4
NF1
AP4E1
CLCA2
IGDCC4
NECAB3
CXorf27
SH3GLB1
CTCF
PLCG2
(a)
STAT1
EGFR
FN1
PLAT
SOD1
PRKCA CDK1
GSK3B
ALB APP
PPP3CA
IFNG
BBC3
SCN5A
KCNMA1
MAP2
ACHE
MAPK3F2
PTPN1
NOS3 BCL2
CAV1
CTNNB1
PRKCB
TGFB1CASP8
TNF
AKT1
F7
MAPK1LDLR
PRKCD
SLC6A3
DRD1
CASP3
CHRNA7
PRSS3
TP53BP2
CASP7
EPHB2
SYNGAP1
CALM1
CTSD
BACE1
ADRA1B
(b)
Figure 4 TBI-related protein interaction network (a) 21 hub proteins (red and green) were identified through the analysis of TherapeuticTargetDatabase (TTD) aswell asOnlineMendelian Inheritance inMan (OMIM) 4 overlapped protein targets (green)were obtained between21 hub proteins in TBI and candidate targets of XFZYD Periwinkle proteins fromHPRD and STRING analyses were not targeted by XFZYD(b) 47 candidate protein targets of XFZYD were screened for treating TBI Yellow candidate protein targets of XFZYD The size of nodes isproportional to the value of degree
10 Evidence-Based Complementary and Alternative Medicine
Table 4 Top 10 proteins of TBI specific proteins according to 2 centrality indicators
Proteins Degree Proteins BetweennessSRC 164 APP 0071814AKT1 157 ALB 0069343ALB 153 SRC 0058568EGFR 139 EGFR 0052938APP 134 AKT1 0045466GAPDH 131 HTT 0037164HSP90AA1 108 GAPDH 0034549BCL2 106 HSP90AA1 0027596MAPK1 105 CTNNB1 0021759HRAS 105 GNB1 0019492Note the centrality indicators identify the important nodes within the network Higher degree centrality and betweenness centrality indicate greaterimportance
Table 5 Top 10 significantly enriched KEGG pathways in TBI-specific proteins
Pathway ID Pathway description Gene count FDR4010 MAPK signaling pathway 44 299E-235200 Pathways in cancer 43 113E-184722 Neurotrophin signaling pathway 41 101E-344151 PI3K-Akt signaling pathway 40 111E-154510 Focal adhesion 39 292E-225205 Proteoglycans in cancer 39 410E-215010 Alzheimer s disease 34 124E-204015 Rap1 signaling pathway 33 769E-174014 Ras signaling pathway 33 646E-164020 Calcium signaling pathway 31 505E-17
0
001
002
003
004
005
006
007
008
0 20 40 60 80 100 120 140 160 180
Betw
eenn
ess
Degree
APP
ALB SRCEGFR
AKT1HTT
Figure 5 Relationship between degree and betweenness centralityin the TBI-specific protein interaction network
for the 119 potential compounds is shown in Table S4Similarly 5 pharmacologically active ingredients in the JunChen and Zuo-Shi groups including quercetin stigmasterolkaempferol baicalin and beta-sitosterol anchored 33 TBI-specific proteins such as CALM SCN5A F2 ACHE F7ADRA1B NOS3 BCL2 CASP3 and AKT1 11 Jun-specificcompounds targeted 2 specific targets (ALB CTNNB1) while12 Chen-specific compounds anchored 3 unique proteins(PRKCD FN1 and BC3) The Zuo-Shi herbs possessed thelargest number of active compounds (89) and targeted 5
unique proteins including EPHB2 BACE1 LDLR MAPK3and PRSS3 GSK3Bwas the common target between theChenandZuo-Shi drugs KCNMA1 CASP7 andAPPwere targetedby the Jun and Zuo-Shi drugs
The top 10 candidate compounds and targets to treat TBIwere showed in Tables 6 and 7 Formost of active compoundsfrom XFZYD each component hit more than one targetTable 6 demonstrated that quercetin had the highest numberof targets (degree =76) followed by kaempferol (degree=43) beta-sitosterol (degree =35) stigmasterol (degree =19)luteolin (degree=18) baicalein (degree=15) 7-Methoxy-2-methyl isoflavone (degree=12) wogonin (degree=12)nobiletin (degree=11) and naringenin (degree=9) Forinstance quercetin (3310158404101584057-pentahydroxyflavone) is anaturally occurring flavonoid commonly found in fruits andvegetables It regulates multiple biological pathways elicitinginduction of apoptosis as well as inhibiting angiogenesisand proliferation [63 64] Quercetin can attenuate neuronalautophagy and apoptosis in rat traumatic brain injury modelvia activation of PI3KAkt signaling pathway [65] It has alsobeen reported to have a protective ability against oxidativestress and mutagenesis in normal cells [66 67] Kaempferol(3 41015840 5 7 tetrahydroxy flavone) is a yellow-colored flavonoidthat is widely distributed in many botanical families[68] It has been shown to possess a variety of biologicalcharacteristics including effects of anti-inflammatory[69] antioxidative [70] tumor growth inhibition [71] and
Evidence-Based Complementary and Alternative Medicine 11
Mol 22Mol 33
Mol 29Mol 32 Mol 23Mol 20
Mol 97Mol 106
Mol 104Mol 100Mol 94
Mol 63Mol 90
Mol 87
Mol 91Mol 61
Mol 19
Mol 109
Mol 161
Mol 118
Mol 111
Mol 158
Mol 114
Mol 149
Mol 141
Mol 152
Mol 14
CASP3
BCL2
NOS3
SCN5A
F7
ADRA1B
ACHE
F2
Mol 4
Mol 49
Mol 127
APP CASP7
Mol 92
Mol 54
Mol 6
Mol 36
KCNMA1
Mol 27
Mol 13Mol 105Mol 140
Mol 17
Mol 39
Mol 2
Mol 21
Mol 12
Mol 142
Mol 126
Mol 135
Mol 121
Mol 119
Mol 124
Mol 120
Mol 117
Mol 133
Mol 131
Mol 134
PlatycodonGrandiforus
Aurantii Fructus
Licorice
Radix Bupleuri
RehmanniaglutinosaLibosch
AngelicaeSinensis Radix
Mol 8
Mol 73
Mol 75
Mol 77
Mol 80
Mol 7
Mol 82
Mol 76
Mol 74
Mol 60
Mol 46
Mol 150
Mol 151
Mol 112
Mol 115
Mol 11
Mol 116
Mol 113
Mol 110
Mol 1
Mol 9Mol 101Mol 10Mol 93
Mol 103Mol 89 Mol 107
Mol 96PRSS3 LDLRMAPK3 BACE1 EPHB2
Mol 35Mol 81
Mol 86
Mol 85
Mol 84
Mol 88
Mol 83Mol 30
Mol 137
ChuanxiongRhizoma
Mol 3
Mol 57
Mol 139
Achyranthis
Bidentatae
RadixMol 102
CASP8
IGHG1AKT1p53
CHRNA7
Mol 40
SLC6A3 PTPN1
SOD1MAPK1
Mol 38
Mol 95
Carthami Flos
Mol 24
Mol 78 Persicae Semen
Mol 122
Mol 25
Mol 98
TGFB1
GSK3B
PRKCA
Radix Paeoniae Rubra
Mol 52
Mol 42
Mol 62
Mol 138
EGFR
STAT1 PPP3CAPLAT
Stigmasterol
MAP2
Beta-sistosterol Quercetin
Kaempferol
SYNGAP1
CALM
Baicalin
PRKCB
CAV1
CTSD
TNF
DRD1
CDK1
IFNG
FN1BBC3 PRKCD
Mol 132
Mol 160
Mol 58
Mol 67
Mol 69 Mol 43ALB
Mol 64CTNNB1
xiongixioChuanxC nxnxioaamaRhihihizoma
Achyranthisiistntnt
aeeaeBidenBidennntatae
RadixadixR diR di
oniae nianiaiaonRadix PaeoPaePRadix P eeoeonRRRRubra dondonddoodPlatycolatycPlatyycPl ycoyccoPlatPlatycoPlatyccocod
rususrusususruGranG anGranGranGranGrGGranndndiforu
ructusuctuc sructusuctusructusruAurantiirA ntaA ntiitiirantAurantintiurantAur iiii Fru
cecececececececeecLiLLiLLiLiLLLLLLLLiLiLiLLiLLLiLicoric
leuririeuuurieurileurleurieurieurieurieurileRadix BBRadRad Bdix Bdixx R i Bdi BBBadix Radix BBuple
RehmanniaRe mRe m nnRehmanniamReRehmhmannnniamanniaRehmehmanniRR nosasasasaosaosglutilutglutgluulutiglgl titinos
LiboschLibLLibo hschib chLiboschboscLLib chschLibos
icaecaecacaeicaeicAngelAnAngeAngelAnAng lAAngeelielicdix dix dixdixdSinensinensSS nennensSinSSiSinensSinensSine sissis Rad
FloslololFlhamaCartharthhaamami Fl
menmennnnmicae Scae SSem
Figure 6 HB-pC-pT network of XFZYD for treating TBI The triangles with circle backgrounds represent the herbs (HB) the squares andcircles represent the potential compounds (pC) and targets (pT)The red triangles squares and circles represent corresponding HB pC andpT in the Junherbs the same is to aqua representing theChen herbs and periwinkle representing the Zuo-Shi herbsThe claybank squares andcircles represent corresponding pC and pT shared by the Chen and Zuo-Shi herbs the same is to blue representing the overlap between theJun and Zuo-Shi herbs and the green representing the overlap between the Chen and Zuo-Shi herbsThe purple squares and circles representthe corresponding pC and pT shared by the 3 groups of herbs
alleviating insulin resistance in type 2 diabetic rats [72]Beta-sitosterol (BS) is a vegetable-derived compound foundin various plants and is suggested to modulate the immunefunction inflammation and pain levels by controlling theproduction of inflammatory cytokines [73]
For targets analysis CALM possesses the largest degree(degree =84) followed by GSK3B (degree =61) SCN5A(degree = 59) F2 (degree = 43) ACHE (degree=28)F7 (degree=28) ADRA1B (degree=28) NOS3 (degree=20)BCL2 (degree=18) and CASP3 (degree=18) which demon-strated their crucial therapeutic effects for treating TBI Forinstance CALM possess an essential position in calciumsignaling pathway and is related to morphological changesmigration proliferation and secretion of cytokines andreactive oxygen species ofMicroglial cells [74]The activationof CaMKII120572 major isoform of Ca2+calmodulin-dependentprotein kinase (CaMK) in brain is directly associated withthe production of proinflammatory cytokines such as TNF-120572and IL-1120573 [75] GSK-3120573 is a serinethreonine-protein kinasewhich is abundant in the central nervous system (CNS)particularly in neurons [76] It can control gene transcrip-tion axonal transport and cytoskeletal dynamics in growthcones [77] The inhibition of GSK-3120573 attenuates apoptotic
signals and prevents neuronal death [78] There is increasingevidence that prothrombin (F2) and its active derivativethrombin are expressed locally in the central nervous systemBeside the central role in the coagulation cascade the gener-ation of thrombin leads to receptor mediated inflammatoryresponses cell proliferationmodulation cell protection andapoptosis [79 80] The role in brain injury depends upon itsconcentration as higher amounts cause neuroinflammationand apoptosis while lower concentrations might even becytoprotective [81]
Direct tissue damage aswell as hypoxic-ischemic increas-ing anaerobic glycolysis of the brain tissue results in theATP-stores depletion and failure of energy-dependent mem-brane ion pumps especially for voltage-dependent Ca2+ andNa+-channels Accumulated Ca2+ activates lipid peroxidasesproteases and phospholipases and caspases at the sametime increasing the intracellular concentration of free fattyacids and free radicals leading to necrosis or apoptosis ofneurocyte [82] At the same time the activation of residentglial cells microglia and astrocytes and the infiltration ofblood leukocytes secrete various immune mediators elicitedinflammatory responses which subsequently intersect withadjacent pathological cascades including oxidative stress
12 Evidence-Based Complementary and Alternative Medicine
Table 6 Top 10 potential candidate compounds of XFZYD for treating TBI according to 2 centrality indicators
Compound Degree Compound Betweennessquercetin 76 quercetin 01682179kaempferol 43 kaempferol 005501511beta-sitosterol 35 wogonin 005141376Stigmasterol 19 beta-sitosterol 004451294luteolin 18 naringenin 003251738baicalein 15 beta-carotene 002977217-Methoxy-2-methyl isoflavone 12 nobiletin 002787992wogonin 12 7-Methoxy-2-methyl isoflavone 002096439nobiletin 11 luteolin 002090223naringenin 9 baicalein 001453488
Table 7 Top 10 potential targets of XFZYD for treating TBI according to 2 centrality indicators
Targets Degree Targets BetweennessCALM 84 CALM 021452046GSK3B 61 SCN5A 011441591SCN5A 59 GSK3B 009553896F2 43 F2 00689171ACHE 28 BCL2 002651903F7 28 CASP3 002372836ADRA1B 28 F7 002032647NOS3 20 ACHE 001845996BCL2 18 ADRA1B 001704505CASP3 18 NOS3 00166699
excitotoxicity or reparative events including angiogenesisscarring and neurogenesis [83] Function analysis of the 47target proteins regulated by XFZYD was mainly associatedwith core pathophysiology process of TBI (Figure 7) 47 targetproteins were connected with 16 key process related to TBIMost of the targets have one or more links to other biolog-ical process such as apoptosis cell proliferation superoxideanion generation nitric oxide biosynthetic process responseto calcium ion I-kappaB kinaseNF-kappaB signaling andregulation of inflammation The above analysis implied themultifunction character of these target proteins regulated byXFZYD Of these target proteins 25 proteins (53 of the 47)such as TGFB1 EGFR CAV1 MAPK1 PRKCB and AKT1were responsible for regulating the apoptosis process 17 forblood coagulation 14 for cell proliferation and axon genesisand 12 for hypoxia andMAPK cascade We found that TGFB1was the crucial protein because it participated in 9 biologicalprocesses related to TBI such as apoptosis blood coagulationcell proliferation and MAPK cascade followed by EGFR (8)CAV1 (7) MAPK1 (6) PRKCB (6) and AKT1 (6)
Overall these observations strongly support the evidencethat the generated HB-pCminuspT network and pT-F networkhave important roles in treating TBI further validating thedrug targeting approach
Molecule docking was used to further validate the bind-ing mode between candidate compounds and their targetproteins We found that 18 TBI-specific target proteins inter-acted with 91 candidate compounds from XFZYD (Figure 8)
Other 29 target proteins were not discussed for the lackof proper protein crystal structure The detailed moleculedocking results are shown in Table S5The 6 essential proteinsincluding GSK3B AKT1 CDK1 F2 NOS3 and ACHEwere used to elucidate the exact binding mode (Figure 9)Quercetin was located within the binding cavity of AKT1 andCDK1 (Figures 9(a) and 9(b) and S1A B) Four conventionalhydrogen bonds were formed between quercetin and AKT1by interacting with the key amino acids including ILE-290 THR-211 and SER 205 Additionally 120587-120587 interactionsbetween quercetin and TRP-90 were found in the activesite which helped the stabilization of the compound at thebinding site (Figure 9(a) S1A) Figure 9(b) and S1B suggestedthat five conventional hydrogen bonds (LEU-83 ASP-146and LYS-33) and 120587-120587 interaction (PHE-80) were formedbetween quercetin and CDK1 The GSK3B-FA complexes(Figure 9(c) S1C) were stabilized by 6 hydrogen-bondinginteractions between FA and LYS-85 GLU-97 TYR-134 andARG-141 Glyasperin Bmainly bonds to F2 through hydrogenbonds by interacting with the key amino acids includingGLY-193 SER-195 and GLY-219 (Figure 9(d) S1D) andan edge-to-face 120587 minus 120587 interaction was also observed withTYR-228 The GLN-247 GLU-351 and GLY-355 from theactive site pocket of NOS3 participated in the hydrogen-bondformationwith 1-Methoxyphaseollidin (Figure 9(e) S1E)The(-)-Medicocarpin formed a total of 6 hydrogen bonds withSER-293 PHE-295 ARG-296 and TRY-341 in the active siteof ACHE (Figure 9(f) S1F) Besides an edge-to-face 120587 minus 120587
Evidence-Based Complementary and Alternative Medicine 13
KCNMA1
P53
IFNG
PPP3CA
CASP8
SCN5A
I-kappaB kinase NF-kappaB signaling
Regulation of inflammation
Response to calcium ion
TGFB1
TNF
PRKCA
CHRNA7
CAV1
STAT1
MAPK cascadeAKT1
BCL2
APP
EGFR
MAPK1
Angiogenesis
SYNGAP1
MAP2
PTPN1
Axonogenesis
GSK3B
EPHB2
CDK1
CASP7FN1
MAPK3
CTNNB1
F2
Autophagy
Cell proliferation
BACE1
SLC6A3
DRD1
ADRA1BACHE
CALMCASP3
PLAT
Apoptotic process
NOS3
PRKCB
Blood coagulation
Response to hypoxia
Superoxide anion generation
Nitric oxide biosynthetic process
Astrocyte activation
Amyloid-beta regulation
Microglial cell activation
LDLR
PRSS3
F7
PRKCD
SOD1ALB
BBC3
Figure 7 pT-F network of XFZYD for treating TBI 16 biological processes (red square) the 47 target proteins (periwinkle circle) of XFZYDparticipate in for treating TBI
interactionwas also observedwith TYR-337 From the resultshydrogen-bonding and edge-to-face 120587 minus 120587 interactions playkey roles in the proteinminusligand recognition and stabilitywhich may be helpful in determining the inhibitor activitiesAnd the C-T network confirmed the potential therapeuticeffects of the candidate compounds from XFZYD to treatTBI through interacting with the relevant proteins The com-putational analysis further elucidated the accurate moleculemechanisms between active compounds and targets
4 Discussion
Traumatic brain injury (TBI) is a growing public healthproblem worldwide and is a leading cause of death anddisability [84] Although major progress has been made in
understanding the pathophysiology of this injury this hasnot yet led to substantial improvements in outcome by alack of treatments which have proven successful during phaseIII trials for modern medicine [85 86] TCM rooted inthousands of years of history may offer an alternative or acomplementary strategy for the treatment of TBI XFZYDa representative formula in TCM has been used for yearsto treat TBI in China and has been demonstrated to beeffective in clinical practice However its ldquomulticomponentsrdquoand ldquomultitargetsrdquo features make it much difficult to decipherthemolecular mechanisms of XFZYD in the treatment of TBIfrom a systematic perspective if employing routine methods
In the present study a network pharmacology-basedmethod was employed to elucidate the pharmacologicalmechanisms of XFZYD to treat TBI according to the drug
14 Evidence-Based Complementary and Alternative Medicine
Figure 8 C-T network through molecule docking validating 119 potential compounds (green triangles) interacting with 18 potential targets(yellow circles) of XFZYDThe size of the nodes is proportional to the value of degree
combination principle of TCM We first proposed a newmodeling system combining OB and DL screening multipledrug targets prediction and validation network constructionand molecule docking to probe the efficiency of a typicalTCM formula XFZYD for the treatment of TBI The 11herbs from XFZYD possessed 162 bioactive compounds andtargeted 285 proteins There were 5 compounds and 189
target proteins overlapped among the Jun Chen and Zuo-Shigroup Furthermore 47 TBI-specific proteins were targetedby 119 (73) bioactive compounds from XFZYD Similarly 5common compounds and 33 (70) common target proteinsamong the 3 groups of drugs were observed Most of thebioactive ingredients targeted more than one protein The 47target proteins regulated several essential pathophysiological
Evidence-Based Complementary and Alternative Medicine 15
(a) (b) (c)
(d) (e) (f)
Figure 9 Hydrogen-bonding networks within the binding site of the compoundminustarget complexes obtained from molecule docking(a) AKT1-quercetin (b) CDK1-quercetin (c) GSK3B-FA (d) F2-glyasperin B (e) NOS3-1-Methoxyphaseollidin and (f) ACHE-(-)-Medicocarpin The molecules are presented as ball and stick models Active site amino acid residues are represented as lines Dotted bluelines in these pictures represent hydrogen bonds with distance unit of A Other O and N atoms are colored as red and blue respectively
processes of TBI that referred to apoptosis inflammationcell proliferation superoxide anion generation nitric oxidebiosynthetic process response to calcium ion etc The aboveanalysis reveals that the synergistic action mechanisms ofXFZYDmay be (1) bioactive compounds overlapping amongthe different group of herbs (2) specific bioactive com-pounds from different groups of herbs targeting the sameproteins (3) specific bioactive compounds from differentgroups of herbs targeting different proteins which participatein the same pathophysiological process of the disease Toa certain degree the 5 compounds including quercetinstigmasterol kaempferol baicalin and beta-sitosterol playedessential role in XFZYD for TBI treatment MAPK3MAPK1AKT1 PRKCA TNF PRKCB EGFR BCL2 GSK3B CASP3PPP3CA and NOS3 were the main target proteins regulatedby XFZYD in the treatment of TBI
Interestingly Beta-carotene (Mol 43) from Carthami Flos(the Jun herb) specifically regulated 120573-Catenin (CTNNB1)and played critical role for curing TBI The 120573-Catenin is acritical downstream component of the Wnt pathway whichplays essential role in the regulation of mammalian neuraldevelopment [87] In vitro and in vivo studies demonstrate
that the Wnt120573-catenin pathway regulates the proliferationand differentiation of neural progenitor cells [88] Neuronaldifferentiation is induced by overexpression of 120573-cateninor the pharmacological inhibition of GSK3120573 (the phospho-rylating enzyme of 120573-catenin) [89 90] This pathway alsopromotes blood vessel formation during vascular develop-ment as well as the vascular repair process after TBI [91]In addition wogonin (Mol 160) from Achyranthis BidentataeRadix (the Chen herb) specifically targeted fibronectin (FN1)Bcl-2-binding component 3 (BBC3) and Protein KinaseC delta type (PRKCD) FN1 an important component ofthe extracellular matrix (ECM) environment promotes cellmigration neurite outgrowth and synapse formation dur-ing neural development [92] It aggregates in the injuredbrain and plays a neuroprotection role through antiapoptosisand anti-inflammation ways following TBI [93 94] BBC3namely p53 upregulated modulator of apoptosis (PUMA) iscritical for the p53-dependent apoptosis pathway which playsan important role in hippocampal neuronal loss and associ-ated cognitive deficits [95] PRKCD one of PKC isoformsactivates signal transduction pathways involved in neuronalregeneration [96] synaptic transmissionplasticity [97] and
16 Evidence-Based Complementary and Alternative Medicine
activation of apoptosis processes [98] as well as higher brainfunctions such as learning andmemory [99]The activators ofPKCare effective for the treatment of TBI [100] FurthermoreMitogen-activated protein kinase 3 (MAPK3) Beta-secretase1 (BACE1) Ephrin type-B receptor 2 (EphB) and low-densitylipoprotein receptor (LDLR) were specifically targeted bythe ingredients in the Zuo-Shi herbs Naringenin (Mol133) targeted LDLR MAPK3 while euchrenone (Mol 60)anchored BACE1 and nobiletin (Mol 134) targeted EphB2MAPK3 is an essential component of the MAP kinase signaltransduction pathway It has been appreciated recently thatthe ERK12 cascade plays a fundamental role in synapticplasticity and memory [101] BACE1 is responsible for pro-duction of A120573 from amyloid precursor protein (APP) A120573can cause cell death activate inflammatory pathways [102]and prime proapoptotic pathways for activation by otherinsults [103] The blocking of BACE1 can ameliorate motorand cognitive deficits and reduce cell loss after experimentalTBI in mice [104] EphB is localized to synaptic sites inhippocampal neurons [105] The interaction between EphBand NMDA receptors regulates excitatory synapse formation[106] LDLR acts as an important receptor that facilitatesbrain A120573 clearance and inhibits amyloid deposition [107]and then ameliorates Alzheimerrsquos disease neuropathologyafter TBI [108] The analysis above indicates the ldquoJunrdquoldquoChenrdquo and ldquoZuo-Shirdquo herbs from XFZYD trigger theirspecific targets regulation respectively for the therapeuticeffects
XFZYD is a very famous traditional Chinese formula inpromoting qi circulation and removing blood stasis accord-ing to TCM theory However several limited researches havedemonstrated its efficacy for treating TBI such as anti-inflammatory and synaptic regulation [30 31] which arein accord with our study However previous studies merelypartially deciphered the molecule mechanism of XFZYD fortreating TBI This study reports 119 bioactive compounds inXFZYD that target 47 TBI-specific proteins such as MAPK3MAPK1 AKT1 PRKCA TNF PRKCB and EGFR Theseproteins regulate several crucial pathophysiological processesof TBI such as apoptosis inflammation blood coagulationand axon genesis Our study demonstrates that the therapeu-tic actions of XFZYD refer to ldquomulticompoundsrdquo ldquomultitar-getsrdquo features rather than only the improvement of bloodcirculation With the help of molecule docking methodwe further validate the interactions between bioactive com-pounds and potential targets of XFZYD The hydrogen-bonding and edge-to-face 120587 minus120587 interactions play key roles inthe proteinminusligand recognition and stability This provides avaluable reference for further experimental investigations ofbioactive ingredients and therapeutic targets of XFZYD fortreating TBI
5 Conclusion
Our work successfully illuminates the efficiency of XFZYDfor the treatment of TBI as well as herb combination ruleof TCM formula Network pharmacology with moleculedocking method confirms the ldquomulticompounds multitar-getsrdquo therapeutic actions of XFZYD in the treatment of TBI
The present work may provide valuable evidence for furtherclinical application of XFZYD for treating TBI
Abbreviations
TBI Traumatic brain injuryXFZYD Xuefu Zhuyu decoctionTCM Traditional Chines medicineDL Drug-likenessOB Oral bioavailabilityHB-cC-cT Herb-candidate compound-candidate
targetHB-pC-pT Herb-potential compound-potential targetCALM CalmodulinGSK3B Glycogen synthase kinase-3 betaSCN5A Sodium channel protein type 5 subunit
alphaF2 ProthrombinACHE AcetylcholinesteraseGO Gene OntologyKEGG Kyoto Encyclopedia of Genes and
Genomes
Data Availability
The data used to support the findings of this study areavailable from the corresponding author upon request
Conflicts of Interest
The authors declare that they have no conflicts of interest
Authorsrsquo Contributions
YangWang and Zebing Huang participated in the conceptionand design of the study YuanyuanZhong YangWang ZebingHuang Jiekun Luo Tao Tang and Pengfei Li acquired andanalyzed the data Yuanyuan Zhong Tao Liu and HanjinCui drafted and revised the manuscript The correspondingauthor and all of the authors have read and approved the finalsubmitted manuscript
Acknowledgments
This work was supported by the National Natural ScienceFoundation of China (Nos 81673719 81874409 81603670and 81803948) Project funded byChina Postdoctoral ScienceFoundation (Nos 2016M600639 and 2017T100614)
Supplementary Materials
Table S1 162 bioactive compounds in XFZYD Table S2 targetproteins of XFZYD Table S3 TBI-specific proteins Table S4119 potential compounds of XFZYD for treating TBI TableS5 docking result of 18 target proteins with 91 potential com-pounds Fig S1 the exact bindingmode between active ingre-dients and protein targets obtained from molecule docking(A) AKT1-quercetin (B) CDK1-quercetin (C) GSK3B-FA
Evidence-Based Complementary and Alternative Medicine 17
(D) F2-glyasperin B (E) NOS3-1-Methoxyphaseollidin and(F)ACHE-(-)-Medicocarpin (Supplementary Materials)
References
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[2] J A Langlois W Rutland-Brown and M M Wald ldquoTheepidemiology and impact of traumatic brain injury a briefoverviewrdquo The Journal of Head Trauma Rehabilitation vol 21no 5 pp 375ndash378 2006
[3] H A Alsulaim B J Smart A O Asemota et al ldquoConsciousstatus predictsmortality among patientswith isolated traumaticbrain injury in administrative datardquo The American Journal ofSurgery vol 214 no 2 pp 207ndash210 2017
[4] M Majdan D Plancikova A Maas et al ldquoYears of life lost dueto traumatic brain injury in Europe A cross-sectional analysisof 16 countriesrdquo PLoS Medicine vol 14 no 7 p e1002331 2017
[5] P Cheng P Yin P Ning et al ldquoTrends in traumatic braininjury mortality in China 2006ndash2013 A population-basedlongitudinal studyrdquo PLoS Medicine vol 14 no 7 Article IDe1002332 2017
[6] M V Russo and D B McGavern ldquoInflammatory neuroprotec-tion following traumatic brain injuryrdquo Science vol 353 no 6301pp 783ndash785 2016
[7] WWang H Li J Yu et al ldquoProtective Effects of ChineseHerbalMedicine Rhizoma drynariae in Rats After Traumatic BrainInjury and Identification of Active Compoundrdquo MolecularNeurobiology vol 53 no 7 pp 4809ndash4820 2016
[8] M Das S Mohapatra and S S Mohapatra ldquoNew perspectiveson central and peripheral immune responses to acute traumaticbrain injuryrdquo Journal of Neuroinflammation vol 9 p 236 2012
[9] J W Finnie ldquoNeuroinflammation Beneficial and detrimentaleffects after traumatic brain injuryrdquo Inflammopharmacologyvol 21 no 4 pp 309ndash320 2013
[10] T Cheng W Wang Q Li et al ldquoCerebroprotection of flavanol(minus)-epicatechin after traumatic brain injury viaNrf2-dependentand -independent pathwaysrdquo Free Radical Biology amp Medicinevol 92 pp 15ndash28 2016
[11] W Young ldquoRole of calcium in central nervous system injuriesrdquoJournal of Neurotrauma vol 9 Suppl 1 pp S9ndashS25 1992
[12] R Bullock A Zauner J S Myseros et al ldquoEvidence forProlonged Release of Excitatory Amino Acids in Severe HumanHead Trauma Relationship to Clinical Eventsrdquo Annals of theNew York Academy of Sciences vol 765 no 1 pp 290ndash297 1995
[13] A T Mazzeo A Beat A Singh and M R Bullock ldquoTherole of mitochondrial transition pore and its modulation intraumatic brain injury and delayed neurodegeneration afterTBIrdquo Experimental Neurology vol 218 no 2 pp 363ndash370 2009
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18 Evidence-Based Complementary and Alternative Medicine
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diseaserdquo International Journal of Molecular Sciences vol 17 no1 2016
[82] M J McGinn and J T Povlishock ldquoPathophysiology of Trau-matic Brain InjuryrdquoNeurosurgery Clinics of North America vol27 no 4 pp 397ndash407 2016
[83] T Woodcock and M C Morganti-Kossmann ldquoThe role ofmarkers of inflammation in traumatic brain injuryrdquo Frontiersin Neurology vol 4 article 18 2013
[84] F Tanriverdi H J Schneider G Aimaretti B E Masel F FCasanueva and F Kelestimur ldquoPituitary dysfunction after trau-matic brain injury A clinical and pathophysiological approachrdquoEndocrine Reviews vol 36 no 3 pp 305ndash342 2015
[85] J V Rosenfeld A I Maas P Bragge M C Morganti-Kossmann G T Manley and R L Gruen ldquoEarly managementof severe traumatic brain injuryrdquoThe Lancet vol 380 no 9847pp 1088ndash1098 2012
[86] B M Aertker S Bedi and C S Cox ldquoStrategies for CNS repairfollowing TBIrdquo Experimental Neurology vol 275 no 3 pp 411ndash426 2016
[87] K Adachi Z Mirzadeh M Sakaguchi et al ldquo120573-catenin signal-ing promotes proliferation of progenitor cells in the adultmousesubventricular zonerdquo Stem Cells vol 25 no 11 pp 2827ndash28362007
[88] Y Hirabayashi and Y Gotoh ldquoStage-dependent fate determina-tion of neural precursor cells in mouse forebrainrdquo NeuroscienceResearch vol 51 no 4 pp 331ndash336 2005
[89] S Ding T Y H Wu A Brinker et al ldquoSynthetic smallmolecules that control stem cell faterdquo Proceedings of theNationalAcadamy of Sciences of the United States of America vol 100 no13 pp 7632ndash7637 2003
[90] N Israsena M Hu W Fu L Kan and J A Kessler ldquoThepresence of FGF2 signaling determines whether 120573-cateninexerts effects on proliferation or neuronal differentiation ofneural stem cellsrdquo Developmental Biology vol 268 no 1 pp220ndash231 2004
[91] A Salehi A Jullienne M Baghchechi et al ldquoUp-regulation ofWnt120573-catenin expression is accompanied with vascular repairafter traumatic brain injuryrdquo Journal of Cerebral Blood Flow ampMetabolism vol 38 no 2 pp 274ndash289 2017
[92] L F Reichardt and K J Tomaselli ldquoExtracellular matrixmolecules and their receptors Functions in neural develop-mentrdquoAnnual Review of Neuroscience vol 14 pp 531ndash570 1991
[93] R M Gibson S E Craig L Heenan C Tournier and M JHumphries ldquoActivation of integrin 12057251205731 delays apoptosis ofNtera2 neuronal cellsrdquoMolecularandCellularNeuroscience vol28 no 3 pp 588ndash598 2005
[94] C C Tate A J Garcıa andMC LaPlaca ldquoPlasma fibronectin isneuroprotective following traumatic brain injuryrdquoExperimentalNeurology vol 207 no 1 pp 13ndash22 2007
[95] C Culmsee and M P Mattson ldquop53 in neuronal apoptosisrdquoBiochemical and Biophysical Research Communications vol 331no 3 pp 761ndash777 2005
[96] M S Geddis and V Rehder ldquoThe phosphorylation state ofneuronal processes determines growth cone formation afterneuronal injuryrdquo Journal of Neuroscience Research vol 74 no2 pp 210ndash220 2003
[97] M L Craske M Fivaz N N Batada and T Meyer ldquoSpines andneurite branches function as geometric attractors that enhanceprotein kinase C actionrdquoThe Journal of Cell Biology vol 170 no7 pp 1147ndash1158 2005
20 Evidence-Based Complementary and Alternative Medicine
[98] A Muscella C Vetrugno L G Cossa et al ldquoApoptosis by[Pt(OOrsquo-acac)(gamma-acac)(DMS)] requires PKC-deltamedi-ated p53 activation in malignant pleural mesotheliomardquo PloSone vol 12 no 7 Article ID e0181114 2017
[99] J S Bonini W C Da Silva L R M Bevilaqua J H MedinaI Izquierdo and M Cammarota ldquoOn the participation ofhippocampal PKC in acquisition consolidation and reconsol-idation of spatial memoryrdquo Neuroscience vol 147 no 1 pp 37ndash45 2007
[100] O Zohar R Lavy X Zi et al ldquoPKC activator therapeutic formild traumatic brain injury in micerdquo Neurobiology of Diseasevol 41 no 2 pp 329ndash337 2011
[101] J David Sweatt ldquoTheneuronalMAPkinase cascade a biochem-ical signal integration system subserving synaptic plasticity andmemoryrdquo Journal ofNeurochemistry vol 76 no 1 pp 1ndash10 2001
[102] Y Matsuoka M Picciano B Maleste et al ldquoInflammatoryresponses to amyloidosis in a transgenic mouse model ofAlzheimerrsquos diseaserdquo The American Journal of Pathology vol158 no 4 pp 1345ndash1354 2001
[103] L Esposito L Gan G-Q Yu C Essrich and L MuckeldquoIntracellularly generated amyloid-120573 peptide counteracts theantiapoptotic function of its precursor protein and primesproapoptotic pathways for activation by other insults in neu-roblastoma cellsrdquo Journal of Neurochemistry vol 91 no 6 pp1260ndash1274 2004
[104] D J Loane A Pocivavsek C E-H Moussa et al ldquoAmyloidprecursor protein secretases as therapeutic targets for traumaticbrain injuryrdquo Nature Medicine vol 15 no 4 pp 377ndash379 2009
[105] R Torres B L Firestein H Dong et al ldquoPDZ proteins bindcluster and synaptically colocalize with Eph receptors and theirephrin ligandsrdquo Neuron vol 21 no 6 pp 1453ndash1463 1998
[106] M B Dalva M A Takasu M Z Lin et al ldquoEphB receptorsinteract with NMDA receptors and regulate excitatory synapseformationrdquo Cell vol 103 no 6 pp 945ndash956 2000
[107] J M Basak P B Verghese H Yoon J Kim and D MHoltzman ldquoLow-density lipoprotein receptor represents anapolipoprotein E-independent pathway of A120573 uptake anddegradation by astrocytesrdquoThe Journal of Biological Chemistryvol 287 no 17 pp 13959ndash13971 2012
[108] L Yao X Gu Q Song et al ldquoNanoformulated alpha-mangostinameliorates Alzheimerrsquos disease neuropathology by elevatingLDLR expression and accelerating amyloid-beta clearancerdquoJournal of Controlled Release vol 226 pp 1ndash14 2016
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Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 5
Table 1 Comparison of molecular properties among the Jun Chen and Zuo-Shi herbs
INDEX MW nHdon nHacc AlogP OB DL(mean plusmn SD) (mean plusmn SD) (mean plusmn SD) (mean plusmn SD) (mean plusmn SD) (mean plusmn SD)
Jun herbs 38028 (9423) 265 (226) 511 (321) 337 (436) 5559 (2391) 049 (018)Chen herbs 38180 (9160) 218 (195) 55 (347) 314 (315) 4564 (1327) 053 (023)Zuo-shi herbs 35745 (8822) 261 (160) 56 (249) 344 (185) 4877 (1505) 044 (019)OB oral bioavailability MW molecular weight DL drug-likeness AlogP partition coefficient between octanol and water nHacc number of hydrogen-bondacceptors nHdon number of hydrogen-bond donors
Table 2 Top 10 candidate compounds according to 2 centrality indicators
Compounds Degree Compounds Betweennessquercetin 153 quercetin 035875838kaempferol 65 naringenin 008768253luteolin 48 kaempferol 007665228wogonin 46 luteolin 0057362047-Methoxy-2-methyl isoflavone 44 baicalein 005542898beta-sitosterol 41 beta-sitosterol 0041007baicalein 40 wogonin 003968963formononetin 39 Stigmasterol 003586788naringenin 39 nobiletin 003057461isorhamnetin 38 formononetin 003051437
showing nodifferencewith the Junherbs (049plusmn018 p=0333)Figure 3(a) indicated that 5 bioactive compounds wereshared by the Jun Chen and Zuo-Shi herbs One compoundoverlapped between the Jun andChen herbs while there were2 compounds shared by the Chen and Zuo-Shi herbs Onecompound overlapped between the Jun and Zuo-Shi herbs
32 Target Prediction of XFZYD A total of 285 potentialtargets from the 162 compounds were generated using thetarget prediction model The amounts of potential targets hitby the Jun Chen and Zuo-Shi drugs were 217 218 and 261respectively The detailed data of the targets is shown in TableS2 As depicted in Figure 3(b) there was a significant targetoverlap among the 3 groups (189 candidate targets) but lessoverlap between the Jun andChenherbs (9 candidate targets)The number of targets shared by the Jun and Zuo-Shi herbswas 14 while 10 targets were overlapped between the Chenand Zuo-Shi herbs
33 HB-cC-cT Network Construction and Analysis We nextestablished aHB-cC-cT network through network analysis toilluminate the relationship among the herbs candidate com-pounds and candidate targets (Figure 3(c)) This networkconsisted of 485 nodes (11 herbs 162 candidate compoundsand 285 candidate targets) and 2585 edges A herb (triangle)and cC (square) are connected if the compound is containedin this herb and the edges between cC and cT represent theinteraction The size of nodes is proportional to the valueof degree The larger size of the node means more phar-macologically important Two centrality indicators degreeand betweenness identify the important nodes within thenetwork Different centralities reflect different importanceof nodes in a network from different angles Interestingly
both of the two types of centrality indicators uniformlyconfirmed themost important 10 candidate compounds fromXFZYD and the top 10 targets anchored by XFZYD (Tables2 and 3) Figure 3(c) demonstrated that licorice possessedthe largest degree (88) compared with other herbs originatedfrom XFZYD This implicated that it contained the mostbioactive compounds (88) including quercetin (Mol 148degree=153) kaempferol (Mol 108 degree=65) 7-Methoxy-2-methyl isoflavone (Mol 33 degree=44) formononetin (Mol63 degree=39) naringenin (Mol 133 degree=39) isorham-netin (Mol 105 degree=38) medicarpin (Mol 131 degree=35)and licochalcone a (Mol 113 degree=33) followed byPersicae Semen (degree=19) Carthami Flos (degree=18)Achyranthis Bidentatae Radix (degree=17) Radix PaeoniaeRubra (degree=14) Radix Bupleuri (degree=12) ChuanxiongRhizoma (degree=6) Aurantii Fructus (degree=4) Platy-codon Grandiforus (degree=4) Rehmannia glutinosa Libosch(degree=3) andAngelicae Sinensis Radix (degree=2) PersicaeSemen (degree=19) and Carthami Flos (degree=18) the Junherbs from XFZYD possessed 29 specific compounds and5 unique target proteins including ALB CTNNB1 MMP10LYZ andNFKB1 Twenty-three Chen-specific potential com-pounds targeted 10 specific proteins including CD14 LBPNR3C1 BBC3 TEP1 PRKCD FN1 PDE10A GSTA1 andGSTA2 The Zuo-Shi herbs possessed the largest number ofspecific compounds (101) and 48 unique proteins such asHTR3A ADRA1D PYGM OLR1 CHRM5 RXRB STAT3MAPK10 OPRD1 and MAPK3 There were 189 proteinsanchored by the Jun Chen and Zuo-Shi drugs
Among the 162 candidate compounds quercetin hadthe largest value of degree (153) implicating its critical rolein XFZYD The four herbs namely Carthami Flos (Jun)Achyranthis Bidentatae Radix (Chen) and licorice and
6 Evidence-Based Complementary and Alternative Medicine
50
40
30
20
10
0
50
40
40
30
20
20
10
0
0
10 30 50 70 90 110
60
60
60
01 02 03 04 05 06 07 08
225 275 325 375 425 475 525 575 625 minus6minus5minus4minus3minus2minus1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
minus2 minus1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
70
minus1 1 3 5 7 9 11 13 15 17
perc
entage
s (
)
perc
entage
s (
)50
40
30
20
10
0
perc
entage
s (
)
perc
entage
s (
)
40
20
0
60
perc
entage
s (
)50
40
30
20
10
0
perc
entage
s (
)
OB
MW
nHdon
DL
nHacc
Distribution
Distribution
Distribution
Distribution
Distribution
Distribution
AlogP
Jun herbsChen herbsZuo-Shi herbs
Jun herbsChen herbsZuo-Shi herbs
Jun herbsChen herbsZuo-Shi herbs
Jun herbsChen herbsZuo-Shi herbs
Jun herbsChen herbsZuo-Shi herbs
Jun herbsChen herbsZuo-Shi herbs
Figure 2 The profile distributions of six important molecular properties for all ingredients from the Jun Chen and Zuo-Shi herbs OBOral bioavailability MW molecular weight DL drug-likeness AlogP partition coefficient between octanol and water nHacc number ofhydrogen-bond acceptors nHdon number of hydrogen-bond donors
Evidence-Based Complementary and Alternative Medicine 7
Jun herbs(29)
Zuo-
Shi h
erbs
(101
)
Chen herbs
(23)
1
2
15
(a)
Jun herbs(5)
Zuo-
Shi h
erbs
(48)
Chen herbs
(10)
9
10
14189
(b)
20Mol 1200000
MMMol 152Mol 15Mol 1521Mol 15Mol 15
Mol 8888
Mol 29Mol 2Mol 2Mol 2Mol 29
Mol 3Mol 3Mol 35Mol 3535
1Mol 121Mol 121Mol 122
Mol 124Mol 124Mol 1Mol 124Mol 124Mol 124
MMol 10Mol 1Mol 10M 00MMMM
MMol 73Mol 77333222
ABATABATA
MAPK3MAPKMAPK
UGT1A1GT1UGT1A1
GOT1GOTT1
SREBF1REBFSREBF1
RXRBRXRBRXRB
CYP19A1CYP19A1YP19A
PLB1PLBPLB1
FASLGFASLGASL
BCHEBCHHE
HSD3B1HSD3
MTTPMTT
CES1CES
TIMP1TIMP
ATP5BATP5
MAPK10MAPK 0
BADBAD
CHRM5CHRM5
EPHBEPHBPHB2
HSD3B2HSD3B2SD3B
SOAT2OATSOAT2
FASNFASNFASN
MT-ND6MT-N
HTR3AHTR3HTR3APLA2G4APLA2G4AA2G
PKIAAIA
ADH1AADHAD
AKR1C1KR1AKR1C1
CREB1CREBB1
HMGCRMGHMGCR
SOAT1OAT
ABCC1ABCC
OPRD1OPRDOPRD1
VCPVCP
LDLRLDLR
ADRA1DDRAA1D
NFATC1FATCFATCATCFATCA
EGLN1GLNGLNGLNGLN
FABP5ABP5ABPABPABPABP5
CYCSCYCSCYCSCYCSYCSFOSL1OSLOSLOSLOSL
ALOX12LOXLOXOXLOX
TDRD7DRDDDRDDRDDRDAPODAPODAPOPOAPOD
NNNNOXNOXNOXNOX5NOXX
Mol 40
NFKB1NFKBFKBFKBFKBLYZLYZLYZZLYZ CTNNB1TNNBTNNNNNN MMP10MMPMPMPMMP1ALBALBALBALBALB
3Mol 433MMol 28Mol 28Mol 2Mol 28
HSPA5SPASPASPA5HSPA5AERBB2ERBB2ERBBRBBB2ERBB2
HSPB1HSPBSPBSPBHSPB1HSPB1B
NQO1NQO1NQONQO1NQONQO1TOP1TOP1TOP1TOPTOPT
PORPPORPORPORPORR ACPPACPPACPPACPPCPP
SLC2ALC2A4LC2A4C2A4LC2A4LC2A4LC
SERPINE1SERPINE1PINRPINERPINPIN
JUNJUNNJUNNN
PTGER3PTGER3TGERTGERGERTGER
COL1A1OL1AOL1AOL1AA1CO
SSLPISLPSLPISLPISLPII
CCCALCCC MMMMMMSS
MMMMP1MMPMMPMMPTNFTNFTNFFTNF
CCND1CCNDCCNDCNDCNDCCND
SMDPSMDPSMDPSMD3PSMD3PSMDNFNFNFFFNF
ELK 1ELKELK LK ELK ELK CCL2CCL2CCLCCL2CCL2CCL2
MMol 14MMol 14Mol 1447
MMol 15MMol 1Mol 115Mol 15M
Mol 122Mol 122Mol 122MMol 12Mol 122222
MMol 24Mol 2Mol 2Mol 2444
Mol 78MMol 78Mol 7Mol 7Mol 788
Mol 38MMMMol 3Mol 3338
Mol 154Mol 154MMol 15ol 1ol 155M
Mol 67MMol 6Mol 6Mol Mol 67Mol 677766
Mol 64MMol 64Mol 6Mol 6Mol 64
Mol 48Mol 48MMMol 4Mol 4448M 4
PPPPARAPARPARPAR
PCOLCEPCOLCECOLCOLCCOLC
SPP1SPP1SPP1PP1SPPP1 AKR1C3AKR1C3KR1CKR1CKR1CKR
NNNOSNN 2222 DUOXDUOXDUOXDUOX2DUOXDUOX2
MCL1MCL1MCLMCLMCLMCL
PARP1ARP1ARPARPPARP1PEEMPOMPOMPOMPOM DDDUDUDUDDU
NGASYNGASYNGAP1NGAPNGAAP1RARAARARA
BAXXXXB X ESR1
GSTM2GSTM2GSTMSTMSTMGSTMSHSAAHSAAHSA1AHSA1AHSAHSAA GABRAGABRA2GABRA2GABRAGABRAGABRAMM2MMM2
CHRM2CHRM2CHRMHRMHRMCHRM2
VDRVDRVDRRVDRVDRARGABRA1GABRA1GABRAA11GABRA1G
IL888IL88
PPPPRSPRSPRSSSSSSSPPARDPARDPARPARDPARDPARDSELESELESELESELEEE
COL3A1COL3A1OL3AOL3AOL3AA1OL3ARB11111
CYP1A2222222 NCOANCOANCOANCOANCOANCOA22222211111 RRRRRR
Quercetin
Mol 9444444
Mol 99
Mol 63
Mol 75555
Mol 37Mol 37Mol 37Mol 37MolMol 33Mol 37MolM
Mol 158
Mol 1Mol 10Mol 1Mol 10000
Mol 49Mol 4Mol 4Mol 4Mol 44Mol 494Mol 4
Mol 6Mol 6666M 66
MMol 60Mol 60Mol 6MM
Mol 101Mol 101Mol 101Mol 10011
Mol 12Mol 12Mol 12Mol 1Mol 12221
Mol 11MMol 1155Mol 11555M6060660
Mol 85Mol 85Mol 85MMol 88558554MMol 114Mol 114444M
Mol 116Mol 116Mol 116MMol 1ol 1Mol 11616M 1
Mol 9000
Mol 100Mol 1000Mol 106MMMol 106Mol 10Mol 1066Mol 322 Mol 30Mol 30Mol 30000MM
Mol 140MMol 14MMol 14Mol 1440Mol 14
PlatycodPlatycodatycodPlatycodP ononoGrandifondifoGrandGrand rususus
RadiRadixR diBupleurBupleuriBupleuriu
RehmanniRehmanniRehmanniiaaaglutinosglutinolutinoglutin aaaLiboschLiboschLiboschLLiLiLicoricececece
AngelicaAngelicaAngelican l eeSinensisSinenS nensisnensis
Radixadixdix
Mol 84
Mol 23 Mol 130Mol 13Mol 13Mol 11Mol 130Mol 13Mol 1MMol 13
Mol 11Mol 1Mol 1Mol 11Mol 11
0Mol 200000
Mol 34Mol 3Mol 3MolMol 3344Mol 3Mol 134
Mol 151
Mol 126Mol 126Mol 12666
Mol 8111AurantiiAurantAuranAurantiitFructusructusFructuructuFr
Mol 77Mol 7Mol 7Mol 77777
Mol 444
MMol 82Mol 8Mol 8222Mol 88
Mol 133
Mol 123MMol 12Mol 12ol 1Mol 121
Mol 142Mol 1422
Mol 36
0Mol 80Mol 88Mol 80M 80
Mol 277Mol 135Mol 13555M 555
Mol 141MMol 144Mol 14111
Mol 104Mol 104MMMol 10Mol 104044M
Mol 922222Mol 22 Mol 2Mol 2Mol 2Mol Mol 2Mol 22M 2
MMol 83333
Mol 110Mol 110
Mol 119MMol 11999999
Mol 88888 Mol 109Mol 10MMol 1Mol 100Mol 109M
Mol 5444
Mol 39999999
Mol 150MMol 155000M 0
Mol 611
21Mol 21MMol 221111
Mol 13
Mol 9Mol 91MMol 9911
Mol 118MMol 11Mol 1Mol 11Mol 1Mol 11ol
Mol 466
Mol 96Mol 96Mol 96Mol 996Mol 9696 Mol 53Mol 5Mol 5MolMol 5Mol 5Mol 535Mol 50Mol 50Mol 5Mol 5Mol 550Mol 5
Mol 107Mol 107MMol 10077Mol 1077
M l 149MMol 1449Mol 149Mol 149
Mol 14Mol 1444
Mol 89Mol 125MMol 1ol 1Mol 12MMMol 1MM
Mol 87
Mol 72MolMol 7Mol 7MolMol 72M 7M Mol 933Mol 112Mol 112Mol 112Mol 1122M
Mol 1Mol MMolMollMol 1Mol Mol 105
MAP2MAP2MAPMAPMAP
IL2I 2L22L22
SLC6A2LC6A2LC6A2LC6A2C6A2S
GABRA5ABRAABRGABRA5AABRCHRNA2CHRNA2HRNA2HRNHRNA2HRNA2
DRD1D1DRDDRDDRDDD1 MMAPKMAPK1MAMADRA1AADDDRADRA1ADRAADRA1A CASP3CASP3CASP3CASP3C
F2
Mol 17 Mol 7666Mol 33
Mol 86Mol 86Mol 8Mol 8Mol 866686
Mol 161Mol 161Mol 161Mol 16Mol 16161MMo
Mol 56Mol 5Mol 556666Mol 113
Mol 97Mol 162MMol 16Mol 1Mol 1ol 111M 1
Mol 131
Mol 99Mol 9MMMolMol 9MMol 9Mol 99Mol 9ol
Mol 111
Mol 128Mol 128Mol 1ol 1Mol 121MolMol 199Mol 117Mol 117777
Mol 744444Mol 77
2Mol 52Mol 52Mol 5MM
MMMol 5Mol 5MMol 5Mol 5Mol 5M
MMol 58Mol 58Mol 58M 8
Mol 138MMol 1388M
Mol 102Mol 102Mol 10Mol 102MMol 10Mol 10
Mol 42MMMol 42M
MMMol 3Mol 3
Mol 137Mol 13Mol 13ol 13Mol 13Mol 13733
Mol 59Mol 59olMol 5Mol 59oMol 5Mol 559
Mol 129lMol 12ol 12ol 12Mol 129o 2ol 12o
Mol 62Mol 62Mol 6Mol 6Mol 6Mol 62Mol 6Mol 6ol
Mol 144Mol 144Mol 14Mol 14ol 14Mol 144ol 1
AchyraAchyraAchyranthisnthisnthisBidentBidentBidentataeataeatae
RadixRadixRadix
Mol 139Mool 13ol 13ol 139Mol 139Mool 13
ChuanxioChuanxioChuanxiongngngRhizomaRhizomaRhizoma
Mol 159Mol 159MoMol 15ol 15ol 15Mol 159o 5
Mol 136Mol 136ol 13ol 13ol 1ol 13ool 13
CXCL11CXCL11XCL1XCL1XCLXCL1p53p53p53p
Mol 47Mol 47Mol 4Mol 4Mol 4ol 47Mol 4o
MMol 156ol 15ol 15ol 1ol 15ooPRKCARKCRKCPRKCACARKC
PPP3CAPP3CAP3CAP3CAP3CAPPP3CAP
CDKN1BDKNDKNDKN1CDKN1BPTENPTENPTENPTENPTENPTENBBB
RadixRadixRadixPaeoniaePaeoniaePaeoniaePae
RubraRubraRubraNR1I3NR1I3NR1INR1INR1INR1I3
HERC5ERCERC5HERC5ERC5OODC1ODCODCODC1CERBB3RBBRBBERBB3RBBRBB3
CTSDCTSDCTSDCTSDCTSDCTSD Mol 18Mol 1Mol 1ol 18Mol 18MolMol 1
PLATPLATPLAPLATPLATL
DIOOO1OO1OEGFEGFEGEGFEGDIODDIODIO
BIL1BIL1BIL1BIL1BB
MMP 3MMP 3MMP 3MMPMMP 3M
THBDTHBDTHBTHBTHBTHBD
CYP1B1YP1BYP1BYP1BYP1BB1
PPPPPARPP GGGGGGPPP
KDR LLAACTACTACTLACTBLACTBACTTT
ESR2
CHEK1CHEK1CHEK1CHEK1CHEK1CHEK111CCHEK1C
CATCATCATCATTTCATCATT
CDK2
CCNA2CCNA2CCNA2CCNA2CCNA2CCNA2CCNA2CCCNA2CC
PIM1 GSK3B
MAPKKK1414MMAPKKKMM KMMM
IL10IL10IL10IL10IL10IL10
S2HAS2HAS2HAS22HAS2ADADDHDH1CADAD HAHAHHHHA
CDKN2ACDKN2CDKNCDKNDKN2ACDKN
Mol 155Mol 155Mol 15Mol 15Mol 15ol 1ol 15ol 15Mol 15MMo
Mol 153Mol 15Mol 15ol 15Mol 153ol 15Mol 15Mol 15
CHEKCHEKCHEKHEK2HEK2HEK
MMP2MMP2MMPMMPMMPMMP2
MGAMMGAMMGAMGAAM
KCNH2222
CXCL2CXCL2CXXCLXCLXCL
GJA1GJA1GJAGJA1GJA1GJA1KK222K2
IKBKBIKBKBIKBKBIKCNH2KCNH2
IFNGIFNGIFNGIFNGIFNGIFNGFNEIF666F6IGHG1IGHG1GHGIGHG1IGGHG
SSStigSStiggmmmaS gg steeerolerol
PPTPNPTPN1PTPN1P 1SCN5ASCN5AASCN5ASCN5A BetaBetaaaa-sata istosssststerolerolllroNR3C1R3CGSTA2STA BBC3BBC3LBPLBP TTEP1TEP1 RKCDPRKCD PDE10ADE10GSTA1STA FN1FN1CD14CD14
BACE1ACEBACE1 SLC6A1LC6AACD163CD16 GSRGSRR ADIPOQDIPOQSTAT3TATOPRM1OPRM1OPRMOPRMOPRMO ADRA1BADRA1BAADRA1BBBB
SLC6A4LC6ALC6ASLC6A44SLCYCYP1A1YP1A1YP1A1CYP1AYP1A1
SLC6A3SLC6A3SLC6ASLC6A3SLC6A3
PDE3APDE3AA
CHRNA7CHRNA7HRNAHRNAHRNAA
PPPTGPP S1S1S1S1SS111NKX3-1NKX3-1NKX3KX3NKX3NKX3-
BIRCBIRC5BIRCBIRCBIRC55
IL6666
HK2HK2HK2HK2HK2HK2
RUNX2UNXUNXUNXRUNX2UNXHSF1HSF1HSF1HSF1HSF1HSF1
IGFBP3GFBPGFBPFBPIGFBP3
STAT1T1TAT1TATSTAT1TATT
NCF1NCFNCFNCF1NCFN
PRSS3RSSSIRT1SIRTGATMGAT OLR1OLRAPOBAPOPYGMPYGM
TGFB1GFBGFBGFBGFBTGFB1F10 MMP9MMPMMPMMPP9PNFE2L2FE2LFE2LFE2LNFE2L2FE2L2L
Mol 160Mol 44Mol 44ol 4Mol 444Mol 444ol 4ol 4ol 44
Mol 55MoMol 5Mol 5Mol 555Mol 55Mol 5Mol 5MoMol 5
MMMol 132
MMMol 57
CRPCRPCRPCRPPHH
RUNX1T1UNX1NX1NX1NX1T1R
BCGABCGABCG2BCGA
CXCL10CXCL10XCXCLCXCLCXCL IGG
CLDN4LDN4LDN4LDN4ABABABAAB RRAHRAHRBBBIB
RARRAF1RAF1RAF11KKKKKKaemKKKKK pffefeferereerf referololol
CASP9CCASPCASP9PMDM2MDMMMDMMDMMDM2MDMMDM
BMAOBMAOBMAOBMAOBMAOBINSRINSRINSRINSRINSRRRNCOA1NCOA11NCOA1
HTR2AHTR2HTR2HTR222A
CHRMCHRMCHRM3MM3BCL2LCL2CL2L1LCL2BCL2L1111 CHCCHCCCCH
BCL2222BCL2CASP8CASP8CASP8CASP8CASP
RELARELARELAAAR
BaicalBaicalBaBaicalcacacalicaBB calcaac ininininininninninnCHUCHUKHUKCHUKHUKUKH AAAAAA
EGFREGFRGFREGFREGFR
NFKBIAFKBIFKBFKBNFKBIAFKBI
E2F2E2F2E2F2E2F2E
APPAPAPPAPPPAPPAPPA
CASP7CASPCASPASPCASPCASPPP IL4IL444L4L44
NUF2NUFNUFNUFNUFNUFNUFNUFN
CDK4CDKCDKCDKCDKCDKCD
XIAPXIAPXIAXIAXIAXIAX MEMEMETMETMETMEETMMME
ADCY2ADCYDCYDCDCYDCD
Mol 127
NNR3C2NR3C2NR3CNR3C2N
DPP4
DCAF5DCACAFDCAF55DCAFAKR1B1AKR1BKKR1BKR1BKR1BCTRB1CTRBTRBCTRBCTRB
LTA4HLTA4LTA4TA4LTA4LTA4H4
XDHXDHXDHHHXDH
PTGS2
Mol 51Mol 51Mol 5MMolMolMol 5Mol 5MMol 5
Mol 145Mol 14Mol 14ol 1ol 1ol 1Mol 1Mo
Mol 146MMMMool 1ol 11461
Mol 143Mol 14Mol 14Mool 1ol 11Mol 1411
Mol 70MMMolMol 7Mol 77Mol 70Mol 7Mol 7
Mol 71Mol 7Mol Mol 777Mol 71Mol 7Mol
Mol 25Mol 25MMMol Mol 22Mol 2
Mol 79Mol 7Mol 7Mol Mol 7Mol 779MM 7
Mol 26Mol 26MMol 2Mol Mol 22Mol 2M 22
CA22222222ADH1BADHADHDHADH1ADH1DH KCNMA1CNMCNMCNMCNMKCNMAKCNMCGABRA6GABRAABRAABRAABRAGABRAGA A6GA RAGRIA2GRIAGRIAGRIAGRIAAAA2GRIA
RASSFRASSF1ASSFASSFASSF
Mol 31MMol 3Mol 3Mol 3311M 3Mol 98Mol 98Mol 98MMol 9Mol 999Mol 98MM
GSTP1GSTP1GSTPGSTPGSTPGSTPF33333
Mol 69Mol 6MMol 6Mol 6Mol 66Mol 69Mol 69M 66
VCAM1VCAMCAMCAMVCAM1C
MAPK8MAPK8MAPKMAPMAPKM
CYP3A4CYP3A4YP3AYP3AYP3AY
Mol 68Mol 68MMol 6Mol 6Mol 66MMol 68Mol 6MMol 16Mol 16Mol 1Mol 1Mol Mol 1Mol 16
MMMMol 6MMol 66Mol 666Mo
CCartCarta hamihamimiiiiiFlosFlosFlosossoss
Mol 9566Mol 65Mol 6Mol 6MMol MolMol 6M 666M
PersPersPerserrr icaeicaeicaesemesemesemeemememeem nnn
ICAM1CAMCAMM1CAMI
E2F1E2FE2F1E2F1E2F1ENPEPPSPEPPSPEPPSEPPSPEPPSNPEPP
IGF2IGF2IGF2IGF2F
FOSFOSFOSFOSSFOSFOSSSS
CCNB1CCNBCNBCNBCCNB1CCNB1CDK1CDK1CDKCDKCDKCC
PRKCBPRKCBRKCRKCRKCR CB
CHRM1CHRM11CHRM11
HHHHSP9HHHH 0ABABAB2B2B2B2PPPPPPPPPPPPTOP2A ACACACACACACACACAACACAACACA
PRKACA
TOP2TOP2
My c FF7FFIRF1IRF1IRF1IRF1IRF1IR
CAV1CCAVCAVCAV1V1CAV1
ACHE
ADRB1ADRBRBADRBADR
ADRADRADRA2A2AA2A
SOD1SSODSOD1D11
NOS33
PLAUPLAUPLAAUUPLAUPRXRA
PGR
HHMOX11MOXMOXHMOX
GSTM1GSTM1GSTMGSTMSTMAAA
HIF1AHIF1HIF1HIF11AHIF1A
FOSL2OSLOSLOSL22FO
ALOX5ALOXALOXALOXALOX5
VEGFAVEGFAVEGFAVEGFAV FFA
PON1PONPONPONN1PON
PIK3CGPIK3CGGG
SULT1E1SULT1E1ULT1ELT1LT1GAGAABRAGABRA3AGG
AKT1AKTT1AKT11A
CHRMCHRMCHRMCHRM4CHRM4RMM
MAOAMAOAMAOAMAOMAOCD40LGCD40LGCD40LGD40LGCD40LGCD40LBRA3RAAABRA3CHRCHHHCHR
ADRB2
(c)
Figure 3 HB-cC-cT network of XFZYD (a) and (b) The distribution of different candidate compounds and targets in the network (red theJun herbs-specific cC (a) and cT (b) aqua the Chen herbs-specific cC (a) and cT (b) periwinkle the Zuo-Shi herbs-specific cC (a) and cT(b) claybank common cC (a) and cT (b) between the Jun and Chen herbs blue common cC (a) and cT (b) between the Jun and Zuo-Shiherbs green common cC (a) and cT (b) between the Chen and Zuo-Shi herbs purple common cC (a) and cT (b) among the 3 group ofherbs (c) The triangles with circle backgrounds represent the herbs (HB) the squares and circles represent the candidate compounds (cC)and candidate targets (cT) The red triangles squares and circles represent corresponding HB cC and cT in the Jun herbs the same is toaqua representing the Chen herbs and periwinkle representing the Zuo-Shi herbs The claybank squares and circles represent correspondingcC and cT overlap between the Jun and Chen herbs the same is to blue representing the overlap between the Jun and Zuo-Shi herbs and thegreen representing the overlap between the Chen and Zuo-Shi herbs The purple squares and circles represent the corresponding cC and cTshared by the 3 group of herbs The size of the node is proportional to the value of degree
Radix Bupleuri (Zuo-Shi) contained quercetin It targeted149 bioactive proteins PTGS2 (degree=126) HSP90AB2P(degree=85) AR (degree=81) NCOA2 (degree=75) PRSS1(degree=68) PTGS1 (degree=67) PPARG (degree=66)and F10 (degree=60) were predicted as the major candidatetargets of quercetin followed by kaempferol (degree=65)which was contained by Carthami Flos (Jun) AchyranthisBidentatae Radix (Chen) and Radix Bupleuri licorice(Zuo-Shi) It targeted 61 bioactive proteins includingPTGS2 (degree=126) HSP90AB2P (degree=85) CALM(degree=81) AR (degree=81) NOS2 (degree=76) andNCOA2 (degree=75) Quercetin stigmasterol kaempferol
baicalin and beta-sitosterol existed in 3 the Jun Chenand Zuo-Shi drugs demonstrating crucial roles of thesecomponents The 5 ingredients in the Jun Chen and Zuo-Shiherbs targeted 186 bioactive proteins which accounted for65 targets of XFZYD Baicalein existed in the Jun andChen herbs Luteolin existed in the Jun and Zuo-Shi herbsSpinasterol and sitosterol existed in the Chen and Zuo-Shiherbs 126 bioactive compounds targeted PTGS2 followedby ESR1 (88) HSP90AB2P (85) AR (81) CALM (81) NOS2(76) NCOA2 (75) PRSS1 (68) PTGS1 (67) and PPARG(66) As depicted in Figure 3(c) these target proteins wereanchored by ingredients in the 3 group drugs
8 Evidence-Based Complementary and Alternative Medicine
Table 3 Top 10 target proteins of XFZYD according to 2 centrality indicators
Proteins Degree Proteins BetweennessPTGS2 126 PTGS2 0128936ESR1 88 NCOA2 0060806HSP90AB2P 85 HSP90AB2P 0049647AR 81 PRKACA 0046484CALM 81 PTGS1 004365NOS2 76 AR 0030252NCOA2 75 PRSS1 0025575PRSS1 68 ESR1 0022212PTGS1 67 PPARG 0021403PPARG 66 PGR 0020685Note the centrality indicators identify the important nodes within the network Higher degree centrality and betweenness centrality indicate greaterimportance
The analysis of the network revealed that quercetin (Mol148) kaempferol (Mol 108) luteolin (Mol 127) wogonin (Mol160) 7-Methoxy-2-methyl (Mol 33) and beta-sitosterol (Mol41) were predicted as the major active compounds of XFZYDTheproteins including F2 NOS2 PTGS1 PTGS2 and CALMwere predicted as essential pharmacological proteins for thetherapeutic effects of XFZYD
34 Analyses on TBI Based Specific Protein Interaction Net-work Network biology integrated with different kinds ofdata including physical or functional networks and diseasegene sets is used to interpret humandiseases Protein-proteininteraction networks (PPI) are fundamental to understandthe cellular organizations biological processes and proteinfunctions [54] From the systematic perspective the analysisof TBI-related PPI will improve the understanding of thecomplicated molecular pathways and the dynamic processesunderlying TBI We screened the TBI-specific genes andprotein targets using Online Mendelian Inheritance in Mandatabase (OMIM) and Therapeutic Target Database (TTD)Figure 4 indicated that 21 TBI-specific genesproteins wereacquired Further these hub-proteins were submitted toHPRD and STRING to establish the TBI-specific proteininteraction network The detailed information of the TBI-specific proteins is shown in Table S3 The results suggestedthat the network consisted of 489 nodes and 5738 edges(Figure 4(a)) We obtained top 10 TBI-related proteinsaccording to 2 centrality indicators generated and summa-rized in Table 4 Interestingly we found that the node withhigher betweenness tends to possess larger degree (Fig-ure 5) Network topology analysis showed that protoonco-gene tyrosine-protein kinase Src (SRC degree=164 between-ness=0059) RAC-alpha serinethreonine-protein kinase(AKT1 degree=157 betweenness=0045) Serum albumin(ALB degree=153 betweenness=0069) Epidermal growthfactor receptor (EGFR degree=139 betweenness=0053)and Amyloid beta A4 protein (APP degree=134 between-ness=0072) contributed to the essential role in the patho-physiology of TBI All of these indicated that the top mutualtarget proteins performed various beneficial functions to treatTBI at the molecular level For example SRC is activatedfollowing engagement of many different classes of cellular
receptors It participates in signal pathways that controla diverse spectrum of biological activities including genetranscription immune response cell adhesion cell cycleprogression apoptosis migration and transformation [55]SRC can result in blood-brain barrier (BBB) disruptionand brain edema at the acute stage the inhibition of SRCfamily kinases can protect hippocampal neurons and improvecognitive function after TBI [56] AKT1 regulates manyprocesses including metabolism proliferation cell survivalgrowth and angiogenesis [57] The PI3KAKTPTEN path-way has been shown to play a pivotal role in neuroprotectionenhancing cell survival by stimulating cell proliferation andinhibiting apoptosis after TBI [58] ALB is the main proteinof plasma and can be a biomarker to predict outcome ofTBI [59] Its main function is the regulation of the colloidalosmotic pressure of blood We found that it may participatein pathological process of TBI
KEGG pathway analysis was also used to determine thefunctions of proteins Table 5 describes the top 10 significantlyenriched KEGG pathways These pathways play crucial rolesin pathophysiology process which have also been widelydiscussed in existing literature For instanceMAPK signalingpathway can promote pathological axonal death throughtriggering a local energy deficit [60] Neurotrophin signalingthrough Trk receptors regulates cell survival proliferationthe fate of neural precursors axon and dendrite growth andpatterning and the expression and activity of functionallyimportant proteins such as ion channels and neurotransmit-ter receptors [61] Engagement of cells with the extracellularmatrix (ECM) proteins is crucial for various biological pro-cesses including cell adhesion differentiation and apoptosiscontributing to maintenance of tissue integrity and woundhealing [62]The 47 TBI-specific proteins targeted byXFZYD(yellow and green) were further discussed below
35 HB-pC-pT Network pT-F Network Construction andMolecule Docking Analyses of XFZYD for the Treatment ofTBI To investigate the therapeutic mechanisms of XFZYDfor the treatment of TBI a HB-pC-pT network of XFZYDfor treating TBI was built (Figure 6) 47 TBI-specific targetproteins (circles) were targeted by 119 potential compounds(squares) from XFZYD (Figure 6) Detailed information
Evidence-Based Complementary and Alternative Medicine 9
BLMH
GPRASP2
GJC2
CAMK2N1
OBSL1
ZNF292
RLFAMPD3
NANOS1CAMTA1HYPKCCS
DLGAP4PF4V1
TRIM2
CHKB
F8A1
TIAM1
BMX
RAPGEF1
APBB1
KCNMA1
XRCC6
TTR
TGM2MAP2
PRNP
RASGRF1 PPP3CADMD SPTAN1
ACE
TGFB2
AP2M1GRAP2
SCN5A
MYH9
PKN1
ACHE
SLC25A13
PHKG1
SPAST
UMOD
PRPF40B
BBC3
TUBA4A TTPALRYR2
STUB1KRT6A
AKAP8L
GFI1BTHRAP3
ZNF232
SOD1
CRKL
CDH2
MBP MAP2K2
DCN
MAP3K5NGFR
PIK3CA
ITGA2
DNM1
RAP1A
GRIN2B
PTPN11
PRKCA
LDLRAP1
DNAJA3INA
SP3
CACNA2D3
CSF1
CNTF
TNFRSF1B
BCL10
KLC1
DCTN1
IDE
SERPINA3
HSPA1A
HSPG2
FN1
LRP2
TUBB
GRIN2A
YWHAB
CAMK2D
HTRA2
PRKCE
CRYAB
APC
GIT1
PIAS4
HPX
TRIP10
PDE4B
MARK4
PLAT
UBE2K
APOC2
LIN7B BGN
MAP3K10MAPK8IP2
CBS
SH3GL3
STAU1
CASP6
PPP5C
KRT16
DAB1
CASP1SHC2
CTSD
UCHL1
HP
SNCACAMK2G
TGFB1
GNB1
PTPN1PIN1
TJP1
CASP8PLCG1
NPHS1
PPP2R2A
DNAJB1
ADRBK1
INADL
NCSTN
MATK
NEDD4L
KAT5IFNGTBP
FRS2
CSNK1A1
CALRKNG1
SGOL2
KRT6B
TRAF3 DLG3
SACS
BDKRB2PRKCB
CAV1
MAPK3F2 SMAD3
APOEAKAP9 RANBP2
CLTC
TSC22D1GAB2PSEN2
MLF1CROT
KRT9
ADRA1B
EPB41
COL4A1
SHC3
ARHGAP32
ATM
PPP2R5A
CAMK2A
NTRK1
KRT18
TNFAPOA2
LAT
GRIN2D
COL4A6
KLK3
VLDLR
COL4A5
COL4A2
NEFH
OR8D2
MTSS1
PDZRN4
PTPN4
OR3A2
OR2T6
DGKG
CASP3
IGF1R
CASK
DRD2
TRAF2
EPHB2
PPM1A
PRKCG
CACNB3
HNRNPUSIN3A
DLG1
CLU
RAP2A
ERBB4CANX
BACE1 GABBR1
KARS
CNOT1
UNG
AGA CDCP1
UTP14A PLAG1
EXOC6
HRAS
CTNNB1
SP1
NOS3 SUMO1
PSEN1
HSPA8
HSP90AA1
BCL2
GNAO1
RPS6KB1
PARK2
DLG4
PTK2B
GSN
NAE1 TLN2LRFN2
PLTP
NUMBLSTX1A
DERL1
NEFM
HMOX2
AATF
PPIAMTRNR2L2AP1M1
OGT
DDB1
AP4M1
MED31
RGS3
TDP2
KRT5
ADAM9
REST
PACSIN1
SHB
CTSL1
LAMA1
SH2B1
PLA2G4F
SMURF2
SLC9A8
KRT14
CIT
TGFB1I1
PPP2R1APIK3R1
NUMB
ADAM17
DLG2
LRP1
APOA4
LTA
IRS1
GSK3B
EGFR
CTSB
TRAF6
SHC1
PPP2CB
NGF
HTT
PPIDLDB3
APOC1HIP1
ZDHHC17COL25A1MAGI3
ITGB5
ACTB
GNA11
ACTN2CAMK2B
CDK1
LIN7C
S100B
PDLIM5
DLGAP1
LIN7A
TPR
SYNGAP1
CFD
GRIN3A
F12
SLC1A3
GRK5
CACNA2D2
TANC1
DUSP4
EXOC4
AHSG
GRIN3B
UCP2
TTN
GPC1
NSF
FGA
DYNLL1
MAPK8IP1
PICALM
PPBP
ITM2B
NID1
STXBP1
CTBP1
SGK1
TRAF1
SQSTM1
TNFRSF1A
ACTN1
OPTN
UBE2D2
AP2A2
FUS
APOA1
DICER1
CACNA1B
CST3
NCOA3
SCARB1
APOC3
SLC6A3
KIAA1551 KRT13
DCD
KIAA0232
ENSG00000258818
SPATA31A7
CTAGE5
CEP44
SH2B2
EPHB4
CAPN1
F7
APBA2
COL4A3
LDLR
GIPC1
GFAP
SERPING1
SRC
MAPK12APP
GAPDH
CDK5
CREBBP
NOS1
YWHAZCALM1
YWHAG
YWHAQGRIN1
RPS6KA3
COL1A2
TCERG1
A2M
CASP7
LRP8
PDK2
AGTR1
FYN
ALBGRB2
MAPK1AKT1
STAT1
MAPT
GSK3A
UBB
PPP2CA
SMAD4
PRKCD
RGS12
SPTB
SH3BP5
CACNA1I
IL16
SLA2
PFN2
NLRC4
TNFRSF14
PRTN3
SNCB
FBLN1
BACE2
SETX
FANCD2
SAP30
RANBP3
PALB2
SLC1A5RASA1
HAP1
LRRC7
CFB
ADRBK2
CDC45
CLSTN3
PCED1B
SCAF1KIAA1377
FAM71E2
FEZ1 ACSBG2
PCDH1
TRAPPC11QTRTD1
KRT1
LRP1B
HADHB
TP53BP2
DRD1
ST13
PRPF40A
TRAIP
KIDINS220
GRK6
MMP17
LTBR
EIF6
PGAM1
CHD3
PRSS3
APBB3
CHRNA7
KRT10
RIMS1
CRMP1
PDIK1L
SORBS3
SYMPK
GCN1L1
RNF19A
HOMER3
GRK4
JARID2
MYLK3
EFS
CRB1
HSD17B10
TST
HOMER2
PHC3
TM2D1 FICD
PEG3
CABLES1
LGALS2
ITIH1
HOXB2
TAF4FLOT1
BABAM1
CFH
HIP1R
LTBMYL4
NCOR1
CASP4
NF1
AP4E1
CLCA2
IGDCC4
NECAB3
CXorf27
SH3GLB1
CTCF
PLCG2
(a)
STAT1
EGFR
FN1
PLAT
SOD1
PRKCA CDK1
GSK3B
ALB APP
PPP3CA
IFNG
BBC3
SCN5A
KCNMA1
MAP2
ACHE
MAPK3F2
PTPN1
NOS3 BCL2
CAV1
CTNNB1
PRKCB
TGFB1CASP8
TNF
AKT1
F7
MAPK1LDLR
PRKCD
SLC6A3
DRD1
CASP3
CHRNA7
PRSS3
TP53BP2
CASP7
EPHB2
SYNGAP1
CALM1
CTSD
BACE1
ADRA1B
(b)
Figure 4 TBI-related protein interaction network (a) 21 hub proteins (red and green) were identified through the analysis of TherapeuticTargetDatabase (TTD) aswell asOnlineMendelian Inheritance inMan (OMIM) 4 overlapped protein targets (green)were obtained between21 hub proteins in TBI and candidate targets of XFZYD Periwinkle proteins fromHPRD and STRING analyses were not targeted by XFZYD(b) 47 candidate protein targets of XFZYD were screened for treating TBI Yellow candidate protein targets of XFZYD The size of nodes isproportional to the value of degree
10 Evidence-Based Complementary and Alternative Medicine
Table 4 Top 10 proteins of TBI specific proteins according to 2 centrality indicators
Proteins Degree Proteins BetweennessSRC 164 APP 0071814AKT1 157 ALB 0069343ALB 153 SRC 0058568EGFR 139 EGFR 0052938APP 134 AKT1 0045466GAPDH 131 HTT 0037164HSP90AA1 108 GAPDH 0034549BCL2 106 HSP90AA1 0027596MAPK1 105 CTNNB1 0021759HRAS 105 GNB1 0019492Note the centrality indicators identify the important nodes within the network Higher degree centrality and betweenness centrality indicate greaterimportance
Table 5 Top 10 significantly enriched KEGG pathways in TBI-specific proteins
Pathway ID Pathway description Gene count FDR4010 MAPK signaling pathway 44 299E-235200 Pathways in cancer 43 113E-184722 Neurotrophin signaling pathway 41 101E-344151 PI3K-Akt signaling pathway 40 111E-154510 Focal adhesion 39 292E-225205 Proteoglycans in cancer 39 410E-215010 Alzheimer s disease 34 124E-204015 Rap1 signaling pathway 33 769E-174014 Ras signaling pathway 33 646E-164020 Calcium signaling pathway 31 505E-17
0
001
002
003
004
005
006
007
008
0 20 40 60 80 100 120 140 160 180
Betw
eenn
ess
Degree
APP
ALB SRCEGFR
AKT1HTT
Figure 5 Relationship between degree and betweenness centralityin the TBI-specific protein interaction network
for the 119 potential compounds is shown in Table S4Similarly 5 pharmacologically active ingredients in the JunChen and Zuo-Shi groups including quercetin stigmasterolkaempferol baicalin and beta-sitosterol anchored 33 TBI-specific proteins such as CALM SCN5A F2 ACHE F7ADRA1B NOS3 BCL2 CASP3 and AKT1 11 Jun-specificcompounds targeted 2 specific targets (ALB CTNNB1) while12 Chen-specific compounds anchored 3 unique proteins(PRKCD FN1 and BC3) The Zuo-Shi herbs possessed thelargest number of active compounds (89) and targeted 5
unique proteins including EPHB2 BACE1 LDLR MAPK3and PRSS3 GSK3Bwas the common target between theChenandZuo-Shi drugs KCNMA1 CASP7 andAPPwere targetedby the Jun and Zuo-Shi drugs
The top 10 candidate compounds and targets to treat TBIwere showed in Tables 6 and 7 Formost of active compoundsfrom XFZYD each component hit more than one targetTable 6 demonstrated that quercetin had the highest numberof targets (degree =76) followed by kaempferol (degree=43) beta-sitosterol (degree =35) stigmasterol (degree =19)luteolin (degree=18) baicalein (degree=15) 7-Methoxy-2-methyl isoflavone (degree=12) wogonin (degree=12)nobiletin (degree=11) and naringenin (degree=9) Forinstance quercetin (3310158404101584057-pentahydroxyflavone) is anaturally occurring flavonoid commonly found in fruits andvegetables It regulates multiple biological pathways elicitinginduction of apoptosis as well as inhibiting angiogenesisand proliferation [63 64] Quercetin can attenuate neuronalautophagy and apoptosis in rat traumatic brain injury modelvia activation of PI3KAkt signaling pathway [65] It has alsobeen reported to have a protective ability against oxidativestress and mutagenesis in normal cells [66 67] Kaempferol(3 41015840 5 7 tetrahydroxy flavone) is a yellow-colored flavonoidthat is widely distributed in many botanical families[68] It has been shown to possess a variety of biologicalcharacteristics including effects of anti-inflammatory[69] antioxidative [70] tumor growth inhibition [71] and
Evidence-Based Complementary and Alternative Medicine 11
Mol 22Mol 33
Mol 29Mol 32 Mol 23Mol 20
Mol 97Mol 106
Mol 104Mol 100Mol 94
Mol 63Mol 90
Mol 87
Mol 91Mol 61
Mol 19
Mol 109
Mol 161
Mol 118
Mol 111
Mol 158
Mol 114
Mol 149
Mol 141
Mol 152
Mol 14
CASP3
BCL2
NOS3
SCN5A
F7
ADRA1B
ACHE
F2
Mol 4
Mol 49
Mol 127
APP CASP7
Mol 92
Mol 54
Mol 6
Mol 36
KCNMA1
Mol 27
Mol 13Mol 105Mol 140
Mol 17
Mol 39
Mol 2
Mol 21
Mol 12
Mol 142
Mol 126
Mol 135
Mol 121
Mol 119
Mol 124
Mol 120
Mol 117
Mol 133
Mol 131
Mol 134
PlatycodonGrandiforus
Aurantii Fructus
Licorice
Radix Bupleuri
RehmanniaglutinosaLibosch
AngelicaeSinensis Radix
Mol 8
Mol 73
Mol 75
Mol 77
Mol 80
Mol 7
Mol 82
Mol 76
Mol 74
Mol 60
Mol 46
Mol 150
Mol 151
Mol 112
Mol 115
Mol 11
Mol 116
Mol 113
Mol 110
Mol 1
Mol 9Mol 101Mol 10Mol 93
Mol 103Mol 89 Mol 107
Mol 96PRSS3 LDLRMAPK3 BACE1 EPHB2
Mol 35Mol 81
Mol 86
Mol 85
Mol 84
Mol 88
Mol 83Mol 30
Mol 137
ChuanxiongRhizoma
Mol 3
Mol 57
Mol 139
Achyranthis
Bidentatae
RadixMol 102
CASP8
IGHG1AKT1p53
CHRNA7
Mol 40
SLC6A3 PTPN1
SOD1MAPK1
Mol 38
Mol 95
Carthami Flos
Mol 24
Mol 78 Persicae Semen
Mol 122
Mol 25
Mol 98
TGFB1
GSK3B
PRKCA
Radix Paeoniae Rubra
Mol 52
Mol 42
Mol 62
Mol 138
EGFR
STAT1 PPP3CAPLAT
Stigmasterol
MAP2
Beta-sistosterol Quercetin
Kaempferol
SYNGAP1
CALM
Baicalin
PRKCB
CAV1
CTSD
TNF
DRD1
CDK1
IFNG
FN1BBC3 PRKCD
Mol 132
Mol 160
Mol 58
Mol 67
Mol 69 Mol 43ALB
Mol 64CTNNB1
xiongixioChuanxC nxnxioaamaRhihihizoma
Achyranthisiistntnt
aeeaeBidenBidennntatae
RadixadixR diR di
oniae nianiaiaonRadix PaeoPaePRadix P eeoeonRRRRubra dondonddoodPlatycolatycPlatyycPl ycoyccoPlatPlatycoPlatyccocod
rususrusususruGranG anGranGranGranGrGGranndndiforu
ructusuctuc sructusuctusructusruAurantiirA ntaA ntiitiirantAurantintiurantAur iiii Fru
cecececececececeecLiLLiLLiLiLLLLLLLLiLiLiLLiLLLiLicoric
leuririeuuurieurileurleurieurieurieurieurileRadix BBRadRad Bdix Bdixx R i Bdi BBBadix Radix BBuple
RehmanniaRe mRe m nnRehmanniamReRehmhmannnniamanniaRehmehmanniRR nosasasasaosaosglutilutglutgluulutiglgl titinos
LiboschLibLLibo hschib chLiboschboscLLib chschLibos
icaecaecacaeicaeicAngelAnAngeAngelAnAng lAAngeelielicdix dix dixdixdSinensinensSS nennensSinSSiSinensSinensSine sissis Rad
FloslololFlhamaCartharthhaamami Fl
menmennnnmicae Scae SSem
Figure 6 HB-pC-pT network of XFZYD for treating TBI The triangles with circle backgrounds represent the herbs (HB) the squares andcircles represent the potential compounds (pC) and targets (pT)The red triangles squares and circles represent corresponding HB pC andpT in the Junherbs the same is to aqua representing theChen herbs and periwinkle representing the Zuo-Shi herbsThe claybank squares andcircles represent corresponding pC and pT shared by the Chen and Zuo-Shi herbs the same is to blue representing the overlap between theJun and Zuo-Shi herbs and the green representing the overlap between the Chen and Zuo-Shi herbsThe purple squares and circles representthe corresponding pC and pT shared by the 3 groups of herbs
alleviating insulin resistance in type 2 diabetic rats [72]Beta-sitosterol (BS) is a vegetable-derived compound foundin various plants and is suggested to modulate the immunefunction inflammation and pain levels by controlling theproduction of inflammatory cytokines [73]
For targets analysis CALM possesses the largest degree(degree =84) followed by GSK3B (degree =61) SCN5A(degree = 59) F2 (degree = 43) ACHE (degree=28)F7 (degree=28) ADRA1B (degree=28) NOS3 (degree=20)BCL2 (degree=18) and CASP3 (degree=18) which demon-strated their crucial therapeutic effects for treating TBI Forinstance CALM possess an essential position in calciumsignaling pathway and is related to morphological changesmigration proliferation and secretion of cytokines andreactive oxygen species ofMicroglial cells [74]The activationof CaMKII120572 major isoform of Ca2+calmodulin-dependentprotein kinase (CaMK) in brain is directly associated withthe production of proinflammatory cytokines such as TNF-120572and IL-1120573 [75] GSK-3120573 is a serinethreonine-protein kinasewhich is abundant in the central nervous system (CNS)particularly in neurons [76] It can control gene transcrip-tion axonal transport and cytoskeletal dynamics in growthcones [77] The inhibition of GSK-3120573 attenuates apoptotic
signals and prevents neuronal death [78] There is increasingevidence that prothrombin (F2) and its active derivativethrombin are expressed locally in the central nervous systemBeside the central role in the coagulation cascade the gener-ation of thrombin leads to receptor mediated inflammatoryresponses cell proliferationmodulation cell protection andapoptosis [79 80] The role in brain injury depends upon itsconcentration as higher amounts cause neuroinflammationand apoptosis while lower concentrations might even becytoprotective [81]
Direct tissue damage aswell as hypoxic-ischemic increas-ing anaerobic glycolysis of the brain tissue results in theATP-stores depletion and failure of energy-dependent mem-brane ion pumps especially for voltage-dependent Ca2+ andNa+-channels Accumulated Ca2+ activates lipid peroxidasesproteases and phospholipases and caspases at the sametime increasing the intracellular concentration of free fattyacids and free radicals leading to necrosis or apoptosis ofneurocyte [82] At the same time the activation of residentglial cells microglia and astrocytes and the infiltration ofblood leukocytes secrete various immune mediators elicitedinflammatory responses which subsequently intersect withadjacent pathological cascades including oxidative stress
12 Evidence-Based Complementary and Alternative Medicine
Table 6 Top 10 potential candidate compounds of XFZYD for treating TBI according to 2 centrality indicators
Compound Degree Compound Betweennessquercetin 76 quercetin 01682179kaempferol 43 kaempferol 005501511beta-sitosterol 35 wogonin 005141376Stigmasterol 19 beta-sitosterol 004451294luteolin 18 naringenin 003251738baicalein 15 beta-carotene 002977217-Methoxy-2-methyl isoflavone 12 nobiletin 002787992wogonin 12 7-Methoxy-2-methyl isoflavone 002096439nobiletin 11 luteolin 002090223naringenin 9 baicalein 001453488
Table 7 Top 10 potential targets of XFZYD for treating TBI according to 2 centrality indicators
Targets Degree Targets BetweennessCALM 84 CALM 021452046GSK3B 61 SCN5A 011441591SCN5A 59 GSK3B 009553896F2 43 F2 00689171ACHE 28 BCL2 002651903F7 28 CASP3 002372836ADRA1B 28 F7 002032647NOS3 20 ACHE 001845996BCL2 18 ADRA1B 001704505CASP3 18 NOS3 00166699
excitotoxicity or reparative events including angiogenesisscarring and neurogenesis [83] Function analysis of the 47target proteins regulated by XFZYD was mainly associatedwith core pathophysiology process of TBI (Figure 7) 47 targetproteins were connected with 16 key process related to TBIMost of the targets have one or more links to other biolog-ical process such as apoptosis cell proliferation superoxideanion generation nitric oxide biosynthetic process responseto calcium ion I-kappaB kinaseNF-kappaB signaling andregulation of inflammation The above analysis implied themultifunction character of these target proteins regulated byXFZYD Of these target proteins 25 proteins (53 of the 47)such as TGFB1 EGFR CAV1 MAPK1 PRKCB and AKT1were responsible for regulating the apoptosis process 17 forblood coagulation 14 for cell proliferation and axon genesisand 12 for hypoxia andMAPK cascade We found that TGFB1was the crucial protein because it participated in 9 biologicalprocesses related to TBI such as apoptosis blood coagulationcell proliferation and MAPK cascade followed by EGFR (8)CAV1 (7) MAPK1 (6) PRKCB (6) and AKT1 (6)
Overall these observations strongly support the evidencethat the generated HB-pCminuspT network and pT-F networkhave important roles in treating TBI further validating thedrug targeting approach
Molecule docking was used to further validate the bind-ing mode between candidate compounds and their targetproteins We found that 18 TBI-specific target proteins inter-acted with 91 candidate compounds from XFZYD (Figure 8)
Other 29 target proteins were not discussed for the lackof proper protein crystal structure The detailed moleculedocking results are shown in Table S5The 6 essential proteinsincluding GSK3B AKT1 CDK1 F2 NOS3 and ACHEwere used to elucidate the exact binding mode (Figure 9)Quercetin was located within the binding cavity of AKT1 andCDK1 (Figures 9(a) and 9(b) and S1A B) Four conventionalhydrogen bonds were formed between quercetin and AKT1by interacting with the key amino acids including ILE-290 THR-211 and SER 205 Additionally 120587-120587 interactionsbetween quercetin and TRP-90 were found in the activesite which helped the stabilization of the compound at thebinding site (Figure 9(a) S1A) Figure 9(b) and S1B suggestedthat five conventional hydrogen bonds (LEU-83 ASP-146and LYS-33) and 120587-120587 interaction (PHE-80) were formedbetween quercetin and CDK1 The GSK3B-FA complexes(Figure 9(c) S1C) were stabilized by 6 hydrogen-bondinginteractions between FA and LYS-85 GLU-97 TYR-134 andARG-141 Glyasperin Bmainly bonds to F2 through hydrogenbonds by interacting with the key amino acids includingGLY-193 SER-195 and GLY-219 (Figure 9(d) S1D) andan edge-to-face 120587 minus 120587 interaction was also observed withTYR-228 The GLN-247 GLU-351 and GLY-355 from theactive site pocket of NOS3 participated in the hydrogen-bondformationwith 1-Methoxyphaseollidin (Figure 9(e) S1E)The(-)-Medicocarpin formed a total of 6 hydrogen bonds withSER-293 PHE-295 ARG-296 and TRY-341 in the active siteof ACHE (Figure 9(f) S1F) Besides an edge-to-face 120587 minus 120587
Evidence-Based Complementary and Alternative Medicine 13
KCNMA1
P53
IFNG
PPP3CA
CASP8
SCN5A
I-kappaB kinase NF-kappaB signaling
Regulation of inflammation
Response to calcium ion
TGFB1
TNF
PRKCA
CHRNA7
CAV1
STAT1
MAPK cascadeAKT1
BCL2
APP
EGFR
MAPK1
Angiogenesis
SYNGAP1
MAP2
PTPN1
Axonogenesis
GSK3B
EPHB2
CDK1
CASP7FN1
MAPK3
CTNNB1
F2
Autophagy
Cell proliferation
BACE1
SLC6A3
DRD1
ADRA1BACHE
CALMCASP3
PLAT
Apoptotic process
NOS3
PRKCB
Blood coagulation
Response to hypoxia
Superoxide anion generation
Nitric oxide biosynthetic process
Astrocyte activation
Amyloid-beta regulation
Microglial cell activation
LDLR
PRSS3
F7
PRKCD
SOD1ALB
BBC3
Figure 7 pT-F network of XFZYD for treating TBI 16 biological processes (red square) the 47 target proteins (periwinkle circle) of XFZYDparticipate in for treating TBI
interactionwas also observedwith TYR-337 From the resultshydrogen-bonding and edge-to-face 120587 minus 120587 interactions playkey roles in the proteinminusligand recognition and stabilitywhich may be helpful in determining the inhibitor activitiesAnd the C-T network confirmed the potential therapeuticeffects of the candidate compounds from XFZYD to treatTBI through interacting with the relevant proteins The com-putational analysis further elucidated the accurate moleculemechanisms between active compounds and targets
4 Discussion
Traumatic brain injury (TBI) is a growing public healthproblem worldwide and is a leading cause of death anddisability [84] Although major progress has been made in
understanding the pathophysiology of this injury this hasnot yet led to substantial improvements in outcome by alack of treatments which have proven successful during phaseIII trials for modern medicine [85 86] TCM rooted inthousands of years of history may offer an alternative or acomplementary strategy for the treatment of TBI XFZYDa representative formula in TCM has been used for yearsto treat TBI in China and has been demonstrated to beeffective in clinical practice However its ldquomulticomponentsrdquoand ldquomultitargetsrdquo features make it much difficult to decipherthemolecular mechanisms of XFZYD in the treatment of TBIfrom a systematic perspective if employing routine methods
In the present study a network pharmacology-basedmethod was employed to elucidate the pharmacologicalmechanisms of XFZYD to treat TBI according to the drug
14 Evidence-Based Complementary and Alternative Medicine
Figure 8 C-T network through molecule docking validating 119 potential compounds (green triangles) interacting with 18 potential targets(yellow circles) of XFZYDThe size of the nodes is proportional to the value of degree
combination principle of TCM We first proposed a newmodeling system combining OB and DL screening multipledrug targets prediction and validation network constructionand molecule docking to probe the efficiency of a typicalTCM formula XFZYD for the treatment of TBI The 11herbs from XFZYD possessed 162 bioactive compounds andtargeted 285 proteins There were 5 compounds and 189
target proteins overlapped among the Jun Chen and Zuo-Shigroup Furthermore 47 TBI-specific proteins were targetedby 119 (73) bioactive compounds from XFZYD Similarly 5common compounds and 33 (70) common target proteinsamong the 3 groups of drugs were observed Most of thebioactive ingredients targeted more than one protein The 47target proteins regulated several essential pathophysiological
Evidence-Based Complementary and Alternative Medicine 15
(a) (b) (c)
(d) (e) (f)
Figure 9 Hydrogen-bonding networks within the binding site of the compoundminustarget complexes obtained from molecule docking(a) AKT1-quercetin (b) CDK1-quercetin (c) GSK3B-FA (d) F2-glyasperin B (e) NOS3-1-Methoxyphaseollidin and (f) ACHE-(-)-Medicocarpin The molecules are presented as ball and stick models Active site amino acid residues are represented as lines Dotted bluelines in these pictures represent hydrogen bonds with distance unit of A Other O and N atoms are colored as red and blue respectively
processes of TBI that referred to apoptosis inflammationcell proliferation superoxide anion generation nitric oxidebiosynthetic process response to calcium ion etc The aboveanalysis reveals that the synergistic action mechanisms ofXFZYDmay be (1) bioactive compounds overlapping amongthe different group of herbs (2) specific bioactive com-pounds from different groups of herbs targeting the sameproteins (3) specific bioactive compounds from differentgroups of herbs targeting different proteins which participatein the same pathophysiological process of the disease Toa certain degree the 5 compounds including quercetinstigmasterol kaempferol baicalin and beta-sitosterol playedessential role in XFZYD for TBI treatment MAPK3MAPK1AKT1 PRKCA TNF PRKCB EGFR BCL2 GSK3B CASP3PPP3CA and NOS3 were the main target proteins regulatedby XFZYD in the treatment of TBI
Interestingly Beta-carotene (Mol 43) from Carthami Flos(the Jun herb) specifically regulated 120573-Catenin (CTNNB1)and played critical role for curing TBI The 120573-Catenin is acritical downstream component of the Wnt pathway whichplays essential role in the regulation of mammalian neuraldevelopment [87] In vitro and in vivo studies demonstrate
that the Wnt120573-catenin pathway regulates the proliferationand differentiation of neural progenitor cells [88] Neuronaldifferentiation is induced by overexpression of 120573-cateninor the pharmacological inhibition of GSK3120573 (the phospho-rylating enzyme of 120573-catenin) [89 90] This pathway alsopromotes blood vessel formation during vascular develop-ment as well as the vascular repair process after TBI [91]In addition wogonin (Mol 160) from Achyranthis BidentataeRadix (the Chen herb) specifically targeted fibronectin (FN1)Bcl-2-binding component 3 (BBC3) and Protein KinaseC delta type (PRKCD) FN1 an important component ofthe extracellular matrix (ECM) environment promotes cellmigration neurite outgrowth and synapse formation dur-ing neural development [92] It aggregates in the injuredbrain and plays a neuroprotection role through antiapoptosisand anti-inflammation ways following TBI [93 94] BBC3namely p53 upregulated modulator of apoptosis (PUMA) iscritical for the p53-dependent apoptosis pathway which playsan important role in hippocampal neuronal loss and associ-ated cognitive deficits [95] PRKCD one of PKC isoformsactivates signal transduction pathways involved in neuronalregeneration [96] synaptic transmissionplasticity [97] and
16 Evidence-Based Complementary and Alternative Medicine
activation of apoptosis processes [98] as well as higher brainfunctions such as learning andmemory [99]The activators ofPKCare effective for the treatment of TBI [100] FurthermoreMitogen-activated protein kinase 3 (MAPK3) Beta-secretase1 (BACE1) Ephrin type-B receptor 2 (EphB) and low-densitylipoprotein receptor (LDLR) were specifically targeted bythe ingredients in the Zuo-Shi herbs Naringenin (Mol133) targeted LDLR MAPK3 while euchrenone (Mol 60)anchored BACE1 and nobiletin (Mol 134) targeted EphB2MAPK3 is an essential component of the MAP kinase signaltransduction pathway It has been appreciated recently thatthe ERK12 cascade plays a fundamental role in synapticplasticity and memory [101] BACE1 is responsible for pro-duction of A120573 from amyloid precursor protein (APP) A120573can cause cell death activate inflammatory pathways [102]and prime proapoptotic pathways for activation by otherinsults [103] The blocking of BACE1 can ameliorate motorand cognitive deficits and reduce cell loss after experimentalTBI in mice [104] EphB is localized to synaptic sites inhippocampal neurons [105] The interaction between EphBand NMDA receptors regulates excitatory synapse formation[106] LDLR acts as an important receptor that facilitatesbrain A120573 clearance and inhibits amyloid deposition [107]and then ameliorates Alzheimerrsquos disease neuropathologyafter TBI [108] The analysis above indicates the ldquoJunrdquoldquoChenrdquo and ldquoZuo-Shirdquo herbs from XFZYD trigger theirspecific targets regulation respectively for the therapeuticeffects
XFZYD is a very famous traditional Chinese formula inpromoting qi circulation and removing blood stasis accord-ing to TCM theory However several limited researches havedemonstrated its efficacy for treating TBI such as anti-inflammatory and synaptic regulation [30 31] which arein accord with our study However previous studies merelypartially deciphered the molecule mechanism of XFZYD fortreating TBI This study reports 119 bioactive compounds inXFZYD that target 47 TBI-specific proteins such as MAPK3MAPK1 AKT1 PRKCA TNF PRKCB and EGFR Theseproteins regulate several crucial pathophysiological processesof TBI such as apoptosis inflammation blood coagulationand axon genesis Our study demonstrates that the therapeu-tic actions of XFZYD refer to ldquomulticompoundsrdquo ldquomultitar-getsrdquo features rather than only the improvement of bloodcirculation With the help of molecule docking methodwe further validate the interactions between bioactive com-pounds and potential targets of XFZYD The hydrogen-bonding and edge-to-face 120587 minus120587 interactions play key roles inthe proteinminusligand recognition and stability This provides avaluable reference for further experimental investigations ofbioactive ingredients and therapeutic targets of XFZYD fortreating TBI
5 Conclusion
Our work successfully illuminates the efficiency of XFZYDfor the treatment of TBI as well as herb combination ruleof TCM formula Network pharmacology with moleculedocking method confirms the ldquomulticompounds multitar-getsrdquo therapeutic actions of XFZYD in the treatment of TBI
The present work may provide valuable evidence for furtherclinical application of XFZYD for treating TBI
Abbreviations
TBI Traumatic brain injuryXFZYD Xuefu Zhuyu decoctionTCM Traditional Chines medicineDL Drug-likenessOB Oral bioavailabilityHB-cC-cT Herb-candidate compound-candidate
targetHB-pC-pT Herb-potential compound-potential targetCALM CalmodulinGSK3B Glycogen synthase kinase-3 betaSCN5A Sodium channel protein type 5 subunit
alphaF2 ProthrombinACHE AcetylcholinesteraseGO Gene OntologyKEGG Kyoto Encyclopedia of Genes and
Genomes
Data Availability
The data used to support the findings of this study areavailable from the corresponding author upon request
Conflicts of Interest
The authors declare that they have no conflicts of interest
Authorsrsquo Contributions
YangWang and Zebing Huang participated in the conceptionand design of the study YuanyuanZhong YangWang ZebingHuang Jiekun Luo Tao Tang and Pengfei Li acquired andanalyzed the data Yuanyuan Zhong Tao Liu and HanjinCui drafted and revised the manuscript The correspondingauthor and all of the authors have read and approved the finalsubmitted manuscript
Acknowledgments
This work was supported by the National Natural ScienceFoundation of China (Nos 81673719 81874409 81603670and 81803948) Project funded byChina Postdoctoral ScienceFoundation (Nos 2016M600639 and 2017T100614)
Supplementary Materials
Table S1 162 bioactive compounds in XFZYD Table S2 targetproteins of XFZYD Table S3 TBI-specific proteins Table S4119 potential compounds of XFZYD for treating TBI TableS5 docking result of 18 target proteins with 91 potential com-pounds Fig S1 the exact bindingmode between active ingre-dients and protein targets obtained from molecule docking(A) AKT1-quercetin (B) CDK1-quercetin (C) GSK3B-FA
Evidence-Based Complementary and Alternative Medicine 17
(D) F2-glyasperin B (E) NOS3-1-Methoxyphaseollidin and(F)ACHE-(-)-Medicocarpin (Supplementary Materials)
References
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[2] J A Langlois W Rutland-Brown and M M Wald ldquoTheepidemiology and impact of traumatic brain injury a briefoverviewrdquo The Journal of Head Trauma Rehabilitation vol 21no 5 pp 375ndash378 2006
[3] H A Alsulaim B J Smart A O Asemota et al ldquoConsciousstatus predictsmortality among patientswith isolated traumaticbrain injury in administrative datardquo The American Journal ofSurgery vol 214 no 2 pp 207ndash210 2017
[4] M Majdan D Plancikova A Maas et al ldquoYears of life lost dueto traumatic brain injury in Europe A cross-sectional analysisof 16 countriesrdquo PLoS Medicine vol 14 no 7 p e1002331 2017
[5] P Cheng P Yin P Ning et al ldquoTrends in traumatic braininjury mortality in China 2006ndash2013 A population-basedlongitudinal studyrdquo PLoS Medicine vol 14 no 7 Article IDe1002332 2017
[6] M V Russo and D B McGavern ldquoInflammatory neuroprotec-tion following traumatic brain injuryrdquo Science vol 353 no 6301pp 783ndash785 2016
[7] WWang H Li J Yu et al ldquoProtective Effects of ChineseHerbalMedicine Rhizoma drynariae in Rats After Traumatic BrainInjury and Identification of Active Compoundrdquo MolecularNeurobiology vol 53 no 7 pp 4809ndash4820 2016
[8] M Das S Mohapatra and S S Mohapatra ldquoNew perspectiveson central and peripheral immune responses to acute traumaticbrain injuryrdquo Journal of Neuroinflammation vol 9 p 236 2012
[9] J W Finnie ldquoNeuroinflammation Beneficial and detrimentaleffects after traumatic brain injuryrdquo Inflammopharmacologyvol 21 no 4 pp 309ndash320 2013
[10] T Cheng W Wang Q Li et al ldquoCerebroprotection of flavanol(minus)-epicatechin after traumatic brain injury viaNrf2-dependentand -independent pathwaysrdquo Free Radical Biology amp Medicinevol 92 pp 15ndash28 2016
[11] W Young ldquoRole of calcium in central nervous system injuriesrdquoJournal of Neurotrauma vol 9 Suppl 1 pp S9ndashS25 1992
[12] R Bullock A Zauner J S Myseros et al ldquoEvidence forProlonged Release of Excitatory Amino Acids in Severe HumanHead Trauma Relationship to Clinical Eventsrdquo Annals of theNew York Academy of Sciences vol 765 no 1 pp 290ndash297 1995
[13] A T Mazzeo A Beat A Singh and M R Bullock ldquoTherole of mitochondrial transition pore and its modulation intraumatic brain injury and delayed neurodegeneration afterTBIrdquo Experimental Neurology vol 218 no 2 pp 363ndash370 2009
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[15] J Ghajar ldquoTraumatic brain injuryrdquo The Lancet vol 356 no9233 pp 923ndash929 2000
[16] D J Loane and A I Faden ldquoNeuroprotection for traumaticbrain injury translational challenges and emerging therapeuticstrategiesrdquoTrends in Pharmacological Sciences vol 31 no 12 pp596ndash604 2010
[17] Y Wang X Fan T Tang et al ldquoRhein and rhubarb simi-larly protect the blood-brain barrier after experimental trau-matic brain injury via gp91phox subunit of NADPH oxidaseROSERKMMP-9 signaling pathwayrdquo Scientific Reports vol 6p 37098 2016
[18] D-X Kong X-J Li and H-Y Zhang ldquoWhere is the hopefor drug discovery Let history tell the futurerdquo Drug DiscoveryTherapy vol 14 no 3-4 pp 115ndash119 2009
[19] F Cheung ldquoTCM made in Chinardquo Nature vol 480 no 7378pp S82ndashS83 2011
[20] C Fu Z Xia Y Liu et al ldquoQualitative analysis of majorconstituents from Xue Fu Zhu Yu Decoction using ultra highperformance liquid chromatographywith hybrid ion trap time-of-flight mass spectrometryrdquo Journal of Separation Science vol39 no 17 pp 3457ndash3468 2016
[21] H-J Zhang and Y-Y Cheng ldquoAn HPLCMS method for iden-tifying major constituents in the hypocholesterolemic extractsof Chinese medicine formula lsquoXue-Fu-Zhu-Yu decoctionrsquordquoBiomedical Chromatography vol 20 no 8 pp 821ndash826 2006
[22] L Zhang L Zhu YWang et al ldquoCharacterization and quantifi-cation of major constituents of Xue Fu Zhu Yu by UPLC-DAD-MSMSrdquo Journal of Pharmaceutical and Biomedical Analysisvol 62 pp 203ndash209 2012
[23] X Yang X Xiong G Yang and J Wang ldquoChinese patentmedicine Xuefu Zhuyu capsule for the treatment of unstableangina pectoris a systematic review of randomized controlledtrialsrdquo Complementary Therapies in Medicine vol 22 no 2 pp391ndash399 2014
[24] J Wang X Yang F Chu et al ldquoThe effects of Xuefu Zhuyuand Shengmai on the evolution of syndromes and inflammatorymarkers in patients with unstable angina pectoris after percuta-neous coronary intervention a randomised controlled clinicaltrialrdquoEvidence-BasedComplementary andAlternativeMedicinevol 2013 Article ID 896467 9 pages 2013
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18 Evidence-Based Complementary and Alternative Medicine
[31] M Sun X-P Zhan C-Y Jin J-Z Shan S Xu and Y-LWang ldquoclinical observation on treatment of post-craniocerebraltraumatic mental disorder by integrative medicinerdquo ChineseJournal of Integrative Medicine vol 14 no 2 pp 137ndash141 2008
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[34] S Li ldquoExploring traditional chinese medicine by a novel thera-peutic concept of network targetrdquo Chinese Journal of IntegrativeMedicine vol 22 no 9 pp 647ndash652 2016
[35] S Li and B Zhang ldquoTraditional Chinese medicine networkpharmacology theory methodology and applicationrdquo ChineseJournal of Natural Medicines vol 11 no 2 pp 110ndash120 2013
[36] A L Hopkins ldquoNetwork pharmacology the next paradigm indrug discoveryrdquoNature Chemical Biology vol 4 no 11 pp 682ndash690 2008
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[39] Bo Zhang Xu Wang and Shao Li ldquoAn Integrative Platform ofTCM Network Pharmacology and Its Application on a HerbalFormula Qing-Luo-Yinrdquo Evidence-Based Complementary andAlternativeMedicine vol 2013 Article ID 456747 12 pages 2013
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[41] J V Turner D J Maddalena and S Agatonovic-KustrinldquoBioavailability Prediction Based on Molecular Structure for aDiverse Series of Drugsrdquo Pharmaceutical Research vol 21 no 1pp 68ndash82 2004
[42] X XuW Zhang CHuang et al ldquoAnovel chemometricmethodfor the prediction of human oral bioavailabilityrdquo InternationalJournal of Molecular Sciences vol 13 no 6 pp 6964ndash6982 2012
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[44] X Wei H Liu X Sun et al ldquoHydroxysafflor yellow A protectsrat brains against ischemia-reperfusion injury by antioxidantactionrdquo Neuroscience Letters vol 386 no 1 pp 58ndash62 2005
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[49] A Hamosh A F Scott J S Amberger C A Bocchini andV AMcKusick ldquoOnline Mendelian Inheritance in Man (OMIM) aknowledgebase of human genes and genetic disordersrdquo NucleicAcids Research vol 33 pp D514ndashD517 2005
[50] T S Keshava Prasad RGoel K Kandasamy et al ldquoHuman pro-tein reference databasemdash2009 updaterdquo Nucleic Acids Researchvol 37 no 1 pp D767ndashD772 2009
[51] A Franceschini D Szklarczyk S Frankild et al ldquoSTRING v91protein-protein interaction networks with increased coverageand integrationrdquoNucleic Acids Research vol 41 no 1 pp D808ndashD815 2013
[52] P Shannon A Markiel O Ozier et al ldquoCytoscape a softwareEnvironment for integratedmodels of biomolecular interactionnetworksrdquo Genome Research vol 13 no 11 pp 2498ndash25042003
[53] G Dennis Jr B T Sherman D A Hosack et al ldquoDAVIDDatabase for Annotation Visualization and IntegratedDiscov-eryrdquo Genome Biology vol 4 no 5 p P3 2003
[54] L Yang X Zhao and X Tang ldquoPredicting disease-related pro-teins based on clique backbone in protein-protein interactionnetworkrdquo International Journal of Biological Sciences vol 10 no7 pp 677ndash688 2014
[55] S MThomas and J S Brugge ldquoCellular functions regulated bySRC family kinasesrdquo Annual Review of Cell and DevelopmentalBiology vol 13 pp 513ndash609 1997
[56] D Z Liu F R Sharp K C Van et al ldquoInhibition of Src familykinases protects hippocampal neurons and improves cognitivefunction after traumatic brain injuryrdquo Journal of Neurotraumavol 31 no 14 pp 1268ndash1276 2014
[57] B D Manning and A Toker ldquoAKTPKB Signaling Navigatingthe Networkrdquo Cell vol 169 no 3 pp 381ndash405 2017
[58] Y Kitagishi and S Matsuda ldquoDiets involved in PPAR andPI3KAKTPTEN pathway may contribute to neuroprotectionin a traumatic brain injuryrdquoAlzheimerrsquos ResearchampTherapy vol5 no 5 p 42 2013
[59] D Chen L Bao S-Q Lu and F Xu ldquoSerum albumin andprealbuminpredict the poor outcomeof traumatic brain injuryrdquoPLoS ONE vol 9 no 3 Article ID e93167 2014
[60] J Yang Z Wu N Renier et al ldquoPathological axonal deaththrough aMapk cascade that triggers a local energy deficitrdquoCellvol 160 no 1-2 pp 161ndash176 2015
[61] E J Huang and L F Reichardt ldquoTrk receptors roles in neuronalsignal transductionrdquoAnnual Review of Biochemistry vol 72 pp609ndash642 2003
[62] J W Lee and R Juliano ldquoMitogenic signal transductionby integrin- and growth factor receptor-mediated pathwaysrdquoMolecules and Cells vol 17 no 2 pp 188ndash202 2004
[63] C S Yang J M Landau M T Huang and H L NewmarkldquoInhibition of carcinogenesis by dietary polyphenolic com-poundsrdquo Annual Review of Nutrition vol 21 pp 381ndash406 2001
[64] A Murakami H Ashida and J Terao ldquoMultitargeted cancerprevention by quercetinrdquoCancer Letters vol 269 no 2 pp 315ndash325 2008
[65] G Du Z Zhao Y Chen et al ldquoQuercetin attenuates neuronalautophagy and apoptosis in rat traumatic brain injury modelvia activation of PI3KAkt signaling pathwayrdquo NeurologicalResearch vol 38 no 11 pp 1ndash8 2016
Evidence-Based Complementary and Alternative Medicine 19
[66] Q Cai R O Rahn and R Zhang ldquoDietary flavonoidsquercetin luteolin andgenistein reduce oxidativeDNAdamageand lipid peroxidation and quench free radicalsrdquoCancer Lettersvol 119 no 1 pp 99ndash107 1997
[67] G A Bongiovanni E A Soria and A R Eynard ldquoEffectsof the plant flavonoids silymarin and quercetin on arsenite-induced oxidative stress in CHO-K1 cellsrdquo Food and ChemicalToxicology vol 45 no 6 pp 971ndash976 2007
[68] H Li L Yang Y Zhang et al ldquoKaempferol inhibits fibroblastcollagen synthesis proliferation and activation in hypertrophicscar via targeting TGF-beta receptor type Irdquo Biomedicine ampPharmacotherapy = Biomedecine amp Pharmacotherapie vol 83pp 967ndash974 2016
[69] K P Devi D S Malar S F Nabavi et al ldquoKaempferol andinflammation From chemistry to medicinerdquo PharmacologicalResearch vol 99 pp 1ndash10 2015
[70] M Zhou H Ren J Han W Wang Q Zheng and DWang ldquoProtective effects of kaempferol against myocardialischemiareperfusion injury in isolated rat heart via antioxidantactivity and inhibition of glycogen synthase kinase-3120573rdquo Oxida-tive Medicine and Cellular Longevity vol 2015 p 481405 2015
[71] C-F Lee J-S Yang F-J Tsai et al ldquoKaempferol inducesATMp53-mediated death receptor and mitochondrial apop-tosis in human umbilical vein endothelial cellsrdquo InternationalJournal of Oncology vol 48 no 5 pp 2007ndash2014 2016
[72] C Luo H Yang C Tang et al ldquoKaempferol alleviates insulinresistance via hepatic IKKNF-120581B signal in type 2 diabetic ratsrdquoInternational Immunopharmacology vol 28 no 1 pp 744ndash7502015
[73] A B Awad and C S Fink ldquoPhytosterols as anticancer dietarycomponents evidence and mechanism of actionrdquo Journal ofNutrition vol 130 no 9 pp 2127ndash2130 2000
[74] K Farber and H Kettenmann ldquoFunctional role of calciumsignals for microglial functionrdquoGlia vol 54 no 7 pp 656ndash6652006
[75] Y Lu Y Gu X Ding et al ldquoIntracellular Ca2+ homeostasisand JAK1STAT3 pathway are involved in the protective effectof propofol on BV2 microglia against hypoxia-induced inflam-mation and apoptosisrdquo PLoS ONE vol 12 no 5 p e01780982017
[76] K Leroy and J-P Brion ldquoDevelopmental expression and local-ization of glycogen synthase kinase-3120573 in rat brainrdquo Journal ofChemical Neuroanatomy vol 16 no 4 pp 279ndash293 1999
[77] C-M Liu E-M Hur and F-Q Zhou ldquoCoordinating geneexpression and axon assembly to control axon growth Potentialrole ofGSK3 signalingrdquo Frontiers inMolecularNeuroscience vol3 p 3 2012
[78] D A E Cross A A Culbert K A Chalmers L Facci S DSkaper and A D Reith ldquoSelective small-molecule inhibitors ofglycogen synthase kinase-3 activity protect primary neuronesfrom deathrdquo Journal of Neurochemistry vol 77 no 1 pp 94ndash102 2001
[79] V S Ossovskaya and NW Bunnett ldquoProtease-activated recep-tors contribution to physiology and diseaserdquo PhysiologicalReviews vol 84 no 2 pp 579ndash621 2004
[80] M Steinhoff J Buddenkotte V Shpacovitch et al ldquoProteinase-activated receptors transducers of proteinase-mediated signal-ing in inflammation and immune responserdquoEndocrine Reviewsvol 26 no 1 pp 1ndash43 2005
[81] H Krenzlin V Lorenz S Danckwardt O Kempski and BAlessandri ldquoThe importance of thrombin in cerebral injury and
diseaserdquo International Journal of Molecular Sciences vol 17 no1 2016
[82] M J McGinn and J T Povlishock ldquoPathophysiology of Trau-matic Brain InjuryrdquoNeurosurgery Clinics of North America vol27 no 4 pp 397ndash407 2016
[83] T Woodcock and M C Morganti-Kossmann ldquoThe role ofmarkers of inflammation in traumatic brain injuryrdquo Frontiersin Neurology vol 4 article 18 2013
[84] F Tanriverdi H J Schneider G Aimaretti B E Masel F FCasanueva and F Kelestimur ldquoPituitary dysfunction after trau-matic brain injury A clinical and pathophysiological approachrdquoEndocrine Reviews vol 36 no 3 pp 305ndash342 2015
[85] J V Rosenfeld A I Maas P Bragge M C Morganti-Kossmann G T Manley and R L Gruen ldquoEarly managementof severe traumatic brain injuryrdquoThe Lancet vol 380 no 9847pp 1088ndash1098 2012
[86] B M Aertker S Bedi and C S Cox ldquoStrategies for CNS repairfollowing TBIrdquo Experimental Neurology vol 275 no 3 pp 411ndash426 2016
[87] K Adachi Z Mirzadeh M Sakaguchi et al ldquo120573-catenin signal-ing promotes proliferation of progenitor cells in the adultmousesubventricular zonerdquo Stem Cells vol 25 no 11 pp 2827ndash28362007
[88] Y Hirabayashi and Y Gotoh ldquoStage-dependent fate determina-tion of neural precursor cells in mouse forebrainrdquo NeuroscienceResearch vol 51 no 4 pp 331ndash336 2005
[89] S Ding T Y H Wu A Brinker et al ldquoSynthetic smallmolecules that control stem cell faterdquo Proceedings of theNationalAcadamy of Sciences of the United States of America vol 100 no13 pp 7632ndash7637 2003
[90] N Israsena M Hu W Fu L Kan and J A Kessler ldquoThepresence of FGF2 signaling determines whether 120573-cateninexerts effects on proliferation or neuronal differentiation ofneural stem cellsrdquo Developmental Biology vol 268 no 1 pp220ndash231 2004
[91] A Salehi A Jullienne M Baghchechi et al ldquoUp-regulation ofWnt120573-catenin expression is accompanied with vascular repairafter traumatic brain injuryrdquo Journal of Cerebral Blood Flow ampMetabolism vol 38 no 2 pp 274ndash289 2017
[92] L F Reichardt and K J Tomaselli ldquoExtracellular matrixmolecules and their receptors Functions in neural develop-mentrdquoAnnual Review of Neuroscience vol 14 pp 531ndash570 1991
[93] R M Gibson S E Craig L Heenan C Tournier and M JHumphries ldquoActivation of integrin 12057251205731 delays apoptosis ofNtera2 neuronal cellsrdquoMolecularandCellularNeuroscience vol28 no 3 pp 588ndash598 2005
[94] C C Tate A J Garcıa andMC LaPlaca ldquoPlasma fibronectin isneuroprotective following traumatic brain injuryrdquoExperimentalNeurology vol 207 no 1 pp 13ndash22 2007
[95] C Culmsee and M P Mattson ldquop53 in neuronal apoptosisrdquoBiochemical and Biophysical Research Communications vol 331no 3 pp 761ndash777 2005
[96] M S Geddis and V Rehder ldquoThe phosphorylation state ofneuronal processes determines growth cone formation afterneuronal injuryrdquo Journal of Neuroscience Research vol 74 no2 pp 210ndash220 2003
[97] M L Craske M Fivaz N N Batada and T Meyer ldquoSpines andneurite branches function as geometric attractors that enhanceprotein kinase C actionrdquoThe Journal of Cell Biology vol 170 no7 pp 1147ndash1158 2005
20 Evidence-Based Complementary and Alternative Medicine
[98] A Muscella C Vetrugno L G Cossa et al ldquoApoptosis by[Pt(OOrsquo-acac)(gamma-acac)(DMS)] requires PKC-deltamedi-ated p53 activation in malignant pleural mesotheliomardquo PloSone vol 12 no 7 Article ID e0181114 2017
[99] J S Bonini W C Da Silva L R M Bevilaqua J H MedinaI Izquierdo and M Cammarota ldquoOn the participation ofhippocampal PKC in acquisition consolidation and reconsol-idation of spatial memoryrdquo Neuroscience vol 147 no 1 pp 37ndash45 2007
[100] O Zohar R Lavy X Zi et al ldquoPKC activator therapeutic formild traumatic brain injury in micerdquo Neurobiology of Diseasevol 41 no 2 pp 329ndash337 2011
[101] J David Sweatt ldquoTheneuronalMAPkinase cascade a biochem-ical signal integration system subserving synaptic plasticity andmemoryrdquo Journal ofNeurochemistry vol 76 no 1 pp 1ndash10 2001
[102] Y Matsuoka M Picciano B Maleste et al ldquoInflammatoryresponses to amyloidosis in a transgenic mouse model ofAlzheimerrsquos diseaserdquo The American Journal of Pathology vol158 no 4 pp 1345ndash1354 2001
[103] L Esposito L Gan G-Q Yu C Essrich and L MuckeldquoIntracellularly generated amyloid-120573 peptide counteracts theantiapoptotic function of its precursor protein and primesproapoptotic pathways for activation by other insults in neu-roblastoma cellsrdquo Journal of Neurochemistry vol 91 no 6 pp1260ndash1274 2004
[104] D J Loane A Pocivavsek C E-H Moussa et al ldquoAmyloidprecursor protein secretases as therapeutic targets for traumaticbrain injuryrdquo Nature Medicine vol 15 no 4 pp 377ndash379 2009
[105] R Torres B L Firestein H Dong et al ldquoPDZ proteins bindcluster and synaptically colocalize with Eph receptors and theirephrin ligandsrdquo Neuron vol 21 no 6 pp 1453ndash1463 1998
[106] M B Dalva M A Takasu M Z Lin et al ldquoEphB receptorsinteract with NMDA receptors and regulate excitatory synapseformationrdquo Cell vol 103 no 6 pp 945ndash956 2000
[107] J M Basak P B Verghese H Yoon J Kim and D MHoltzman ldquoLow-density lipoprotein receptor represents anapolipoprotein E-independent pathway of A120573 uptake anddegradation by astrocytesrdquoThe Journal of Biological Chemistryvol 287 no 17 pp 13959ndash13971 2012
[108] L Yao X Gu Q Song et al ldquoNanoformulated alpha-mangostinameliorates Alzheimerrsquos disease neuropathology by elevatingLDLR expression and accelerating amyloid-beta clearancerdquoJournal of Controlled Release vol 226 pp 1ndash14 2016
Stem Cells International
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Disease Markers
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Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
6 Evidence-Based Complementary and Alternative Medicine
50
40
30
20
10
0
50
40
40
30
20
20
10
0
0
10 30 50 70 90 110
60
60
60
01 02 03 04 05 06 07 08
225 275 325 375 425 475 525 575 625 minus6minus5minus4minus3minus2minus1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
minus2 minus1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
70
minus1 1 3 5 7 9 11 13 15 17
perc
entage
s (
)
perc
entage
s (
)50
40
30
20
10
0
perc
entage
s (
)
perc
entage
s (
)
40
20
0
60
perc
entage
s (
)50
40
30
20
10
0
perc
entage
s (
)
OB
MW
nHdon
DL
nHacc
Distribution
Distribution
Distribution
Distribution
Distribution
Distribution
AlogP
Jun herbsChen herbsZuo-Shi herbs
Jun herbsChen herbsZuo-Shi herbs
Jun herbsChen herbsZuo-Shi herbs
Jun herbsChen herbsZuo-Shi herbs
Jun herbsChen herbsZuo-Shi herbs
Jun herbsChen herbsZuo-Shi herbs
Figure 2 The profile distributions of six important molecular properties for all ingredients from the Jun Chen and Zuo-Shi herbs OBOral bioavailability MW molecular weight DL drug-likeness AlogP partition coefficient between octanol and water nHacc number ofhydrogen-bond acceptors nHdon number of hydrogen-bond donors
Evidence-Based Complementary and Alternative Medicine 7
Jun herbs(29)
Zuo-
Shi h
erbs
(101
)
Chen herbs
(23)
1
2
15
(a)
Jun herbs(5)
Zuo-
Shi h
erbs
(48)
Chen herbs
(10)
9
10
14189
(b)
20Mol 1200000
MMMol 152Mol 15Mol 1521Mol 15Mol 15
Mol 8888
Mol 29Mol 2Mol 2Mol 2Mol 29
Mol 3Mol 3Mol 35Mol 3535
1Mol 121Mol 121Mol 122
Mol 124Mol 124Mol 1Mol 124Mol 124Mol 124
MMol 10Mol 1Mol 10M 00MMMM
MMol 73Mol 77333222
ABATABATA
MAPK3MAPKMAPK
UGT1A1GT1UGT1A1
GOT1GOTT1
SREBF1REBFSREBF1
RXRBRXRBRXRB
CYP19A1CYP19A1YP19A
PLB1PLBPLB1
FASLGFASLGASL
BCHEBCHHE
HSD3B1HSD3
MTTPMTT
CES1CES
TIMP1TIMP
ATP5BATP5
MAPK10MAPK 0
BADBAD
CHRM5CHRM5
EPHBEPHBPHB2
HSD3B2HSD3B2SD3B
SOAT2OATSOAT2
FASNFASNFASN
MT-ND6MT-N
HTR3AHTR3HTR3APLA2G4APLA2G4AA2G
PKIAAIA
ADH1AADHAD
AKR1C1KR1AKR1C1
CREB1CREBB1
HMGCRMGHMGCR
SOAT1OAT
ABCC1ABCC
OPRD1OPRDOPRD1
VCPVCP
LDLRLDLR
ADRA1DDRAA1D
NFATC1FATCFATCATCFATCA
EGLN1GLNGLNGLNGLN
FABP5ABP5ABPABPABPABP5
CYCSCYCSCYCSCYCSYCSFOSL1OSLOSLOSLOSL
ALOX12LOXLOXOXLOX
TDRD7DRDDDRDDRDDRDAPODAPODAPOPOAPOD
NNNNOXNOXNOXNOX5NOXX
Mol 40
NFKB1NFKBFKBFKBFKBLYZLYZLYZZLYZ CTNNB1TNNBTNNNNNN MMP10MMPMPMPMMP1ALBALBALBALBALB
3Mol 433MMol 28Mol 28Mol 2Mol 28
HSPA5SPASPASPA5HSPA5AERBB2ERBB2ERBBRBBB2ERBB2
HSPB1HSPBSPBSPBHSPB1HSPB1B
NQO1NQO1NQONQO1NQONQO1TOP1TOP1TOP1TOPTOPT
PORPPORPORPORPORR ACPPACPPACPPACPPCPP
SLC2ALC2A4LC2A4C2A4LC2A4LC2A4LC
SERPINE1SERPINE1PINRPINERPINPIN
JUNJUNNJUNNN
PTGER3PTGER3TGERTGERGERTGER
COL1A1OL1AOL1AOL1AA1CO
SSLPISLPSLPISLPISLPII
CCCALCCC MMMMMMSS
MMMMP1MMPMMPMMPTNFTNFTNFFTNF
CCND1CCNDCCNDCNDCNDCCND
SMDPSMDPSMDPSMD3PSMD3PSMDNFNFNFFFNF
ELK 1ELKELK LK ELK ELK CCL2CCL2CCLCCL2CCL2CCL2
MMol 14MMol 14Mol 1447
MMol 15MMol 1Mol 115Mol 15M
Mol 122Mol 122Mol 122MMol 12Mol 122222
MMol 24Mol 2Mol 2Mol 2444
Mol 78MMol 78Mol 7Mol 7Mol 788
Mol 38MMMMol 3Mol 3338
Mol 154Mol 154MMol 15ol 1ol 155M
Mol 67MMol 6Mol 6Mol Mol 67Mol 677766
Mol 64MMol 64Mol 6Mol 6Mol 64
Mol 48Mol 48MMMol 4Mol 4448M 4
PPPPARAPARPARPAR
PCOLCEPCOLCECOLCOLCCOLC
SPP1SPP1SPP1PP1SPPP1 AKR1C3AKR1C3KR1CKR1CKR1CKR
NNNOSNN 2222 DUOXDUOXDUOXDUOX2DUOXDUOX2
MCL1MCL1MCLMCLMCLMCL
PARP1ARP1ARPARPPARP1PEEMPOMPOMPOMPOM DDDUDUDUDDU
NGASYNGASYNGAP1NGAPNGAAP1RARAARARA
BAXXXXB X ESR1
GSTM2GSTM2GSTMSTMSTMGSTMSHSAAHSAAHSA1AHSA1AHSAHSAA GABRAGABRA2GABRA2GABRAGABRAGABRAMM2MMM2
CHRM2CHRM2CHRMHRMHRMCHRM2
VDRVDRVDRRVDRVDRARGABRA1GABRA1GABRAA11GABRA1G
IL888IL88
PPPPRSPRSPRSSSSSSSPPARDPARDPARPARDPARDPARDSELESELESELESELEEE
COL3A1COL3A1OL3AOL3AOL3AA1OL3ARB11111
CYP1A2222222 NCOANCOANCOANCOANCOANCOA22222211111 RRRRRR
Quercetin
Mol 9444444
Mol 99
Mol 63
Mol 75555
Mol 37Mol 37Mol 37Mol 37MolMol 33Mol 37MolM
Mol 158
Mol 1Mol 10Mol 1Mol 10000
Mol 49Mol 4Mol 4Mol 4Mol 44Mol 494Mol 4
Mol 6Mol 6666M 66
MMol 60Mol 60Mol 6MM
Mol 101Mol 101Mol 101Mol 10011
Mol 12Mol 12Mol 12Mol 1Mol 12221
Mol 11MMol 1155Mol 11555M6060660
Mol 85Mol 85Mol 85MMol 88558554MMol 114Mol 114444M
Mol 116Mol 116Mol 116MMol 1ol 1Mol 11616M 1
Mol 9000
Mol 100Mol 1000Mol 106MMMol 106Mol 10Mol 1066Mol 322 Mol 30Mol 30Mol 30000MM
Mol 140MMol 14MMol 14Mol 1440Mol 14
PlatycodPlatycodatycodPlatycodP ononoGrandifondifoGrandGrand rususus
RadiRadixR diBupleurBupleuriBupleuriu
RehmanniRehmanniRehmanniiaaaglutinosglutinolutinoglutin aaaLiboschLiboschLiboschLLiLiLicoricececece
AngelicaAngelicaAngelican l eeSinensisSinenS nensisnensis
Radixadixdix
Mol 84
Mol 23 Mol 130Mol 13Mol 13Mol 11Mol 130Mol 13Mol 1MMol 13
Mol 11Mol 1Mol 1Mol 11Mol 11
0Mol 200000
Mol 34Mol 3Mol 3MolMol 3344Mol 3Mol 134
Mol 151
Mol 126Mol 126Mol 12666
Mol 8111AurantiiAurantAuranAurantiitFructusructusFructuructuFr
Mol 77Mol 7Mol 7Mol 77777
Mol 444
MMol 82Mol 8Mol 8222Mol 88
Mol 133
Mol 123MMol 12Mol 12ol 1Mol 121
Mol 142Mol 1422
Mol 36
0Mol 80Mol 88Mol 80M 80
Mol 277Mol 135Mol 13555M 555
Mol 141MMol 144Mol 14111
Mol 104Mol 104MMMol 10Mol 104044M
Mol 922222Mol 22 Mol 2Mol 2Mol 2Mol Mol 2Mol 22M 2
MMol 83333
Mol 110Mol 110
Mol 119MMol 11999999
Mol 88888 Mol 109Mol 10MMol 1Mol 100Mol 109M
Mol 5444
Mol 39999999
Mol 150MMol 155000M 0
Mol 611
21Mol 21MMol 221111
Mol 13
Mol 9Mol 91MMol 9911
Mol 118MMol 11Mol 1Mol 11Mol 1Mol 11ol
Mol 466
Mol 96Mol 96Mol 96Mol 996Mol 9696 Mol 53Mol 5Mol 5MolMol 5Mol 5Mol 535Mol 50Mol 50Mol 5Mol 5Mol 550Mol 5
Mol 107Mol 107MMol 10077Mol 1077
M l 149MMol 1449Mol 149Mol 149
Mol 14Mol 1444
Mol 89Mol 125MMol 1ol 1Mol 12MMMol 1MM
Mol 87
Mol 72MolMol 7Mol 7MolMol 72M 7M Mol 933Mol 112Mol 112Mol 112Mol 1122M
Mol 1Mol MMolMollMol 1Mol Mol 105
MAP2MAP2MAPMAPMAP
IL2I 2L22L22
SLC6A2LC6A2LC6A2LC6A2C6A2S
GABRA5ABRAABRGABRA5AABRCHRNA2CHRNA2HRNA2HRNHRNA2HRNA2
DRD1D1DRDDRDDRDDD1 MMAPKMAPK1MAMADRA1AADDDRADRA1ADRAADRA1A CASP3CASP3CASP3CASP3C
F2
Mol 17 Mol 7666Mol 33
Mol 86Mol 86Mol 8Mol 8Mol 866686
Mol 161Mol 161Mol 161Mol 16Mol 16161MMo
Mol 56Mol 5Mol 556666Mol 113
Mol 97Mol 162MMol 16Mol 1Mol 1ol 111M 1
Mol 131
Mol 99Mol 9MMMolMol 9MMol 9Mol 99Mol 9ol
Mol 111
Mol 128Mol 128Mol 1ol 1Mol 121MolMol 199Mol 117Mol 117777
Mol 744444Mol 77
2Mol 52Mol 52Mol 5MM
MMMol 5Mol 5MMol 5Mol 5Mol 5M
MMol 58Mol 58Mol 58M 8
Mol 138MMol 1388M
Mol 102Mol 102Mol 10Mol 102MMol 10Mol 10
Mol 42MMMol 42M
MMMol 3Mol 3
Mol 137Mol 13Mol 13ol 13Mol 13Mol 13733
Mol 59Mol 59olMol 5Mol 59oMol 5Mol 559
Mol 129lMol 12ol 12ol 12Mol 129o 2ol 12o
Mol 62Mol 62Mol 6Mol 6Mol 6Mol 62Mol 6Mol 6ol
Mol 144Mol 144Mol 14Mol 14ol 14Mol 144ol 1
AchyraAchyraAchyranthisnthisnthisBidentBidentBidentataeataeatae
RadixRadixRadix
Mol 139Mool 13ol 13ol 139Mol 139Mool 13
ChuanxioChuanxioChuanxiongngngRhizomaRhizomaRhizoma
Mol 159Mol 159MoMol 15ol 15ol 15Mol 159o 5
Mol 136Mol 136ol 13ol 13ol 1ol 13ool 13
CXCL11CXCL11XCL1XCL1XCLXCL1p53p53p53p
Mol 47Mol 47Mol 4Mol 4Mol 4ol 47Mol 4o
MMol 156ol 15ol 15ol 1ol 15ooPRKCARKCRKCPRKCACARKC
PPP3CAPP3CAP3CAP3CAP3CAPPP3CAP
CDKN1BDKNDKNDKN1CDKN1BPTENPTENPTENPTENPTENPTENBBB
RadixRadixRadixPaeoniaePaeoniaePaeoniaePae
RubraRubraRubraNR1I3NR1I3NR1INR1INR1INR1I3
HERC5ERCERC5HERC5ERC5OODC1ODCODCODC1CERBB3RBBRBBERBB3RBBRBB3
CTSDCTSDCTSDCTSDCTSDCTSD Mol 18Mol 1Mol 1ol 18Mol 18MolMol 1
PLATPLATPLAPLATPLATL
DIOOO1OO1OEGFEGFEGEGFEGDIODDIODIO
BIL1BIL1BIL1BIL1BB
MMP 3MMP 3MMP 3MMPMMP 3M
THBDTHBDTHBTHBTHBTHBD
CYP1B1YP1BYP1BYP1BYP1BB1
PPPPPARPP GGGGGGPPP
KDR LLAACTACTACTLACTBLACTBACTTT
ESR2
CHEK1CHEK1CHEK1CHEK1CHEK1CHEK111CCHEK1C
CATCATCATCATTTCATCATT
CDK2
CCNA2CCNA2CCNA2CCNA2CCNA2CCNA2CCNA2CCCNA2CC
PIM1 GSK3B
MAPKKK1414MMAPKKKMM KMMM
IL10IL10IL10IL10IL10IL10
S2HAS2HAS2HAS22HAS2ADADDHDH1CADAD HAHAHHHHA
CDKN2ACDKN2CDKNCDKNDKN2ACDKN
Mol 155Mol 155Mol 15Mol 15Mol 15ol 1ol 15ol 15Mol 15MMo
Mol 153Mol 15Mol 15ol 15Mol 153ol 15Mol 15Mol 15
CHEKCHEKCHEKHEK2HEK2HEK
MMP2MMP2MMPMMPMMPMMP2
MGAMMGAMMGAMGAAM
KCNH2222
CXCL2CXCL2CXXCLXCLXCL
GJA1GJA1GJAGJA1GJA1GJA1KK222K2
IKBKBIKBKBIKBKBIKCNH2KCNH2
IFNGIFNGIFNGIFNGIFNGIFNGFNEIF666F6IGHG1IGHG1GHGIGHG1IGGHG
SSStigSStiggmmmaS gg steeerolerol
PPTPNPTPN1PTPN1P 1SCN5ASCN5AASCN5ASCN5A BetaBetaaaa-sata istosssststerolerolllroNR3C1R3CGSTA2STA BBC3BBC3LBPLBP TTEP1TEP1 RKCDPRKCD PDE10ADE10GSTA1STA FN1FN1CD14CD14
BACE1ACEBACE1 SLC6A1LC6AACD163CD16 GSRGSRR ADIPOQDIPOQSTAT3TATOPRM1OPRM1OPRMOPRMOPRMO ADRA1BADRA1BAADRA1BBBB
SLC6A4LC6ALC6ASLC6A44SLCYCYP1A1YP1A1YP1A1CYP1AYP1A1
SLC6A3SLC6A3SLC6ASLC6A3SLC6A3
PDE3APDE3AA
CHRNA7CHRNA7HRNAHRNAHRNAA
PPPTGPP S1S1S1S1SS111NKX3-1NKX3-1NKX3KX3NKX3NKX3-
BIRCBIRC5BIRCBIRCBIRC55
IL6666
HK2HK2HK2HK2HK2HK2
RUNX2UNXUNXUNXRUNX2UNXHSF1HSF1HSF1HSF1HSF1HSF1
IGFBP3GFBPGFBPFBPIGFBP3
STAT1T1TAT1TATSTAT1TATT
NCF1NCFNCFNCF1NCFN
PRSS3RSSSIRT1SIRTGATMGAT OLR1OLRAPOBAPOPYGMPYGM
TGFB1GFBGFBGFBGFBTGFB1F10 MMP9MMPMMPMMPP9PNFE2L2FE2LFE2LFE2LNFE2L2FE2L2L
Mol 160Mol 44Mol 44ol 4Mol 444Mol 444ol 4ol 4ol 44
Mol 55MoMol 5Mol 5Mol 555Mol 55Mol 5Mol 5MoMol 5
MMMol 132
MMMol 57
CRPCRPCRPCRPPHH
RUNX1T1UNX1NX1NX1NX1T1R
BCGABCGABCG2BCGA
CXCL10CXCL10XCXCLCXCLCXCL IGG
CLDN4LDN4LDN4LDN4ABABABAAB RRAHRAHRBBBIB
RARRAF1RAF1RAF11KKKKKKaemKKKKK pffefeferereerf referololol
CASP9CCASPCASP9PMDM2MDMMMDMMDMMDM2MDMMDM
BMAOBMAOBMAOBMAOBMAOBINSRINSRINSRINSRINSRRRNCOA1NCOA11NCOA1
HTR2AHTR2HTR2HTR222A
CHRMCHRMCHRM3MM3BCL2LCL2CL2L1LCL2BCL2L1111 CHCCHCCCCH
BCL2222BCL2CASP8CASP8CASP8CASP8CASP
RELARELARELAAAR
BaicalBaicalBaBaicalcacacalicaBB calcaac ininininininninninnCHUCHUKHUKCHUKHUKUKH AAAAAA
EGFREGFRGFREGFREGFR
NFKBIAFKBIFKBFKBNFKBIAFKBI
E2F2E2F2E2F2E2F2E
APPAPAPPAPPPAPPAPPA
CASP7CASPCASPASPCASPCASPPP IL4IL444L4L44
NUF2NUFNUFNUFNUFNUFNUFNUFN
CDK4CDKCDKCDKCDKCDKCD
XIAPXIAPXIAXIAXIAXIAX MEMEMETMETMETMEETMMME
ADCY2ADCYDCYDCDCYDCD
Mol 127
NNR3C2NR3C2NR3CNR3C2N
DPP4
DCAF5DCACAFDCAF55DCAFAKR1B1AKR1BKKR1BKR1BKR1BCTRB1CTRBTRBCTRBCTRB
LTA4HLTA4LTA4TA4LTA4LTA4H4
XDHXDHXDHHHXDH
PTGS2
Mol 51Mol 51Mol 5MMolMolMol 5Mol 5MMol 5
Mol 145Mol 14Mol 14ol 1ol 1ol 1Mol 1Mo
Mol 146MMMMool 1ol 11461
Mol 143Mol 14Mol 14Mool 1ol 11Mol 1411
Mol 70MMMolMol 7Mol 77Mol 70Mol 7Mol 7
Mol 71Mol 7Mol Mol 777Mol 71Mol 7Mol
Mol 25Mol 25MMMol Mol 22Mol 2
Mol 79Mol 7Mol 7Mol Mol 7Mol 779MM 7
Mol 26Mol 26MMol 2Mol Mol 22Mol 2M 22
CA22222222ADH1BADHADHDHADH1ADH1DH KCNMA1CNMCNMCNMCNMKCNMAKCNMCGABRA6GABRAABRAABRAABRAGABRAGA A6GA RAGRIA2GRIAGRIAGRIAGRIAAAA2GRIA
RASSFRASSF1ASSFASSFASSF
Mol 31MMol 3Mol 3Mol 3311M 3Mol 98Mol 98Mol 98MMol 9Mol 999Mol 98MM
GSTP1GSTP1GSTPGSTPGSTPGSTPF33333
Mol 69Mol 6MMol 6Mol 6Mol 66Mol 69Mol 69M 66
VCAM1VCAMCAMCAMVCAM1C
MAPK8MAPK8MAPKMAPMAPKM
CYP3A4CYP3A4YP3AYP3AYP3AY
Mol 68Mol 68MMol 6Mol 6Mol 66MMol 68Mol 6MMol 16Mol 16Mol 1Mol 1Mol Mol 1Mol 16
MMMMol 6MMol 66Mol 666Mo
CCartCarta hamihamimiiiiiFlosFlosFlosossoss
Mol 9566Mol 65Mol 6Mol 6MMol MolMol 6M 666M
PersPersPerserrr icaeicaeicaesemesemesemeemememeem nnn
ICAM1CAMCAMM1CAMI
E2F1E2FE2F1E2F1E2F1ENPEPPSPEPPSPEPPSEPPSPEPPSNPEPP
IGF2IGF2IGF2IGF2F
FOSFOSFOSFOSSFOSFOSSSS
CCNB1CCNBCNBCNBCCNB1CCNB1CDK1CDK1CDKCDKCDKCC
PRKCBPRKCBRKCRKCRKCR CB
CHRM1CHRM11CHRM11
HHHHSP9HHHH 0ABABAB2B2B2B2PPPPPPPPPPPPTOP2A ACACACACACACACACAACACAACACA
PRKACA
TOP2TOP2
My c FF7FFIRF1IRF1IRF1IRF1IRF1IR
CAV1CCAVCAVCAV1V1CAV1
ACHE
ADRB1ADRBRBADRBADR
ADRADRADRA2A2AA2A
SOD1SSODSOD1D11
NOS33
PLAUPLAUPLAAUUPLAUPRXRA
PGR
HHMOX11MOXMOXHMOX
GSTM1GSTM1GSTMGSTMSTMAAA
HIF1AHIF1HIF1HIF11AHIF1A
FOSL2OSLOSLOSL22FO
ALOX5ALOXALOXALOXALOX5
VEGFAVEGFAVEGFAVEGFAV FFA
PON1PONPONPONN1PON
PIK3CGPIK3CGGG
SULT1E1SULT1E1ULT1ELT1LT1GAGAABRAGABRA3AGG
AKT1AKTT1AKT11A
CHRMCHRMCHRMCHRM4CHRM4RMM
MAOAMAOAMAOAMAOMAOCD40LGCD40LGCD40LGD40LGCD40LGCD40LBRA3RAAABRA3CHRCHHHCHR
ADRB2
(c)
Figure 3 HB-cC-cT network of XFZYD (a) and (b) The distribution of different candidate compounds and targets in the network (red theJun herbs-specific cC (a) and cT (b) aqua the Chen herbs-specific cC (a) and cT (b) periwinkle the Zuo-Shi herbs-specific cC (a) and cT(b) claybank common cC (a) and cT (b) between the Jun and Chen herbs blue common cC (a) and cT (b) between the Jun and Zuo-Shiherbs green common cC (a) and cT (b) between the Chen and Zuo-Shi herbs purple common cC (a) and cT (b) among the 3 group ofherbs (c) The triangles with circle backgrounds represent the herbs (HB) the squares and circles represent the candidate compounds (cC)and candidate targets (cT) The red triangles squares and circles represent corresponding HB cC and cT in the Jun herbs the same is toaqua representing the Chen herbs and periwinkle representing the Zuo-Shi herbs The claybank squares and circles represent correspondingcC and cT overlap between the Jun and Chen herbs the same is to blue representing the overlap between the Jun and Zuo-Shi herbs and thegreen representing the overlap between the Chen and Zuo-Shi herbs The purple squares and circles represent the corresponding cC and cTshared by the 3 group of herbs The size of the node is proportional to the value of degree
Radix Bupleuri (Zuo-Shi) contained quercetin It targeted149 bioactive proteins PTGS2 (degree=126) HSP90AB2P(degree=85) AR (degree=81) NCOA2 (degree=75) PRSS1(degree=68) PTGS1 (degree=67) PPARG (degree=66)and F10 (degree=60) were predicted as the major candidatetargets of quercetin followed by kaempferol (degree=65)which was contained by Carthami Flos (Jun) AchyranthisBidentatae Radix (Chen) and Radix Bupleuri licorice(Zuo-Shi) It targeted 61 bioactive proteins includingPTGS2 (degree=126) HSP90AB2P (degree=85) CALM(degree=81) AR (degree=81) NOS2 (degree=76) andNCOA2 (degree=75) Quercetin stigmasterol kaempferol
baicalin and beta-sitosterol existed in 3 the Jun Chenand Zuo-Shi drugs demonstrating crucial roles of thesecomponents The 5 ingredients in the Jun Chen and Zuo-Shiherbs targeted 186 bioactive proteins which accounted for65 targets of XFZYD Baicalein existed in the Jun andChen herbs Luteolin existed in the Jun and Zuo-Shi herbsSpinasterol and sitosterol existed in the Chen and Zuo-Shiherbs 126 bioactive compounds targeted PTGS2 followedby ESR1 (88) HSP90AB2P (85) AR (81) CALM (81) NOS2(76) NCOA2 (75) PRSS1 (68) PTGS1 (67) and PPARG(66) As depicted in Figure 3(c) these target proteins wereanchored by ingredients in the 3 group drugs
8 Evidence-Based Complementary and Alternative Medicine
Table 3 Top 10 target proteins of XFZYD according to 2 centrality indicators
Proteins Degree Proteins BetweennessPTGS2 126 PTGS2 0128936ESR1 88 NCOA2 0060806HSP90AB2P 85 HSP90AB2P 0049647AR 81 PRKACA 0046484CALM 81 PTGS1 004365NOS2 76 AR 0030252NCOA2 75 PRSS1 0025575PRSS1 68 ESR1 0022212PTGS1 67 PPARG 0021403PPARG 66 PGR 0020685Note the centrality indicators identify the important nodes within the network Higher degree centrality and betweenness centrality indicate greaterimportance
The analysis of the network revealed that quercetin (Mol148) kaempferol (Mol 108) luteolin (Mol 127) wogonin (Mol160) 7-Methoxy-2-methyl (Mol 33) and beta-sitosterol (Mol41) were predicted as the major active compounds of XFZYDTheproteins including F2 NOS2 PTGS1 PTGS2 and CALMwere predicted as essential pharmacological proteins for thetherapeutic effects of XFZYD
34 Analyses on TBI Based Specific Protein Interaction Net-work Network biology integrated with different kinds ofdata including physical or functional networks and diseasegene sets is used to interpret humandiseases Protein-proteininteraction networks (PPI) are fundamental to understandthe cellular organizations biological processes and proteinfunctions [54] From the systematic perspective the analysisof TBI-related PPI will improve the understanding of thecomplicated molecular pathways and the dynamic processesunderlying TBI We screened the TBI-specific genes andprotein targets using Online Mendelian Inheritance in Mandatabase (OMIM) and Therapeutic Target Database (TTD)Figure 4 indicated that 21 TBI-specific genesproteins wereacquired Further these hub-proteins were submitted toHPRD and STRING to establish the TBI-specific proteininteraction network The detailed information of the TBI-specific proteins is shown in Table S3 The results suggestedthat the network consisted of 489 nodes and 5738 edges(Figure 4(a)) We obtained top 10 TBI-related proteinsaccording to 2 centrality indicators generated and summa-rized in Table 4 Interestingly we found that the node withhigher betweenness tends to possess larger degree (Fig-ure 5) Network topology analysis showed that protoonco-gene tyrosine-protein kinase Src (SRC degree=164 between-ness=0059) RAC-alpha serinethreonine-protein kinase(AKT1 degree=157 betweenness=0045) Serum albumin(ALB degree=153 betweenness=0069) Epidermal growthfactor receptor (EGFR degree=139 betweenness=0053)and Amyloid beta A4 protein (APP degree=134 between-ness=0072) contributed to the essential role in the patho-physiology of TBI All of these indicated that the top mutualtarget proteins performed various beneficial functions to treatTBI at the molecular level For example SRC is activatedfollowing engagement of many different classes of cellular
receptors It participates in signal pathways that controla diverse spectrum of biological activities including genetranscription immune response cell adhesion cell cycleprogression apoptosis migration and transformation [55]SRC can result in blood-brain barrier (BBB) disruptionand brain edema at the acute stage the inhibition of SRCfamily kinases can protect hippocampal neurons and improvecognitive function after TBI [56] AKT1 regulates manyprocesses including metabolism proliferation cell survivalgrowth and angiogenesis [57] The PI3KAKTPTEN path-way has been shown to play a pivotal role in neuroprotectionenhancing cell survival by stimulating cell proliferation andinhibiting apoptosis after TBI [58] ALB is the main proteinof plasma and can be a biomarker to predict outcome ofTBI [59] Its main function is the regulation of the colloidalosmotic pressure of blood We found that it may participatein pathological process of TBI
KEGG pathway analysis was also used to determine thefunctions of proteins Table 5 describes the top 10 significantlyenriched KEGG pathways These pathways play crucial rolesin pathophysiology process which have also been widelydiscussed in existing literature For instanceMAPK signalingpathway can promote pathological axonal death throughtriggering a local energy deficit [60] Neurotrophin signalingthrough Trk receptors regulates cell survival proliferationthe fate of neural precursors axon and dendrite growth andpatterning and the expression and activity of functionallyimportant proteins such as ion channels and neurotransmit-ter receptors [61] Engagement of cells with the extracellularmatrix (ECM) proteins is crucial for various biological pro-cesses including cell adhesion differentiation and apoptosiscontributing to maintenance of tissue integrity and woundhealing [62]The 47 TBI-specific proteins targeted byXFZYD(yellow and green) were further discussed below
35 HB-pC-pT Network pT-F Network Construction andMolecule Docking Analyses of XFZYD for the Treatment ofTBI To investigate the therapeutic mechanisms of XFZYDfor the treatment of TBI a HB-pC-pT network of XFZYDfor treating TBI was built (Figure 6) 47 TBI-specific targetproteins (circles) were targeted by 119 potential compounds(squares) from XFZYD (Figure 6) Detailed information
Evidence-Based Complementary and Alternative Medicine 9
BLMH
GPRASP2
GJC2
CAMK2N1
OBSL1
ZNF292
RLFAMPD3
NANOS1CAMTA1HYPKCCS
DLGAP4PF4V1
TRIM2
CHKB
F8A1
TIAM1
BMX
RAPGEF1
APBB1
KCNMA1
XRCC6
TTR
TGM2MAP2
PRNP
RASGRF1 PPP3CADMD SPTAN1
ACE
TGFB2
AP2M1GRAP2
SCN5A
MYH9
PKN1
ACHE
SLC25A13
PHKG1
SPAST
UMOD
PRPF40B
BBC3
TUBA4A TTPALRYR2
STUB1KRT6A
AKAP8L
GFI1BTHRAP3
ZNF232
SOD1
CRKL
CDH2
MBP MAP2K2
DCN
MAP3K5NGFR
PIK3CA
ITGA2
DNM1
RAP1A
GRIN2B
PTPN11
PRKCA
LDLRAP1
DNAJA3INA
SP3
CACNA2D3
CSF1
CNTF
TNFRSF1B
BCL10
KLC1
DCTN1
IDE
SERPINA3
HSPA1A
HSPG2
FN1
LRP2
TUBB
GRIN2A
YWHAB
CAMK2D
HTRA2
PRKCE
CRYAB
APC
GIT1
PIAS4
HPX
TRIP10
PDE4B
MARK4
PLAT
UBE2K
APOC2
LIN7B BGN
MAP3K10MAPK8IP2
CBS
SH3GL3
STAU1
CASP6
PPP5C
KRT16
DAB1
CASP1SHC2
CTSD
UCHL1
HP
SNCACAMK2G
TGFB1
GNB1
PTPN1PIN1
TJP1
CASP8PLCG1
NPHS1
PPP2R2A
DNAJB1
ADRBK1
INADL
NCSTN
MATK
NEDD4L
KAT5IFNGTBP
FRS2
CSNK1A1
CALRKNG1
SGOL2
KRT6B
TRAF3 DLG3
SACS
BDKRB2PRKCB
CAV1
MAPK3F2 SMAD3
APOEAKAP9 RANBP2
CLTC
TSC22D1GAB2PSEN2
MLF1CROT
KRT9
ADRA1B
EPB41
COL4A1
SHC3
ARHGAP32
ATM
PPP2R5A
CAMK2A
NTRK1
KRT18
TNFAPOA2
LAT
GRIN2D
COL4A6
KLK3
VLDLR
COL4A5
COL4A2
NEFH
OR8D2
MTSS1
PDZRN4
PTPN4
OR3A2
OR2T6
DGKG
CASP3
IGF1R
CASK
DRD2
TRAF2
EPHB2
PPM1A
PRKCG
CACNB3
HNRNPUSIN3A
DLG1
CLU
RAP2A
ERBB4CANX
BACE1 GABBR1
KARS
CNOT1
UNG
AGA CDCP1
UTP14A PLAG1
EXOC6
HRAS
CTNNB1
SP1
NOS3 SUMO1
PSEN1
HSPA8
HSP90AA1
BCL2
GNAO1
RPS6KB1
PARK2
DLG4
PTK2B
GSN
NAE1 TLN2LRFN2
PLTP
NUMBLSTX1A
DERL1
NEFM
HMOX2
AATF
PPIAMTRNR2L2AP1M1
OGT
DDB1
AP4M1
MED31
RGS3
TDP2
KRT5
ADAM9
REST
PACSIN1
SHB
CTSL1
LAMA1
SH2B1
PLA2G4F
SMURF2
SLC9A8
KRT14
CIT
TGFB1I1
PPP2R1APIK3R1
NUMB
ADAM17
DLG2
LRP1
APOA4
LTA
IRS1
GSK3B
EGFR
CTSB
TRAF6
SHC1
PPP2CB
NGF
HTT
PPIDLDB3
APOC1HIP1
ZDHHC17COL25A1MAGI3
ITGB5
ACTB
GNA11
ACTN2CAMK2B
CDK1
LIN7C
S100B
PDLIM5
DLGAP1
LIN7A
TPR
SYNGAP1
CFD
GRIN3A
F12
SLC1A3
GRK5
CACNA2D2
TANC1
DUSP4
EXOC4
AHSG
GRIN3B
UCP2
TTN
GPC1
NSF
FGA
DYNLL1
MAPK8IP1
PICALM
PPBP
ITM2B
NID1
STXBP1
CTBP1
SGK1
TRAF1
SQSTM1
TNFRSF1A
ACTN1
OPTN
UBE2D2
AP2A2
FUS
APOA1
DICER1
CACNA1B
CST3
NCOA3
SCARB1
APOC3
SLC6A3
KIAA1551 KRT13
DCD
KIAA0232
ENSG00000258818
SPATA31A7
CTAGE5
CEP44
SH2B2
EPHB4
CAPN1
F7
APBA2
COL4A3
LDLR
GIPC1
GFAP
SERPING1
SRC
MAPK12APP
GAPDH
CDK5
CREBBP
NOS1
YWHAZCALM1
YWHAG
YWHAQGRIN1
RPS6KA3
COL1A2
TCERG1
A2M
CASP7
LRP8
PDK2
AGTR1
FYN
ALBGRB2
MAPK1AKT1
STAT1
MAPT
GSK3A
UBB
PPP2CA
SMAD4
PRKCD
RGS12
SPTB
SH3BP5
CACNA1I
IL16
SLA2
PFN2
NLRC4
TNFRSF14
PRTN3
SNCB
FBLN1
BACE2
SETX
FANCD2
SAP30
RANBP3
PALB2
SLC1A5RASA1
HAP1
LRRC7
CFB
ADRBK2
CDC45
CLSTN3
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SCAF1KIAA1377
FAM71E2
FEZ1 ACSBG2
PCDH1
TRAPPC11QTRTD1
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TP53BP2
DRD1
ST13
PRPF40A
TRAIP
KIDINS220
GRK6
MMP17
LTBR
EIF6
PGAM1
CHD3
PRSS3
APBB3
CHRNA7
KRT10
RIMS1
CRMP1
PDIK1L
SORBS3
SYMPK
GCN1L1
RNF19A
HOMER3
GRK4
JARID2
MYLK3
EFS
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HSD17B10
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PHC3
TM2D1 FICD
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CABLES1
LGALS2
ITIH1
HOXB2
TAF4FLOT1
BABAM1
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HIP1R
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NCOR1
CASP4
NF1
AP4E1
CLCA2
IGDCC4
NECAB3
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SH3GLB1
CTCF
PLCG2
(a)
STAT1
EGFR
FN1
PLAT
SOD1
PRKCA CDK1
GSK3B
ALB APP
PPP3CA
IFNG
BBC3
SCN5A
KCNMA1
MAP2
ACHE
MAPK3F2
PTPN1
NOS3 BCL2
CAV1
CTNNB1
PRKCB
TGFB1CASP8
TNF
AKT1
F7
MAPK1LDLR
PRKCD
SLC6A3
DRD1
CASP3
CHRNA7
PRSS3
TP53BP2
CASP7
EPHB2
SYNGAP1
CALM1
CTSD
BACE1
ADRA1B
(b)
Figure 4 TBI-related protein interaction network (a) 21 hub proteins (red and green) were identified through the analysis of TherapeuticTargetDatabase (TTD) aswell asOnlineMendelian Inheritance inMan (OMIM) 4 overlapped protein targets (green)were obtained between21 hub proteins in TBI and candidate targets of XFZYD Periwinkle proteins fromHPRD and STRING analyses were not targeted by XFZYD(b) 47 candidate protein targets of XFZYD were screened for treating TBI Yellow candidate protein targets of XFZYD The size of nodes isproportional to the value of degree
10 Evidence-Based Complementary and Alternative Medicine
Table 4 Top 10 proteins of TBI specific proteins according to 2 centrality indicators
Proteins Degree Proteins BetweennessSRC 164 APP 0071814AKT1 157 ALB 0069343ALB 153 SRC 0058568EGFR 139 EGFR 0052938APP 134 AKT1 0045466GAPDH 131 HTT 0037164HSP90AA1 108 GAPDH 0034549BCL2 106 HSP90AA1 0027596MAPK1 105 CTNNB1 0021759HRAS 105 GNB1 0019492Note the centrality indicators identify the important nodes within the network Higher degree centrality and betweenness centrality indicate greaterimportance
Table 5 Top 10 significantly enriched KEGG pathways in TBI-specific proteins
Pathway ID Pathway description Gene count FDR4010 MAPK signaling pathway 44 299E-235200 Pathways in cancer 43 113E-184722 Neurotrophin signaling pathway 41 101E-344151 PI3K-Akt signaling pathway 40 111E-154510 Focal adhesion 39 292E-225205 Proteoglycans in cancer 39 410E-215010 Alzheimer s disease 34 124E-204015 Rap1 signaling pathway 33 769E-174014 Ras signaling pathway 33 646E-164020 Calcium signaling pathway 31 505E-17
0
001
002
003
004
005
006
007
008
0 20 40 60 80 100 120 140 160 180
Betw
eenn
ess
Degree
APP
ALB SRCEGFR
AKT1HTT
Figure 5 Relationship between degree and betweenness centralityin the TBI-specific protein interaction network
for the 119 potential compounds is shown in Table S4Similarly 5 pharmacologically active ingredients in the JunChen and Zuo-Shi groups including quercetin stigmasterolkaempferol baicalin and beta-sitosterol anchored 33 TBI-specific proteins such as CALM SCN5A F2 ACHE F7ADRA1B NOS3 BCL2 CASP3 and AKT1 11 Jun-specificcompounds targeted 2 specific targets (ALB CTNNB1) while12 Chen-specific compounds anchored 3 unique proteins(PRKCD FN1 and BC3) The Zuo-Shi herbs possessed thelargest number of active compounds (89) and targeted 5
unique proteins including EPHB2 BACE1 LDLR MAPK3and PRSS3 GSK3Bwas the common target between theChenandZuo-Shi drugs KCNMA1 CASP7 andAPPwere targetedby the Jun and Zuo-Shi drugs
The top 10 candidate compounds and targets to treat TBIwere showed in Tables 6 and 7 Formost of active compoundsfrom XFZYD each component hit more than one targetTable 6 demonstrated that quercetin had the highest numberof targets (degree =76) followed by kaempferol (degree=43) beta-sitosterol (degree =35) stigmasterol (degree =19)luteolin (degree=18) baicalein (degree=15) 7-Methoxy-2-methyl isoflavone (degree=12) wogonin (degree=12)nobiletin (degree=11) and naringenin (degree=9) Forinstance quercetin (3310158404101584057-pentahydroxyflavone) is anaturally occurring flavonoid commonly found in fruits andvegetables It regulates multiple biological pathways elicitinginduction of apoptosis as well as inhibiting angiogenesisand proliferation [63 64] Quercetin can attenuate neuronalautophagy and apoptosis in rat traumatic brain injury modelvia activation of PI3KAkt signaling pathway [65] It has alsobeen reported to have a protective ability against oxidativestress and mutagenesis in normal cells [66 67] Kaempferol(3 41015840 5 7 tetrahydroxy flavone) is a yellow-colored flavonoidthat is widely distributed in many botanical families[68] It has been shown to possess a variety of biologicalcharacteristics including effects of anti-inflammatory[69] antioxidative [70] tumor growth inhibition [71] and
Evidence-Based Complementary and Alternative Medicine 11
Mol 22Mol 33
Mol 29Mol 32 Mol 23Mol 20
Mol 97Mol 106
Mol 104Mol 100Mol 94
Mol 63Mol 90
Mol 87
Mol 91Mol 61
Mol 19
Mol 109
Mol 161
Mol 118
Mol 111
Mol 158
Mol 114
Mol 149
Mol 141
Mol 152
Mol 14
CASP3
BCL2
NOS3
SCN5A
F7
ADRA1B
ACHE
F2
Mol 4
Mol 49
Mol 127
APP CASP7
Mol 92
Mol 54
Mol 6
Mol 36
KCNMA1
Mol 27
Mol 13Mol 105Mol 140
Mol 17
Mol 39
Mol 2
Mol 21
Mol 12
Mol 142
Mol 126
Mol 135
Mol 121
Mol 119
Mol 124
Mol 120
Mol 117
Mol 133
Mol 131
Mol 134
PlatycodonGrandiforus
Aurantii Fructus
Licorice
Radix Bupleuri
RehmanniaglutinosaLibosch
AngelicaeSinensis Radix
Mol 8
Mol 73
Mol 75
Mol 77
Mol 80
Mol 7
Mol 82
Mol 76
Mol 74
Mol 60
Mol 46
Mol 150
Mol 151
Mol 112
Mol 115
Mol 11
Mol 116
Mol 113
Mol 110
Mol 1
Mol 9Mol 101Mol 10Mol 93
Mol 103Mol 89 Mol 107
Mol 96PRSS3 LDLRMAPK3 BACE1 EPHB2
Mol 35Mol 81
Mol 86
Mol 85
Mol 84
Mol 88
Mol 83Mol 30
Mol 137
ChuanxiongRhizoma
Mol 3
Mol 57
Mol 139
Achyranthis
Bidentatae
RadixMol 102
CASP8
IGHG1AKT1p53
CHRNA7
Mol 40
SLC6A3 PTPN1
SOD1MAPK1
Mol 38
Mol 95
Carthami Flos
Mol 24
Mol 78 Persicae Semen
Mol 122
Mol 25
Mol 98
TGFB1
GSK3B
PRKCA
Radix Paeoniae Rubra
Mol 52
Mol 42
Mol 62
Mol 138
EGFR
STAT1 PPP3CAPLAT
Stigmasterol
MAP2
Beta-sistosterol Quercetin
Kaempferol
SYNGAP1
CALM
Baicalin
PRKCB
CAV1
CTSD
TNF
DRD1
CDK1
IFNG
FN1BBC3 PRKCD
Mol 132
Mol 160
Mol 58
Mol 67
Mol 69 Mol 43ALB
Mol 64CTNNB1
xiongixioChuanxC nxnxioaamaRhihihizoma
Achyranthisiistntnt
aeeaeBidenBidennntatae
RadixadixR diR di
oniae nianiaiaonRadix PaeoPaePRadix P eeoeonRRRRubra dondonddoodPlatycolatycPlatyycPl ycoyccoPlatPlatycoPlatyccocod
rususrusususruGranG anGranGranGranGrGGranndndiforu
ructusuctuc sructusuctusructusruAurantiirA ntaA ntiitiirantAurantintiurantAur iiii Fru
cecececececececeecLiLLiLLiLiLLLLLLLLiLiLiLLiLLLiLicoric
leuririeuuurieurileurleurieurieurieurieurileRadix BBRadRad Bdix Bdixx R i Bdi BBBadix Radix BBuple
RehmanniaRe mRe m nnRehmanniamReRehmhmannnniamanniaRehmehmanniRR nosasasasaosaosglutilutglutgluulutiglgl titinos
LiboschLibLLibo hschib chLiboschboscLLib chschLibos
icaecaecacaeicaeicAngelAnAngeAngelAnAng lAAngeelielicdix dix dixdixdSinensinensSS nennensSinSSiSinensSinensSine sissis Rad
FloslololFlhamaCartharthhaamami Fl
menmennnnmicae Scae SSem
Figure 6 HB-pC-pT network of XFZYD for treating TBI The triangles with circle backgrounds represent the herbs (HB) the squares andcircles represent the potential compounds (pC) and targets (pT)The red triangles squares and circles represent corresponding HB pC andpT in the Junherbs the same is to aqua representing theChen herbs and periwinkle representing the Zuo-Shi herbsThe claybank squares andcircles represent corresponding pC and pT shared by the Chen and Zuo-Shi herbs the same is to blue representing the overlap between theJun and Zuo-Shi herbs and the green representing the overlap between the Chen and Zuo-Shi herbsThe purple squares and circles representthe corresponding pC and pT shared by the 3 groups of herbs
alleviating insulin resistance in type 2 diabetic rats [72]Beta-sitosterol (BS) is a vegetable-derived compound foundin various plants and is suggested to modulate the immunefunction inflammation and pain levels by controlling theproduction of inflammatory cytokines [73]
For targets analysis CALM possesses the largest degree(degree =84) followed by GSK3B (degree =61) SCN5A(degree = 59) F2 (degree = 43) ACHE (degree=28)F7 (degree=28) ADRA1B (degree=28) NOS3 (degree=20)BCL2 (degree=18) and CASP3 (degree=18) which demon-strated their crucial therapeutic effects for treating TBI Forinstance CALM possess an essential position in calciumsignaling pathway and is related to morphological changesmigration proliferation and secretion of cytokines andreactive oxygen species ofMicroglial cells [74]The activationof CaMKII120572 major isoform of Ca2+calmodulin-dependentprotein kinase (CaMK) in brain is directly associated withthe production of proinflammatory cytokines such as TNF-120572and IL-1120573 [75] GSK-3120573 is a serinethreonine-protein kinasewhich is abundant in the central nervous system (CNS)particularly in neurons [76] It can control gene transcrip-tion axonal transport and cytoskeletal dynamics in growthcones [77] The inhibition of GSK-3120573 attenuates apoptotic
signals and prevents neuronal death [78] There is increasingevidence that prothrombin (F2) and its active derivativethrombin are expressed locally in the central nervous systemBeside the central role in the coagulation cascade the gener-ation of thrombin leads to receptor mediated inflammatoryresponses cell proliferationmodulation cell protection andapoptosis [79 80] The role in brain injury depends upon itsconcentration as higher amounts cause neuroinflammationand apoptosis while lower concentrations might even becytoprotective [81]
Direct tissue damage aswell as hypoxic-ischemic increas-ing anaerobic glycolysis of the brain tissue results in theATP-stores depletion and failure of energy-dependent mem-brane ion pumps especially for voltage-dependent Ca2+ andNa+-channels Accumulated Ca2+ activates lipid peroxidasesproteases and phospholipases and caspases at the sametime increasing the intracellular concentration of free fattyacids and free radicals leading to necrosis or apoptosis ofneurocyte [82] At the same time the activation of residentglial cells microglia and astrocytes and the infiltration ofblood leukocytes secrete various immune mediators elicitedinflammatory responses which subsequently intersect withadjacent pathological cascades including oxidative stress
12 Evidence-Based Complementary and Alternative Medicine
Table 6 Top 10 potential candidate compounds of XFZYD for treating TBI according to 2 centrality indicators
Compound Degree Compound Betweennessquercetin 76 quercetin 01682179kaempferol 43 kaempferol 005501511beta-sitosterol 35 wogonin 005141376Stigmasterol 19 beta-sitosterol 004451294luteolin 18 naringenin 003251738baicalein 15 beta-carotene 002977217-Methoxy-2-methyl isoflavone 12 nobiletin 002787992wogonin 12 7-Methoxy-2-methyl isoflavone 002096439nobiletin 11 luteolin 002090223naringenin 9 baicalein 001453488
Table 7 Top 10 potential targets of XFZYD for treating TBI according to 2 centrality indicators
Targets Degree Targets BetweennessCALM 84 CALM 021452046GSK3B 61 SCN5A 011441591SCN5A 59 GSK3B 009553896F2 43 F2 00689171ACHE 28 BCL2 002651903F7 28 CASP3 002372836ADRA1B 28 F7 002032647NOS3 20 ACHE 001845996BCL2 18 ADRA1B 001704505CASP3 18 NOS3 00166699
excitotoxicity or reparative events including angiogenesisscarring and neurogenesis [83] Function analysis of the 47target proteins regulated by XFZYD was mainly associatedwith core pathophysiology process of TBI (Figure 7) 47 targetproteins were connected with 16 key process related to TBIMost of the targets have one or more links to other biolog-ical process such as apoptosis cell proliferation superoxideanion generation nitric oxide biosynthetic process responseto calcium ion I-kappaB kinaseNF-kappaB signaling andregulation of inflammation The above analysis implied themultifunction character of these target proteins regulated byXFZYD Of these target proteins 25 proteins (53 of the 47)such as TGFB1 EGFR CAV1 MAPK1 PRKCB and AKT1were responsible for regulating the apoptosis process 17 forblood coagulation 14 for cell proliferation and axon genesisand 12 for hypoxia andMAPK cascade We found that TGFB1was the crucial protein because it participated in 9 biologicalprocesses related to TBI such as apoptosis blood coagulationcell proliferation and MAPK cascade followed by EGFR (8)CAV1 (7) MAPK1 (6) PRKCB (6) and AKT1 (6)
Overall these observations strongly support the evidencethat the generated HB-pCminuspT network and pT-F networkhave important roles in treating TBI further validating thedrug targeting approach
Molecule docking was used to further validate the bind-ing mode between candidate compounds and their targetproteins We found that 18 TBI-specific target proteins inter-acted with 91 candidate compounds from XFZYD (Figure 8)
Other 29 target proteins were not discussed for the lackof proper protein crystal structure The detailed moleculedocking results are shown in Table S5The 6 essential proteinsincluding GSK3B AKT1 CDK1 F2 NOS3 and ACHEwere used to elucidate the exact binding mode (Figure 9)Quercetin was located within the binding cavity of AKT1 andCDK1 (Figures 9(a) and 9(b) and S1A B) Four conventionalhydrogen bonds were formed between quercetin and AKT1by interacting with the key amino acids including ILE-290 THR-211 and SER 205 Additionally 120587-120587 interactionsbetween quercetin and TRP-90 were found in the activesite which helped the stabilization of the compound at thebinding site (Figure 9(a) S1A) Figure 9(b) and S1B suggestedthat five conventional hydrogen bonds (LEU-83 ASP-146and LYS-33) and 120587-120587 interaction (PHE-80) were formedbetween quercetin and CDK1 The GSK3B-FA complexes(Figure 9(c) S1C) were stabilized by 6 hydrogen-bondinginteractions between FA and LYS-85 GLU-97 TYR-134 andARG-141 Glyasperin Bmainly bonds to F2 through hydrogenbonds by interacting with the key amino acids includingGLY-193 SER-195 and GLY-219 (Figure 9(d) S1D) andan edge-to-face 120587 minus 120587 interaction was also observed withTYR-228 The GLN-247 GLU-351 and GLY-355 from theactive site pocket of NOS3 participated in the hydrogen-bondformationwith 1-Methoxyphaseollidin (Figure 9(e) S1E)The(-)-Medicocarpin formed a total of 6 hydrogen bonds withSER-293 PHE-295 ARG-296 and TRY-341 in the active siteof ACHE (Figure 9(f) S1F) Besides an edge-to-face 120587 minus 120587
Evidence-Based Complementary and Alternative Medicine 13
KCNMA1
P53
IFNG
PPP3CA
CASP8
SCN5A
I-kappaB kinase NF-kappaB signaling
Regulation of inflammation
Response to calcium ion
TGFB1
TNF
PRKCA
CHRNA7
CAV1
STAT1
MAPK cascadeAKT1
BCL2
APP
EGFR
MAPK1
Angiogenesis
SYNGAP1
MAP2
PTPN1
Axonogenesis
GSK3B
EPHB2
CDK1
CASP7FN1
MAPK3
CTNNB1
F2
Autophagy
Cell proliferation
BACE1
SLC6A3
DRD1
ADRA1BACHE
CALMCASP3
PLAT
Apoptotic process
NOS3
PRKCB
Blood coagulation
Response to hypoxia
Superoxide anion generation
Nitric oxide biosynthetic process
Astrocyte activation
Amyloid-beta regulation
Microglial cell activation
LDLR
PRSS3
F7
PRKCD
SOD1ALB
BBC3
Figure 7 pT-F network of XFZYD for treating TBI 16 biological processes (red square) the 47 target proteins (periwinkle circle) of XFZYDparticipate in for treating TBI
interactionwas also observedwith TYR-337 From the resultshydrogen-bonding and edge-to-face 120587 minus 120587 interactions playkey roles in the proteinminusligand recognition and stabilitywhich may be helpful in determining the inhibitor activitiesAnd the C-T network confirmed the potential therapeuticeffects of the candidate compounds from XFZYD to treatTBI through interacting with the relevant proteins The com-putational analysis further elucidated the accurate moleculemechanisms between active compounds and targets
4 Discussion
Traumatic brain injury (TBI) is a growing public healthproblem worldwide and is a leading cause of death anddisability [84] Although major progress has been made in
understanding the pathophysiology of this injury this hasnot yet led to substantial improvements in outcome by alack of treatments which have proven successful during phaseIII trials for modern medicine [85 86] TCM rooted inthousands of years of history may offer an alternative or acomplementary strategy for the treatment of TBI XFZYDa representative formula in TCM has been used for yearsto treat TBI in China and has been demonstrated to beeffective in clinical practice However its ldquomulticomponentsrdquoand ldquomultitargetsrdquo features make it much difficult to decipherthemolecular mechanisms of XFZYD in the treatment of TBIfrom a systematic perspective if employing routine methods
In the present study a network pharmacology-basedmethod was employed to elucidate the pharmacologicalmechanisms of XFZYD to treat TBI according to the drug
14 Evidence-Based Complementary and Alternative Medicine
Figure 8 C-T network through molecule docking validating 119 potential compounds (green triangles) interacting with 18 potential targets(yellow circles) of XFZYDThe size of the nodes is proportional to the value of degree
combination principle of TCM We first proposed a newmodeling system combining OB and DL screening multipledrug targets prediction and validation network constructionand molecule docking to probe the efficiency of a typicalTCM formula XFZYD for the treatment of TBI The 11herbs from XFZYD possessed 162 bioactive compounds andtargeted 285 proteins There were 5 compounds and 189
target proteins overlapped among the Jun Chen and Zuo-Shigroup Furthermore 47 TBI-specific proteins were targetedby 119 (73) bioactive compounds from XFZYD Similarly 5common compounds and 33 (70) common target proteinsamong the 3 groups of drugs were observed Most of thebioactive ingredients targeted more than one protein The 47target proteins regulated several essential pathophysiological
Evidence-Based Complementary and Alternative Medicine 15
(a) (b) (c)
(d) (e) (f)
Figure 9 Hydrogen-bonding networks within the binding site of the compoundminustarget complexes obtained from molecule docking(a) AKT1-quercetin (b) CDK1-quercetin (c) GSK3B-FA (d) F2-glyasperin B (e) NOS3-1-Methoxyphaseollidin and (f) ACHE-(-)-Medicocarpin The molecules are presented as ball and stick models Active site amino acid residues are represented as lines Dotted bluelines in these pictures represent hydrogen bonds with distance unit of A Other O and N atoms are colored as red and blue respectively
processes of TBI that referred to apoptosis inflammationcell proliferation superoxide anion generation nitric oxidebiosynthetic process response to calcium ion etc The aboveanalysis reveals that the synergistic action mechanisms ofXFZYDmay be (1) bioactive compounds overlapping amongthe different group of herbs (2) specific bioactive com-pounds from different groups of herbs targeting the sameproteins (3) specific bioactive compounds from differentgroups of herbs targeting different proteins which participatein the same pathophysiological process of the disease Toa certain degree the 5 compounds including quercetinstigmasterol kaempferol baicalin and beta-sitosterol playedessential role in XFZYD for TBI treatment MAPK3MAPK1AKT1 PRKCA TNF PRKCB EGFR BCL2 GSK3B CASP3PPP3CA and NOS3 were the main target proteins regulatedby XFZYD in the treatment of TBI
Interestingly Beta-carotene (Mol 43) from Carthami Flos(the Jun herb) specifically regulated 120573-Catenin (CTNNB1)and played critical role for curing TBI The 120573-Catenin is acritical downstream component of the Wnt pathway whichplays essential role in the regulation of mammalian neuraldevelopment [87] In vitro and in vivo studies demonstrate
that the Wnt120573-catenin pathway regulates the proliferationand differentiation of neural progenitor cells [88] Neuronaldifferentiation is induced by overexpression of 120573-cateninor the pharmacological inhibition of GSK3120573 (the phospho-rylating enzyme of 120573-catenin) [89 90] This pathway alsopromotes blood vessel formation during vascular develop-ment as well as the vascular repair process after TBI [91]In addition wogonin (Mol 160) from Achyranthis BidentataeRadix (the Chen herb) specifically targeted fibronectin (FN1)Bcl-2-binding component 3 (BBC3) and Protein KinaseC delta type (PRKCD) FN1 an important component ofthe extracellular matrix (ECM) environment promotes cellmigration neurite outgrowth and synapse formation dur-ing neural development [92] It aggregates in the injuredbrain and plays a neuroprotection role through antiapoptosisand anti-inflammation ways following TBI [93 94] BBC3namely p53 upregulated modulator of apoptosis (PUMA) iscritical for the p53-dependent apoptosis pathway which playsan important role in hippocampal neuronal loss and associ-ated cognitive deficits [95] PRKCD one of PKC isoformsactivates signal transduction pathways involved in neuronalregeneration [96] synaptic transmissionplasticity [97] and
16 Evidence-Based Complementary and Alternative Medicine
activation of apoptosis processes [98] as well as higher brainfunctions such as learning andmemory [99]The activators ofPKCare effective for the treatment of TBI [100] FurthermoreMitogen-activated protein kinase 3 (MAPK3) Beta-secretase1 (BACE1) Ephrin type-B receptor 2 (EphB) and low-densitylipoprotein receptor (LDLR) were specifically targeted bythe ingredients in the Zuo-Shi herbs Naringenin (Mol133) targeted LDLR MAPK3 while euchrenone (Mol 60)anchored BACE1 and nobiletin (Mol 134) targeted EphB2MAPK3 is an essential component of the MAP kinase signaltransduction pathway It has been appreciated recently thatthe ERK12 cascade plays a fundamental role in synapticplasticity and memory [101] BACE1 is responsible for pro-duction of A120573 from amyloid precursor protein (APP) A120573can cause cell death activate inflammatory pathways [102]and prime proapoptotic pathways for activation by otherinsults [103] The blocking of BACE1 can ameliorate motorand cognitive deficits and reduce cell loss after experimentalTBI in mice [104] EphB is localized to synaptic sites inhippocampal neurons [105] The interaction between EphBand NMDA receptors regulates excitatory synapse formation[106] LDLR acts as an important receptor that facilitatesbrain A120573 clearance and inhibits amyloid deposition [107]and then ameliorates Alzheimerrsquos disease neuropathologyafter TBI [108] The analysis above indicates the ldquoJunrdquoldquoChenrdquo and ldquoZuo-Shirdquo herbs from XFZYD trigger theirspecific targets regulation respectively for the therapeuticeffects
XFZYD is a very famous traditional Chinese formula inpromoting qi circulation and removing blood stasis accord-ing to TCM theory However several limited researches havedemonstrated its efficacy for treating TBI such as anti-inflammatory and synaptic regulation [30 31] which arein accord with our study However previous studies merelypartially deciphered the molecule mechanism of XFZYD fortreating TBI This study reports 119 bioactive compounds inXFZYD that target 47 TBI-specific proteins such as MAPK3MAPK1 AKT1 PRKCA TNF PRKCB and EGFR Theseproteins regulate several crucial pathophysiological processesof TBI such as apoptosis inflammation blood coagulationand axon genesis Our study demonstrates that the therapeu-tic actions of XFZYD refer to ldquomulticompoundsrdquo ldquomultitar-getsrdquo features rather than only the improvement of bloodcirculation With the help of molecule docking methodwe further validate the interactions between bioactive com-pounds and potential targets of XFZYD The hydrogen-bonding and edge-to-face 120587 minus120587 interactions play key roles inthe proteinminusligand recognition and stability This provides avaluable reference for further experimental investigations ofbioactive ingredients and therapeutic targets of XFZYD fortreating TBI
5 Conclusion
Our work successfully illuminates the efficiency of XFZYDfor the treatment of TBI as well as herb combination ruleof TCM formula Network pharmacology with moleculedocking method confirms the ldquomulticompounds multitar-getsrdquo therapeutic actions of XFZYD in the treatment of TBI
The present work may provide valuable evidence for furtherclinical application of XFZYD for treating TBI
Abbreviations
TBI Traumatic brain injuryXFZYD Xuefu Zhuyu decoctionTCM Traditional Chines medicineDL Drug-likenessOB Oral bioavailabilityHB-cC-cT Herb-candidate compound-candidate
targetHB-pC-pT Herb-potential compound-potential targetCALM CalmodulinGSK3B Glycogen synthase kinase-3 betaSCN5A Sodium channel protein type 5 subunit
alphaF2 ProthrombinACHE AcetylcholinesteraseGO Gene OntologyKEGG Kyoto Encyclopedia of Genes and
Genomes
Data Availability
The data used to support the findings of this study areavailable from the corresponding author upon request
Conflicts of Interest
The authors declare that they have no conflicts of interest
Authorsrsquo Contributions
YangWang and Zebing Huang participated in the conceptionand design of the study YuanyuanZhong YangWang ZebingHuang Jiekun Luo Tao Tang and Pengfei Li acquired andanalyzed the data Yuanyuan Zhong Tao Liu and HanjinCui drafted and revised the manuscript The correspondingauthor and all of the authors have read and approved the finalsubmitted manuscript
Acknowledgments
This work was supported by the National Natural ScienceFoundation of China (Nos 81673719 81874409 81603670and 81803948) Project funded byChina Postdoctoral ScienceFoundation (Nos 2016M600639 and 2017T100614)
Supplementary Materials
Table S1 162 bioactive compounds in XFZYD Table S2 targetproteins of XFZYD Table S3 TBI-specific proteins Table S4119 potential compounds of XFZYD for treating TBI TableS5 docking result of 18 target proteins with 91 potential com-pounds Fig S1 the exact bindingmode between active ingre-dients and protein targets obtained from molecule docking(A) AKT1-quercetin (B) CDK1-quercetin (C) GSK3B-FA
Evidence-Based Complementary and Alternative Medicine 17
(D) F2-glyasperin B (E) NOS3-1-Methoxyphaseollidin and(F)ACHE-(-)-Medicocarpin (Supplementary Materials)
References
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[2] J A Langlois W Rutland-Brown and M M Wald ldquoTheepidemiology and impact of traumatic brain injury a briefoverviewrdquo The Journal of Head Trauma Rehabilitation vol 21no 5 pp 375ndash378 2006
[3] H A Alsulaim B J Smart A O Asemota et al ldquoConsciousstatus predictsmortality among patientswith isolated traumaticbrain injury in administrative datardquo The American Journal ofSurgery vol 214 no 2 pp 207ndash210 2017
[4] M Majdan D Plancikova A Maas et al ldquoYears of life lost dueto traumatic brain injury in Europe A cross-sectional analysisof 16 countriesrdquo PLoS Medicine vol 14 no 7 p e1002331 2017
[5] P Cheng P Yin P Ning et al ldquoTrends in traumatic braininjury mortality in China 2006ndash2013 A population-basedlongitudinal studyrdquo PLoS Medicine vol 14 no 7 Article IDe1002332 2017
[6] M V Russo and D B McGavern ldquoInflammatory neuroprotec-tion following traumatic brain injuryrdquo Science vol 353 no 6301pp 783ndash785 2016
[7] WWang H Li J Yu et al ldquoProtective Effects of ChineseHerbalMedicine Rhizoma drynariae in Rats After Traumatic BrainInjury and Identification of Active Compoundrdquo MolecularNeurobiology vol 53 no 7 pp 4809ndash4820 2016
[8] M Das S Mohapatra and S S Mohapatra ldquoNew perspectiveson central and peripheral immune responses to acute traumaticbrain injuryrdquo Journal of Neuroinflammation vol 9 p 236 2012
[9] J W Finnie ldquoNeuroinflammation Beneficial and detrimentaleffects after traumatic brain injuryrdquo Inflammopharmacologyvol 21 no 4 pp 309ndash320 2013
[10] T Cheng W Wang Q Li et al ldquoCerebroprotection of flavanol(minus)-epicatechin after traumatic brain injury viaNrf2-dependentand -independent pathwaysrdquo Free Radical Biology amp Medicinevol 92 pp 15ndash28 2016
[11] W Young ldquoRole of calcium in central nervous system injuriesrdquoJournal of Neurotrauma vol 9 Suppl 1 pp S9ndashS25 1992
[12] R Bullock A Zauner J S Myseros et al ldquoEvidence forProlonged Release of Excitatory Amino Acids in Severe HumanHead Trauma Relationship to Clinical Eventsrdquo Annals of theNew York Academy of Sciences vol 765 no 1 pp 290ndash297 1995
[13] A T Mazzeo A Beat A Singh and M R Bullock ldquoTherole of mitochondrial transition pore and its modulation intraumatic brain injury and delayed neurodegeneration afterTBIrdquo Experimental Neurology vol 218 no 2 pp 363ndash370 2009
[14] S Gennai A Monsel Q Hao et al ldquoCell-Based therapy fortraumatic brain injuryrdquo British Journal of Anaesthesia vol 115no 2 pp 203ndash212 2015
[15] J Ghajar ldquoTraumatic brain injuryrdquo The Lancet vol 356 no9233 pp 923ndash929 2000
[16] D J Loane and A I Faden ldquoNeuroprotection for traumaticbrain injury translational challenges and emerging therapeuticstrategiesrdquoTrends in Pharmacological Sciences vol 31 no 12 pp596ndash604 2010
[17] Y Wang X Fan T Tang et al ldquoRhein and rhubarb simi-larly protect the blood-brain barrier after experimental trau-matic brain injury via gp91phox subunit of NADPH oxidaseROSERKMMP-9 signaling pathwayrdquo Scientific Reports vol 6p 37098 2016
[18] D-X Kong X-J Li and H-Y Zhang ldquoWhere is the hopefor drug discovery Let history tell the futurerdquo Drug DiscoveryTherapy vol 14 no 3-4 pp 115ndash119 2009
[19] F Cheung ldquoTCM made in Chinardquo Nature vol 480 no 7378pp S82ndashS83 2011
[20] C Fu Z Xia Y Liu et al ldquoQualitative analysis of majorconstituents from Xue Fu Zhu Yu Decoction using ultra highperformance liquid chromatographywith hybrid ion trap time-of-flight mass spectrometryrdquo Journal of Separation Science vol39 no 17 pp 3457ndash3468 2016
[21] H-J Zhang and Y-Y Cheng ldquoAn HPLCMS method for iden-tifying major constituents in the hypocholesterolemic extractsof Chinese medicine formula lsquoXue-Fu-Zhu-Yu decoctionrsquordquoBiomedical Chromatography vol 20 no 8 pp 821ndash826 2006
[22] L Zhang L Zhu YWang et al ldquoCharacterization and quantifi-cation of major constituents of Xue Fu Zhu Yu by UPLC-DAD-MSMSrdquo Journal of Pharmaceutical and Biomedical Analysisvol 62 pp 203ndash209 2012
[23] X Yang X Xiong G Yang and J Wang ldquoChinese patentmedicine Xuefu Zhuyu capsule for the treatment of unstableangina pectoris a systematic review of randomized controlledtrialsrdquo Complementary Therapies in Medicine vol 22 no 2 pp391ndash399 2014
[24] J Wang X Yang F Chu et al ldquoThe effects of Xuefu Zhuyuand Shengmai on the evolution of syndromes and inflammatorymarkers in patients with unstable angina pectoris after percuta-neous coronary intervention a randomised controlled clinicaltrialrdquoEvidence-BasedComplementary andAlternativeMedicinevol 2013 Article ID 896467 9 pages 2013
[25] Q Zhang H Yu J Qi et al ldquoNatural formulas and the natureof formulas Exploring potential therapeutic targets based ontraditional Chinese herbal formulasrdquo PLoS ONE vol 12 no 2p e0171628 2017
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[27] Y-C Shen C-K Lu K-T Liou et al ldquoCommon and uniquemechanisms of Chinese herbal remedies on ischemic strokemice revealed by transcriptome analysesrdquo Journal of Ethnophar-macology vol 173 pp 370ndash382 2015
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18 Evidence-Based Complementary and Alternative Medicine
[31] M Sun X-P Zhan C-Y Jin J-Z Shan S Xu and Y-LWang ldquoclinical observation on treatment of post-craniocerebraltraumatic mental disorder by integrative medicinerdquo ChineseJournal of Integrative Medicine vol 14 no 2 pp 137ndash141 2008
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[34] S Li ldquoExploring traditional chinese medicine by a novel thera-peutic concept of network targetrdquo Chinese Journal of IntegrativeMedicine vol 22 no 9 pp 647ndash652 2016
[35] S Li and B Zhang ldquoTraditional Chinese medicine networkpharmacology theory methodology and applicationrdquo ChineseJournal of Natural Medicines vol 11 no 2 pp 110ndash120 2013
[36] A L Hopkins ldquoNetwork pharmacology the next paradigm indrug discoveryrdquoNature Chemical Biology vol 4 no 11 pp 682ndash690 2008
[37] Y Li C Han J Wang et al ldquoInvestigation into the mechanismof Eucommia ulmoides Oliv based on a systems pharmacologyapproachrdquo Journal of Ethnopharmacology vol 151 no 1 pp452ndash460 2014
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[39] Bo Zhang Xu Wang and Shao Li ldquoAn Integrative Platform ofTCM Network Pharmacology and Its Application on a HerbalFormula Qing-Luo-Yinrdquo Evidence-Based Complementary andAlternativeMedicine vol 2013 Article ID 456747 12 pages 2013
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[41] J V Turner D J Maddalena and S Agatonovic-KustrinldquoBioavailability Prediction Based on Molecular Structure for aDiverse Series of Drugsrdquo Pharmaceutical Research vol 21 no 1pp 68ndash82 2004
[42] X XuW Zhang CHuang et al ldquoAnovel chemometricmethodfor the prediction of human oral bioavailabilityrdquo InternationalJournal of Molecular Sciences vol 13 no 6 pp 6964ndash6982 2012
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[44] X Wei H Liu X Sun et al ldquoHydroxysafflor yellow A protectsrat brains against ischemia-reperfusion injury by antioxidantactionrdquo Neuroscience Letters vol 386 no 1 pp 58ndash62 2005
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[46] Y Yamanishi M Kotera M Kanehisa and S Goto ldquoDrug-target interactionprediction from chemical genomic and phar-macological data in an integrated frameworkrdquo Bioinformaticsvol 26 no 12 pp i246ndashi254 2010
[47] H Yu J Chen X Xu et al ldquoA systematic prediction ofmultiple drug-target interactions from chemical genomic andpharmacological datardquo PLoS ONE vol 7 no 5 Article IDe37608 2012
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[50] T S Keshava Prasad RGoel K Kandasamy et al ldquoHuman pro-tein reference databasemdash2009 updaterdquo Nucleic Acids Researchvol 37 no 1 pp D767ndashD772 2009
[51] A Franceschini D Szklarczyk S Frankild et al ldquoSTRING v91protein-protein interaction networks with increased coverageand integrationrdquoNucleic Acids Research vol 41 no 1 pp D808ndashD815 2013
[52] P Shannon A Markiel O Ozier et al ldquoCytoscape a softwareEnvironment for integratedmodels of biomolecular interactionnetworksrdquo Genome Research vol 13 no 11 pp 2498ndash25042003
[53] G Dennis Jr B T Sherman D A Hosack et al ldquoDAVIDDatabase for Annotation Visualization and IntegratedDiscov-eryrdquo Genome Biology vol 4 no 5 p P3 2003
[54] L Yang X Zhao and X Tang ldquoPredicting disease-related pro-teins based on clique backbone in protein-protein interactionnetworkrdquo International Journal of Biological Sciences vol 10 no7 pp 677ndash688 2014
[55] S MThomas and J S Brugge ldquoCellular functions regulated bySRC family kinasesrdquo Annual Review of Cell and DevelopmentalBiology vol 13 pp 513ndash609 1997
[56] D Z Liu F R Sharp K C Van et al ldquoInhibition of Src familykinases protects hippocampal neurons and improves cognitivefunction after traumatic brain injuryrdquo Journal of Neurotraumavol 31 no 14 pp 1268ndash1276 2014
[57] B D Manning and A Toker ldquoAKTPKB Signaling Navigatingthe Networkrdquo Cell vol 169 no 3 pp 381ndash405 2017
[58] Y Kitagishi and S Matsuda ldquoDiets involved in PPAR andPI3KAKTPTEN pathway may contribute to neuroprotectionin a traumatic brain injuryrdquoAlzheimerrsquos ResearchampTherapy vol5 no 5 p 42 2013
[59] D Chen L Bao S-Q Lu and F Xu ldquoSerum albumin andprealbuminpredict the poor outcomeof traumatic brain injuryrdquoPLoS ONE vol 9 no 3 Article ID e93167 2014
[60] J Yang Z Wu N Renier et al ldquoPathological axonal deaththrough aMapk cascade that triggers a local energy deficitrdquoCellvol 160 no 1-2 pp 161ndash176 2015
[61] E J Huang and L F Reichardt ldquoTrk receptors roles in neuronalsignal transductionrdquoAnnual Review of Biochemistry vol 72 pp609ndash642 2003
[62] J W Lee and R Juliano ldquoMitogenic signal transductionby integrin- and growth factor receptor-mediated pathwaysrdquoMolecules and Cells vol 17 no 2 pp 188ndash202 2004
[63] C S Yang J M Landau M T Huang and H L NewmarkldquoInhibition of carcinogenesis by dietary polyphenolic com-poundsrdquo Annual Review of Nutrition vol 21 pp 381ndash406 2001
[64] A Murakami H Ashida and J Terao ldquoMultitargeted cancerprevention by quercetinrdquoCancer Letters vol 269 no 2 pp 315ndash325 2008
[65] G Du Z Zhao Y Chen et al ldquoQuercetin attenuates neuronalautophagy and apoptosis in rat traumatic brain injury modelvia activation of PI3KAkt signaling pathwayrdquo NeurologicalResearch vol 38 no 11 pp 1ndash8 2016
Evidence-Based Complementary and Alternative Medicine 19
[66] Q Cai R O Rahn and R Zhang ldquoDietary flavonoidsquercetin luteolin andgenistein reduce oxidativeDNAdamageand lipid peroxidation and quench free radicalsrdquoCancer Lettersvol 119 no 1 pp 99ndash107 1997
[67] G A Bongiovanni E A Soria and A R Eynard ldquoEffectsof the plant flavonoids silymarin and quercetin on arsenite-induced oxidative stress in CHO-K1 cellsrdquo Food and ChemicalToxicology vol 45 no 6 pp 971ndash976 2007
[68] H Li L Yang Y Zhang et al ldquoKaempferol inhibits fibroblastcollagen synthesis proliferation and activation in hypertrophicscar via targeting TGF-beta receptor type Irdquo Biomedicine ampPharmacotherapy = Biomedecine amp Pharmacotherapie vol 83pp 967ndash974 2016
[69] K P Devi D S Malar S F Nabavi et al ldquoKaempferol andinflammation From chemistry to medicinerdquo PharmacologicalResearch vol 99 pp 1ndash10 2015
[70] M Zhou H Ren J Han W Wang Q Zheng and DWang ldquoProtective effects of kaempferol against myocardialischemiareperfusion injury in isolated rat heart via antioxidantactivity and inhibition of glycogen synthase kinase-3120573rdquo Oxida-tive Medicine and Cellular Longevity vol 2015 p 481405 2015
[71] C-F Lee J-S Yang F-J Tsai et al ldquoKaempferol inducesATMp53-mediated death receptor and mitochondrial apop-tosis in human umbilical vein endothelial cellsrdquo InternationalJournal of Oncology vol 48 no 5 pp 2007ndash2014 2016
[72] C Luo H Yang C Tang et al ldquoKaempferol alleviates insulinresistance via hepatic IKKNF-120581B signal in type 2 diabetic ratsrdquoInternational Immunopharmacology vol 28 no 1 pp 744ndash7502015
[73] A B Awad and C S Fink ldquoPhytosterols as anticancer dietarycomponents evidence and mechanism of actionrdquo Journal ofNutrition vol 130 no 9 pp 2127ndash2130 2000
[74] K Farber and H Kettenmann ldquoFunctional role of calciumsignals for microglial functionrdquoGlia vol 54 no 7 pp 656ndash6652006
[75] Y Lu Y Gu X Ding et al ldquoIntracellular Ca2+ homeostasisand JAK1STAT3 pathway are involved in the protective effectof propofol on BV2 microglia against hypoxia-induced inflam-mation and apoptosisrdquo PLoS ONE vol 12 no 5 p e01780982017
[76] K Leroy and J-P Brion ldquoDevelopmental expression and local-ization of glycogen synthase kinase-3120573 in rat brainrdquo Journal ofChemical Neuroanatomy vol 16 no 4 pp 279ndash293 1999
[77] C-M Liu E-M Hur and F-Q Zhou ldquoCoordinating geneexpression and axon assembly to control axon growth Potentialrole ofGSK3 signalingrdquo Frontiers inMolecularNeuroscience vol3 p 3 2012
[78] D A E Cross A A Culbert K A Chalmers L Facci S DSkaper and A D Reith ldquoSelective small-molecule inhibitors ofglycogen synthase kinase-3 activity protect primary neuronesfrom deathrdquo Journal of Neurochemistry vol 77 no 1 pp 94ndash102 2001
[79] V S Ossovskaya and NW Bunnett ldquoProtease-activated recep-tors contribution to physiology and diseaserdquo PhysiologicalReviews vol 84 no 2 pp 579ndash621 2004
[80] M Steinhoff J Buddenkotte V Shpacovitch et al ldquoProteinase-activated receptors transducers of proteinase-mediated signal-ing in inflammation and immune responserdquoEndocrine Reviewsvol 26 no 1 pp 1ndash43 2005
[81] H Krenzlin V Lorenz S Danckwardt O Kempski and BAlessandri ldquoThe importance of thrombin in cerebral injury and
diseaserdquo International Journal of Molecular Sciences vol 17 no1 2016
[82] M J McGinn and J T Povlishock ldquoPathophysiology of Trau-matic Brain InjuryrdquoNeurosurgery Clinics of North America vol27 no 4 pp 397ndash407 2016
[83] T Woodcock and M C Morganti-Kossmann ldquoThe role ofmarkers of inflammation in traumatic brain injuryrdquo Frontiersin Neurology vol 4 article 18 2013
[84] F Tanriverdi H J Schneider G Aimaretti B E Masel F FCasanueva and F Kelestimur ldquoPituitary dysfunction after trau-matic brain injury A clinical and pathophysiological approachrdquoEndocrine Reviews vol 36 no 3 pp 305ndash342 2015
[85] J V Rosenfeld A I Maas P Bragge M C Morganti-Kossmann G T Manley and R L Gruen ldquoEarly managementof severe traumatic brain injuryrdquoThe Lancet vol 380 no 9847pp 1088ndash1098 2012
[86] B M Aertker S Bedi and C S Cox ldquoStrategies for CNS repairfollowing TBIrdquo Experimental Neurology vol 275 no 3 pp 411ndash426 2016
[87] K Adachi Z Mirzadeh M Sakaguchi et al ldquo120573-catenin signal-ing promotes proliferation of progenitor cells in the adultmousesubventricular zonerdquo Stem Cells vol 25 no 11 pp 2827ndash28362007
[88] Y Hirabayashi and Y Gotoh ldquoStage-dependent fate determina-tion of neural precursor cells in mouse forebrainrdquo NeuroscienceResearch vol 51 no 4 pp 331ndash336 2005
[89] S Ding T Y H Wu A Brinker et al ldquoSynthetic smallmolecules that control stem cell faterdquo Proceedings of theNationalAcadamy of Sciences of the United States of America vol 100 no13 pp 7632ndash7637 2003
[90] N Israsena M Hu W Fu L Kan and J A Kessler ldquoThepresence of FGF2 signaling determines whether 120573-cateninexerts effects on proliferation or neuronal differentiation ofneural stem cellsrdquo Developmental Biology vol 268 no 1 pp220ndash231 2004
[91] A Salehi A Jullienne M Baghchechi et al ldquoUp-regulation ofWnt120573-catenin expression is accompanied with vascular repairafter traumatic brain injuryrdquo Journal of Cerebral Blood Flow ampMetabolism vol 38 no 2 pp 274ndash289 2017
[92] L F Reichardt and K J Tomaselli ldquoExtracellular matrixmolecules and their receptors Functions in neural develop-mentrdquoAnnual Review of Neuroscience vol 14 pp 531ndash570 1991
[93] R M Gibson S E Craig L Heenan C Tournier and M JHumphries ldquoActivation of integrin 12057251205731 delays apoptosis ofNtera2 neuronal cellsrdquoMolecularandCellularNeuroscience vol28 no 3 pp 588ndash598 2005
[94] C C Tate A J Garcıa andMC LaPlaca ldquoPlasma fibronectin isneuroprotective following traumatic brain injuryrdquoExperimentalNeurology vol 207 no 1 pp 13ndash22 2007
[95] C Culmsee and M P Mattson ldquop53 in neuronal apoptosisrdquoBiochemical and Biophysical Research Communications vol 331no 3 pp 761ndash777 2005
[96] M S Geddis and V Rehder ldquoThe phosphorylation state ofneuronal processes determines growth cone formation afterneuronal injuryrdquo Journal of Neuroscience Research vol 74 no2 pp 210ndash220 2003
[97] M L Craske M Fivaz N N Batada and T Meyer ldquoSpines andneurite branches function as geometric attractors that enhanceprotein kinase C actionrdquoThe Journal of Cell Biology vol 170 no7 pp 1147ndash1158 2005
20 Evidence-Based Complementary and Alternative Medicine
[98] A Muscella C Vetrugno L G Cossa et al ldquoApoptosis by[Pt(OOrsquo-acac)(gamma-acac)(DMS)] requires PKC-deltamedi-ated p53 activation in malignant pleural mesotheliomardquo PloSone vol 12 no 7 Article ID e0181114 2017
[99] J S Bonini W C Da Silva L R M Bevilaqua J H MedinaI Izquierdo and M Cammarota ldquoOn the participation ofhippocampal PKC in acquisition consolidation and reconsol-idation of spatial memoryrdquo Neuroscience vol 147 no 1 pp 37ndash45 2007
[100] O Zohar R Lavy X Zi et al ldquoPKC activator therapeutic formild traumatic brain injury in micerdquo Neurobiology of Diseasevol 41 no 2 pp 329ndash337 2011
[101] J David Sweatt ldquoTheneuronalMAPkinase cascade a biochem-ical signal integration system subserving synaptic plasticity andmemoryrdquo Journal ofNeurochemistry vol 76 no 1 pp 1ndash10 2001
[102] Y Matsuoka M Picciano B Maleste et al ldquoInflammatoryresponses to amyloidosis in a transgenic mouse model ofAlzheimerrsquos diseaserdquo The American Journal of Pathology vol158 no 4 pp 1345ndash1354 2001
[103] L Esposito L Gan G-Q Yu C Essrich and L MuckeldquoIntracellularly generated amyloid-120573 peptide counteracts theantiapoptotic function of its precursor protein and primesproapoptotic pathways for activation by other insults in neu-roblastoma cellsrdquo Journal of Neurochemistry vol 91 no 6 pp1260ndash1274 2004
[104] D J Loane A Pocivavsek C E-H Moussa et al ldquoAmyloidprecursor protein secretases as therapeutic targets for traumaticbrain injuryrdquo Nature Medicine vol 15 no 4 pp 377ndash379 2009
[105] R Torres B L Firestein H Dong et al ldquoPDZ proteins bindcluster and synaptically colocalize with Eph receptors and theirephrin ligandsrdquo Neuron vol 21 no 6 pp 1453ndash1463 1998
[106] M B Dalva M A Takasu M Z Lin et al ldquoEphB receptorsinteract with NMDA receptors and regulate excitatory synapseformationrdquo Cell vol 103 no 6 pp 945ndash956 2000
[107] J M Basak P B Verghese H Yoon J Kim and D MHoltzman ldquoLow-density lipoprotein receptor represents anapolipoprotein E-independent pathway of A120573 uptake anddegradation by astrocytesrdquoThe Journal of Biological Chemistryvol 287 no 17 pp 13959ndash13971 2012
[108] L Yao X Gu Q Song et al ldquoNanoformulated alpha-mangostinameliorates Alzheimerrsquos disease neuropathology by elevatingLDLR expression and accelerating amyloid-beta clearancerdquoJournal of Controlled Release vol 226 pp 1ndash14 2016
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
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Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
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Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
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Volume 2018
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Hindawiwwwhindawicom Volume 2018
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Hindawiwwwhindawicom Volume 2018
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Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 7
Jun herbs(29)
Zuo-
Shi h
erbs
(101
)
Chen herbs
(23)
1
2
15
(a)
Jun herbs(5)
Zuo-
Shi h
erbs
(48)
Chen herbs
(10)
9
10
14189
(b)
20Mol 1200000
MMMol 152Mol 15Mol 1521Mol 15Mol 15
Mol 8888
Mol 29Mol 2Mol 2Mol 2Mol 29
Mol 3Mol 3Mol 35Mol 3535
1Mol 121Mol 121Mol 122
Mol 124Mol 124Mol 1Mol 124Mol 124Mol 124
MMol 10Mol 1Mol 10M 00MMMM
MMol 73Mol 77333222
ABATABATA
MAPK3MAPKMAPK
UGT1A1GT1UGT1A1
GOT1GOTT1
SREBF1REBFSREBF1
RXRBRXRBRXRB
CYP19A1CYP19A1YP19A
PLB1PLBPLB1
FASLGFASLGASL
BCHEBCHHE
HSD3B1HSD3
MTTPMTT
CES1CES
TIMP1TIMP
ATP5BATP5
MAPK10MAPK 0
BADBAD
CHRM5CHRM5
EPHBEPHBPHB2
HSD3B2HSD3B2SD3B
SOAT2OATSOAT2
FASNFASNFASN
MT-ND6MT-N
HTR3AHTR3HTR3APLA2G4APLA2G4AA2G
PKIAAIA
ADH1AADHAD
AKR1C1KR1AKR1C1
CREB1CREBB1
HMGCRMGHMGCR
SOAT1OAT
ABCC1ABCC
OPRD1OPRDOPRD1
VCPVCP
LDLRLDLR
ADRA1DDRAA1D
NFATC1FATCFATCATCFATCA
EGLN1GLNGLNGLNGLN
FABP5ABP5ABPABPABPABP5
CYCSCYCSCYCSCYCSYCSFOSL1OSLOSLOSLOSL
ALOX12LOXLOXOXLOX
TDRD7DRDDDRDDRDDRDAPODAPODAPOPOAPOD
NNNNOXNOXNOXNOX5NOXX
Mol 40
NFKB1NFKBFKBFKBFKBLYZLYZLYZZLYZ CTNNB1TNNBTNNNNNN MMP10MMPMPMPMMP1ALBALBALBALBALB
3Mol 433MMol 28Mol 28Mol 2Mol 28
HSPA5SPASPASPA5HSPA5AERBB2ERBB2ERBBRBBB2ERBB2
HSPB1HSPBSPBSPBHSPB1HSPB1B
NQO1NQO1NQONQO1NQONQO1TOP1TOP1TOP1TOPTOPT
PORPPORPORPORPORR ACPPACPPACPPACPPCPP
SLC2ALC2A4LC2A4C2A4LC2A4LC2A4LC
SERPINE1SERPINE1PINRPINERPINPIN
JUNJUNNJUNNN
PTGER3PTGER3TGERTGERGERTGER
COL1A1OL1AOL1AOL1AA1CO
SSLPISLPSLPISLPISLPII
CCCALCCC MMMMMMSS
MMMMP1MMPMMPMMPTNFTNFTNFFTNF
CCND1CCNDCCNDCNDCNDCCND
SMDPSMDPSMDPSMD3PSMD3PSMDNFNFNFFFNF
ELK 1ELKELK LK ELK ELK CCL2CCL2CCLCCL2CCL2CCL2
MMol 14MMol 14Mol 1447
MMol 15MMol 1Mol 115Mol 15M
Mol 122Mol 122Mol 122MMol 12Mol 122222
MMol 24Mol 2Mol 2Mol 2444
Mol 78MMol 78Mol 7Mol 7Mol 788
Mol 38MMMMol 3Mol 3338
Mol 154Mol 154MMol 15ol 1ol 155M
Mol 67MMol 6Mol 6Mol Mol 67Mol 677766
Mol 64MMol 64Mol 6Mol 6Mol 64
Mol 48Mol 48MMMol 4Mol 4448M 4
PPPPARAPARPARPAR
PCOLCEPCOLCECOLCOLCCOLC
SPP1SPP1SPP1PP1SPPP1 AKR1C3AKR1C3KR1CKR1CKR1CKR
NNNOSNN 2222 DUOXDUOXDUOXDUOX2DUOXDUOX2
MCL1MCL1MCLMCLMCLMCL
PARP1ARP1ARPARPPARP1PEEMPOMPOMPOMPOM DDDUDUDUDDU
NGASYNGASYNGAP1NGAPNGAAP1RARAARARA
BAXXXXB X ESR1
GSTM2GSTM2GSTMSTMSTMGSTMSHSAAHSAAHSA1AHSA1AHSAHSAA GABRAGABRA2GABRA2GABRAGABRAGABRAMM2MMM2
CHRM2CHRM2CHRMHRMHRMCHRM2
VDRVDRVDRRVDRVDRARGABRA1GABRA1GABRAA11GABRA1G
IL888IL88
PPPPRSPRSPRSSSSSSSPPARDPARDPARPARDPARDPARDSELESELESELESELEEE
COL3A1COL3A1OL3AOL3AOL3AA1OL3ARB11111
CYP1A2222222 NCOANCOANCOANCOANCOANCOA22222211111 RRRRRR
Quercetin
Mol 9444444
Mol 99
Mol 63
Mol 75555
Mol 37Mol 37Mol 37Mol 37MolMol 33Mol 37MolM
Mol 158
Mol 1Mol 10Mol 1Mol 10000
Mol 49Mol 4Mol 4Mol 4Mol 44Mol 494Mol 4
Mol 6Mol 6666M 66
MMol 60Mol 60Mol 6MM
Mol 101Mol 101Mol 101Mol 10011
Mol 12Mol 12Mol 12Mol 1Mol 12221
Mol 11MMol 1155Mol 11555M6060660
Mol 85Mol 85Mol 85MMol 88558554MMol 114Mol 114444M
Mol 116Mol 116Mol 116MMol 1ol 1Mol 11616M 1
Mol 9000
Mol 100Mol 1000Mol 106MMMol 106Mol 10Mol 1066Mol 322 Mol 30Mol 30Mol 30000MM
Mol 140MMol 14MMol 14Mol 1440Mol 14
PlatycodPlatycodatycodPlatycodP ononoGrandifondifoGrandGrand rususus
RadiRadixR diBupleurBupleuriBupleuriu
RehmanniRehmanniRehmanniiaaaglutinosglutinolutinoglutin aaaLiboschLiboschLiboschLLiLiLicoricececece
AngelicaAngelicaAngelican l eeSinensisSinenS nensisnensis
Radixadixdix
Mol 84
Mol 23 Mol 130Mol 13Mol 13Mol 11Mol 130Mol 13Mol 1MMol 13
Mol 11Mol 1Mol 1Mol 11Mol 11
0Mol 200000
Mol 34Mol 3Mol 3MolMol 3344Mol 3Mol 134
Mol 151
Mol 126Mol 126Mol 12666
Mol 8111AurantiiAurantAuranAurantiitFructusructusFructuructuFr
Mol 77Mol 7Mol 7Mol 77777
Mol 444
MMol 82Mol 8Mol 8222Mol 88
Mol 133
Mol 123MMol 12Mol 12ol 1Mol 121
Mol 142Mol 1422
Mol 36
0Mol 80Mol 88Mol 80M 80
Mol 277Mol 135Mol 13555M 555
Mol 141MMol 144Mol 14111
Mol 104Mol 104MMMol 10Mol 104044M
Mol 922222Mol 22 Mol 2Mol 2Mol 2Mol Mol 2Mol 22M 2
MMol 83333
Mol 110Mol 110
Mol 119MMol 11999999
Mol 88888 Mol 109Mol 10MMol 1Mol 100Mol 109M
Mol 5444
Mol 39999999
Mol 150MMol 155000M 0
Mol 611
21Mol 21MMol 221111
Mol 13
Mol 9Mol 91MMol 9911
Mol 118MMol 11Mol 1Mol 11Mol 1Mol 11ol
Mol 466
Mol 96Mol 96Mol 96Mol 996Mol 9696 Mol 53Mol 5Mol 5MolMol 5Mol 5Mol 535Mol 50Mol 50Mol 5Mol 5Mol 550Mol 5
Mol 107Mol 107MMol 10077Mol 1077
M l 149MMol 1449Mol 149Mol 149
Mol 14Mol 1444
Mol 89Mol 125MMol 1ol 1Mol 12MMMol 1MM
Mol 87
Mol 72MolMol 7Mol 7MolMol 72M 7M Mol 933Mol 112Mol 112Mol 112Mol 1122M
Mol 1Mol MMolMollMol 1Mol Mol 105
MAP2MAP2MAPMAPMAP
IL2I 2L22L22
SLC6A2LC6A2LC6A2LC6A2C6A2S
GABRA5ABRAABRGABRA5AABRCHRNA2CHRNA2HRNA2HRNHRNA2HRNA2
DRD1D1DRDDRDDRDDD1 MMAPKMAPK1MAMADRA1AADDDRADRA1ADRAADRA1A CASP3CASP3CASP3CASP3C
F2
Mol 17 Mol 7666Mol 33
Mol 86Mol 86Mol 8Mol 8Mol 866686
Mol 161Mol 161Mol 161Mol 16Mol 16161MMo
Mol 56Mol 5Mol 556666Mol 113
Mol 97Mol 162MMol 16Mol 1Mol 1ol 111M 1
Mol 131
Mol 99Mol 9MMMolMol 9MMol 9Mol 99Mol 9ol
Mol 111
Mol 128Mol 128Mol 1ol 1Mol 121MolMol 199Mol 117Mol 117777
Mol 744444Mol 77
2Mol 52Mol 52Mol 5MM
MMMol 5Mol 5MMol 5Mol 5Mol 5M
MMol 58Mol 58Mol 58M 8
Mol 138MMol 1388M
Mol 102Mol 102Mol 10Mol 102MMol 10Mol 10
Mol 42MMMol 42M
MMMol 3Mol 3
Mol 137Mol 13Mol 13ol 13Mol 13Mol 13733
Mol 59Mol 59olMol 5Mol 59oMol 5Mol 559
Mol 129lMol 12ol 12ol 12Mol 129o 2ol 12o
Mol 62Mol 62Mol 6Mol 6Mol 6Mol 62Mol 6Mol 6ol
Mol 144Mol 144Mol 14Mol 14ol 14Mol 144ol 1
AchyraAchyraAchyranthisnthisnthisBidentBidentBidentataeataeatae
RadixRadixRadix
Mol 139Mool 13ol 13ol 139Mol 139Mool 13
ChuanxioChuanxioChuanxiongngngRhizomaRhizomaRhizoma
Mol 159Mol 159MoMol 15ol 15ol 15Mol 159o 5
Mol 136Mol 136ol 13ol 13ol 1ol 13ool 13
CXCL11CXCL11XCL1XCL1XCLXCL1p53p53p53p
Mol 47Mol 47Mol 4Mol 4Mol 4ol 47Mol 4o
MMol 156ol 15ol 15ol 1ol 15ooPRKCARKCRKCPRKCACARKC
PPP3CAPP3CAP3CAP3CAP3CAPPP3CAP
CDKN1BDKNDKNDKN1CDKN1BPTENPTENPTENPTENPTENPTENBBB
RadixRadixRadixPaeoniaePaeoniaePaeoniaePae
RubraRubraRubraNR1I3NR1I3NR1INR1INR1INR1I3
HERC5ERCERC5HERC5ERC5OODC1ODCODCODC1CERBB3RBBRBBERBB3RBBRBB3
CTSDCTSDCTSDCTSDCTSDCTSD Mol 18Mol 1Mol 1ol 18Mol 18MolMol 1
PLATPLATPLAPLATPLATL
DIOOO1OO1OEGFEGFEGEGFEGDIODDIODIO
BIL1BIL1BIL1BIL1BB
MMP 3MMP 3MMP 3MMPMMP 3M
THBDTHBDTHBTHBTHBTHBD
CYP1B1YP1BYP1BYP1BYP1BB1
PPPPPARPP GGGGGGPPP
KDR LLAACTACTACTLACTBLACTBACTTT
ESR2
CHEK1CHEK1CHEK1CHEK1CHEK1CHEK111CCHEK1C
CATCATCATCATTTCATCATT
CDK2
CCNA2CCNA2CCNA2CCNA2CCNA2CCNA2CCNA2CCCNA2CC
PIM1 GSK3B
MAPKKK1414MMAPKKKMM KMMM
IL10IL10IL10IL10IL10IL10
S2HAS2HAS2HAS22HAS2ADADDHDH1CADAD HAHAHHHHA
CDKN2ACDKN2CDKNCDKNDKN2ACDKN
Mol 155Mol 155Mol 15Mol 15Mol 15ol 1ol 15ol 15Mol 15MMo
Mol 153Mol 15Mol 15ol 15Mol 153ol 15Mol 15Mol 15
CHEKCHEKCHEKHEK2HEK2HEK
MMP2MMP2MMPMMPMMPMMP2
MGAMMGAMMGAMGAAM
KCNH2222
CXCL2CXCL2CXXCLXCLXCL
GJA1GJA1GJAGJA1GJA1GJA1KK222K2
IKBKBIKBKBIKBKBIKCNH2KCNH2
IFNGIFNGIFNGIFNGIFNGIFNGFNEIF666F6IGHG1IGHG1GHGIGHG1IGGHG
SSStigSStiggmmmaS gg steeerolerol
PPTPNPTPN1PTPN1P 1SCN5ASCN5AASCN5ASCN5A BetaBetaaaa-sata istosssststerolerolllroNR3C1R3CGSTA2STA BBC3BBC3LBPLBP TTEP1TEP1 RKCDPRKCD PDE10ADE10GSTA1STA FN1FN1CD14CD14
BACE1ACEBACE1 SLC6A1LC6AACD163CD16 GSRGSRR ADIPOQDIPOQSTAT3TATOPRM1OPRM1OPRMOPRMOPRMO ADRA1BADRA1BAADRA1BBBB
SLC6A4LC6ALC6ASLC6A44SLCYCYP1A1YP1A1YP1A1CYP1AYP1A1
SLC6A3SLC6A3SLC6ASLC6A3SLC6A3
PDE3APDE3AA
CHRNA7CHRNA7HRNAHRNAHRNAA
PPPTGPP S1S1S1S1SS111NKX3-1NKX3-1NKX3KX3NKX3NKX3-
BIRCBIRC5BIRCBIRCBIRC55
IL6666
HK2HK2HK2HK2HK2HK2
RUNX2UNXUNXUNXRUNX2UNXHSF1HSF1HSF1HSF1HSF1HSF1
IGFBP3GFBPGFBPFBPIGFBP3
STAT1T1TAT1TATSTAT1TATT
NCF1NCFNCFNCF1NCFN
PRSS3RSSSIRT1SIRTGATMGAT OLR1OLRAPOBAPOPYGMPYGM
TGFB1GFBGFBGFBGFBTGFB1F10 MMP9MMPMMPMMPP9PNFE2L2FE2LFE2LFE2LNFE2L2FE2L2L
Mol 160Mol 44Mol 44ol 4Mol 444Mol 444ol 4ol 4ol 44
Mol 55MoMol 5Mol 5Mol 555Mol 55Mol 5Mol 5MoMol 5
MMMol 132
MMMol 57
CRPCRPCRPCRPPHH
RUNX1T1UNX1NX1NX1NX1T1R
BCGABCGABCG2BCGA
CXCL10CXCL10XCXCLCXCLCXCL IGG
CLDN4LDN4LDN4LDN4ABABABAAB RRAHRAHRBBBIB
RARRAF1RAF1RAF11KKKKKKaemKKKKK pffefeferereerf referololol
CASP9CCASPCASP9PMDM2MDMMMDMMDMMDM2MDMMDM
BMAOBMAOBMAOBMAOBMAOBINSRINSRINSRINSRINSRRRNCOA1NCOA11NCOA1
HTR2AHTR2HTR2HTR222A
CHRMCHRMCHRM3MM3BCL2LCL2CL2L1LCL2BCL2L1111 CHCCHCCCCH
BCL2222BCL2CASP8CASP8CASP8CASP8CASP
RELARELARELAAAR
BaicalBaicalBaBaicalcacacalicaBB calcaac ininininininninninnCHUCHUKHUKCHUKHUKUKH AAAAAA
EGFREGFRGFREGFREGFR
NFKBIAFKBIFKBFKBNFKBIAFKBI
E2F2E2F2E2F2E2F2E
APPAPAPPAPPPAPPAPPA
CASP7CASPCASPASPCASPCASPPP IL4IL444L4L44
NUF2NUFNUFNUFNUFNUFNUFNUFN
CDK4CDKCDKCDKCDKCDKCD
XIAPXIAPXIAXIAXIAXIAX MEMEMETMETMETMEETMMME
ADCY2ADCYDCYDCDCYDCD
Mol 127
NNR3C2NR3C2NR3CNR3C2N
DPP4
DCAF5DCACAFDCAF55DCAFAKR1B1AKR1BKKR1BKR1BKR1BCTRB1CTRBTRBCTRBCTRB
LTA4HLTA4LTA4TA4LTA4LTA4H4
XDHXDHXDHHHXDH
PTGS2
Mol 51Mol 51Mol 5MMolMolMol 5Mol 5MMol 5
Mol 145Mol 14Mol 14ol 1ol 1ol 1Mol 1Mo
Mol 146MMMMool 1ol 11461
Mol 143Mol 14Mol 14Mool 1ol 11Mol 1411
Mol 70MMMolMol 7Mol 77Mol 70Mol 7Mol 7
Mol 71Mol 7Mol Mol 777Mol 71Mol 7Mol
Mol 25Mol 25MMMol Mol 22Mol 2
Mol 79Mol 7Mol 7Mol Mol 7Mol 779MM 7
Mol 26Mol 26MMol 2Mol Mol 22Mol 2M 22
CA22222222ADH1BADHADHDHADH1ADH1DH KCNMA1CNMCNMCNMCNMKCNMAKCNMCGABRA6GABRAABRAABRAABRAGABRAGA A6GA RAGRIA2GRIAGRIAGRIAGRIAAAA2GRIA
RASSFRASSF1ASSFASSFASSF
Mol 31MMol 3Mol 3Mol 3311M 3Mol 98Mol 98Mol 98MMol 9Mol 999Mol 98MM
GSTP1GSTP1GSTPGSTPGSTPGSTPF33333
Mol 69Mol 6MMol 6Mol 6Mol 66Mol 69Mol 69M 66
VCAM1VCAMCAMCAMVCAM1C
MAPK8MAPK8MAPKMAPMAPKM
CYP3A4CYP3A4YP3AYP3AYP3AY
Mol 68Mol 68MMol 6Mol 6Mol 66MMol 68Mol 6MMol 16Mol 16Mol 1Mol 1Mol Mol 1Mol 16
MMMMol 6MMol 66Mol 666Mo
CCartCarta hamihamimiiiiiFlosFlosFlosossoss
Mol 9566Mol 65Mol 6Mol 6MMol MolMol 6M 666M
PersPersPerserrr icaeicaeicaesemesemesemeemememeem nnn
ICAM1CAMCAMM1CAMI
E2F1E2FE2F1E2F1E2F1ENPEPPSPEPPSPEPPSEPPSPEPPSNPEPP
IGF2IGF2IGF2IGF2F
FOSFOSFOSFOSSFOSFOSSSS
CCNB1CCNBCNBCNBCCNB1CCNB1CDK1CDK1CDKCDKCDKCC
PRKCBPRKCBRKCRKCRKCR CB
CHRM1CHRM11CHRM11
HHHHSP9HHHH 0ABABAB2B2B2B2PPPPPPPPPPPPTOP2A ACACACACACACACACAACACAACACA
PRKACA
TOP2TOP2
My c FF7FFIRF1IRF1IRF1IRF1IRF1IR
CAV1CCAVCAVCAV1V1CAV1
ACHE
ADRB1ADRBRBADRBADR
ADRADRADRA2A2AA2A
SOD1SSODSOD1D11
NOS33
PLAUPLAUPLAAUUPLAUPRXRA
PGR
HHMOX11MOXMOXHMOX
GSTM1GSTM1GSTMGSTMSTMAAA
HIF1AHIF1HIF1HIF11AHIF1A
FOSL2OSLOSLOSL22FO
ALOX5ALOXALOXALOXALOX5
VEGFAVEGFAVEGFAVEGFAV FFA
PON1PONPONPONN1PON
PIK3CGPIK3CGGG
SULT1E1SULT1E1ULT1ELT1LT1GAGAABRAGABRA3AGG
AKT1AKTT1AKT11A
CHRMCHRMCHRMCHRM4CHRM4RMM
MAOAMAOAMAOAMAOMAOCD40LGCD40LGCD40LGD40LGCD40LGCD40LBRA3RAAABRA3CHRCHHHCHR
ADRB2
(c)
Figure 3 HB-cC-cT network of XFZYD (a) and (b) The distribution of different candidate compounds and targets in the network (red theJun herbs-specific cC (a) and cT (b) aqua the Chen herbs-specific cC (a) and cT (b) periwinkle the Zuo-Shi herbs-specific cC (a) and cT(b) claybank common cC (a) and cT (b) between the Jun and Chen herbs blue common cC (a) and cT (b) between the Jun and Zuo-Shiherbs green common cC (a) and cT (b) between the Chen and Zuo-Shi herbs purple common cC (a) and cT (b) among the 3 group ofherbs (c) The triangles with circle backgrounds represent the herbs (HB) the squares and circles represent the candidate compounds (cC)and candidate targets (cT) The red triangles squares and circles represent corresponding HB cC and cT in the Jun herbs the same is toaqua representing the Chen herbs and periwinkle representing the Zuo-Shi herbs The claybank squares and circles represent correspondingcC and cT overlap between the Jun and Chen herbs the same is to blue representing the overlap between the Jun and Zuo-Shi herbs and thegreen representing the overlap between the Chen and Zuo-Shi herbs The purple squares and circles represent the corresponding cC and cTshared by the 3 group of herbs The size of the node is proportional to the value of degree
Radix Bupleuri (Zuo-Shi) contained quercetin It targeted149 bioactive proteins PTGS2 (degree=126) HSP90AB2P(degree=85) AR (degree=81) NCOA2 (degree=75) PRSS1(degree=68) PTGS1 (degree=67) PPARG (degree=66)and F10 (degree=60) were predicted as the major candidatetargets of quercetin followed by kaempferol (degree=65)which was contained by Carthami Flos (Jun) AchyranthisBidentatae Radix (Chen) and Radix Bupleuri licorice(Zuo-Shi) It targeted 61 bioactive proteins includingPTGS2 (degree=126) HSP90AB2P (degree=85) CALM(degree=81) AR (degree=81) NOS2 (degree=76) andNCOA2 (degree=75) Quercetin stigmasterol kaempferol
baicalin and beta-sitosterol existed in 3 the Jun Chenand Zuo-Shi drugs demonstrating crucial roles of thesecomponents The 5 ingredients in the Jun Chen and Zuo-Shiherbs targeted 186 bioactive proteins which accounted for65 targets of XFZYD Baicalein existed in the Jun andChen herbs Luteolin existed in the Jun and Zuo-Shi herbsSpinasterol and sitosterol existed in the Chen and Zuo-Shiherbs 126 bioactive compounds targeted PTGS2 followedby ESR1 (88) HSP90AB2P (85) AR (81) CALM (81) NOS2(76) NCOA2 (75) PRSS1 (68) PTGS1 (67) and PPARG(66) As depicted in Figure 3(c) these target proteins wereanchored by ingredients in the 3 group drugs
8 Evidence-Based Complementary and Alternative Medicine
Table 3 Top 10 target proteins of XFZYD according to 2 centrality indicators
Proteins Degree Proteins BetweennessPTGS2 126 PTGS2 0128936ESR1 88 NCOA2 0060806HSP90AB2P 85 HSP90AB2P 0049647AR 81 PRKACA 0046484CALM 81 PTGS1 004365NOS2 76 AR 0030252NCOA2 75 PRSS1 0025575PRSS1 68 ESR1 0022212PTGS1 67 PPARG 0021403PPARG 66 PGR 0020685Note the centrality indicators identify the important nodes within the network Higher degree centrality and betweenness centrality indicate greaterimportance
The analysis of the network revealed that quercetin (Mol148) kaempferol (Mol 108) luteolin (Mol 127) wogonin (Mol160) 7-Methoxy-2-methyl (Mol 33) and beta-sitosterol (Mol41) were predicted as the major active compounds of XFZYDTheproteins including F2 NOS2 PTGS1 PTGS2 and CALMwere predicted as essential pharmacological proteins for thetherapeutic effects of XFZYD
34 Analyses on TBI Based Specific Protein Interaction Net-work Network biology integrated with different kinds ofdata including physical or functional networks and diseasegene sets is used to interpret humandiseases Protein-proteininteraction networks (PPI) are fundamental to understandthe cellular organizations biological processes and proteinfunctions [54] From the systematic perspective the analysisof TBI-related PPI will improve the understanding of thecomplicated molecular pathways and the dynamic processesunderlying TBI We screened the TBI-specific genes andprotein targets using Online Mendelian Inheritance in Mandatabase (OMIM) and Therapeutic Target Database (TTD)Figure 4 indicated that 21 TBI-specific genesproteins wereacquired Further these hub-proteins were submitted toHPRD and STRING to establish the TBI-specific proteininteraction network The detailed information of the TBI-specific proteins is shown in Table S3 The results suggestedthat the network consisted of 489 nodes and 5738 edges(Figure 4(a)) We obtained top 10 TBI-related proteinsaccording to 2 centrality indicators generated and summa-rized in Table 4 Interestingly we found that the node withhigher betweenness tends to possess larger degree (Fig-ure 5) Network topology analysis showed that protoonco-gene tyrosine-protein kinase Src (SRC degree=164 between-ness=0059) RAC-alpha serinethreonine-protein kinase(AKT1 degree=157 betweenness=0045) Serum albumin(ALB degree=153 betweenness=0069) Epidermal growthfactor receptor (EGFR degree=139 betweenness=0053)and Amyloid beta A4 protein (APP degree=134 between-ness=0072) contributed to the essential role in the patho-physiology of TBI All of these indicated that the top mutualtarget proteins performed various beneficial functions to treatTBI at the molecular level For example SRC is activatedfollowing engagement of many different classes of cellular
receptors It participates in signal pathways that controla diverse spectrum of biological activities including genetranscription immune response cell adhesion cell cycleprogression apoptosis migration and transformation [55]SRC can result in blood-brain barrier (BBB) disruptionand brain edema at the acute stage the inhibition of SRCfamily kinases can protect hippocampal neurons and improvecognitive function after TBI [56] AKT1 regulates manyprocesses including metabolism proliferation cell survivalgrowth and angiogenesis [57] The PI3KAKTPTEN path-way has been shown to play a pivotal role in neuroprotectionenhancing cell survival by stimulating cell proliferation andinhibiting apoptosis after TBI [58] ALB is the main proteinof plasma and can be a biomarker to predict outcome ofTBI [59] Its main function is the regulation of the colloidalosmotic pressure of blood We found that it may participatein pathological process of TBI
KEGG pathway analysis was also used to determine thefunctions of proteins Table 5 describes the top 10 significantlyenriched KEGG pathways These pathways play crucial rolesin pathophysiology process which have also been widelydiscussed in existing literature For instanceMAPK signalingpathway can promote pathological axonal death throughtriggering a local energy deficit [60] Neurotrophin signalingthrough Trk receptors regulates cell survival proliferationthe fate of neural precursors axon and dendrite growth andpatterning and the expression and activity of functionallyimportant proteins such as ion channels and neurotransmit-ter receptors [61] Engagement of cells with the extracellularmatrix (ECM) proteins is crucial for various biological pro-cesses including cell adhesion differentiation and apoptosiscontributing to maintenance of tissue integrity and woundhealing [62]The 47 TBI-specific proteins targeted byXFZYD(yellow and green) were further discussed below
35 HB-pC-pT Network pT-F Network Construction andMolecule Docking Analyses of XFZYD for the Treatment ofTBI To investigate the therapeutic mechanisms of XFZYDfor the treatment of TBI a HB-pC-pT network of XFZYDfor treating TBI was built (Figure 6) 47 TBI-specific targetproteins (circles) were targeted by 119 potential compounds(squares) from XFZYD (Figure 6) Detailed information
Evidence-Based Complementary and Alternative Medicine 9
BLMH
GPRASP2
GJC2
CAMK2N1
OBSL1
ZNF292
RLFAMPD3
NANOS1CAMTA1HYPKCCS
DLGAP4PF4V1
TRIM2
CHKB
F8A1
TIAM1
BMX
RAPGEF1
APBB1
KCNMA1
XRCC6
TTR
TGM2MAP2
PRNP
RASGRF1 PPP3CADMD SPTAN1
ACE
TGFB2
AP2M1GRAP2
SCN5A
MYH9
PKN1
ACHE
SLC25A13
PHKG1
SPAST
UMOD
PRPF40B
BBC3
TUBA4A TTPALRYR2
STUB1KRT6A
AKAP8L
GFI1BTHRAP3
ZNF232
SOD1
CRKL
CDH2
MBP MAP2K2
DCN
MAP3K5NGFR
PIK3CA
ITGA2
DNM1
RAP1A
GRIN2B
PTPN11
PRKCA
LDLRAP1
DNAJA3INA
SP3
CACNA2D3
CSF1
CNTF
TNFRSF1B
BCL10
KLC1
DCTN1
IDE
SERPINA3
HSPA1A
HSPG2
FN1
LRP2
TUBB
GRIN2A
YWHAB
CAMK2D
HTRA2
PRKCE
CRYAB
APC
GIT1
PIAS4
HPX
TRIP10
PDE4B
MARK4
PLAT
UBE2K
APOC2
LIN7B BGN
MAP3K10MAPK8IP2
CBS
SH3GL3
STAU1
CASP6
PPP5C
KRT16
DAB1
CASP1SHC2
CTSD
UCHL1
HP
SNCACAMK2G
TGFB1
GNB1
PTPN1PIN1
TJP1
CASP8PLCG1
NPHS1
PPP2R2A
DNAJB1
ADRBK1
INADL
NCSTN
MATK
NEDD4L
KAT5IFNGTBP
FRS2
CSNK1A1
CALRKNG1
SGOL2
KRT6B
TRAF3 DLG3
SACS
BDKRB2PRKCB
CAV1
MAPK3F2 SMAD3
APOEAKAP9 RANBP2
CLTC
TSC22D1GAB2PSEN2
MLF1CROT
KRT9
ADRA1B
EPB41
COL4A1
SHC3
ARHGAP32
ATM
PPP2R5A
CAMK2A
NTRK1
KRT18
TNFAPOA2
LAT
GRIN2D
COL4A6
KLK3
VLDLR
COL4A5
COL4A2
NEFH
OR8D2
MTSS1
PDZRN4
PTPN4
OR3A2
OR2T6
DGKG
CASP3
IGF1R
CASK
DRD2
TRAF2
EPHB2
PPM1A
PRKCG
CACNB3
HNRNPUSIN3A
DLG1
CLU
RAP2A
ERBB4CANX
BACE1 GABBR1
KARS
CNOT1
UNG
AGA CDCP1
UTP14A PLAG1
EXOC6
HRAS
CTNNB1
SP1
NOS3 SUMO1
PSEN1
HSPA8
HSP90AA1
BCL2
GNAO1
RPS6KB1
PARK2
DLG4
PTK2B
GSN
NAE1 TLN2LRFN2
PLTP
NUMBLSTX1A
DERL1
NEFM
HMOX2
AATF
PPIAMTRNR2L2AP1M1
OGT
DDB1
AP4M1
MED31
RGS3
TDP2
KRT5
ADAM9
REST
PACSIN1
SHB
CTSL1
LAMA1
SH2B1
PLA2G4F
SMURF2
SLC9A8
KRT14
CIT
TGFB1I1
PPP2R1APIK3R1
NUMB
ADAM17
DLG2
LRP1
APOA4
LTA
IRS1
GSK3B
EGFR
CTSB
TRAF6
SHC1
PPP2CB
NGF
HTT
PPIDLDB3
APOC1HIP1
ZDHHC17COL25A1MAGI3
ITGB5
ACTB
GNA11
ACTN2CAMK2B
CDK1
LIN7C
S100B
PDLIM5
DLGAP1
LIN7A
TPR
SYNGAP1
CFD
GRIN3A
F12
SLC1A3
GRK5
CACNA2D2
TANC1
DUSP4
EXOC4
AHSG
GRIN3B
UCP2
TTN
GPC1
NSF
FGA
DYNLL1
MAPK8IP1
PICALM
PPBP
ITM2B
NID1
STXBP1
CTBP1
SGK1
TRAF1
SQSTM1
TNFRSF1A
ACTN1
OPTN
UBE2D2
AP2A2
FUS
APOA1
DICER1
CACNA1B
CST3
NCOA3
SCARB1
APOC3
SLC6A3
KIAA1551 KRT13
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KIAA0232
ENSG00000258818
SPATA31A7
CTAGE5
CEP44
SH2B2
EPHB4
CAPN1
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APBA2
COL4A3
LDLR
GIPC1
GFAP
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SRC
MAPK12APP
GAPDH
CDK5
CREBBP
NOS1
YWHAZCALM1
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YWHAQGRIN1
RPS6KA3
COL1A2
TCERG1
A2M
CASP7
LRP8
PDK2
AGTR1
FYN
ALBGRB2
MAPK1AKT1
STAT1
MAPT
GSK3A
UBB
PPP2CA
SMAD4
PRKCD
RGS12
SPTB
SH3BP5
CACNA1I
IL16
SLA2
PFN2
NLRC4
TNFRSF14
PRTN3
SNCB
FBLN1
BACE2
SETX
FANCD2
SAP30
RANBP3
PALB2
SLC1A5RASA1
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CFB
ADRBK2
CDC45
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DRD1
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GRK6
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CHD3
PRSS3
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CHRNA7
KRT10
RIMS1
CRMP1
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SORBS3
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GCN1L1
RNF19A
HOMER3
GRK4
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MYLK3
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HOXB2
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NCOR1
CASP4
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AP4E1
CLCA2
IGDCC4
NECAB3
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SH3GLB1
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PLCG2
(a)
STAT1
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FN1
PLAT
SOD1
PRKCA CDK1
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ALB APP
PPP3CA
IFNG
BBC3
SCN5A
KCNMA1
MAP2
ACHE
MAPK3F2
PTPN1
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PRKCB
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TNF
AKT1
F7
MAPK1LDLR
PRKCD
SLC6A3
DRD1
CASP3
CHRNA7
PRSS3
TP53BP2
CASP7
EPHB2
SYNGAP1
CALM1
CTSD
BACE1
ADRA1B
(b)
Figure 4 TBI-related protein interaction network (a) 21 hub proteins (red and green) were identified through the analysis of TherapeuticTargetDatabase (TTD) aswell asOnlineMendelian Inheritance inMan (OMIM) 4 overlapped protein targets (green)were obtained between21 hub proteins in TBI and candidate targets of XFZYD Periwinkle proteins fromHPRD and STRING analyses were not targeted by XFZYD(b) 47 candidate protein targets of XFZYD were screened for treating TBI Yellow candidate protein targets of XFZYD The size of nodes isproportional to the value of degree
10 Evidence-Based Complementary and Alternative Medicine
Table 4 Top 10 proteins of TBI specific proteins according to 2 centrality indicators
Proteins Degree Proteins BetweennessSRC 164 APP 0071814AKT1 157 ALB 0069343ALB 153 SRC 0058568EGFR 139 EGFR 0052938APP 134 AKT1 0045466GAPDH 131 HTT 0037164HSP90AA1 108 GAPDH 0034549BCL2 106 HSP90AA1 0027596MAPK1 105 CTNNB1 0021759HRAS 105 GNB1 0019492Note the centrality indicators identify the important nodes within the network Higher degree centrality and betweenness centrality indicate greaterimportance
Table 5 Top 10 significantly enriched KEGG pathways in TBI-specific proteins
Pathway ID Pathway description Gene count FDR4010 MAPK signaling pathway 44 299E-235200 Pathways in cancer 43 113E-184722 Neurotrophin signaling pathway 41 101E-344151 PI3K-Akt signaling pathway 40 111E-154510 Focal adhesion 39 292E-225205 Proteoglycans in cancer 39 410E-215010 Alzheimer s disease 34 124E-204015 Rap1 signaling pathway 33 769E-174014 Ras signaling pathway 33 646E-164020 Calcium signaling pathway 31 505E-17
0
001
002
003
004
005
006
007
008
0 20 40 60 80 100 120 140 160 180
Betw
eenn
ess
Degree
APP
ALB SRCEGFR
AKT1HTT
Figure 5 Relationship between degree and betweenness centralityin the TBI-specific protein interaction network
for the 119 potential compounds is shown in Table S4Similarly 5 pharmacologically active ingredients in the JunChen and Zuo-Shi groups including quercetin stigmasterolkaempferol baicalin and beta-sitosterol anchored 33 TBI-specific proteins such as CALM SCN5A F2 ACHE F7ADRA1B NOS3 BCL2 CASP3 and AKT1 11 Jun-specificcompounds targeted 2 specific targets (ALB CTNNB1) while12 Chen-specific compounds anchored 3 unique proteins(PRKCD FN1 and BC3) The Zuo-Shi herbs possessed thelargest number of active compounds (89) and targeted 5
unique proteins including EPHB2 BACE1 LDLR MAPK3and PRSS3 GSK3Bwas the common target between theChenandZuo-Shi drugs KCNMA1 CASP7 andAPPwere targetedby the Jun and Zuo-Shi drugs
The top 10 candidate compounds and targets to treat TBIwere showed in Tables 6 and 7 Formost of active compoundsfrom XFZYD each component hit more than one targetTable 6 demonstrated that quercetin had the highest numberof targets (degree =76) followed by kaempferol (degree=43) beta-sitosterol (degree =35) stigmasterol (degree =19)luteolin (degree=18) baicalein (degree=15) 7-Methoxy-2-methyl isoflavone (degree=12) wogonin (degree=12)nobiletin (degree=11) and naringenin (degree=9) Forinstance quercetin (3310158404101584057-pentahydroxyflavone) is anaturally occurring flavonoid commonly found in fruits andvegetables It regulates multiple biological pathways elicitinginduction of apoptosis as well as inhibiting angiogenesisand proliferation [63 64] Quercetin can attenuate neuronalautophagy and apoptosis in rat traumatic brain injury modelvia activation of PI3KAkt signaling pathway [65] It has alsobeen reported to have a protective ability against oxidativestress and mutagenesis in normal cells [66 67] Kaempferol(3 41015840 5 7 tetrahydroxy flavone) is a yellow-colored flavonoidthat is widely distributed in many botanical families[68] It has been shown to possess a variety of biologicalcharacteristics including effects of anti-inflammatory[69] antioxidative [70] tumor growth inhibition [71] and
Evidence-Based Complementary and Alternative Medicine 11
Mol 22Mol 33
Mol 29Mol 32 Mol 23Mol 20
Mol 97Mol 106
Mol 104Mol 100Mol 94
Mol 63Mol 90
Mol 87
Mol 91Mol 61
Mol 19
Mol 109
Mol 161
Mol 118
Mol 111
Mol 158
Mol 114
Mol 149
Mol 141
Mol 152
Mol 14
CASP3
BCL2
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ACHE
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Mol 4
Mol 49
Mol 127
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Mol 92
Mol 54
Mol 6
Mol 36
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Mol 27
Mol 13Mol 105Mol 140
Mol 17
Mol 39
Mol 2
Mol 21
Mol 12
Mol 142
Mol 126
Mol 135
Mol 121
Mol 119
Mol 124
Mol 120
Mol 117
Mol 133
Mol 131
Mol 134
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Licorice
Radix Bupleuri
RehmanniaglutinosaLibosch
AngelicaeSinensis Radix
Mol 8
Mol 73
Mol 75
Mol 77
Mol 80
Mol 7
Mol 82
Mol 76
Mol 74
Mol 60
Mol 46
Mol 150
Mol 151
Mol 112
Mol 115
Mol 11
Mol 116
Mol 113
Mol 110
Mol 1
Mol 9Mol 101Mol 10Mol 93
Mol 103Mol 89 Mol 107
Mol 96PRSS3 LDLRMAPK3 BACE1 EPHB2
Mol 35Mol 81
Mol 86
Mol 85
Mol 84
Mol 88
Mol 83Mol 30
Mol 137
ChuanxiongRhizoma
Mol 3
Mol 57
Mol 139
Achyranthis
Bidentatae
RadixMol 102
CASP8
IGHG1AKT1p53
CHRNA7
Mol 40
SLC6A3 PTPN1
SOD1MAPK1
Mol 38
Mol 95
Carthami Flos
Mol 24
Mol 78 Persicae Semen
Mol 122
Mol 25
Mol 98
TGFB1
GSK3B
PRKCA
Radix Paeoniae Rubra
Mol 52
Mol 42
Mol 62
Mol 138
EGFR
STAT1 PPP3CAPLAT
Stigmasterol
MAP2
Beta-sistosterol Quercetin
Kaempferol
SYNGAP1
CALM
Baicalin
PRKCB
CAV1
CTSD
TNF
DRD1
CDK1
IFNG
FN1BBC3 PRKCD
Mol 132
Mol 160
Mol 58
Mol 67
Mol 69 Mol 43ALB
Mol 64CTNNB1
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FloslololFlhamaCartharthhaamami Fl
menmennnnmicae Scae SSem
Figure 6 HB-pC-pT network of XFZYD for treating TBI The triangles with circle backgrounds represent the herbs (HB) the squares andcircles represent the potential compounds (pC) and targets (pT)The red triangles squares and circles represent corresponding HB pC andpT in the Junherbs the same is to aqua representing theChen herbs and periwinkle representing the Zuo-Shi herbsThe claybank squares andcircles represent corresponding pC and pT shared by the Chen and Zuo-Shi herbs the same is to blue representing the overlap between theJun and Zuo-Shi herbs and the green representing the overlap between the Chen and Zuo-Shi herbsThe purple squares and circles representthe corresponding pC and pT shared by the 3 groups of herbs
alleviating insulin resistance in type 2 diabetic rats [72]Beta-sitosterol (BS) is a vegetable-derived compound foundin various plants and is suggested to modulate the immunefunction inflammation and pain levels by controlling theproduction of inflammatory cytokines [73]
For targets analysis CALM possesses the largest degree(degree =84) followed by GSK3B (degree =61) SCN5A(degree = 59) F2 (degree = 43) ACHE (degree=28)F7 (degree=28) ADRA1B (degree=28) NOS3 (degree=20)BCL2 (degree=18) and CASP3 (degree=18) which demon-strated their crucial therapeutic effects for treating TBI Forinstance CALM possess an essential position in calciumsignaling pathway and is related to morphological changesmigration proliferation and secretion of cytokines andreactive oxygen species ofMicroglial cells [74]The activationof CaMKII120572 major isoform of Ca2+calmodulin-dependentprotein kinase (CaMK) in brain is directly associated withthe production of proinflammatory cytokines such as TNF-120572and IL-1120573 [75] GSK-3120573 is a serinethreonine-protein kinasewhich is abundant in the central nervous system (CNS)particularly in neurons [76] It can control gene transcrip-tion axonal transport and cytoskeletal dynamics in growthcones [77] The inhibition of GSK-3120573 attenuates apoptotic
signals and prevents neuronal death [78] There is increasingevidence that prothrombin (F2) and its active derivativethrombin are expressed locally in the central nervous systemBeside the central role in the coagulation cascade the gener-ation of thrombin leads to receptor mediated inflammatoryresponses cell proliferationmodulation cell protection andapoptosis [79 80] The role in brain injury depends upon itsconcentration as higher amounts cause neuroinflammationand apoptosis while lower concentrations might even becytoprotective [81]
Direct tissue damage aswell as hypoxic-ischemic increas-ing anaerobic glycolysis of the brain tissue results in theATP-stores depletion and failure of energy-dependent mem-brane ion pumps especially for voltage-dependent Ca2+ andNa+-channels Accumulated Ca2+ activates lipid peroxidasesproteases and phospholipases and caspases at the sametime increasing the intracellular concentration of free fattyacids and free radicals leading to necrosis or apoptosis ofneurocyte [82] At the same time the activation of residentglial cells microglia and astrocytes and the infiltration ofblood leukocytes secrete various immune mediators elicitedinflammatory responses which subsequently intersect withadjacent pathological cascades including oxidative stress
12 Evidence-Based Complementary and Alternative Medicine
Table 6 Top 10 potential candidate compounds of XFZYD for treating TBI according to 2 centrality indicators
Compound Degree Compound Betweennessquercetin 76 quercetin 01682179kaempferol 43 kaempferol 005501511beta-sitosterol 35 wogonin 005141376Stigmasterol 19 beta-sitosterol 004451294luteolin 18 naringenin 003251738baicalein 15 beta-carotene 002977217-Methoxy-2-methyl isoflavone 12 nobiletin 002787992wogonin 12 7-Methoxy-2-methyl isoflavone 002096439nobiletin 11 luteolin 002090223naringenin 9 baicalein 001453488
Table 7 Top 10 potential targets of XFZYD for treating TBI according to 2 centrality indicators
Targets Degree Targets BetweennessCALM 84 CALM 021452046GSK3B 61 SCN5A 011441591SCN5A 59 GSK3B 009553896F2 43 F2 00689171ACHE 28 BCL2 002651903F7 28 CASP3 002372836ADRA1B 28 F7 002032647NOS3 20 ACHE 001845996BCL2 18 ADRA1B 001704505CASP3 18 NOS3 00166699
excitotoxicity or reparative events including angiogenesisscarring and neurogenesis [83] Function analysis of the 47target proteins regulated by XFZYD was mainly associatedwith core pathophysiology process of TBI (Figure 7) 47 targetproteins were connected with 16 key process related to TBIMost of the targets have one or more links to other biolog-ical process such as apoptosis cell proliferation superoxideanion generation nitric oxide biosynthetic process responseto calcium ion I-kappaB kinaseNF-kappaB signaling andregulation of inflammation The above analysis implied themultifunction character of these target proteins regulated byXFZYD Of these target proteins 25 proteins (53 of the 47)such as TGFB1 EGFR CAV1 MAPK1 PRKCB and AKT1were responsible for regulating the apoptosis process 17 forblood coagulation 14 for cell proliferation and axon genesisand 12 for hypoxia andMAPK cascade We found that TGFB1was the crucial protein because it participated in 9 biologicalprocesses related to TBI such as apoptosis blood coagulationcell proliferation and MAPK cascade followed by EGFR (8)CAV1 (7) MAPK1 (6) PRKCB (6) and AKT1 (6)
Overall these observations strongly support the evidencethat the generated HB-pCminuspT network and pT-F networkhave important roles in treating TBI further validating thedrug targeting approach
Molecule docking was used to further validate the bind-ing mode between candidate compounds and their targetproteins We found that 18 TBI-specific target proteins inter-acted with 91 candidate compounds from XFZYD (Figure 8)
Other 29 target proteins were not discussed for the lackof proper protein crystal structure The detailed moleculedocking results are shown in Table S5The 6 essential proteinsincluding GSK3B AKT1 CDK1 F2 NOS3 and ACHEwere used to elucidate the exact binding mode (Figure 9)Quercetin was located within the binding cavity of AKT1 andCDK1 (Figures 9(a) and 9(b) and S1A B) Four conventionalhydrogen bonds were formed between quercetin and AKT1by interacting with the key amino acids including ILE-290 THR-211 and SER 205 Additionally 120587-120587 interactionsbetween quercetin and TRP-90 were found in the activesite which helped the stabilization of the compound at thebinding site (Figure 9(a) S1A) Figure 9(b) and S1B suggestedthat five conventional hydrogen bonds (LEU-83 ASP-146and LYS-33) and 120587-120587 interaction (PHE-80) were formedbetween quercetin and CDK1 The GSK3B-FA complexes(Figure 9(c) S1C) were stabilized by 6 hydrogen-bondinginteractions between FA and LYS-85 GLU-97 TYR-134 andARG-141 Glyasperin Bmainly bonds to F2 through hydrogenbonds by interacting with the key amino acids includingGLY-193 SER-195 and GLY-219 (Figure 9(d) S1D) andan edge-to-face 120587 minus 120587 interaction was also observed withTYR-228 The GLN-247 GLU-351 and GLY-355 from theactive site pocket of NOS3 participated in the hydrogen-bondformationwith 1-Methoxyphaseollidin (Figure 9(e) S1E)The(-)-Medicocarpin formed a total of 6 hydrogen bonds withSER-293 PHE-295 ARG-296 and TRY-341 in the active siteof ACHE (Figure 9(f) S1F) Besides an edge-to-face 120587 minus 120587
Evidence-Based Complementary and Alternative Medicine 13
KCNMA1
P53
IFNG
PPP3CA
CASP8
SCN5A
I-kappaB kinase NF-kappaB signaling
Regulation of inflammation
Response to calcium ion
TGFB1
TNF
PRKCA
CHRNA7
CAV1
STAT1
MAPK cascadeAKT1
BCL2
APP
EGFR
MAPK1
Angiogenesis
SYNGAP1
MAP2
PTPN1
Axonogenesis
GSK3B
EPHB2
CDK1
CASP7FN1
MAPK3
CTNNB1
F2
Autophagy
Cell proliferation
BACE1
SLC6A3
DRD1
ADRA1BACHE
CALMCASP3
PLAT
Apoptotic process
NOS3
PRKCB
Blood coagulation
Response to hypoxia
Superoxide anion generation
Nitric oxide biosynthetic process
Astrocyte activation
Amyloid-beta regulation
Microglial cell activation
LDLR
PRSS3
F7
PRKCD
SOD1ALB
BBC3
Figure 7 pT-F network of XFZYD for treating TBI 16 biological processes (red square) the 47 target proteins (periwinkle circle) of XFZYDparticipate in for treating TBI
interactionwas also observedwith TYR-337 From the resultshydrogen-bonding and edge-to-face 120587 minus 120587 interactions playkey roles in the proteinminusligand recognition and stabilitywhich may be helpful in determining the inhibitor activitiesAnd the C-T network confirmed the potential therapeuticeffects of the candidate compounds from XFZYD to treatTBI through interacting with the relevant proteins The com-putational analysis further elucidated the accurate moleculemechanisms between active compounds and targets
4 Discussion
Traumatic brain injury (TBI) is a growing public healthproblem worldwide and is a leading cause of death anddisability [84] Although major progress has been made in
understanding the pathophysiology of this injury this hasnot yet led to substantial improvements in outcome by alack of treatments which have proven successful during phaseIII trials for modern medicine [85 86] TCM rooted inthousands of years of history may offer an alternative or acomplementary strategy for the treatment of TBI XFZYDa representative formula in TCM has been used for yearsto treat TBI in China and has been demonstrated to beeffective in clinical practice However its ldquomulticomponentsrdquoand ldquomultitargetsrdquo features make it much difficult to decipherthemolecular mechanisms of XFZYD in the treatment of TBIfrom a systematic perspective if employing routine methods
In the present study a network pharmacology-basedmethod was employed to elucidate the pharmacologicalmechanisms of XFZYD to treat TBI according to the drug
14 Evidence-Based Complementary and Alternative Medicine
Figure 8 C-T network through molecule docking validating 119 potential compounds (green triangles) interacting with 18 potential targets(yellow circles) of XFZYDThe size of the nodes is proportional to the value of degree
combination principle of TCM We first proposed a newmodeling system combining OB and DL screening multipledrug targets prediction and validation network constructionand molecule docking to probe the efficiency of a typicalTCM formula XFZYD for the treatment of TBI The 11herbs from XFZYD possessed 162 bioactive compounds andtargeted 285 proteins There were 5 compounds and 189
target proteins overlapped among the Jun Chen and Zuo-Shigroup Furthermore 47 TBI-specific proteins were targetedby 119 (73) bioactive compounds from XFZYD Similarly 5common compounds and 33 (70) common target proteinsamong the 3 groups of drugs were observed Most of thebioactive ingredients targeted more than one protein The 47target proteins regulated several essential pathophysiological
Evidence-Based Complementary and Alternative Medicine 15
(a) (b) (c)
(d) (e) (f)
Figure 9 Hydrogen-bonding networks within the binding site of the compoundminustarget complexes obtained from molecule docking(a) AKT1-quercetin (b) CDK1-quercetin (c) GSK3B-FA (d) F2-glyasperin B (e) NOS3-1-Methoxyphaseollidin and (f) ACHE-(-)-Medicocarpin The molecules are presented as ball and stick models Active site amino acid residues are represented as lines Dotted bluelines in these pictures represent hydrogen bonds with distance unit of A Other O and N atoms are colored as red and blue respectively
processes of TBI that referred to apoptosis inflammationcell proliferation superoxide anion generation nitric oxidebiosynthetic process response to calcium ion etc The aboveanalysis reveals that the synergistic action mechanisms ofXFZYDmay be (1) bioactive compounds overlapping amongthe different group of herbs (2) specific bioactive com-pounds from different groups of herbs targeting the sameproteins (3) specific bioactive compounds from differentgroups of herbs targeting different proteins which participatein the same pathophysiological process of the disease Toa certain degree the 5 compounds including quercetinstigmasterol kaempferol baicalin and beta-sitosterol playedessential role in XFZYD for TBI treatment MAPK3MAPK1AKT1 PRKCA TNF PRKCB EGFR BCL2 GSK3B CASP3PPP3CA and NOS3 were the main target proteins regulatedby XFZYD in the treatment of TBI
Interestingly Beta-carotene (Mol 43) from Carthami Flos(the Jun herb) specifically regulated 120573-Catenin (CTNNB1)and played critical role for curing TBI The 120573-Catenin is acritical downstream component of the Wnt pathway whichplays essential role in the regulation of mammalian neuraldevelopment [87] In vitro and in vivo studies demonstrate
that the Wnt120573-catenin pathway regulates the proliferationand differentiation of neural progenitor cells [88] Neuronaldifferentiation is induced by overexpression of 120573-cateninor the pharmacological inhibition of GSK3120573 (the phospho-rylating enzyme of 120573-catenin) [89 90] This pathway alsopromotes blood vessel formation during vascular develop-ment as well as the vascular repair process after TBI [91]In addition wogonin (Mol 160) from Achyranthis BidentataeRadix (the Chen herb) specifically targeted fibronectin (FN1)Bcl-2-binding component 3 (BBC3) and Protein KinaseC delta type (PRKCD) FN1 an important component ofthe extracellular matrix (ECM) environment promotes cellmigration neurite outgrowth and synapse formation dur-ing neural development [92] It aggregates in the injuredbrain and plays a neuroprotection role through antiapoptosisand anti-inflammation ways following TBI [93 94] BBC3namely p53 upregulated modulator of apoptosis (PUMA) iscritical for the p53-dependent apoptosis pathway which playsan important role in hippocampal neuronal loss and associ-ated cognitive deficits [95] PRKCD one of PKC isoformsactivates signal transduction pathways involved in neuronalregeneration [96] synaptic transmissionplasticity [97] and
16 Evidence-Based Complementary and Alternative Medicine
activation of apoptosis processes [98] as well as higher brainfunctions such as learning andmemory [99]The activators ofPKCare effective for the treatment of TBI [100] FurthermoreMitogen-activated protein kinase 3 (MAPK3) Beta-secretase1 (BACE1) Ephrin type-B receptor 2 (EphB) and low-densitylipoprotein receptor (LDLR) were specifically targeted bythe ingredients in the Zuo-Shi herbs Naringenin (Mol133) targeted LDLR MAPK3 while euchrenone (Mol 60)anchored BACE1 and nobiletin (Mol 134) targeted EphB2MAPK3 is an essential component of the MAP kinase signaltransduction pathway It has been appreciated recently thatthe ERK12 cascade plays a fundamental role in synapticplasticity and memory [101] BACE1 is responsible for pro-duction of A120573 from amyloid precursor protein (APP) A120573can cause cell death activate inflammatory pathways [102]and prime proapoptotic pathways for activation by otherinsults [103] The blocking of BACE1 can ameliorate motorand cognitive deficits and reduce cell loss after experimentalTBI in mice [104] EphB is localized to synaptic sites inhippocampal neurons [105] The interaction between EphBand NMDA receptors regulates excitatory synapse formation[106] LDLR acts as an important receptor that facilitatesbrain A120573 clearance and inhibits amyloid deposition [107]and then ameliorates Alzheimerrsquos disease neuropathologyafter TBI [108] The analysis above indicates the ldquoJunrdquoldquoChenrdquo and ldquoZuo-Shirdquo herbs from XFZYD trigger theirspecific targets regulation respectively for the therapeuticeffects
XFZYD is a very famous traditional Chinese formula inpromoting qi circulation and removing blood stasis accord-ing to TCM theory However several limited researches havedemonstrated its efficacy for treating TBI such as anti-inflammatory and synaptic regulation [30 31] which arein accord with our study However previous studies merelypartially deciphered the molecule mechanism of XFZYD fortreating TBI This study reports 119 bioactive compounds inXFZYD that target 47 TBI-specific proteins such as MAPK3MAPK1 AKT1 PRKCA TNF PRKCB and EGFR Theseproteins regulate several crucial pathophysiological processesof TBI such as apoptosis inflammation blood coagulationand axon genesis Our study demonstrates that the therapeu-tic actions of XFZYD refer to ldquomulticompoundsrdquo ldquomultitar-getsrdquo features rather than only the improvement of bloodcirculation With the help of molecule docking methodwe further validate the interactions between bioactive com-pounds and potential targets of XFZYD The hydrogen-bonding and edge-to-face 120587 minus120587 interactions play key roles inthe proteinminusligand recognition and stability This provides avaluable reference for further experimental investigations ofbioactive ingredients and therapeutic targets of XFZYD fortreating TBI
5 Conclusion
Our work successfully illuminates the efficiency of XFZYDfor the treatment of TBI as well as herb combination ruleof TCM formula Network pharmacology with moleculedocking method confirms the ldquomulticompounds multitar-getsrdquo therapeutic actions of XFZYD in the treatment of TBI
The present work may provide valuable evidence for furtherclinical application of XFZYD for treating TBI
Abbreviations
TBI Traumatic brain injuryXFZYD Xuefu Zhuyu decoctionTCM Traditional Chines medicineDL Drug-likenessOB Oral bioavailabilityHB-cC-cT Herb-candidate compound-candidate
targetHB-pC-pT Herb-potential compound-potential targetCALM CalmodulinGSK3B Glycogen synthase kinase-3 betaSCN5A Sodium channel protein type 5 subunit
alphaF2 ProthrombinACHE AcetylcholinesteraseGO Gene OntologyKEGG Kyoto Encyclopedia of Genes and
Genomes
Data Availability
The data used to support the findings of this study areavailable from the corresponding author upon request
Conflicts of Interest
The authors declare that they have no conflicts of interest
Authorsrsquo Contributions
YangWang and Zebing Huang participated in the conceptionand design of the study YuanyuanZhong YangWang ZebingHuang Jiekun Luo Tao Tang and Pengfei Li acquired andanalyzed the data Yuanyuan Zhong Tao Liu and HanjinCui drafted and revised the manuscript The correspondingauthor and all of the authors have read and approved the finalsubmitted manuscript
Acknowledgments
This work was supported by the National Natural ScienceFoundation of China (Nos 81673719 81874409 81603670and 81803948) Project funded byChina Postdoctoral ScienceFoundation (Nos 2016M600639 and 2017T100614)
Supplementary Materials
Table S1 162 bioactive compounds in XFZYD Table S2 targetproteins of XFZYD Table S3 TBI-specific proteins Table S4119 potential compounds of XFZYD for treating TBI TableS5 docking result of 18 target proteins with 91 potential com-pounds Fig S1 the exact bindingmode between active ingre-dients and protein targets obtained from molecule docking(A) AKT1-quercetin (B) CDK1-quercetin (C) GSK3B-FA
Evidence-Based Complementary and Alternative Medicine 17
(D) F2-glyasperin B (E) NOS3-1-Methoxyphaseollidin and(F)ACHE-(-)-Medicocarpin (Supplementary Materials)
References
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[2] J A Langlois W Rutland-Brown and M M Wald ldquoTheepidemiology and impact of traumatic brain injury a briefoverviewrdquo The Journal of Head Trauma Rehabilitation vol 21no 5 pp 375ndash378 2006
[3] H A Alsulaim B J Smart A O Asemota et al ldquoConsciousstatus predictsmortality among patientswith isolated traumaticbrain injury in administrative datardquo The American Journal ofSurgery vol 214 no 2 pp 207ndash210 2017
[4] M Majdan D Plancikova A Maas et al ldquoYears of life lost dueto traumatic brain injury in Europe A cross-sectional analysisof 16 countriesrdquo PLoS Medicine vol 14 no 7 p e1002331 2017
[5] P Cheng P Yin P Ning et al ldquoTrends in traumatic braininjury mortality in China 2006ndash2013 A population-basedlongitudinal studyrdquo PLoS Medicine vol 14 no 7 Article IDe1002332 2017
[6] M V Russo and D B McGavern ldquoInflammatory neuroprotec-tion following traumatic brain injuryrdquo Science vol 353 no 6301pp 783ndash785 2016
[7] WWang H Li J Yu et al ldquoProtective Effects of ChineseHerbalMedicine Rhizoma drynariae in Rats After Traumatic BrainInjury and Identification of Active Compoundrdquo MolecularNeurobiology vol 53 no 7 pp 4809ndash4820 2016
[8] M Das S Mohapatra and S S Mohapatra ldquoNew perspectiveson central and peripheral immune responses to acute traumaticbrain injuryrdquo Journal of Neuroinflammation vol 9 p 236 2012
[9] J W Finnie ldquoNeuroinflammation Beneficial and detrimentaleffects after traumatic brain injuryrdquo Inflammopharmacologyvol 21 no 4 pp 309ndash320 2013
[10] T Cheng W Wang Q Li et al ldquoCerebroprotection of flavanol(minus)-epicatechin after traumatic brain injury viaNrf2-dependentand -independent pathwaysrdquo Free Radical Biology amp Medicinevol 92 pp 15ndash28 2016
[11] W Young ldquoRole of calcium in central nervous system injuriesrdquoJournal of Neurotrauma vol 9 Suppl 1 pp S9ndashS25 1992
[12] R Bullock A Zauner J S Myseros et al ldquoEvidence forProlonged Release of Excitatory Amino Acids in Severe HumanHead Trauma Relationship to Clinical Eventsrdquo Annals of theNew York Academy of Sciences vol 765 no 1 pp 290ndash297 1995
[13] A T Mazzeo A Beat A Singh and M R Bullock ldquoTherole of mitochondrial transition pore and its modulation intraumatic brain injury and delayed neurodegeneration afterTBIrdquo Experimental Neurology vol 218 no 2 pp 363ndash370 2009
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[17] Y Wang X Fan T Tang et al ldquoRhein and rhubarb simi-larly protect the blood-brain barrier after experimental trau-matic brain injury via gp91phox subunit of NADPH oxidaseROSERKMMP-9 signaling pathwayrdquo Scientific Reports vol 6p 37098 2016
[18] D-X Kong X-J Li and H-Y Zhang ldquoWhere is the hopefor drug discovery Let history tell the futurerdquo Drug DiscoveryTherapy vol 14 no 3-4 pp 115ndash119 2009
[19] F Cheung ldquoTCM made in Chinardquo Nature vol 480 no 7378pp S82ndashS83 2011
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18 Evidence-Based Complementary and Alternative Medicine
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[63] C S Yang J M Landau M T Huang and H L NewmarkldquoInhibition of carcinogenesis by dietary polyphenolic com-poundsrdquo Annual Review of Nutrition vol 21 pp 381ndash406 2001
[64] A Murakami H Ashida and J Terao ldquoMultitargeted cancerprevention by quercetinrdquoCancer Letters vol 269 no 2 pp 315ndash325 2008
[65] G Du Z Zhao Y Chen et al ldquoQuercetin attenuates neuronalautophagy and apoptosis in rat traumatic brain injury modelvia activation of PI3KAkt signaling pathwayrdquo NeurologicalResearch vol 38 no 11 pp 1ndash8 2016
Evidence-Based Complementary and Alternative Medicine 19
[66] Q Cai R O Rahn and R Zhang ldquoDietary flavonoidsquercetin luteolin andgenistein reduce oxidativeDNAdamageand lipid peroxidation and quench free radicalsrdquoCancer Lettersvol 119 no 1 pp 99ndash107 1997
[67] G A Bongiovanni E A Soria and A R Eynard ldquoEffectsof the plant flavonoids silymarin and quercetin on arsenite-induced oxidative stress in CHO-K1 cellsrdquo Food and ChemicalToxicology vol 45 no 6 pp 971ndash976 2007
[68] H Li L Yang Y Zhang et al ldquoKaempferol inhibits fibroblastcollagen synthesis proliferation and activation in hypertrophicscar via targeting TGF-beta receptor type Irdquo Biomedicine ampPharmacotherapy = Biomedecine amp Pharmacotherapie vol 83pp 967ndash974 2016
[69] K P Devi D S Malar S F Nabavi et al ldquoKaempferol andinflammation From chemistry to medicinerdquo PharmacologicalResearch vol 99 pp 1ndash10 2015
[70] M Zhou H Ren J Han W Wang Q Zheng and DWang ldquoProtective effects of kaempferol against myocardialischemiareperfusion injury in isolated rat heart via antioxidantactivity and inhibition of glycogen synthase kinase-3120573rdquo Oxida-tive Medicine and Cellular Longevity vol 2015 p 481405 2015
[71] C-F Lee J-S Yang F-J Tsai et al ldquoKaempferol inducesATMp53-mediated death receptor and mitochondrial apop-tosis in human umbilical vein endothelial cellsrdquo InternationalJournal of Oncology vol 48 no 5 pp 2007ndash2014 2016
[72] C Luo H Yang C Tang et al ldquoKaempferol alleviates insulinresistance via hepatic IKKNF-120581B signal in type 2 diabetic ratsrdquoInternational Immunopharmacology vol 28 no 1 pp 744ndash7502015
[73] A B Awad and C S Fink ldquoPhytosterols as anticancer dietarycomponents evidence and mechanism of actionrdquo Journal ofNutrition vol 130 no 9 pp 2127ndash2130 2000
[74] K Farber and H Kettenmann ldquoFunctional role of calciumsignals for microglial functionrdquoGlia vol 54 no 7 pp 656ndash6652006
[75] Y Lu Y Gu X Ding et al ldquoIntracellular Ca2+ homeostasisand JAK1STAT3 pathway are involved in the protective effectof propofol on BV2 microglia against hypoxia-induced inflam-mation and apoptosisrdquo PLoS ONE vol 12 no 5 p e01780982017
[76] K Leroy and J-P Brion ldquoDevelopmental expression and local-ization of glycogen synthase kinase-3120573 in rat brainrdquo Journal ofChemical Neuroanatomy vol 16 no 4 pp 279ndash293 1999
[77] C-M Liu E-M Hur and F-Q Zhou ldquoCoordinating geneexpression and axon assembly to control axon growth Potentialrole ofGSK3 signalingrdquo Frontiers inMolecularNeuroscience vol3 p 3 2012
[78] D A E Cross A A Culbert K A Chalmers L Facci S DSkaper and A D Reith ldquoSelective small-molecule inhibitors ofglycogen synthase kinase-3 activity protect primary neuronesfrom deathrdquo Journal of Neurochemistry vol 77 no 1 pp 94ndash102 2001
[79] V S Ossovskaya and NW Bunnett ldquoProtease-activated recep-tors contribution to physiology and diseaserdquo PhysiologicalReviews vol 84 no 2 pp 579ndash621 2004
[80] M Steinhoff J Buddenkotte V Shpacovitch et al ldquoProteinase-activated receptors transducers of proteinase-mediated signal-ing in inflammation and immune responserdquoEndocrine Reviewsvol 26 no 1 pp 1ndash43 2005
[81] H Krenzlin V Lorenz S Danckwardt O Kempski and BAlessandri ldquoThe importance of thrombin in cerebral injury and
diseaserdquo International Journal of Molecular Sciences vol 17 no1 2016
[82] M J McGinn and J T Povlishock ldquoPathophysiology of Trau-matic Brain InjuryrdquoNeurosurgery Clinics of North America vol27 no 4 pp 397ndash407 2016
[83] T Woodcock and M C Morganti-Kossmann ldquoThe role ofmarkers of inflammation in traumatic brain injuryrdquo Frontiersin Neurology vol 4 article 18 2013
[84] F Tanriverdi H J Schneider G Aimaretti B E Masel F FCasanueva and F Kelestimur ldquoPituitary dysfunction after trau-matic brain injury A clinical and pathophysiological approachrdquoEndocrine Reviews vol 36 no 3 pp 305ndash342 2015
[85] J V Rosenfeld A I Maas P Bragge M C Morganti-Kossmann G T Manley and R L Gruen ldquoEarly managementof severe traumatic brain injuryrdquoThe Lancet vol 380 no 9847pp 1088ndash1098 2012
[86] B M Aertker S Bedi and C S Cox ldquoStrategies for CNS repairfollowing TBIrdquo Experimental Neurology vol 275 no 3 pp 411ndash426 2016
[87] K Adachi Z Mirzadeh M Sakaguchi et al ldquo120573-catenin signal-ing promotes proliferation of progenitor cells in the adultmousesubventricular zonerdquo Stem Cells vol 25 no 11 pp 2827ndash28362007
[88] Y Hirabayashi and Y Gotoh ldquoStage-dependent fate determina-tion of neural precursor cells in mouse forebrainrdquo NeuroscienceResearch vol 51 no 4 pp 331ndash336 2005
[89] S Ding T Y H Wu A Brinker et al ldquoSynthetic smallmolecules that control stem cell faterdquo Proceedings of theNationalAcadamy of Sciences of the United States of America vol 100 no13 pp 7632ndash7637 2003
[90] N Israsena M Hu W Fu L Kan and J A Kessler ldquoThepresence of FGF2 signaling determines whether 120573-cateninexerts effects on proliferation or neuronal differentiation ofneural stem cellsrdquo Developmental Biology vol 268 no 1 pp220ndash231 2004
[91] A Salehi A Jullienne M Baghchechi et al ldquoUp-regulation ofWnt120573-catenin expression is accompanied with vascular repairafter traumatic brain injuryrdquo Journal of Cerebral Blood Flow ampMetabolism vol 38 no 2 pp 274ndash289 2017
[92] L F Reichardt and K J Tomaselli ldquoExtracellular matrixmolecules and their receptors Functions in neural develop-mentrdquoAnnual Review of Neuroscience vol 14 pp 531ndash570 1991
[93] R M Gibson S E Craig L Heenan C Tournier and M JHumphries ldquoActivation of integrin 12057251205731 delays apoptosis ofNtera2 neuronal cellsrdquoMolecularandCellularNeuroscience vol28 no 3 pp 588ndash598 2005
[94] C C Tate A J Garcıa andMC LaPlaca ldquoPlasma fibronectin isneuroprotective following traumatic brain injuryrdquoExperimentalNeurology vol 207 no 1 pp 13ndash22 2007
[95] C Culmsee and M P Mattson ldquop53 in neuronal apoptosisrdquoBiochemical and Biophysical Research Communications vol 331no 3 pp 761ndash777 2005
[96] M S Geddis and V Rehder ldquoThe phosphorylation state ofneuronal processes determines growth cone formation afterneuronal injuryrdquo Journal of Neuroscience Research vol 74 no2 pp 210ndash220 2003
[97] M L Craske M Fivaz N N Batada and T Meyer ldquoSpines andneurite branches function as geometric attractors that enhanceprotein kinase C actionrdquoThe Journal of Cell Biology vol 170 no7 pp 1147ndash1158 2005
20 Evidence-Based Complementary and Alternative Medicine
[98] A Muscella C Vetrugno L G Cossa et al ldquoApoptosis by[Pt(OOrsquo-acac)(gamma-acac)(DMS)] requires PKC-deltamedi-ated p53 activation in malignant pleural mesotheliomardquo PloSone vol 12 no 7 Article ID e0181114 2017
[99] J S Bonini W C Da Silva L R M Bevilaqua J H MedinaI Izquierdo and M Cammarota ldquoOn the participation ofhippocampal PKC in acquisition consolidation and reconsol-idation of spatial memoryrdquo Neuroscience vol 147 no 1 pp 37ndash45 2007
[100] O Zohar R Lavy X Zi et al ldquoPKC activator therapeutic formild traumatic brain injury in micerdquo Neurobiology of Diseasevol 41 no 2 pp 329ndash337 2011
[101] J David Sweatt ldquoTheneuronalMAPkinase cascade a biochem-ical signal integration system subserving synaptic plasticity andmemoryrdquo Journal ofNeurochemistry vol 76 no 1 pp 1ndash10 2001
[102] Y Matsuoka M Picciano B Maleste et al ldquoInflammatoryresponses to amyloidosis in a transgenic mouse model ofAlzheimerrsquos diseaserdquo The American Journal of Pathology vol158 no 4 pp 1345ndash1354 2001
[103] L Esposito L Gan G-Q Yu C Essrich and L MuckeldquoIntracellularly generated amyloid-120573 peptide counteracts theantiapoptotic function of its precursor protein and primesproapoptotic pathways for activation by other insults in neu-roblastoma cellsrdquo Journal of Neurochemistry vol 91 no 6 pp1260ndash1274 2004
[104] D J Loane A Pocivavsek C E-H Moussa et al ldquoAmyloidprecursor protein secretases as therapeutic targets for traumaticbrain injuryrdquo Nature Medicine vol 15 no 4 pp 377ndash379 2009
[105] R Torres B L Firestein H Dong et al ldquoPDZ proteins bindcluster and synaptically colocalize with Eph receptors and theirephrin ligandsrdquo Neuron vol 21 no 6 pp 1453ndash1463 1998
[106] M B Dalva M A Takasu M Z Lin et al ldquoEphB receptorsinteract with NMDA receptors and regulate excitatory synapseformationrdquo Cell vol 103 no 6 pp 945ndash956 2000
[107] J M Basak P B Verghese H Yoon J Kim and D MHoltzman ldquoLow-density lipoprotein receptor represents anapolipoprotein E-independent pathway of A120573 uptake anddegradation by astrocytesrdquoThe Journal of Biological Chemistryvol 287 no 17 pp 13959ndash13971 2012
[108] L Yao X Gu Q Song et al ldquoNanoformulated alpha-mangostinameliorates Alzheimerrsquos disease neuropathology by elevatingLDLR expression and accelerating amyloid-beta clearancerdquoJournal of Controlled Release vol 226 pp 1ndash14 2016
Stem Cells International
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Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
8 Evidence-Based Complementary and Alternative Medicine
Table 3 Top 10 target proteins of XFZYD according to 2 centrality indicators
Proteins Degree Proteins BetweennessPTGS2 126 PTGS2 0128936ESR1 88 NCOA2 0060806HSP90AB2P 85 HSP90AB2P 0049647AR 81 PRKACA 0046484CALM 81 PTGS1 004365NOS2 76 AR 0030252NCOA2 75 PRSS1 0025575PRSS1 68 ESR1 0022212PTGS1 67 PPARG 0021403PPARG 66 PGR 0020685Note the centrality indicators identify the important nodes within the network Higher degree centrality and betweenness centrality indicate greaterimportance
The analysis of the network revealed that quercetin (Mol148) kaempferol (Mol 108) luteolin (Mol 127) wogonin (Mol160) 7-Methoxy-2-methyl (Mol 33) and beta-sitosterol (Mol41) were predicted as the major active compounds of XFZYDTheproteins including F2 NOS2 PTGS1 PTGS2 and CALMwere predicted as essential pharmacological proteins for thetherapeutic effects of XFZYD
34 Analyses on TBI Based Specific Protein Interaction Net-work Network biology integrated with different kinds ofdata including physical or functional networks and diseasegene sets is used to interpret humandiseases Protein-proteininteraction networks (PPI) are fundamental to understandthe cellular organizations biological processes and proteinfunctions [54] From the systematic perspective the analysisof TBI-related PPI will improve the understanding of thecomplicated molecular pathways and the dynamic processesunderlying TBI We screened the TBI-specific genes andprotein targets using Online Mendelian Inheritance in Mandatabase (OMIM) and Therapeutic Target Database (TTD)Figure 4 indicated that 21 TBI-specific genesproteins wereacquired Further these hub-proteins were submitted toHPRD and STRING to establish the TBI-specific proteininteraction network The detailed information of the TBI-specific proteins is shown in Table S3 The results suggestedthat the network consisted of 489 nodes and 5738 edges(Figure 4(a)) We obtained top 10 TBI-related proteinsaccording to 2 centrality indicators generated and summa-rized in Table 4 Interestingly we found that the node withhigher betweenness tends to possess larger degree (Fig-ure 5) Network topology analysis showed that protoonco-gene tyrosine-protein kinase Src (SRC degree=164 between-ness=0059) RAC-alpha serinethreonine-protein kinase(AKT1 degree=157 betweenness=0045) Serum albumin(ALB degree=153 betweenness=0069) Epidermal growthfactor receptor (EGFR degree=139 betweenness=0053)and Amyloid beta A4 protein (APP degree=134 between-ness=0072) contributed to the essential role in the patho-physiology of TBI All of these indicated that the top mutualtarget proteins performed various beneficial functions to treatTBI at the molecular level For example SRC is activatedfollowing engagement of many different classes of cellular
receptors It participates in signal pathways that controla diverse spectrum of biological activities including genetranscription immune response cell adhesion cell cycleprogression apoptosis migration and transformation [55]SRC can result in blood-brain barrier (BBB) disruptionand brain edema at the acute stage the inhibition of SRCfamily kinases can protect hippocampal neurons and improvecognitive function after TBI [56] AKT1 regulates manyprocesses including metabolism proliferation cell survivalgrowth and angiogenesis [57] The PI3KAKTPTEN path-way has been shown to play a pivotal role in neuroprotectionenhancing cell survival by stimulating cell proliferation andinhibiting apoptosis after TBI [58] ALB is the main proteinof plasma and can be a biomarker to predict outcome ofTBI [59] Its main function is the regulation of the colloidalosmotic pressure of blood We found that it may participatein pathological process of TBI
KEGG pathway analysis was also used to determine thefunctions of proteins Table 5 describes the top 10 significantlyenriched KEGG pathways These pathways play crucial rolesin pathophysiology process which have also been widelydiscussed in existing literature For instanceMAPK signalingpathway can promote pathological axonal death throughtriggering a local energy deficit [60] Neurotrophin signalingthrough Trk receptors regulates cell survival proliferationthe fate of neural precursors axon and dendrite growth andpatterning and the expression and activity of functionallyimportant proteins such as ion channels and neurotransmit-ter receptors [61] Engagement of cells with the extracellularmatrix (ECM) proteins is crucial for various biological pro-cesses including cell adhesion differentiation and apoptosiscontributing to maintenance of tissue integrity and woundhealing [62]The 47 TBI-specific proteins targeted byXFZYD(yellow and green) were further discussed below
35 HB-pC-pT Network pT-F Network Construction andMolecule Docking Analyses of XFZYD for the Treatment ofTBI To investigate the therapeutic mechanisms of XFZYDfor the treatment of TBI a HB-pC-pT network of XFZYDfor treating TBI was built (Figure 6) 47 TBI-specific targetproteins (circles) were targeted by 119 potential compounds(squares) from XFZYD (Figure 6) Detailed information
Evidence-Based Complementary and Alternative Medicine 9
BLMH
GPRASP2
GJC2
CAMK2N1
OBSL1
ZNF292
RLFAMPD3
NANOS1CAMTA1HYPKCCS
DLGAP4PF4V1
TRIM2
CHKB
F8A1
TIAM1
BMX
RAPGEF1
APBB1
KCNMA1
XRCC6
TTR
TGM2MAP2
PRNP
RASGRF1 PPP3CADMD SPTAN1
ACE
TGFB2
AP2M1GRAP2
SCN5A
MYH9
PKN1
ACHE
SLC25A13
PHKG1
SPAST
UMOD
PRPF40B
BBC3
TUBA4A TTPALRYR2
STUB1KRT6A
AKAP8L
GFI1BTHRAP3
ZNF232
SOD1
CRKL
CDH2
MBP MAP2K2
DCN
MAP3K5NGFR
PIK3CA
ITGA2
DNM1
RAP1A
GRIN2B
PTPN11
PRKCA
LDLRAP1
DNAJA3INA
SP3
CACNA2D3
CSF1
CNTF
TNFRSF1B
BCL10
KLC1
DCTN1
IDE
SERPINA3
HSPA1A
HSPG2
FN1
LRP2
TUBB
GRIN2A
YWHAB
CAMK2D
HTRA2
PRKCE
CRYAB
APC
GIT1
PIAS4
HPX
TRIP10
PDE4B
MARK4
PLAT
UBE2K
APOC2
LIN7B BGN
MAP3K10MAPK8IP2
CBS
SH3GL3
STAU1
CASP6
PPP5C
KRT16
DAB1
CASP1SHC2
CTSD
UCHL1
HP
SNCACAMK2G
TGFB1
GNB1
PTPN1PIN1
TJP1
CASP8PLCG1
NPHS1
PPP2R2A
DNAJB1
ADRBK1
INADL
NCSTN
MATK
NEDD4L
KAT5IFNGTBP
FRS2
CSNK1A1
CALRKNG1
SGOL2
KRT6B
TRAF3 DLG3
SACS
BDKRB2PRKCB
CAV1
MAPK3F2 SMAD3
APOEAKAP9 RANBP2
CLTC
TSC22D1GAB2PSEN2
MLF1CROT
KRT9
ADRA1B
EPB41
COL4A1
SHC3
ARHGAP32
ATM
PPP2R5A
CAMK2A
NTRK1
KRT18
TNFAPOA2
LAT
GRIN2D
COL4A6
KLK3
VLDLR
COL4A5
COL4A2
NEFH
OR8D2
MTSS1
PDZRN4
PTPN4
OR3A2
OR2T6
DGKG
CASP3
IGF1R
CASK
DRD2
TRAF2
EPHB2
PPM1A
PRKCG
CACNB3
HNRNPUSIN3A
DLG1
CLU
RAP2A
ERBB4CANX
BACE1 GABBR1
KARS
CNOT1
UNG
AGA CDCP1
UTP14A PLAG1
EXOC6
HRAS
CTNNB1
SP1
NOS3 SUMO1
PSEN1
HSPA8
HSP90AA1
BCL2
GNAO1
RPS6KB1
PARK2
DLG4
PTK2B
GSN
NAE1 TLN2LRFN2
PLTP
NUMBLSTX1A
DERL1
NEFM
HMOX2
AATF
PPIAMTRNR2L2AP1M1
OGT
DDB1
AP4M1
MED31
RGS3
TDP2
KRT5
ADAM9
REST
PACSIN1
SHB
CTSL1
LAMA1
SH2B1
PLA2G4F
SMURF2
SLC9A8
KRT14
CIT
TGFB1I1
PPP2R1APIK3R1
NUMB
ADAM17
DLG2
LRP1
APOA4
LTA
IRS1
GSK3B
EGFR
CTSB
TRAF6
SHC1
PPP2CB
NGF
HTT
PPIDLDB3
APOC1HIP1
ZDHHC17COL25A1MAGI3
ITGB5
ACTB
GNA11
ACTN2CAMK2B
CDK1
LIN7C
S100B
PDLIM5
DLGAP1
LIN7A
TPR
SYNGAP1
CFD
GRIN3A
F12
SLC1A3
GRK5
CACNA2D2
TANC1
DUSP4
EXOC4
AHSG
GRIN3B
UCP2
TTN
GPC1
NSF
FGA
DYNLL1
MAPK8IP1
PICALM
PPBP
ITM2B
NID1
STXBP1
CTBP1
SGK1
TRAF1
SQSTM1
TNFRSF1A
ACTN1
OPTN
UBE2D2
AP2A2
FUS
APOA1
DICER1
CACNA1B
CST3
NCOA3
SCARB1
APOC3
SLC6A3
KIAA1551 KRT13
DCD
KIAA0232
ENSG00000258818
SPATA31A7
CTAGE5
CEP44
SH2B2
EPHB4
CAPN1
F7
APBA2
COL4A3
LDLR
GIPC1
GFAP
SERPING1
SRC
MAPK12APP
GAPDH
CDK5
CREBBP
NOS1
YWHAZCALM1
YWHAG
YWHAQGRIN1
RPS6KA3
COL1A2
TCERG1
A2M
CASP7
LRP8
PDK2
AGTR1
FYN
ALBGRB2
MAPK1AKT1
STAT1
MAPT
GSK3A
UBB
PPP2CA
SMAD4
PRKCD
RGS12
SPTB
SH3BP5
CACNA1I
IL16
SLA2
PFN2
NLRC4
TNFRSF14
PRTN3
SNCB
FBLN1
BACE2
SETX
FANCD2
SAP30
RANBP3
PALB2
SLC1A5RASA1
HAP1
LRRC7
CFB
ADRBK2
CDC45
CLSTN3
PCED1B
SCAF1KIAA1377
FAM71E2
FEZ1 ACSBG2
PCDH1
TRAPPC11QTRTD1
KRT1
LRP1B
HADHB
TP53BP2
DRD1
ST13
PRPF40A
TRAIP
KIDINS220
GRK6
MMP17
LTBR
EIF6
PGAM1
CHD3
PRSS3
APBB3
CHRNA7
KRT10
RIMS1
CRMP1
PDIK1L
SORBS3
SYMPK
GCN1L1
RNF19A
HOMER3
GRK4
JARID2
MYLK3
EFS
CRB1
HSD17B10
TST
HOMER2
PHC3
TM2D1 FICD
PEG3
CABLES1
LGALS2
ITIH1
HOXB2
TAF4FLOT1
BABAM1
CFH
HIP1R
LTBMYL4
NCOR1
CASP4
NF1
AP4E1
CLCA2
IGDCC4
NECAB3
CXorf27
SH3GLB1
CTCF
PLCG2
(a)
STAT1
EGFR
FN1
PLAT
SOD1
PRKCA CDK1
GSK3B
ALB APP
PPP3CA
IFNG
BBC3
SCN5A
KCNMA1
MAP2
ACHE
MAPK3F2
PTPN1
NOS3 BCL2
CAV1
CTNNB1
PRKCB
TGFB1CASP8
TNF
AKT1
F7
MAPK1LDLR
PRKCD
SLC6A3
DRD1
CASP3
CHRNA7
PRSS3
TP53BP2
CASP7
EPHB2
SYNGAP1
CALM1
CTSD
BACE1
ADRA1B
(b)
Figure 4 TBI-related protein interaction network (a) 21 hub proteins (red and green) were identified through the analysis of TherapeuticTargetDatabase (TTD) aswell asOnlineMendelian Inheritance inMan (OMIM) 4 overlapped protein targets (green)were obtained between21 hub proteins in TBI and candidate targets of XFZYD Periwinkle proteins fromHPRD and STRING analyses were not targeted by XFZYD(b) 47 candidate protein targets of XFZYD were screened for treating TBI Yellow candidate protein targets of XFZYD The size of nodes isproportional to the value of degree
10 Evidence-Based Complementary and Alternative Medicine
Table 4 Top 10 proteins of TBI specific proteins according to 2 centrality indicators
Proteins Degree Proteins BetweennessSRC 164 APP 0071814AKT1 157 ALB 0069343ALB 153 SRC 0058568EGFR 139 EGFR 0052938APP 134 AKT1 0045466GAPDH 131 HTT 0037164HSP90AA1 108 GAPDH 0034549BCL2 106 HSP90AA1 0027596MAPK1 105 CTNNB1 0021759HRAS 105 GNB1 0019492Note the centrality indicators identify the important nodes within the network Higher degree centrality and betweenness centrality indicate greaterimportance
Table 5 Top 10 significantly enriched KEGG pathways in TBI-specific proteins
Pathway ID Pathway description Gene count FDR4010 MAPK signaling pathway 44 299E-235200 Pathways in cancer 43 113E-184722 Neurotrophin signaling pathway 41 101E-344151 PI3K-Akt signaling pathway 40 111E-154510 Focal adhesion 39 292E-225205 Proteoglycans in cancer 39 410E-215010 Alzheimer s disease 34 124E-204015 Rap1 signaling pathway 33 769E-174014 Ras signaling pathway 33 646E-164020 Calcium signaling pathway 31 505E-17
0
001
002
003
004
005
006
007
008
0 20 40 60 80 100 120 140 160 180
Betw
eenn
ess
Degree
APP
ALB SRCEGFR
AKT1HTT
Figure 5 Relationship between degree and betweenness centralityin the TBI-specific protein interaction network
for the 119 potential compounds is shown in Table S4Similarly 5 pharmacologically active ingredients in the JunChen and Zuo-Shi groups including quercetin stigmasterolkaempferol baicalin and beta-sitosterol anchored 33 TBI-specific proteins such as CALM SCN5A F2 ACHE F7ADRA1B NOS3 BCL2 CASP3 and AKT1 11 Jun-specificcompounds targeted 2 specific targets (ALB CTNNB1) while12 Chen-specific compounds anchored 3 unique proteins(PRKCD FN1 and BC3) The Zuo-Shi herbs possessed thelargest number of active compounds (89) and targeted 5
unique proteins including EPHB2 BACE1 LDLR MAPK3and PRSS3 GSK3Bwas the common target between theChenandZuo-Shi drugs KCNMA1 CASP7 andAPPwere targetedby the Jun and Zuo-Shi drugs
The top 10 candidate compounds and targets to treat TBIwere showed in Tables 6 and 7 Formost of active compoundsfrom XFZYD each component hit more than one targetTable 6 demonstrated that quercetin had the highest numberof targets (degree =76) followed by kaempferol (degree=43) beta-sitosterol (degree =35) stigmasterol (degree =19)luteolin (degree=18) baicalein (degree=15) 7-Methoxy-2-methyl isoflavone (degree=12) wogonin (degree=12)nobiletin (degree=11) and naringenin (degree=9) Forinstance quercetin (3310158404101584057-pentahydroxyflavone) is anaturally occurring flavonoid commonly found in fruits andvegetables It regulates multiple biological pathways elicitinginduction of apoptosis as well as inhibiting angiogenesisand proliferation [63 64] Quercetin can attenuate neuronalautophagy and apoptosis in rat traumatic brain injury modelvia activation of PI3KAkt signaling pathway [65] It has alsobeen reported to have a protective ability against oxidativestress and mutagenesis in normal cells [66 67] Kaempferol(3 41015840 5 7 tetrahydroxy flavone) is a yellow-colored flavonoidthat is widely distributed in many botanical families[68] It has been shown to possess a variety of biologicalcharacteristics including effects of anti-inflammatory[69] antioxidative [70] tumor growth inhibition [71] and
Evidence-Based Complementary and Alternative Medicine 11
Mol 22Mol 33
Mol 29Mol 32 Mol 23Mol 20
Mol 97Mol 106
Mol 104Mol 100Mol 94
Mol 63Mol 90
Mol 87
Mol 91Mol 61
Mol 19
Mol 109
Mol 161
Mol 118
Mol 111
Mol 158
Mol 114
Mol 149
Mol 141
Mol 152
Mol 14
CASP3
BCL2
NOS3
SCN5A
F7
ADRA1B
ACHE
F2
Mol 4
Mol 49
Mol 127
APP CASP7
Mol 92
Mol 54
Mol 6
Mol 36
KCNMA1
Mol 27
Mol 13Mol 105Mol 140
Mol 17
Mol 39
Mol 2
Mol 21
Mol 12
Mol 142
Mol 126
Mol 135
Mol 121
Mol 119
Mol 124
Mol 120
Mol 117
Mol 133
Mol 131
Mol 134
PlatycodonGrandiforus
Aurantii Fructus
Licorice
Radix Bupleuri
RehmanniaglutinosaLibosch
AngelicaeSinensis Radix
Mol 8
Mol 73
Mol 75
Mol 77
Mol 80
Mol 7
Mol 82
Mol 76
Mol 74
Mol 60
Mol 46
Mol 150
Mol 151
Mol 112
Mol 115
Mol 11
Mol 116
Mol 113
Mol 110
Mol 1
Mol 9Mol 101Mol 10Mol 93
Mol 103Mol 89 Mol 107
Mol 96PRSS3 LDLRMAPK3 BACE1 EPHB2
Mol 35Mol 81
Mol 86
Mol 85
Mol 84
Mol 88
Mol 83Mol 30
Mol 137
ChuanxiongRhizoma
Mol 3
Mol 57
Mol 139
Achyranthis
Bidentatae
RadixMol 102
CASP8
IGHG1AKT1p53
CHRNA7
Mol 40
SLC6A3 PTPN1
SOD1MAPK1
Mol 38
Mol 95
Carthami Flos
Mol 24
Mol 78 Persicae Semen
Mol 122
Mol 25
Mol 98
TGFB1
GSK3B
PRKCA
Radix Paeoniae Rubra
Mol 52
Mol 42
Mol 62
Mol 138
EGFR
STAT1 PPP3CAPLAT
Stigmasterol
MAP2
Beta-sistosterol Quercetin
Kaempferol
SYNGAP1
CALM
Baicalin
PRKCB
CAV1
CTSD
TNF
DRD1
CDK1
IFNG
FN1BBC3 PRKCD
Mol 132
Mol 160
Mol 58
Mol 67
Mol 69 Mol 43ALB
Mol 64CTNNB1
xiongixioChuanxC nxnxioaamaRhihihizoma
Achyranthisiistntnt
aeeaeBidenBidennntatae
RadixadixR diR di
oniae nianiaiaonRadix PaeoPaePRadix P eeoeonRRRRubra dondonddoodPlatycolatycPlatyycPl ycoyccoPlatPlatycoPlatyccocod
rususrusususruGranG anGranGranGranGrGGranndndiforu
ructusuctuc sructusuctusructusruAurantiirA ntaA ntiitiirantAurantintiurantAur iiii Fru
cecececececececeecLiLLiLLiLiLLLLLLLLiLiLiLLiLLLiLicoric
leuririeuuurieurileurleurieurieurieurieurileRadix BBRadRad Bdix Bdixx R i Bdi BBBadix Radix BBuple
RehmanniaRe mRe m nnRehmanniamReRehmhmannnniamanniaRehmehmanniRR nosasasasaosaosglutilutglutgluulutiglgl titinos
LiboschLibLLibo hschib chLiboschboscLLib chschLibos
icaecaecacaeicaeicAngelAnAngeAngelAnAng lAAngeelielicdix dix dixdixdSinensinensSS nennensSinSSiSinensSinensSine sissis Rad
FloslololFlhamaCartharthhaamami Fl
menmennnnmicae Scae SSem
Figure 6 HB-pC-pT network of XFZYD for treating TBI The triangles with circle backgrounds represent the herbs (HB) the squares andcircles represent the potential compounds (pC) and targets (pT)The red triangles squares and circles represent corresponding HB pC andpT in the Junherbs the same is to aqua representing theChen herbs and periwinkle representing the Zuo-Shi herbsThe claybank squares andcircles represent corresponding pC and pT shared by the Chen and Zuo-Shi herbs the same is to blue representing the overlap between theJun and Zuo-Shi herbs and the green representing the overlap between the Chen and Zuo-Shi herbsThe purple squares and circles representthe corresponding pC and pT shared by the 3 groups of herbs
alleviating insulin resistance in type 2 diabetic rats [72]Beta-sitosterol (BS) is a vegetable-derived compound foundin various plants and is suggested to modulate the immunefunction inflammation and pain levels by controlling theproduction of inflammatory cytokines [73]
For targets analysis CALM possesses the largest degree(degree =84) followed by GSK3B (degree =61) SCN5A(degree = 59) F2 (degree = 43) ACHE (degree=28)F7 (degree=28) ADRA1B (degree=28) NOS3 (degree=20)BCL2 (degree=18) and CASP3 (degree=18) which demon-strated their crucial therapeutic effects for treating TBI Forinstance CALM possess an essential position in calciumsignaling pathway and is related to morphological changesmigration proliferation and secretion of cytokines andreactive oxygen species ofMicroglial cells [74]The activationof CaMKII120572 major isoform of Ca2+calmodulin-dependentprotein kinase (CaMK) in brain is directly associated withthe production of proinflammatory cytokines such as TNF-120572and IL-1120573 [75] GSK-3120573 is a serinethreonine-protein kinasewhich is abundant in the central nervous system (CNS)particularly in neurons [76] It can control gene transcrip-tion axonal transport and cytoskeletal dynamics in growthcones [77] The inhibition of GSK-3120573 attenuates apoptotic
signals and prevents neuronal death [78] There is increasingevidence that prothrombin (F2) and its active derivativethrombin are expressed locally in the central nervous systemBeside the central role in the coagulation cascade the gener-ation of thrombin leads to receptor mediated inflammatoryresponses cell proliferationmodulation cell protection andapoptosis [79 80] The role in brain injury depends upon itsconcentration as higher amounts cause neuroinflammationand apoptosis while lower concentrations might even becytoprotective [81]
Direct tissue damage aswell as hypoxic-ischemic increas-ing anaerobic glycolysis of the brain tissue results in theATP-stores depletion and failure of energy-dependent mem-brane ion pumps especially for voltage-dependent Ca2+ andNa+-channels Accumulated Ca2+ activates lipid peroxidasesproteases and phospholipases and caspases at the sametime increasing the intracellular concentration of free fattyacids and free radicals leading to necrosis or apoptosis ofneurocyte [82] At the same time the activation of residentglial cells microglia and astrocytes and the infiltration ofblood leukocytes secrete various immune mediators elicitedinflammatory responses which subsequently intersect withadjacent pathological cascades including oxidative stress
12 Evidence-Based Complementary and Alternative Medicine
Table 6 Top 10 potential candidate compounds of XFZYD for treating TBI according to 2 centrality indicators
Compound Degree Compound Betweennessquercetin 76 quercetin 01682179kaempferol 43 kaempferol 005501511beta-sitosterol 35 wogonin 005141376Stigmasterol 19 beta-sitosterol 004451294luteolin 18 naringenin 003251738baicalein 15 beta-carotene 002977217-Methoxy-2-methyl isoflavone 12 nobiletin 002787992wogonin 12 7-Methoxy-2-methyl isoflavone 002096439nobiletin 11 luteolin 002090223naringenin 9 baicalein 001453488
Table 7 Top 10 potential targets of XFZYD for treating TBI according to 2 centrality indicators
Targets Degree Targets BetweennessCALM 84 CALM 021452046GSK3B 61 SCN5A 011441591SCN5A 59 GSK3B 009553896F2 43 F2 00689171ACHE 28 BCL2 002651903F7 28 CASP3 002372836ADRA1B 28 F7 002032647NOS3 20 ACHE 001845996BCL2 18 ADRA1B 001704505CASP3 18 NOS3 00166699
excitotoxicity or reparative events including angiogenesisscarring and neurogenesis [83] Function analysis of the 47target proteins regulated by XFZYD was mainly associatedwith core pathophysiology process of TBI (Figure 7) 47 targetproteins were connected with 16 key process related to TBIMost of the targets have one or more links to other biolog-ical process such as apoptosis cell proliferation superoxideanion generation nitric oxide biosynthetic process responseto calcium ion I-kappaB kinaseNF-kappaB signaling andregulation of inflammation The above analysis implied themultifunction character of these target proteins regulated byXFZYD Of these target proteins 25 proteins (53 of the 47)such as TGFB1 EGFR CAV1 MAPK1 PRKCB and AKT1were responsible for regulating the apoptosis process 17 forblood coagulation 14 for cell proliferation and axon genesisand 12 for hypoxia andMAPK cascade We found that TGFB1was the crucial protein because it participated in 9 biologicalprocesses related to TBI such as apoptosis blood coagulationcell proliferation and MAPK cascade followed by EGFR (8)CAV1 (7) MAPK1 (6) PRKCB (6) and AKT1 (6)
Overall these observations strongly support the evidencethat the generated HB-pCminuspT network and pT-F networkhave important roles in treating TBI further validating thedrug targeting approach
Molecule docking was used to further validate the bind-ing mode between candidate compounds and their targetproteins We found that 18 TBI-specific target proteins inter-acted with 91 candidate compounds from XFZYD (Figure 8)
Other 29 target proteins were not discussed for the lackof proper protein crystal structure The detailed moleculedocking results are shown in Table S5The 6 essential proteinsincluding GSK3B AKT1 CDK1 F2 NOS3 and ACHEwere used to elucidate the exact binding mode (Figure 9)Quercetin was located within the binding cavity of AKT1 andCDK1 (Figures 9(a) and 9(b) and S1A B) Four conventionalhydrogen bonds were formed between quercetin and AKT1by interacting with the key amino acids including ILE-290 THR-211 and SER 205 Additionally 120587-120587 interactionsbetween quercetin and TRP-90 were found in the activesite which helped the stabilization of the compound at thebinding site (Figure 9(a) S1A) Figure 9(b) and S1B suggestedthat five conventional hydrogen bonds (LEU-83 ASP-146and LYS-33) and 120587-120587 interaction (PHE-80) were formedbetween quercetin and CDK1 The GSK3B-FA complexes(Figure 9(c) S1C) were stabilized by 6 hydrogen-bondinginteractions between FA and LYS-85 GLU-97 TYR-134 andARG-141 Glyasperin Bmainly bonds to F2 through hydrogenbonds by interacting with the key amino acids includingGLY-193 SER-195 and GLY-219 (Figure 9(d) S1D) andan edge-to-face 120587 minus 120587 interaction was also observed withTYR-228 The GLN-247 GLU-351 and GLY-355 from theactive site pocket of NOS3 participated in the hydrogen-bondformationwith 1-Methoxyphaseollidin (Figure 9(e) S1E)The(-)-Medicocarpin formed a total of 6 hydrogen bonds withSER-293 PHE-295 ARG-296 and TRY-341 in the active siteof ACHE (Figure 9(f) S1F) Besides an edge-to-face 120587 minus 120587
Evidence-Based Complementary and Alternative Medicine 13
KCNMA1
P53
IFNG
PPP3CA
CASP8
SCN5A
I-kappaB kinase NF-kappaB signaling
Regulation of inflammation
Response to calcium ion
TGFB1
TNF
PRKCA
CHRNA7
CAV1
STAT1
MAPK cascadeAKT1
BCL2
APP
EGFR
MAPK1
Angiogenesis
SYNGAP1
MAP2
PTPN1
Axonogenesis
GSK3B
EPHB2
CDK1
CASP7FN1
MAPK3
CTNNB1
F2
Autophagy
Cell proliferation
BACE1
SLC6A3
DRD1
ADRA1BACHE
CALMCASP3
PLAT
Apoptotic process
NOS3
PRKCB
Blood coagulation
Response to hypoxia
Superoxide anion generation
Nitric oxide biosynthetic process
Astrocyte activation
Amyloid-beta regulation
Microglial cell activation
LDLR
PRSS3
F7
PRKCD
SOD1ALB
BBC3
Figure 7 pT-F network of XFZYD for treating TBI 16 biological processes (red square) the 47 target proteins (periwinkle circle) of XFZYDparticipate in for treating TBI
interactionwas also observedwith TYR-337 From the resultshydrogen-bonding and edge-to-face 120587 minus 120587 interactions playkey roles in the proteinminusligand recognition and stabilitywhich may be helpful in determining the inhibitor activitiesAnd the C-T network confirmed the potential therapeuticeffects of the candidate compounds from XFZYD to treatTBI through interacting with the relevant proteins The com-putational analysis further elucidated the accurate moleculemechanisms between active compounds and targets
4 Discussion
Traumatic brain injury (TBI) is a growing public healthproblem worldwide and is a leading cause of death anddisability [84] Although major progress has been made in
understanding the pathophysiology of this injury this hasnot yet led to substantial improvements in outcome by alack of treatments which have proven successful during phaseIII trials for modern medicine [85 86] TCM rooted inthousands of years of history may offer an alternative or acomplementary strategy for the treatment of TBI XFZYDa representative formula in TCM has been used for yearsto treat TBI in China and has been demonstrated to beeffective in clinical practice However its ldquomulticomponentsrdquoand ldquomultitargetsrdquo features make it much difficult to decipherthemolecular mechanisms of XFZYD in the treatment of TBIfrom a systematic perspective if employing routine methods
In the present study a network pharmacology-basedmethod was employed to elucidate the pharmacologicalmechanisms of XFZYD to treat TBI according to the drug
14 Evidence-Based Complementary and Alternative Medicine
Figure 8 C-T network through molecule docking validating 119 potential compounds (green triangles) interacting with 18 potential targets(yellow circles) of XFZYDThe size of the nodes is proportional to the value of degree
combination principle of TCM We first proposed a newmodeling system combining OB and DL screening multipledrug targets prediction and validation network constructionand molecule docking to probe the efficiency of a typicalTCM formula XFZYD for the treatment of TBI The 11herbs from XFZYD possessed 162 bioactive compounds andtargeted 285 proteins There were 5 compounds and 189
target proteins overlapped among the Jun Chen and Zuo-Shigroup Furthermore 47 TBI-specific proteins were targetedby 119 (73) bioactive compounds from XFZYD Similarly 5common compounds and 33 (70) common target proteinsamong the 3 groups of drugs were observed Most of thebioactive ingredients targeted more than one protein The 47target proteins regulated several essential pathophysiological
Evidence-Based Complementary and Alternative Medicine 15
(a) (b) (c)
(d) (e) (f)
Figure 9 Hydrogen-bonding networks within the binding site of the compoundminustarget complexes obtained from molecule docking(a) AKT1-quercetin (b) CDK1-quercetin (c) GSK3B-FA (d) F2-glyasperin B (e) NOS3-1-Methoxyphaseollidin and (f) ACHE-(-)-Medicocarpin The molecules are presented as ball and stick models Active site amino acid residues are represented as lines Dotted bluelines in these pictures represent hydrogen bonds with distance unit of A Other O and N atoms are colored as red and blue respectively
processes of TBI that referred to apoptosis inflammationcell proliferation superoxide anion generation nitric oxidebiosynthetic process response to calcium ion etc The aboveanalysis reveals that the synergistic action mechanisms ofXFZYDmay be (1) bioactive compounds overlapping amongthe different group of herbs (2) specific bioactive com-pounds from different groups of herbs targeting the sameproteins (3) specific bioactive compounds from differentgroups of herbs targeting different proteins which participatein the same pathophysiological process of the disease Toa certain degree the 5 compounds including quercetinstigmasterol kaempferol baicalin and beta-sitosterol playedessential role in XFZYD for TBI treatment MAPK3MAPK1AKT1 PRKCA TNF PRKCB EGFR BCL2 GSK3B CASP3PPP3CA and NOS3 were the main target proteins regulatedby XFZYD in the treatment of TBI
Interestingly Beta-carotene (Mol 43) from Carthami Flos(the Jun herb) specifically regulated 120573-Catenin (CTNNB1)and played critical role for curing TBI The 120573-Catenin is acritical downstream component of the Wnt pathway whichplays essential role in the regulation of mammalian neuraldevelopment [87] In vitro and in vivo studies demonstrate
that the Wnt120573-catenin pathway regulates the proliferationand differentiation of neural progenitor cells [88] Neuronaldifferentiation is induced by overexpression of 120573-cateninor the pharmacological inhibition of GSK3120573 (the phospho-rylating enzyme of 120573-catenin) [89 90] This pathway alsopromotes blood vessel formation during vascular develop-ment as well as the vascular repair process after TBI [91]In addition wogonin (Mol 160) from Achyranthis BidentataeRadix (the Chen herb) specifically targeted fibronectin (FN1)Bcl-2-binding component 3 (BBC3) and Protein KinaseC delta type (PRKCD) FN1 an important component ofthe extracellular matrix (ECM) environment promotes cellmigration neurite outgrowth and synapse formation dur-ing neural development [92] It aggregates in the injuredbrain and plays a neuroprotection role through antiapoptosisand anti-inflammation ways following TBI [93 94] BBC3namely p53 upregulated modulator of apoptosis (PUMA) iscritical for the p53-dependent apoptosis pathway which playsan important role in hippocampal neuronal loss and associ-ated cognitive deficits [95] PRKCD one of PKC isoformsactivates signal transduction pathways involved in neuronalregeneration [96] synaptic transmissionplasticity [97] and
16 Evidence-Based Complementary and Alternative Medicine
activation of apoptosis processes [98] as well as higher brainfunctions such as learning andmemory [99]The activators ofPKCare effective for the treatment of TBI [100] FurthermoreMitogen-activated protein kinase 3 (MAPK3) Beta-secretase1 (BACE1) Ephrin type-B receptor 2 (EphB) and low-densitylipoprotein receptor (LDLR) were specifically targeted bythe ingredients in the Zuo-Shi herbs Naringenin (Mol133) targeted LDLR MAPK3 while euchrenone (Mol 60)anchored BACE1 and nobiletin (Mol 134) targeted EphB2MAPK3 is an essential component of the MAP kinase signaltransduction pathway It has been appreciated recently thatthe ERK12 cascade plays a fundamental role in synapticplasticity and memory [101] BACE1 is responsible for pro-duction of A120573 from amyloid precursor protein (APP) A120573can cause cell death activate inflammatory pathways [102]and prime proapoptotic pathways for activation by otherinsults [103] The blocking of BACE1 can ameliorate motorand cognitive deficits and reduce cell loss after experimentalTBI in mice [104] EphB is localized to synaptic sites inhippocampal neurons [105] The interaction between EphBand NMDA receptors regulates excitatory synapse formation[106] LDLR acts as an important receptor that facilitatesbrain A120573 clearance and inhibits amyloid deposition [107]and then ameliorates Alzheimerrsquos disease neuropathologyafter TBI [108] The analysis above indicates the ldquoJunrdquoldquoChenrdquo and ldquoZuo-Shirdquo herbs from XFZYD trigger theirspecific targets regulation respectively for the therapeuticeffects
XFZYD is a very famous traditional Chinese formula inpromoting qi circulation and removing blood stasis accord-ing to TCM theory However several limited researches havedemonstrated its efficacy for treating TBI such as anti-inflammatory and synaptic regulation [30 31] which arein accord with our study However previous studies merelypartially deciphered the molecule mechanism of XFZYD fortreating TBI This study reports 119 bioactive compounds inXFZYD that target 47 TBI-specific proteins such as MAPK3MAPK1 AKT1 PRKCA TNF PRKCB and EGFR Theseproteins regulate several crucial pathophysiological processesof TBI such as apoptosis inflammation blood coagulationand axon genesis Our study demonstrates that the therapeu-tic actions of XFZYD refer to ldquomulticompoundsrdquo ldquomultitar-getsrdquo features rather than only the improvement of bloodcirculation With the help of molecule docking methodwe further validate the interactions between bioactive com-pounds and potential targets of XFZYD The hydrogen-bonding and edge-to-face 120587 minus120587 interactions play key roles inthe proteinminusligand recognition and stability This provides avaluable reference for further experimental investigations ofbioactive ingredients and therapeutic targets of XFZYD fortreating TBI
5 Conclusion
Our work successfully illuminates the efficiency of XFZYDfor the treatment of TBI as well as herb combination ruleof TCM formula Network pharmacology with moleculedocking method confirms the ldquomulticompounds multitar-getsrdquo therapeutic actions of XFZYD in the treatment of TBI
The present work may provide valuable evidence for furtherclinical application of XFZYD for treating TBI
Abbreviations
TBI Traumatic brain injuryXFZYD Xuefu Zhuyu decoctionTCM Traditional Chines medicineDL Drug-likenessOB Oral bioavailabilityHB-cC-cT Herb-candidate compound-candidate
targetHB-pC-pT Herb-potential compound-potential targetCALM CalmodulinGSK3B Glycogen synthase kinase-3 betaSCN5A Sodium channel protein type 5 subunit
alphaF2 ProthrombinACHE AcetylcholinesteraseGO Gene OntologyKEGG Kyoto Encyclopedia of Genes and
Genomes
Data Availability
The data used to support the findings of this study areavailable from the corresponding author upon request
Conflicts of Interest
The authors declare that they have no conflicts of interest
Authorsrsquo Contributions
YangWang and Zebing Huang participated in the conceptionand design of the study YuanyuanZhong YangWang ZebingHuang Jiekun Luo Tao Tang and Pengfei Li acquired andanalyzed the data Yuanyuan Zhong Tao Liu and HanjinCui drafted and revised the manuscript The correspondingauthor and all of the authors have read and approved the finalsubmitted manuscript
Acknowledgments
This work was supported by the National Natural ScienceFoundation of China (Nos 81673719 81874409 81603670and 81803948) Project funded byChina Postdoctoral ScienceFoundation (Nos 2016M600639 and 2017T100614)
Supplementary Materials
Table S1 162 bioactive compounds in XFZYD Table S2 targetproteins of XFZYD Table S3 TBI-specific proteins Table S4119 potential compounds of XFZYD for treating TBI TableS5 docking result of 18 target proteins with 91 potential com-pounds Fig S1 the exact bindingmode between active ingre-dients and protein targets obtained from molecule docking(A) AKT1-quercetin (B) CDK1-quercetin (C) GSK3B-FA
Evidence-Based Complementary and Alternative Medicine 17
(D) F2-glyasperin B (E) NOS3-1-Methoxyphaseollidin and(F)ACHE-(-)-Medicocarpin (Supplementary Materials)
References
[1] L Yang Y Chu D Tweedie et al ldquoPost-trauma administrationof the pifithrin-120572 oxygen analog improves histological andfunctional outcomes after experimental traumatic brain injuryrdquoExperimental Neurology vol 269 pp 56ndash66 2015
[2] J A Langlois W Rutland-Brown and M M Wald ldquoTheepidemiology and impact of traumatic brain injury a briefoverviewrdquo The Journal of Head Trauma Rehabilitation vol 21no 5 pp 375ndash378 2006
[3] H A Alsulaim B J Smart A O Asemota et al ldquoConsciousstatus predictsmortality among patientswith isolated traumaticbrain injury in administrative datardquo The American Journal ofSurgery vol 214 no 2 pp 207ndash210 2017
[4] M Majdan D Plancikova A Maas et al ldquoYears of life lost dueto traumatic brain injury in Europe A cross-sectional analysisof 16 countriesrdquo PLoS Medicine vol 14 no 7 p e1002331 2017
[5] P Cheng P Yin P Ning et al ldquoTrends in traumatic braininjury mortality in China 2006ndash2013 A population-basedlongitudinal studyrdquo PLoS Medicine vol 14 no 7 Article IDe1002332 2017
[6] M V Russo and D B McGavern ldquoInflammatory neuroprotec-tion following traumatic brain injuryrdquo Science vol 353 no 6301pp 783ndash785 2016
[7] WWang H Li J Yu et al ldquoProtective Effects of ChineseHerbalMedicine Rhizoma drynariae in Rats After Traumatic BrainInjury and Identification of Active Compoundrdquo MolecularNeurobiology vol 53 no 7 pp 4809ndash4820 2016
[8] M Das S Mohapatra and S S Mohapatra ldquoNew perspectiveson central and peripheral immune responses to acute traumaticbrain injuryrdquo Journal of Neuroinflammation vol 9 p 236 2012
[9] J W Finnie ldquoNeuroinflammation Beneficial and detrimentaleffects after traumatic brain injuryrdquo Inflammopharmacologyvol 21 no 4 pp 309ndash320 2013
[10] T Cheng W Wang Q Li et al ldquoCerebroprotection of flavanol(minus)-epicatechin after traumatic brain injury viaNrf2-dependentand -independent pathwaysrdquo Free Radical Biology amp Medicinevol 92 pp 15ndash28 2016
[11] W Young ldquoRole of calcium in central nervous system injuriesrdquoJournal of Neurotrauma vol 9 Suppl 1 pp S9ndashS25 1992
[12] R Bullock A Zauner J S Myseros et al ldquoEvidence forProlonged Release of Excitatory Amino Acids in Severe HumanHead Trauma Relationship to Clinical Eventsrdquo Annals of theNew York Academy of Sciences vol 765 no 1 pp 290ndash297 1995
[13] A T Mazzeo A Beat A Singh and M R Bullock ldquoTherole of mitochondrial transition pore and its modulation intraumatic brain injury and delayed neurodegeneration afterTBIrdquo Experimental Neurology vol 218 no 2 pp 363ndash370 2009
[14] S Gennai A Monsel Q Hao et al ldquoCell-Based therapy fortraumatic brain injuryrdquo British Journal of Anaesthesia vol 115no 2 pp 203ndash212 2015
[15] J Ghajar ldquoTraumatic brain injuryrdquo The Lancet vol 356 no9233 pp 923ndash929 2000
[16] D J Loane and A I Faden ldquoNeuroprotection for traumaticbrain injury translational challenges and emerging therapeuticstrategiesrdquoTrends in Pharmacological Sciences vol 31 no 12 pp596ndash604 2010
[17] Y Wang X Fan T Tang et al ldquoRhein and rhubarb simi-larly protect the blood-brain barrier after experimental trau-matic brain injury via gp91phox subunit of NADPH oxidaseROSERKMMP-9 signaling pathwayrdquo Scientific Reports vol 6p 37098 2016
[18] D-X Kong X-J Li and H-Y Zhang ldquoWhere is the hopefor drug discovery Let history tell the futurerdquo Drug DiscoveryTherapy vol 14 no 3-4 pp 115ndash119 2009
[19] F Cheung ldquoTCM made in Chinardquo Nature vol 480 no 7378pp S82ndashS83 2011
[20] C Fu Z Xia Y Liu et al ldquoQualitative analysis of majorconstituents from Xue Fu Zhu Yu Decoction using ultra highperformance liquid chromatographywith hybrid ion trap time-of-flight mass spectrometryrdquo Journal of Separation Science vol39 no 17 pp 3457ndash3468 2016
[21] H-J Zhang and Y-Y Cheng ldquoAn HPLCMS method for iden-tifying major constituents in the hypocholesterolemic extractsof Chinese medicine formula lsquoXue-Fu-Zhu-Yu decoctionrsquordquoBiomedical Chromatography vol 20 no 8 pp 821ndash826 2006
[22] L Zhang L Zhu YWang et al ldquoCharacterization and quantifi-cation of major constituents of Xue Fu Zhu Yu by UPLC-DAD-MSMSrdquo Journal of Pharmaceutical and Biomedical Analysisvol 62 pp 203ndash209 2012
[23] X Yang X Xiong G Yang and J Wang ldquoChinese patentmedicine Xuefu Zhuyu capsule for the treatment of unstableangina pectoris a systematic review of randomized controlledtrialsrdquo Complementary Therapies in Medicine vol 22 no 2 pp391ndash399 2014
[24] J Wang X Yang F Chu et al ldquoThe effects of Xuefu Zhuyuand Shengmai on the evolution of syndromes and inflammatorymarkers in patients with unstable angina pectoris after percuta-neous coronary intervention a randomised controlled clinicaltrialrdquoEvidence-BasedComplementary andAlternativeMedicinevol 2013 Article ID 896467 9 pages 2013
[25] Q Zhang H Yu J Qi et al ldquoNatural formulas and the natureof formulas Exploring potential therapeutic targets based ontraditional Chinese herbal formulasrdquo PLoS ONE vol 12 no 2p e0171628 2017
[26] J LeeWHsu T Yen et al ldquoTraditional ChinesemedicineXue-Fu-Zhu-Yu decoction potentiates tissue plasminogen activatoragainst thromboembolic stroke in ratsrdquo Journal of Ethnophar-macology vol 134 no 3 pp 824ndash830 2011
[27] Y-C Shen C-K Lu K-T Liou et al ldquoCommon and uniquemechanisms of Chinese herbal remedies on ischemic strokemice revealed by transcriptome analysesrdquo Journal of Ethnophar-macology vol 173 pp 370ndash382 2015
[28] F Xue-hai G Yan and L Jian-tao ldquoTherapeutic effect of XiefuZhuyu decoction joint brain protein hydrolysat injection intraumatic brain injury and its effect on cerebrospinal fluid ofET-1rdquo Chinese Journal of Biochemical Pharmaceutics no 02 pp55ndash58 2017
[29] L Shuxiang W Jingchun C Jie et al ldquoEffect of Xuefu Zhuyudecoction on the neural functional recovery and living abilityin patients with craniocerebral injuryrdquo Modern Journal ofIntegrated Traditional Chinese andWesternMedicine no 04 pp350ndash352 2015
[30] L Zhong W Ning and W Dong-pi ldquoModel Study of theTherapy of Replenishing Qi and Activating Blood Circulationon Protecting the Brain Impairment Caused by Hypoxic -ischemic Encephalopathy in SD Ratsrdquo Journal of TraditionalChinese Medicine vol 07 pp 1552ndash1555 2011
18 Evidence-Based Complementary and Alternative Medicine
[31] M Sun X-P Zhan C-Y Jin J-Z Shan S Xu and Y-LWang ldquoclinical observation on treatment of post-craniocerebraltraumatic mental disorder by integrative medicinerdquo ChineseJournal of Integrative Medicine vol 14 no 2 pp 137ndash141 2008
[32] Z Xing Z Xia W Peng et al ldquoXuefu Zhuyu decoction atraditional Chinese medicine provides neuroprotection in arat model of traumatic brain injury via an anti-inflammatorypathwayrdquo Scientific Reports vol 6 Article ID 20040 2016
[33] J Zhou T Liu H Cui et al ldquoXuefu zhuyu decoction improvescognitive impairment in experimental traumatic brain injuryvia synaptic regulationrdquo Oncotarget vol 8 no 42 2017
[34] S Li ldquoExploring traditional chinese medicine by a novel thera-peutic concept of network targetrdquo Chinese Journal of IntegrativeMedicine vol 22 no 9 pp 647ndash652 2016
[35] S Li and B Zhang ldquoTraditional Chinese medicine networkpharmacology theory methodology and applicationrdquo ChineseJournal of Natural Medicines vol 11 no 2 pp 110ndash120 2013
[36] A L Hopkins ldquoNetwork pharmacology the next paradigm indrug discoveryrdquoNature Chemical Biology vol 4 no 11 pp 682ndash690 2008
[37] Y Li C Han J Wang et al ldquoInvestigation into the mechanismof Eucommia ulmoides Oliv based on a systems pharmacologyapproachrdquo Journal of Ethnopharmacology vol 151 no 1 pp452ndash460 2014
[38] Y Yao X Zhang Z Wang et al ldquoDeciphering the combinationprinciples of Traditional Chinese Medicine from a systemspharmacology perspective based on Ma-huang DecoctionrdquoJournal of Ethnopharmacology vol 150 no 2 pp 619ndash638 2013
[39] Bo Zhang Xu Wang and Shao Li ldquoAn Integrative Platform ofTCM Network Pharmacology and Its Application on a HerbalFormula Qing-Luo-Yinrdquo Evidence-Based Complementary andAlternativeMedicine vol 2013 Article ID 456747 12 pages 2013
[40] J Ru P Li J Wang et al ldquoTCMSP a database of systemspharmacology for drug discovery from herbal medicinesrdquoJournal of Cheminformatics vol 6 no 1 p 13 2014
[41] J V Turner D J Maddalena and S Agatonovic-KustrinldquoBioavailability Prediction Based on Molecular Structure for aDiverse Series of Drugsrdquo Pharmaceutical Research vol 21 no 1pp 68ndash82 2004
[42] X XuW Zhang CHuang et al ldquoAnovel chemometricmethodfor the prediction of human oral bioavailabilityrdquo InternationalJournal of Molecular Sciences vol 13 no 6 pp 6964ndash6982 2012
[43] A Zhang W Pan J Lv and H Wu ldquoProtective Effect ofAmygdalin on LPS-Induced Acute Lung Injury by InhibitingNF-120581B and NLRP3 Signaling Pathwaysrdquo Inflammation vol 40no 3 pp 745ndash751 2017
[44] X Wei H Liu X Sun et al ldquoHydroxysafflor yellow A protectsrat brains against ischemia-reperfusion injury by antioxidantactionrdquo Neuroscience Letters vol 386 no 1 pp 58ndash62 2005
[45] W PWalters andM A Murcko ldquoPrediction of lsquodrug-likenessrsquordquoAdvanced Drug Delivery Reviews vol 54 no 3 pp 255ndash2712002
[46] Y Yamanishi M Kotera M Kanehisa and S Goto ldquoDrug-target interactionprediction from chemical genomic and phar-macological data in an integrated frameworkrdquo Bioinformaticsvol 26 no 12 pp i246ndashi254 2010
[47] H Yu J Chen X Xu et al ldquoA systematic prediction ofmultiple drug-target interactions from chemical genomic andpharmacological datardquo PLoS ONE vol 7 no 5 Article IDe37608 2012
[48] X Chen Z L Ji and Y Z Chen ldquoTTD therapeutic targetdatabaserdquo Nucleic Acids Research vol 30 no 1 pp 412ndash4152002
[49] A Hamosh A F Scott J S Amberger C A Bocchini andV AMcKusick ldquoOnline Mendelian Inheritance in Man (OMIM) aknowledgebase of human genes and genetic disordersrdquo NucleicAcids Research vol 33 pp D514ndashD517 2005
[50] T S Keshava Prasad RGoel K Kandasamy et al ldquoHuman pro-tein reference databasemdash2009 updaterdquo Nucleic Acids Researchvol 37 no 1 pp D767ndashD772 2009
[51] A Franceschini D Szklarczyk S Frankild et al ldquoSTRING v91protein-protein interaction networks with increased coverageand integrationrdquoNucleic Acids Research vol 41 no 1 pp D808ndashD815 2013
[52] P Shannon A Markiel O Ozier et al ldquoCytoscape a softwareEnvironment for integratedmodels of biomolecular interactionnetworksrdquo Genome Research vol 13 no 11 pp 2498ndash25042003
[53] G Dennis Jr B T Sherman D A Hosack et al ldquoDAVIDDatabase for Annotation Visualization and IntegratedDiscov-eryrdquo Genome Biology vol 4 no 5 p P3 2003
[54] L Yang X Zhao and X Tang ldquoPredicting disease-related pro-teins based on clique backbone in protein-protein interactionnetworkrdquo International Journal of Biological Sciences vol 10 no7 pp 677ndash688 2014
[55] S MThomas and J S Brugge ldquoCellular functions regulated bySRC family kinasesrdquo Annual Review of Cell and DevelopmentalBiology vol 13 pp 513ndash609 1997
[56] D Z Liu F R Sharp K C Van et al ldquoInhibition of Src familykinases protects hippocampal neurons and improves cognitivefunction after traumatic brain injuryrdquo Journal of Neurotraumavol 31 no 14 pp 1268ndash1276 2014
[57] B D Manning and A Toker ldquoAKTPKB Signaling Navigatingthe Networkrdquo Cell vol 169 no 3 pp 381ndash405 2017
[58] Y Kitagishi and S Matsuda ldquoDiets involved in PPAR andPI3KAKTPTEN pathway may contribute to neuroprotectionin a traumatic brain injuryrdquoAlzheimerrsquos ResearchampTherapy vol5 no 5 p 42 2013
[59] D Chen L Bao S-Q Lu and F Xu ldquoSerum albumin andprealbuminpredict the poor outcomeof traumatic brain injuryrdquoPLoS ONE vol 9 no 3 Article ID e93167 2014
[60] J Yang Z Wu N Renier et al ldquoPathological axonal deaththrough aMapk cascade that triggers a local energy deficitrdquoCellvol 160 no 1-2 pp 161ndash176 2015
[61] E J Huang and L F Reichardt ldquoTrk receptors roles in neuronalsignal transductionrdquoAnnual Review of Biochemistry vol 72 pp609ndash642 2003
[62] J W Lee and R Juliano ldquoMitogenic signal transductionby integrin- and growth factor receptor-mediated pathwaysrdquoMolecules and Cells vol 17 no 2 pp 188ndash202 2004
[63] C S Yang J M Landau M T Huang and H L NewmarkldquoInhibition of carcinogenesis by dietary polyphenolic com-poundsrdquo Annual Review of Nutrition vol 21 pp 381ndash406 2001
[64] A Murakami H Ashida and J Terao ldquoMultitargeted cancerprevention by quercetinrdquoCancer Letters vol 269 no 2 pp 315ndash325 2008
[65] G Du Z Zhao Y Chen et al ldquoQuercetin attenuates neuronalautophagy and apoptosis in rat traumatic brain injury modelvia activation of PI3KAkt signaling pathwayrdquo NeurologicalResearch vol 38 no 11 pp 1ndash8 2016
Evidence-Based Complementary and Alternative Medicine 19
[66] Q Cai R O Rahn and R Zhang ldquoDietary flavonoidsquercetin luteolin andgenistein reduce oxidativeDNAdamageand lipid peroxidation and quench free radicalsrdquoCancer Lettersvol 119 no 1 pp 99ndash107 1997
[67] G A Bongiovanni E A Soria and A R Eynard ldquoEffectsof the plant flavonoids silymarin and quercetin on arsenite-induced oxidative stress in CHO-K1 cellsrdquo Food and ChemicalToxicology vol 45 no 6 pp 971ndash976 2007
[68] H Li L Yang Y Zhang et al ldquoKaempferol inhibits fibroblastcollagen synthesis proliferation and activation in hypertrophicscar via targeting TGF-beta receptor type Irdquo Biomedicine ampPharmacotherapy = Biomedecine amp Pharmacotherapie vol 83pp 967ndash974 2016
[69] K P Devi D S Malar S F Nabavi et al ldquoKaempferol andinflammation From chemistry to medicinerdquo PharmacologicalResearch vol 99 pp 1ndash10 2015
[70] M Zhou H Ren J Han W Wang Q Zheng and DWang ldquoProtective effects of kaempferol against myocardialischemiareperfusion injury in isolated rat heart via antioxidantactivity and inhibition of glycogen synthase kinase-3120573rdquo Oxida-tive Medicine and Cellular Longevity vol 2015 p 481405 2015
[71] C-F Lee J-S Yang F-J Tsai et al ldquoKaempferol inducesATMp53-mediated death receptor and mitochondrial apop-tosis in human umbilical vein endothelial cellsrdquo InternationalJournal of Oncology vol 48 no 5 pp 2007ndash2014 2016
[72] C Luo H Yang C Tang et al ldquoKaempferol alleviates insulinresistance via hepatic IKKNF-120581B signal in type 2 diabetic ratsrdquoInternational Immunopharmacology vol 28 no 1 pp 744ndash7502015
[73] A B Awad and C S Fink ldquoPhytosterols as anticancer dietarycomponents evidence and mechanism of actionrdquo Journal ofNutrition vol 130 no 9 pp 2127ndash2130 2000
[74] K Farber and H Kettenmann ldquoFunctional role of calciumsignals for microglial functionrdquoGlia vol 54 no 7 pp 656ndash6652006
[75] Y Lu Y Gu X Ding et al ldquoIntracellular Ca2+ homeostasisand JAK1STAT3 pathway are involved in the protective effectof propofol on BV2 microglia against hypoxia-induced inflam-mation and apoptosisrdquo PLoS ONE vol 12 no 5 p e01780982017
[76] K Leroy and J-P Brion ldquoDevelopmental expression and local-ization of glycogen synthase kinase-3120573 in rat brainrdquo Journal ofChemical Neuroanatomy vol 16 no 4 pp 279ndash293 1999
[77] C-M Liu E-M Hur and F-Q Zhou ldquoCoordinating geneexpression and axon assembly to control axon growth Potentialrole ofGSK3 signalingrdquo Frontiers inMolecularNeuroscience vol3 p 3 2012
[78] D A E Cross A A Culbert K A Chalmers L Facci S DSkaper and A D Reith ldquoSelective small-molecule inhibitors ofglycogen synthase kinase-3 activity protect primary neuronesfrom deathrdquo Journal of Neurochemistry vol 77 no 1 pp 94ndash102 2001
[79] V S Ossovskaya and NW Bunnett ldquoProtease-activated recep-tors contribution to physiology and diseaserdquo PhysiologicalReviews vol 84 no 2 pp 579ndash621 2004
[80] M Steinhoff J Buddenkotte V Shpacovitch et al ldquoProteinase-activated receptors transducers of proteinase-mediated signal-ing in inflammation and immune responserdquoEndocrine Reviewsvol 26 no 1 pp 1ndash43 2005
[81] H Krenzlin V Lorenz S Danckwardt O Kempski and BAlessandri ldquoThe importance of thrombin in cerebral injury and
diseaserdquo International Journal of Molecular Sciences vol 17 no1 2016
[82] M J McGinn and J T Povlishock ldquoPathophysiology of Trau-matic Brain InjuryrdquoNeurosurgery Clinics of North America vol27 no 4 pp 397ndash407 2016
[83] T Woodcock and M C Morganti-Kossmann ldquoThe role ofmarkers of inflammation in traumatic brain injuryrdquo Frontiersin Neurology vol 4 article 18 2013
[84] F Tanriverdi H J Schneider G Aimaretti B E Masel F FCasanueva and F Kelestimur ldquoPituitary dysfunction after trau-matic brain injury A clinical and pathophysiological approachrdquoEndocrine Reviews vol 36 no 3 pp 305ndash342 2015
[85] J V Rosenfeld A I Maas P Bragge M C Morganti-Kossmann G T Manley and R L Gruen ldquoEarly managementof severe traumatic brain injuryrdquoThe Lancet vol 380 no 9847pp 1088ndash1098 2012
[86] B M Aertker S Bedi and C S Cox ldquoStrategies for CNS repairfollowing TBIrdquo Experimental Neurology vol 275 no 3 pp 411ndash426 2016
[87] K Adachi Z Mirzadeh M Sakaguchi et al ldquo120573-catenin signal-ing promotes proliferation of progenitor cells in the adultmousesubventricular zonerdquo Stem Cells vol 25 no 11 pp 2827ndash28362007
[88] Y Hirabayashi and Y Gotoh ldquoStage-dependent fate determina-tion of neural precursor cells in mouse forebrainrdquo NeuroscienceResearch vol 51 no 4 pp 331ndash336 2005
[89] S Ding T Y H Wu A Brinker et al ldquoSynthetic smallmolecules that control stem cell faterdquo Proceedings of theNationalAcadamy of Sciences of the United States of America vol 100 no13 pp 7632ndash7637 2003
[90] N Israsena M Hu W Fu L Kan and J A Kessler ldquoThepresence of FGF2 signaling determines whether 120573-cateninexerts effects on proliferation or neuronal differentiation ofneural stem cellsrdquo Developmental Biology vol 268 no 1 pp220ndash231 2004
[91] A Salehi A Jullienne M Baghchechi et al ldquoUp-regulation ofWnt120573-catenin expression is accompanied with vascular repairafter traumatic brain injuryrdquo Journal of Cerebral Blood Flow ampMetabolism vol 38 no 2 pp 274ndash289 2017
[92] L F Reichardt and K J Tomaselli ldquoExtracellular matrixmolecules and their receptors Functions in neural develop-mentrdquoAnnual Review of Neuroscience vol 14 pp 531ndash570 1991
[93] R M Gibson S E Craig L Heenan C Tournier and M JHumphries ldquoActivation of integrin 12057251205731 delays apoptosis ofNtera2 neuronal cellsrdquoMolecularandCellularNeuroscience vol28 no 3 pp 588ndash598 2005
[94] C C Tate A J Garcıa andMC LaPlaca ldquoPlasma fibronectin isneuroprotective following traumatic brain injuryrdquoExperimentalNeurology vol 207 no 1 pp 13ndash22 2007
[95] C Culmsee and M P Mattson ldquop53 in neuronal apoptosisrdquoBiochemical and Biophysical Research Communications vol 331no 3 pp 761ndash777 2005
[96] M S Geddis and V Rehder ldquoThe phosphorylation state ofneuronal processes determines growth cone formation afterneuronal injuryrdquo Journal of Neuroscience Research vol 74 no2 pp 210ndash220 2003
[97] M L Craske M Fivaz N N Batada and T Meyer ldquoSpines andneurite branches function as geometric attractors that enhanceprotein kinase C actionrdquoThe Journal of Cell Biology vol 170 no7 pp 1147ndash1158 2005
20 Evidence-Based Complementary and Alternative Medicine
[98] A Muscella C Vetrugno L G Cossa et al ldquoApoptosis by[Pt(OOrsquo-acac)(gamma-acac)(DMS)] requires PKC-deltamedi-ated p53 activation in malignant pleural mesotheliomardquo PloSone vol 12 no 7 Article ID e0181114 2017
[99] J S Bonini W C Da Silva L R M Bevilaqua J H MedinaI Izquierdo and M Cammarota ldquoOn the participation ofhippocampal PKC in acquisition consolidation and reconsol-idation of spatial memoryrdquo Neuroscience vol 147 no 1 pp 37ndash45 2007
[100] O Zohar R Lavy X Zi et al ldquoPKC activator therapeutic formild traumatic brain injury in micerdquo Neurobiology of Diseasevol 41 no 2 pp 329ndash337 2011
[101] J David Sweatt ldquoTheneuronalMAPkinase cascade a biochem-ical signal integration system subserving synaptic plasticity andmemoryrdquo Journal ofNeurochemistry vol 76 no 1 pp 1ndash10 2001
[102] Y Matsuoka M Picciano B Maleste et al ldquoInflammatoryresponses to amyloidosis in a transgenic mouse model ofAlzheimerrsquos diseaserdquo The American Journal of Pathology vol158 no 4 pp 1345ndash1354 2001
[103] L Esposito L Gan G-Q Yu C Essrich and L MuckeldquoIntracellularly generated amyloid-120573 peptide counteracts theantiapoptotic function of its precursor protein and primesproapoptotic pathways for activation by other insults in neu-roblastoma cellsrdquo Journal of Neurochemistry vol 91 no 6 pp1260ndash1274 2004
[104] D J Loane A Pocivavsek C E-H Moussa et al ldquoAmyloidprecursor protein secretases as therapeutic targets for traumaticbrain injuryrdquo Nature Medicine vol 15 no 4 pp 377ndash379 2009
[105] R Torres B L Firestein H Dong et al ldquoPDZ proteins bindcluster and synaptically colocalize with Eph receptors and theirephrin ligandsrdquo Neuron vol 21 no 6 pp 1453ndash1463 1998
[106] M B Dalva M A Takasu M Z Lin et al ldquoEphB receptorsinteract with NMDA receptors and regulate excitatory synapseformationrdquo Cell vol 103 no 6 pp 945ndash956 2000
[107] J M Basak P B Verghese H Yoon J Kim and D MHoltzman ldquoLow-density lipoprotein receptor represents anapolipoprotein E-independent pathway of A120573 uptake anddegradation by astrocytesrdquoThe Journal of Biological Chemistryvol 287 no 17 pp 13959ndash13971 2012
[108] L Yao X Gu Q Song et al ldquoNanoformulated alpha-mangostinameliorates Alzheimerrsquos disease neuropathology by elevatingLDLR expression and accelerating amyloid-beta clearancerdquoJournal of Controlled Release vol 226 pp 1ndash14 2016
Stem Cells International
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Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 9
BLMH
GPRASP2
GJC2
CAMK2N1
OBSL1
ZNF292
RLFAMPD3
NANOS1CAMTA1HYPKCCS
DLGAP4PF4V1
TRIM2
CHKB
F8A1
TIAM1
BMX
RAPGEF1
APBB1
KCNMA1
XRCC6
TTR
TGM2MAP2
PRNP
RASGRF1 PPP3CADMD SPTAN1
ACE
TGFB2
AP2M1GRAP2
SCN5A
MYH9
PKN1
ACHE
SLC25A13
PHKG1
SPAST
UMOD
PRPF40B
BBC3
TUBA4A TTPALRYR2
STUB1KRT6A
AKAP8L
GFI1BTHRAP3
ZNF232
SOD1
CRKL
CDH2
MBP MAP2K2
DCN
MAP3K5NGFR
PIK3CA
ITGA2
DNM1
RAP1A
GRIN2B
PTPN11
PRKCA
LDLRAP1
DNAJA3INA
SP3
CACNA2D3
CSF1
CNTF
TNFRSF1B
BCL10
KLC1
DCTN1
IDE
SERPINA3
HSPA1A
HSPG2
FN1
LRP2
TUBB
GRIN2A
YWHAB
CAMK2D
HTRA2
PRKCE
CRYAB
APC
GIT1
PIAS4
HPX
TRIP10
PDE4B
MARK4
PLAT
UBE2K
APOC2
LIN7B BGN
MAP3K10MAPK8IP2
CBS
SH3GL3
STAU1
CASP6
PPP5C
KRT16
DAB1
CASP1SHC2
CTSD
UCHL1
HP
SNCACAMK2G
TGFB1
GNB1
PTPN1PIN1
TJP1
CASP8PLCG1
NPHS1
PPP2R2A
DNAJB1
ADRBK1
INADL
NCSTN
MATK
NEDD4L
KAT5IFNGTBP
FRS2
CSNK1A1
CALRKNG1
SGOL2
KRT6B
TRAF3 DLG3
SACS
BDKRB2PRKCB
CAV1
MAPK3F2 SMAD3
APOEAKAP9 RANBP2
CLTC
TSC22D1GAB2PSEN2
MLF1CROT
KRT9
ADRA1B
EPB41
COL4A1
SHC3
ARHGAP32
ATM
PPP2R5A
CAMK2A
NTRK1
KRT18
TNFAPOA2
LAT
GRIN2D
COL4A6
KLK3
VLDLR
COL4A5
COL4A2
NEFH
OR8D2
MTSS1
PDZRN4
PTPN4
OR3A2
OR2T6
DGKG
CASP3
IGF1R
CASK
DRD2
TRAF2
EPHB2
PPM1A
PRKCG
CACNB3
HNRNPUSIN3A
DLG1
CLU
RAP2A
ERBB4CANX
BACE1 GABBR1
KARS
CNOT1
UNG
AGA CDCP1
UTP14A PLAG1
EXOC6
HRAS
CTNNB1
SP1
NOS3 SUMO1
PSEN1
HSPA8
HSP90AA1
BCL2
GNAO1
RPS6KB1
PARK2
DLG4
PTK2B
GSN
NAE1 TLN2LRFN2
PLTP
NUMBLSTX1A
DERL1
NEFM
HMOX2
AATF
PPIAMTRNR2L2AP1M1
OGT
DDB1
AP4M1
MED31
RGS3
TDP2
KRT5
ADAM9
REST
PACSIN1
SHB
CTSL1
LAMA1
SH2B1
PLA2G4F
SMURF2
SLC9A8
KRT14
CIT
TGFB1I1
PPP2R1APIK3R1
NUMB
ADAM17
DLG2
LRP1
APOA4
LTA
IRS1
GSK3B
EGFR
CTSB
TRAF6
SHC1
PPP2CB
NGF
HTT
PPIDLDB3
APOC1HIP1
ZDHHC17COL25A1MAGI3
ITGB5
ACTB
GNA11
ACTN2CAMK2B
CDK1
LIN7C
S100B
PDLIM5
DLGAP1
LIN7A
TPR
SYNGAP1
CFD
GRIN3A
F12
SLC1A3
GRK5
CACNA2D2
TANC1
DUSP4
EXOC4
AHSG
GRIN3B
UCP2
TTN
GPC1
NSF
FGA
DYNLL1
MAPK8IP1
PICALM
PPBP
ITM2B
NID1
STXBP1
CTBP1
SGK1
TRAF1
SQSTM1
TNFRSF1A
ACTN1
OPTN
UBE2D2
AP2A2
FUS
APOA1
DICER1
CACNA1B
CST3
NCOA3
SCARB1
APOC3
SLC6A3
KIAA1551 KRT13
DCD
KIAA0232
ENSG00000258818
SPATA31A7
CTAGE5
CEP44
SH2B2
EPHB4
CAPN1
F7
APBA2
COL4A3
LDLR
GIPC1
GFAP
SERPING1
SRC
MAPK12APP
GAPDH
CDK5
CREBBP
NOS1
YWHAZCALM1
YWHAG
YWHAQGRIN1
RPS6KA3
COL1A2
TCERG1
A2M
CASP7
LRP8
PDK2
AGTR1
FYN
ALBGRB2
MAPK1AKT1
STAT1
MAPT
GSK3A
UBB
PPP2CA
SMAD4
PRKCD
RGS12
SPTB
SH3BP5
CACNA1I
IL16
SLA2
PFN2
NLRC4
TNFRSF14
PRTN3
SNCB
FBLN1
BACE2
SETX
FANCD2
SAP30
RANBP3
PALB2
SLC1A5RASA1
HAP1
LRRC7
CFB
ADRBK2
CDC45
CLSTN3
PCED1B
SCAF1KIAA1377
FAM71E2
FEZ1 ACSBG2
PCDH1
TRAPPC11QTRTD1
KRT1
LRP1B
HADHB
TP53BP2
DRD1
ST13
PRPF40A
TRAIP
KIDINS220
GRK6
MMP17
LTBR
EIF6
PGAM1
CHD3
PRSS3
APBB3
CHRNA7
KRT10
RIMS1
CRMP1
PDIK1L
SORBS3
SYMPK
GCN1L1
RNF19A
HOMER3
GRK4
JARID2
MYLK3
EFS
CRB1
HSD17B10
TST
HOMER2
PHC3
TM2D1 FICD
PEG3
CABLES1
LGALS2
ITIH1
HOXB2
TAF4FLOT1
BABAM1
CFH
HIP1R
LTBMYL4
NCOR1
CASP4
NF1
AP4E1
CLCA2
IGDCC4
NECAB3
CXorf27
SH3GLB1
CTCF
PLCG2
(a)
STAT1
EGFR
FN1
PLAT
SOD1
PRKCA CDK1
GSK3B
ALB APP
PPP3CA
IFNG
BBC3
SCN5A
KCNMA1
MAP2
ACHE
MAPK3F2
PTPN1
NOS3 BCL2
CAV1
CTNNB1
PRKCB
TGFB1CASP8
TNF
AKT1
F7
MAPK1LDLR
PRKCD
SLC6A3
DRD1
CASP3
CHRNA7
PRSS3
TP53BP2
CASP7
EPHB2
SYNGAP1
CALM1
CTSD
BACE1
ADRA1B
(b)
Figure 4 TBI-related protein interaction network (a) 21 hub proteins (red and green) were identified through the analysis of TherapeuticTargetDatabase (TTD) aswell asOnlineMendelian Inheritance inMan (OMIM) 4 overlapped protein targets (green)were obtained between21 hub proteins in TBI and candidate targets of XFZYD Periwinkle proteins fromHPRD and STRING analyses were not targeted by XFZYD(b) 47 candidate protein targets of XFZYD were screened for treating TBI Yellow candidate protein targets of XFZYD The size of nodes isproportional to the value of degree
10 Evidence-Based Complementary and Alternative Medicine
Table 4 Top 10 proteins of TBI specific proteins according to 2 centrality indicators
Proteins Degree Proteins BetweennessSRC 164 APP 0071814AKT1 157 ALB 0069343ALB 153 SRC 0058568EGFR 139 EGFR 0052938APP 134 AKT1 0045466GAPDH 131 HTT 0037164HSP90AA1 108 GAPDH 0034549BCL2 106 HSP90AA1 0027596MAPK1 105 CTNNB1 0021759HRAS 105 GNB1 0019492Note the centrality indicators identify the important nodes within the network Higher degree centrality and betweenness centrality indicate greaterimportance
Table 5 Top 10 significantly enriched KEGG pathways in TBI-specific proteins
Pathway ID Pathway description Gene count FDR4010 MAPK signaling pathway 44 299E-235200 Pathways in cancer 43 113E-184722 Neurotrophin signaling pathway 41 101E-344151 PI3K-Akt signaling pathway 40 111E-154510 Focal adhesion 39 292E-225205 Proteoglycans in cancer 39 410E-215010 Alzheimer s disease 34 124E-204015 Rap1 signaling pathway 33 769E-174014 Ras signaling pathway 33 646E-164020 Calcium signaling pathway 31 505E-17
0
001
002
003
004
005
006
007
008
0 20 40 60 80 100 120 140 160 180
Betw
eenn
ess
Degree
APP
ALB SRCEGFR
AKT1HTT
Figure 5 Relationship between degree and betweenness centralityin the TBI-specific protein interaction network
for the 119 potential compounds is shown in Table S4Similarly 5 pharmacologically active ingredients in the JunChen and Zuo-Shi groups including quercetin stigmasterolkaempferol baicalin and beta-sitosterol anchored 33 TBI-specific proteins such as CALM SCN5A F2 ACHE F7ADRA1B NOS3 BCL2 CASP3 and AKT1 11 Jun-specificcompounds targeted 2 specific targets (ALB CTNNB1) while12 Chen-specific compounds anchored 3 unique proteins(PRKCD FN1 and BC3) The Zuo-Shi herbs possessed thelargest number of active compounds (89) and targeted 5
unique proteins including EPHB2 BACE1 LDLR MAPK3and PRSS3 GSK3Bwas the common target between theChenandZuo-Shi drugs KCNMA1 CASP7 andAPPwere targetedby the Jun and Zuo-Shi drugs
The top 10 candidate compounds and targets to treat TBIwere showed in Tables 6 and 7 Formost of active compoundsfrom XFZYD each component hit more than one targetTable 6 demonstrated that quercetin had the highest numberof targets (degree =76) followed by kaempferol (degree=43) beta-sitosterol (degree =35) stigmasterol (degree =19)luteolin (degree=18) baicalein (degree=15) 7-Methoxy-2-methyl isoflavone (degree=12) wogonin (degree=12)nobiletin (degree=11) and naringenin (degree=9) Forinstance quercetin (3310158404101584057-pentahydroxyflavone) is anaturally occurring flavonoid commonly found in fruits andvegetables It regulates multiple biological pathways elicitinginduction of apoptosis as well as inhibiting angiogenesisand proliferation [63 64] Quercetin can attenuate neuronalautophagy and apoptosis in rat traumatic brain injury modelvia activation of PI3KAkt signaling pathway [65] It has alsobeen reported to have a protective ability against oxidativestress and mutagenesis in normal cells [66 67] Kaempferol(3 41015840 5 7 tetrahydroxy flavone) is a yellow-colored flavonoidthat is widely distributed in many botanical families[68] It has been shown to possess a variety of biologicalcharacteristics including effects of anti-inflammatory[69] antioxidative [70] tumor growth inhibition [71] and
Evidence-Based Complementary and Alternative Medicine 11
Mol 22Mol 33
Mol 29Mol 32 Mol 23Mol 20
Mol 97Mol 106
Mol 104Mol 100Mol 94
Mol 63Mol 90
Mol 87
Mol 91Mol 61
Mol 19
Mol 109
Mol 161
Mol 118
Mol 111
Mol 158
Mol 114
Mol 149
Mol 141
Mol 152
Mol 14
CASP3
BCL2
NOS3
SCN5A
F7
ADRA1B
ACHE
F2
Mol 4
Mol 49
Mol 127
APP CASP7
Mol 92
Mol 54
Mol 6
Mol 36
KCNMA1
Mol 27
Mol 13Mol 105Mol 140
Mol 17
Mol 39
Mol 2
Mol 21
Mol 12
Mol 142
Mol 126
Mol 135
Mol 121
Mol 119
Mol 124
Mol 120
Mol 117
Mol 133
Mol 131
Mol 134
PlatycodonGrandiforus
Aurantii Fructus
Licorice
Radix Bupleuri
RehmanniaglutinosaLibosch
AngelicaeSinensis Radix
Mol 8
Mol 73
Mol 75
Mol 77
Mol 80
Mol 7
Mol 82
Mol 76
Mol 74
Mol 60
Mol 46
Mol 150
Mol 151
Mol 112
Mol 115
Mol 11
Mol 116
Mol 113
Mol 110
Mol 1
Mol 9Mol 101Mol 10Mol 93
Mol 103Mol 89 Mol 107
Mol 96PRSS3 LDLRMAPK3 BACE1 EPHB2
Mol 35Mol 81
Mol 86
Mol 85
Mol 84
Mol 88
Mol 83Mol 30
Mol 137
ChuanxiongRhizoma
Mol 3
Mol 57
Mol 139
Achyranthis
Bidentatae
RadixMol 102
CASP8
IGHG1AKT1p53
CHRNA7
Mol 40
SLC6A3 PTPN1
SOD1MAPK1
Mol 38
Mol 95
Carthami Flos
Mol 24
Mol 78 Persicae Semen
Mol 122
Mol 25
Mol 98
TGFB1
GSK3B
PRKCA
Radix Paeoniae Rubra
Mol 52
Mol 42
Mol 62
Mol 138
EGFR
STAT1 PPP3CAPLAT
Stigmasterol
MAP2
Beta-sistosterol Quercetin
Kaempferol
SYNGAP1
CALM
Baicalin
PRKCB
CAV1
CTSD
TNF
DRD1
CDK1
IFNG
FN1BBC3 PRKCD
Mol 132
Mol 160
Mol 58
Mol 67
Mol 69 Mol 43ALB
Mol 64CTNNB1
xiongixioChuanxC nxnxioaamaRhihihizoma
Achyranthisiistntnt
aeeaeBidenBidennntatae
RadixadixR diR di
oniae nianiaiaonRadix PaeoPaePRadix P eeoeonRRRRubra dondonddoodPlatycolatycPlatyycPl ycoyccoPlatPlatycoPlatyccocod
rususrusususruGranG anGranGranGranGrGGranndndiforu
ructusuctuc sructusuctusructusruAurantiirA ntaA ntiitiirantAurantintiurantAur iiii Fru
cecececececececeecLiLLiLLiLiLLLLLLLLiLiLiLLiLLLiLicoric
leuririeuuurieurileurleurieurieurieurieurileRadix BBRadRad Bdix Bdixx R i Bdi BBBadix Radix BBuple
RehmanniaRe mRe m nnRehmanniamReRehmhmannnniamanniaRehmehmanniRR nosasasasaosaosglutilutglutgluulutiglgl titinos
LiboschLibLLibo hschib chLiboschboscLLib chschLibos
icaecaecacaeicaeicAngelAnAngeAngelAnAng lAAngeelielicdix dix dixdixdSinensinensSS nennensSinSSiSinensSinensSine sissis Rad
FloslololFlhamaCartharthhaamami Fl
menmennnnmicae Scae SSem
Figure 6 HB-pC-pT network of XFZYD for treating TBI The triangles with circle backgrounds represent the herbs (HB) the squares andcircles represent the potential compounds (pC) and targets (pT)The red triangles squares and circles represent corresponding HB pC andpT in the Junherbs the same is to aqua representing theChen herbs and periwinkle representing the Zuo-Shi herbsThe claybank squares andcircles represent corresponding pC and pT shared by the Chen and Zuo-Shi herbs the same is to blue representing the overlap between theJun and Zuo-Shi herbs and the green representing the overlap between the Chen and Zuo-Shi herbsThe purple squares and circles representthe corresponding pC and pT shared by the 3 groups of herbs
alleviating insulin resistance in type 2 diabetic rats [72]Beta-sitosterol (BS) is a vegetable-derived compound foundin various plants and is suggested to modulate the immunefunction inflammation and pain levels by controlling theproduction of inflammatory cytokines [73]
For targets analysis CALM possesses the largest degree(degree =84) followed by GSK3B (degree =61) SCN5A(degree = 59) F2 (degree = 43) ACHE (degree=28)F7 (degree=28) ADRA1B (degree=28) NOS3 (degree=20)BCL2 (degree=18) and CASP3 (degree=18) which demon-strated their crucial therapeutic effects for treating TBI Forinstance CALM possess an essential position in calciumsignaling pathway and is related to morphological changesmigration proliferation and secretion of cytokines andreactive oxygen species ofMicroglial cells [74]The activationof CaMKII120572 major isoform of Ca2+calmodulin-dependentprotein kinase (CaMK) in brain is directly associated withthe production of proinflammatory cytokines such as TNF-120572and IL-1120573 [75] GSK-3120573 is a serinethreonine-protein kinasewhich is abundant in the central nervous system (CNS)particularly in neurons [76] It can control gene transcrip-tion axonal transport and cytoskeletal dynamics in growthcones [77] The inhibition of GSK-3120573 attenuates apoptotic
signals and prevents neuronal death [78] There is increasingevidence that prothrombin (F2) and its active derivativethrombin are expressed locally in the central nervous systemBeside the central role in the coagulation cascade the gener-ation of thrombin leads to receptor mediated inflammatoryresponses cell proliferationmodulation cell protection andapoptosis [79 80] The role in brain injury depends upon itsconcentration as higher amounts cause neuroinflammationand apoptosis while lower concentrations might even becytoprotective [81]
Direct tissue damage aswell as hypoxic-ischemic increas-ing anaerobic glycolysis of the brain tissue results in theATP-stores depletion and failure of energy-dependent mem-brane ion pumps especially for voltage-dependent Ca2+ andNa+-channels Accumulated Ca2+ activates lipid peroxidasesproteases and phospholipases and caspases at the sametime increasing the intracellular concentration of free fattyacids and free radicals leading to necrosis or apoptosis ofneurocyte [82] At the same time the activation of residentglial cells microglia and astrocytes and the infiltration ofblood leukocytes secrete various immune mediators elicitedinflammatory responses which subsequently intersect withadjacent pathological cascades including oxidative stress
12 Evidence-Based Complementary and Alternative Medicine
Table 6 Top 10 potential candidate compounds of XFZYD for treating TBI according to 2 centrality indicators
Compound Degree Compound Betweennessquercetin 76 quercetin 01682179kaempferol 43 kaempferol 005501511beta-sitosterol 35 wogonin 005141376Stigmasterol 19 beta-sitosterol 004451294luteolin 18 naringenin 003251738baicalein 15 beta-carotene 002977217-Methoxy-2-methyl isoflavone 12 nobiletin 002787992wogonin 12 7-Methoxy-2-methyl isoflavone 002096439nobiletin 11 luteolin 002090223naringenin 9 baicalein 001453488
Table 7 Top 10 potential targets of XFZYD for treating TBI according to 2 centrality indicators
Targets Degree Targets BetweennessCALM 84 CALM 021452046GSK3B 61 SCN5A 011441591SCN5A 59 GSK3B 009553896F2 43 F2 00689171ACHE 28 BCL2 002651903F7 28 CASP3 002372836ADRA1B 28 F7 002032647NOS3 20 ACHE 001845996BCL2 18 ADRA1B 001704505CASP3 18 NOS3 00166699
excitotoxicity or reparative events including angiogenesisscarring and neurogenesis [83] Function analysis of the 47target proteins regulated by XFZYD was mainly associatedwith core pathophysiology process of TBI (Figure 7) 47 targetproteins were connected with 16 key process related to TBIMost of the targets have one or more links to other biolog-ical process such as apoptosis cell proliferation superoxideanion generation nitric oxide biosynthetic process responseto calcium ion I-kappaB kinaseNF-kappaB signaling andregulation of inflammation The above analysis implied themultifunction character of these target proteins regulated byXFZYD Of these target proteins 25 proteins (53 of the 47)such as TGFB1 EGFR CAV1 MAPK1 PRKCB and AKT1were responsible for regulating the apoptosis process 17 forblood coagulation 14 for cell proliferation and axon genesisand 12 for hypoxia andMAPK cascade We found that TGFB1was the crucial protein because it participated in 9 biologicalprocesses related to TBI such as apoptosis blood coagulationcell proliferation and MAPK cascade followed by EGFR (8)CAV1 (7) MAPK1 (6) PRKCB (6) and AKT1 (6)
Overall these observations strongly support the evidencethat the generated HB-pCminuspT network and pT-F networkhave important roles in treating TBI further validating thedrug targeting approach
Molecule docking was used to further validate the bind-ing mode between candidate compounds and their targetproteins We found that 18 TBI-specific target proteins inter-acted with 91 candidate compounds from XFZYD (Figure 8)
Other 29 target proteins were not discussed for the lackof proper protein crystal structure The detailed moleculedocking results are shown in Table S5The 6 essential proteinsincluding GSK3B AKT1 CDK1 F2 NOS3 and ACHEwere used to elucidate the exact binding mode (Figure 9)Quercetin was located within the binding cavity of AKT1 andCDK1 (Figures 9(a) and 9(b) and S1A B) Four conventionalhydrogen bonds were formed between quercetin and AKT1by interacting with the key amino acids including ILE-290 THR-211 and SER 205 Additionally 120587-120587 interactionsbetween quercetin and TRP-90 were found in the activesite which helped the stabilization of the compound at thebinding site (Figure 9(a) S1A) Figure 9(b) and S1B suggestedthat five conventional hydrogen bonds (LEU-83 ASP-146and LYS-33) and 120587-120587 interaction (PHE-80) were formedbetween quercetin and CDK1 The GSK3B-FA complexes(Figure 9(c) S1C) were stabilized by 6 hydrogen-bondinginteractions between FA and LYS-85 GLU-97 TYR-134 andARG-141 Glyasperin Bmainly bonds to F2 through hydrogenbonds by interacting with the key amino acids includingGLY-193 SER-195 and GLY-219 (Figure 9(d) S1D) andan edge-to-face 120587 minus 120587 interaction was also observed withTYR-228 The GLN-247 GLU-351 and GLY-355 from theactive site pocket of NOS3 participated in the hydrogen-bondformationwith 1-Methoxyphaseollidin (Figure 9(e) S1E)The(-)-Medicocarpin formed a total of 6 hydrogen bonds withSER-293 PHE-295 ARG-296 and TRY-341 in the active siteof ACHE (Figure 9(f) S1F) Besides an edge-to-face 120587 minus 120587
Evidence-Based Complementary and Alternative Medicine 13
KCNMA1
P53
IFNG
PPP3CA
CASP8
SCN5A
I-kappaB kinase NF-kappaB signaling
Regulation of inflammation
Response to calcium ion
TGFB1
TNF
PRKCA
CHRNA7
CAV1
STAT1
MAPK cascadeAKT1
BCL2
APP
EGFR
MAPK1
Angiogenesis
SYNGAP1
MAP2
PTPN1
Axonogenesis
GSK3B
EPHB2
CDK1
CASP7FN1
MAPK3
CTNNB1
F2
Autophagy
Cell proliferation
BACE1
SLC6A3
DRD1
ADRA1BACHE
CALMCASP3
PLAT
Apoptotic process
NOS3
PRKCB
Blood coagulation
Response to hypoxia
Superoxide anion generation
Nitric oxide biosynthetic process
Astrocyte activation
Amyloid-beta regulation
Microglial cell activation
LDLR
PRSS3
F7
PRKCD
SOD1ALB
BBC3
Figure 7 pT-F network of XFZYD for treating TBI 16 biological processes (red square) the 47 target proteins (periwinkle circle) of XFZYDparticipate in for treating TBI
interactionwas also observedwith TYR-337 From the resultshydrogen-bonding and edge-to-face 120587 minus 120587 interactions playkey roles in the proteinminusligand recognition and stabilitywhich may be helpful in determining the inhibitor activitiesAnd the C-T network confirmed the potential therapeuticeffects of the candidate compounds from XFZYD to treatTBI through interacting with the relevant proteins The com-putational analysis further elucidated the accurate moleculemechanisms between active compounds and targets
4 Discussion
Traumatic brain injury (TBI) is a growing public healthproblem worldwide and is a leading cause of death anddisability [84] Although major progress has been made in
understanding the pathophysiology of this injury this hasnot yet led to substantial improvements in outcome by alack of treatments which have proven successful during phaseIII trials for modern medicine [85 86] TCM rooted inthousands of years of history may offer an alternative or acomplementary strategy for the treatment of TBI XFZYDa representative formula in TCM has been used for yearsto treat TBI in China and has been demonstrated to beeffective in clinical practice However its ldquomulticomponentsrdquoand ldquomultitargetsrdquo features make it much difficult to decipherthemolecular mechanisms of XFZYD in the treatment of TBIfrom a systematic perspective if employing routine methods
In the present study a network pharmacology-basedmethod was employed to elucidate the pharmacologicalmechanisms of XFZYD to treat TBI according to the drug
14 Evidence-Based Complementary and Alternative Medicine
Figure 8 C-T network through molecule docking validating 119 potential compounds (green triangles) interacting with 18 potential targets(yellow circles) of XFZYDThe size of the nodes is proportional to the value of degree
combination principle of TCM We first proposed a newmodeling system combining OB and DL screening multipledrug targets prediction and validation network constructionand molecule docking to probe the efficiency of a typicalTCM formula XFZYD for the treatment of TBI The 11herbs from XFZYD possessed 162 bioactive compounds andtargeted 285 proteins There were 5 compounds and 189
target proteins overlapped among the Jun Chen and Zuo-Shigroup Furthermore 47 TBI-specific proteins were targetedby 119 (73) bioactive compounds from XFZYD Similarly 5common compounds and 33 (70) common target proteinsamong the 3 groups of drugs were observed Most of thebioactive ingredients targeted more than one protein The 47target proteins regulated several essential pathophysiological
Evidence-Based Complementary and Alternative Medicine 15
(a) (b) (c)
(d) (e) (f)
Figure 9 Hydrogen-bonding networks within the binding site of the compoundminustarget complexes obtained from molecule docking(a) AKT1-quercetin (b) CDK1-quercetin (c) GSK3B-FA (d) F2-glyasperin B (e) NOS3-1-Methoxyphaseollidin and (f) ACHE-(-)-Medicocarpin The molecules are presented as ball and stick models Active site amino acid residues are represented as lines Dotted bluelines in these pictures represent hydrogen bonds with distance unit of A Other O and N atoms are colored as red and blue respectively
processes of TBI that referred to apoptosis inflammationcell proliferation superoxide anion generation nitric oxidebiosynthetic process response to calcium ion etc The aboveanalysis reveals that the synergistic action mechanisms ofXFZYDmay be (1) bioactive compounds overlapping amongthe different group of herbs (2) specific bioactive com-pounds from different groups of herbs targeting the sameproteins (3) specific bioactive compounds from differentgroups of herbs targeting different proteins which participatein the same pathophysiological process of the disease Toa certain degree the 5 compounds including quercetinstigmasterol kaempferol baicalin and beta-sitosterol playedessential role in XFZYD for TBI treatment MAPK3MAPK1AKT1 PRKCA TNF PRKCB EGFR BCL2 GSK3B CASP3PPP3CA and NOS3 were the main target proteins regulatedby XFZYD in the treatment of TBI
Interestingly Beta-carotene (Mol 43) from Carthami Flos(the Jun herb) specifically regulated 120573-Catenin (CTNNB1)and played critical role for curing TBI The 120573-Catenin is acritical downstream component of the Wnt pathway whichplays essential role in the regulation of mammalian neuraldevelopment [87] In vitro and in vivo studies demonstrate
that the Wnt120573-catenin pathway regulates the proliferationand differentiation of neural progenitor cells [88] Neuronaldifferentiation is induced by overexpression of 120573-cateninor the pharmacological inhibition of GSK3120573 (the phospho-rylating enzyme of 120573-catenin) [89 90] This pathway alsopromotes blood vessel formation during vascular develop-ment as well as the vascular repair process after TBI [91]In addition wogonin (Mol 160) from Achyranthis BidentataeRadix (the Chen herb) specifically targeted fibronectin (FN1)Bcl-2-binding component 3 (BBC3) and Protein KinaseC delta type (PRKCD) FN1 an important component ofthe extracellular matrix (ECM) environment promotes cellmigration neurite outgrowth and synapse formation dur-ing neural development [92] It aggregates in the injuredbrain and plays a neuroprotection role through antiapoptosisand anti-inflammation ways following TBI [93 94] BBC3namely p53 upregulated modulator of apoptosis (PUMA) iscritical for the p53-dependent apoptosis pathway which playsan important role in hippocampal neuronal loss and associ-ated cognitive deficits [95] PRKCD one of PKC isoformsactivates signal transduction pathways involved in neuronalregeneration [96] synaptic transmissionplasticity [97] and
16 Evidence-Based Complementary and Alternative Medicine
activation of apoptosis processes [98] as well as higher brainfunctions such as learning andmemory [99]The activators ofPKCare effective for the treatment of TBI [100] FurthermoreMitogen-activated protein kinase 3 (MAPK3) Beta-secretase1 (BACE1) Ephrin type-B receptor 2 (EphB) and low-densitylipoprotein receptor (LDLR) were specifically targeted bythe ingredients in the Zuo-Shi herbs Naringenin (Mol133) targeted LDLR MAPK3 while euchrenone (Mol 60)anchored BACE1 and nobiletin (Mol 134) targeted EphB2MAPK3 is an essential component of the MAP kinase signaltransduction pathway It has been appreciated recently thatthe ERK12 cascade plays a fundamental role in synapticplasticity and memory [101] BACE1 is responsible for pro-duction of A120573 from amyloid precursor protein (APP) A120573can cause cell death activate inflammatory pathways [102]and prime proapoptotic pathways for activation by otherinsults [103] The blocking of BACE1 can ameliorate motorand cognitive deficits and reduce cell loss after experimentalTBI in mice [104] EphB is localized to synaptic sites inhippocampal neurons [105] The interaction between EphBand NMDA receptors regulates excitatory synapse formation[106] LDLR acts as an important receptor that facilitatesbrain A120573 clearance and inhibits amyloid deposition [107]and then ameliorates Alzheimerrsquos disease neuropathologyafter TBI [108] The analysis above indicates the ldquoJunrdquoldquoChenrdquo and ldquoZuo-Shirdquo herbs from XFZYD trigger theirspecific targets regulation respectively for the therapeuticeffects
XFZYD is a very famous traditional Chinese formula inpromoting qi circulation and removing blood stasis accord-ing to TCM theory However several limited researches havedemonstrated its efficacy for treating TBI such as anti-inflammatory and synaptic regulation [30 31] which arein accord with our study However previous studies merelypartially deciphered the molecule mechanism of XFZYD fortreating TBI This study reports 119 bioactive compounds inXFZYD that target 47 TBI-specific proteins such as MAPK3MAPK1 AKT1 PRKCA TNF PRKCB and EGFR Theseproteins regulate several crucial pathophysiological processesof TBI such as apoptosis inflammation blood coagulationand axon genesis Our study demonstrates that the therapeu-tic actions of XFZYD refer to ldquomulticompoundsrdquo ldquomultitar-getsrdquo features rather than only the improvement of bloodcirculation With the help of molecule docking methodwe further validate the interactions between bioactive com-pounds and potential targets of XFZYD The hydrogen-bonding and edge-to-face 120587 minus120587 interactions play key roles inthe proteinminusligand recognition and stability This provides avaluable reference for further experimental investigations ofbioactive ingredients and therapeutic targets of XFZYD fortreating TBI
5 Conclusion
Our work successfully illuminates the efficiency of XFZYDfor the treatment of TBI as well as herb combination ruleof TCM formula Network pharmacology with moleculedocking method confirms the ldquomulticompounds multitar-getsrdquo therapeutic actions of XFZYD in the treatment of TBI
The present work may provide valuable evidence for furtherclinical application of XFZYD for treating TBI
Abbreviations
TBI Traumatic brain injuryXFZYD Xuefu Zhuyu decoctionTCM Traditional Chines medicineDL Drug-likenessOB Oral bioavailabilityHB-cC-cT Herb-candidate compound-candidate
targetHB-pC-pT Herb-potential compound-potential targetCALM CalmodulinGSK3B Glycogen synthase kinase-3 betaSCN5A Sodium channel protein type 5 subunit
alphaF2 ProthrombinACHE AcetylcholinesteraseGO Gene OntologyKEGG Kyoto Encyclopedia of Genes and
Genomes
Data Availability
The data used to support the findings of this study areavailable from the corresponding author upon request
Conflicts of Interest
The authors declare that they have no conflicts of interest
Authorsrsquo Contributions
YangWang and Zebing Huang participated in the conceptionand design of the study YuanyuanZhong YangWang ZebingHuang Jiekun Luo Tao Tang and Pengfei Li acquired andanalyzed the data Yuanyuan Zhong Tao Liu and HanjinCui drafted and revised the manuscript The correspondingauthor and all of the authors have read and approved the finalsubmitted manuscript
Acknowledgments
This work was supported by the National Natural ScienceFoundation of China (Nos 81673719 81874409 81603670and 81803948) Project funded byChina Postdoctoral ScienceFoundation (Nos 2016M600639 and 2017T100614)
Supplementary Materials
Table S1 162 bioactive compounds in XFZYD Table S2 targetproteins of XFZYD Table S3 TBI-specific proteins Table S4119 potential compounds of XFZYD for treating TBI TableS5 docking result of 18 target proteins with 91 potential com-pounds Fig S1 the exact bindingmode between active ingre-dients and protein targets obtained from molecule docking(A) AKT1-quercetin (B) CDK1-quercetin (C) GSK3B-FA
Evidence-Based Complementary and Alternative Medicine 17
(D) F2-glyasperin B (E) NOS3-1-Methoxyphaseollidin and(F)ACHE-(-)-Medicocarpin (Supplementary Materials)
References
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[2] J A Langlois W Rutland-Brown and M M Wald ldquoTheepidemiology and impact of traumatic brain injury a briefoverviewrdquo The Journal of Head Trauma Rehabilitation vol 21no 5 pp 375ndash378 2006
[3] H A Alsulaim B J Smart A O Asemota et al ldquoConsciousstatus predictsmortality among patientswith isolated traumaticbrain injury in administrative datardquo The American Journal ofSurgery vol 214 no 2 pp 207ndash210 2017
[4] M Majdan D Plancikova A Maas et al ldquoYears of life lost dueto traumatic brain injury in Europe A cross-sectional analysisof 16 countriesrdquo PLoS Medicine vol 14 no 7 p e1002331 2017
[5] P Cheng P Yin P Ning et al ldquoTrends in traumatic braininjury mortality in China 2006ndash2013 A population-basedlongitudinal studyrdquo PLoS Medicine vol 14 no 7 Article IDe1002332 2017
[6] M V Russo and D B McGavern ldquoInflammatory neuroprotec-tion following traumatic brain injuryrdquo Science vol 353 no 6301pp 783ndash785 2016
[7] WWang H Li J Yu et al ldquoProtective Effects of ChineseHerbalMedicine Rhizoma drynariae in Rats After Traumatic BrainInjury and Identification of Active Compoundrdquo MolecularNeurobiology vol 53 no 7 pp 4809ndash4820 2016
[8] M Das S Mohapatra and S S Mohapatra ldquoNew perspectiveson central and peripheral immune responses to acute traumaticbrain injuryrdquo Journal of Neuroinflammation vol 9 p 236 2012
[9] J W Finnie ldquoNeuroinflammation Beneficial and detrimentaleffects after traumatic brain injuryrdquo Inflammopharmacologyvol 21 no 4 pp 309ndash320 2013
[10] T Cheng W Wang Q Li et al ldquoCerebroprotection of flavanol(minus)-epicatechin after traumatic brain injury viaNrf2-dependentand -independent pathwaysrdquo Free Radical Biology amp Medicinevol 92 pp 15ndash28 2016
[11] W Young ldquoRole of calcium in central nervous system injuriesrdquoJournal of Neurotrauma vol 9 Suppl 1 pp S9ndashS25 1992
[12] R Bullock A Zauner J S Myseros et al ldquoEvidence forProlonged Release of Excitatory Amino Acids in Severe HumanHead Trauma Relationship to Clinical Eventsrdquo Annals of theNew York Academy of Sciences vol 765 no 1 pp 290ndash297 1995
[13] A T Mazzeo A Beat A Singh and M R Bullock ldquoTherole of mitochondrial transition pore and its modulation intraumatic brain injury and delayed neurodegeneration afterTBIrdquo Experimental Neurology vol 218 no 2 pp 363ndash370 2009
[14] S Gennai A Monsel Q Hao et al ldquoCell-Based therapy fortraumatic brain injuryrdquo British Journal of Anaesthesia vol 115no 2 pp 203ndash212 2015
[15] J Ghajar ldquoTraumatic brain injuryrdquo The Lancet vol 356 no9233 pp 923ndash929 2000
[16] D J Loane and A I Faden ldquoNeuroprotection for traumaticbrain injury translational challenges and emerging therapeuticstrategiesrdquoTrends in Pharmacological Sciences vol 31 no 12 pp596ndash604 2010
[17] Y Wang X Fan T Tang et al ldquoRhein and rhubarb simi-larly protect the blood-brain barrier after experimental trau-matic brain injury via gp91phox subunit of NADPH oxidaseROSERKMMP-9 signaling pathwayrdquo Scientific Reports vol 6p 37098 2016
[18] D-X Kong X-J Li and H-Y Zhang ldquoWhere is the hopefor drug discovery Let history tell the futurerdquo Drug DiscoveryTherapy vol 14 no 3-4 pp 115ndash119 2009
[19] F Cheung ldquoTCM made in Chinardquo Nature vol 480 no 7378pp S82ndashS83 2011
[20] C Fu Z Xia Y Liu et al ldquoQualitative analysis of majorconstituents from Xue Fu Zhu Yu Decoction using ultra highperformance liquid chromatographywith hybrid ion trap time-of-flight mass spectrometryrdquo Journal of Separation Science vol39 no 17 pp 3457ndash3468 2016
[21] H-J Zhang and Y-Y Cheng ldquoAn HPLCMS method for iden-tifying major constituents in the hypocholesterolemic extractsof Chinese medicine formula lsquoXue-Fu-Zhu-Yu decoctionrsquordquoBiomedical Chromatography vol 20 no 8 pp 821ndash826 2006
[22] L Zhang L Zhu YWang et al ldquoCharacterization and quantifi-cation of major constituents of Xue Fu Zhu Yu by UPLC-DAD-MSMSrdquo Journal of Pharmaceutical and Biomedical Analysisvol 62 pp 203ndash209 2012
[23] X Yang X Xiong G Yang and J Wang ldquoChinese patentmedicine Xuefu Zhuyu capsule for the treatment of unstableangina pectoris a systematic review of randomized controlledtrialsrdquo Complementary Therapies in Medicine vol 22 no 2 pp391ndash399 2014
[24] J Wang X Yang F Chu et al ldquoThe effects of Xuefu Zhuyuand Shengmai on the evolution of syndromes and inflammatorymarkers in patients with unstable angina pectoris after percuta-neous coronary intervention a randomised controlled clinicaltrialrdquoEvidence-BasedComplementary andAlternativeMedicinevol 2013 Article ID 896467 9 pages 2013
[25] Q Zhang H Yu J Qi et al ldquoNatural formulas and the natureof formulas Exploring potential therapeutic targets based ontraditional Chinese herbal formulasrdquo PLoS ONE vol 12 no 2p e0171628 2017
[26] J LeeWHsu T Yen et al ldquoTraditional ChinesemedicineXue-Fu-Zhu-Yu decoction potentiates tissue plasminogen activatoragainst thromboembolic stroke in ratsrdquo Journal of Ethnophar-macology vol 134 no 3 pp 824ndash830 2011
[27] Y-C Shen C-K Lu K-T Liou et al ldquoCommon and uniquemechanisms of Chinese herbal remedies on ischemic strokemice revealed by transcriptome analysesrdquo Journal of Ethnophar-macology vol 173 pp 370ndash382 2015
[28] F Xue-hai G Yan and L Jian-tao ldquoTherapeutic effect of XiefuZhuyu decoction joint brain protein hydrolysat injection intraumatic brain injury and its effect on cerebrospinal fluid ofET-1rdquo Chinese Journal of Biochemical Pharmaceutics no 02 pp55ndash58 2017
[29] L Shuxiang W Jingchun C Jie et al ldquoEffect of Xuefu Zhuyudecoction on the neural functional recovery and living abilityin patients with craniocerebral injuryrdquo Modern Journal ofIntegrated Traditional Chinese andWesternMedicine no 04 pp350ndash352 2015
[30] L Zhong W Ning and W Dong-pi ldquoModel Study of theTherapy of Replenishing Qi and Activating Blood Circulationon Protecting the Brain Impairment Caused by Hypoxic -ischemic Encephalopathy in SD Ratsrdquo Journal of TraditionalChinese Medicine vol 07 pp 1552ndash1555 2011
18 Evidence-Based Complementary and Alternative Medicine
[31] M Sun X-P Zhan C-Y Jin J-Z Shan S Xu and Y-LWang ldquoclinical observation on treatment of post-craniocerebraltraumatic mental disorder by integrative medicinerdquo ChineseJournal of Integrative Medicine vol 14 no 2 pp 137ndash141 2008
[32] Z Xing Z Xia W Peng et al ldquoXuefu Zhuyu decoction atraditional Chinese medicine provides neuroprotection in arat model of traumatic brain injury via an anti-inflammatorypathwayrdquo Scientific Reports vol 6 Article ID 20040 2016
[33] J Zhou T Liu H Cui et al ldquoXuefu zhuyu decoction improvescognitive impairment in experimental traumatic brain injuryvia synaptic regulationrdquo Oncotarget vol 8 no 42 2017
[34] S Li ldquoExploring traditional chinese medicine by a novel thera-peutic concept of network targetrdquo Chinese Journal of IntegrativeMedicine vol 22 no 9 pp 647ndash652 2016
[35] S Li and B Zhang ldquoTraditional Chinese medicine networkpharmacology theory methodology and applicationrdquo ChineseJournal of Natural Medicines vol 11 no 2 pp 110ndash120 2013
[36] A L Hopkins ldquoNetwork pharmacology the next paradigm indrug discoveryrdquoNature Chemical Biology vol 4 no 11 pp 682ndash690 2008
[37] Y Li C Han J Wang et al ldquoInvestigation into the mechanismof Eucommia ulmoides Oliv based on a systems pharmacologyapproachrdquo Journal of Ethnopharmacology vol 151 no 1 pp452ndash460 2014
[38] Y Yao X Zhang Z Wang et al ldquoDeciphering the combinationprinciples of Traditional Chinese Medicine from a systemspharmacology perspective based on Ma-huang DecoctionrdquoJournal of Ethnopharmacology vol 150 no 2 pp 619ndash638 2013
[39] Bo Zhang Xu Wang and Shao Li ldquoAn Integrative Platform ofTCM Network Pharmacology and Its Application on a HerbalFormula Qing-Luo-Yinrdquo Evidence-Based Complementary andAlternativeMedicine vol 2013 Article ID 456747 12 pages 2013
[40] J Ru P Li J Wang et al ldquoTCMSP a database of systemspharmacology for drug discovery from herbal medicinesrdquoJournal of Cheminformatics vol 6 no 1 p 13 2014
[41] J V Turner D J Maddalena and S Agatonovic-KustrinldquoBioavailability Prediction Based on Molecular Structure for aDiverse Series of Drugsrdquo Pharmaceutical Research vol 21 no 1pp 68ndash82 2004
[42] X XuW Zhang CHuang et al ldquoAnovel chemometricmethodfor the prediction of human oral bioavailabilityrdquo InternationalJournal of Molecular Sciences vol 13 no 6 pp 6964ndash6982 2012
[43] A Zhang W Pan J Lv and H Wu ldquoProtective Effect ofAmygdalin on LPS-Induced Acute Lung Injury by InhibitingNF-120581B and NLRP3 Signaling Pathwaysrdquo Inflammation vol 40no 3 pp 745ndash751 2017
[44] X Wei H Liu X Sun et al ldquoHydroxysafflor yellow A protectsrat brains against ischemia-reperfusion injury by antioxidantactionrdquo Neuroscience Letters vol 386 no 1 pp 58ndash62 2005
[45] W PWalters andM A Murcko ldquoPrediction of lsquodrug-likenessrsquordquoAdvanced Drug Delivery Reviews vol 54 no 3 pp 255ndash2712002
[46] Y Yamanishi M Kotera M Kanehisa and S Goto ldquoDrug-target interactionprediction from chemical genomic and phar-macological data in an integrated frameworkrdquo Bioinformaticsvol 26 no 12 pp i246ndashi254 2010
[47] H Yu J Chen X Xu et al ldquoA systematic prediction ofmultiple drug-target interactions from chemical genomic andpharmacological datardquo PLoS ONE vol 7 no 5 Article IDe37608 2012
[48] X Chen Z L Ji and Y Z Chen ldquoTTD therapeutic targetdatabaserdquo Nucleic Acids Research vol 30 no 1 pp 412ndash4152002
[49] A Hamosh A F Scott J S Amberger C A Bocchini andV AMcKusick ldquoOnline Mendelian Inheritance in Man (OMIM) aknowledgebase of human genes and genetic disordersrdquo NucleicAcids Research vol 33 pp D514ndashD517 2005
[50] T S Keshava Prasad RGoel K Kandasamy et al ldquoHuman pro-tein reference databasemdash2009 updaterdquo Nucleic Acids Researchvol 37 no 1 pp D767ndashD772 2009
[51] A Franceschini D Szklarczyk S Frankild et al ldquoSTRING v91protein-protein interaction networks with increased coverageand integrationrdquoNucleic Acids Research vol 41 no 1 pp D808ndashD815 2013
[52] P Shannon A Markiel O Ozier et al ldquoCytoscape a softwareEnvironment for integratedmodels of biomolecular interactionnetworksrdquo Genome Research vol 13 no 11 pp 2498ndash25042003
[53] G Dennis Jr B T Sherman D A Hosack et al ldquoDAVIDDatabase for Annotation Visualization and IntegratedDiscov-eryrdquo Genome Biology vol 4 no 5 p P3 2003
[54] L Yang X Zhao and X Tang ldquoPredicting disease-related pro-teins based on clique backbone in protein-protein interactionnetworkrdquo International Journal of Biological Sciences vol 10 no7 pp 677ndash688 2014
[55] S MThomas and J S Brugge ldquoCellular functions regulated bySRC family kinasesrdquo Annual Review of Cell and DevelopmentalBiology vol 13 pp 513ndash609 1997
[56] D Z Liu F R Sharp K C Van et al ldquoInhibition of Src familykinases protects hippocampal neurons and improves cognitivefunction after traumatic brain injuryrdquo Journal of Neurotraumavol 31 no 14 pp 1268ndash1276 2014
[57] B D Manning and A Toker ldquoAKTPKB Signaling Navigatingthe Networkrdquo Cell vol 169 no 3 pp 381ndash405 2017
[58] Y Kitagishi and S Matsuda ldquoDiets involved in PPAR andPI3KAKTPTEN pathway may contribute to neuroprotectionin a traumatic brain injuryrdquoAlzheimerrsquos ResearchampTherapy vol5 no 5 p 42 2013
[59] D Chen L Bao S-Q Lu and F Xu ldquoSerum albumin andprealbuminpredict the poor outcomeof traumatic brain injuryrdquoPLoS ONE vol 9 no 3 Article ID e93167 2014
[60] J Yang Z Wu N Renier et al ldquoPathological axonal deaththrough aMapk cascade that triggers a local energy deficitrdquoCellvol 160 no 1-2 pp 161ndash176 2015
[61] E J Huang and L F Reichardt ldquoTrk receptors roles in neuronalsignal transductionrdquoAnnual Review of Biochemistry vol 72 pp609ndash642 2003
[62] J W Lee and R Juliano ldquoMitogenic signal transductionby integrin- and growth factor receptor-mediated pathwaysrdquoMolecules and Cells vol 17 no 2 pp 188ndash202 2004
[63] C S Yang J M Landau M T Huang and H L NewmarkldquoInhibition of carcinogenesis by dietary polyphenolic com-poundsrdquo Annual Review of Nutrition vol 21 pp 381ndash406 2001
[64] A Murakami H Ashida and J Terao ldquoMultitargeted cancerprevention by quercetinrdquoCancer Letters vol 269 no 2 pp 315ndash325 2008
[65] G Du Z Zhao Y Chen et al ldquoQuercetin attenuates neuronalautophagy and apoptosis in rat traumatic brain injury modelvia activation of PI3KAkt signaling pathwayrdquo NeurologicalResearch vol 38 no 11 pp 1ndash8 2016
Evidence-Based Complementary and Alternative Medicine 19
[66] Q Cai R O Rahn and R Zhang ldquoDietary flavonoidsquercetin luteolin andgenistein reduce oxidativeDNAdamageand lipid peroxidation and quench free radicalsrdquoCancer Lettersvol 119 no 1 pp 99ndash107 1997
[67] G A Bongiovanni E A Soria and A R Eynard ldquoEffectsof the plant flavonoids silymarin and quercetin on arsenite-induced oxidative stress in CHO-K1 cellsrdquo Food and ChemicalToxicology vol 45 no 6 pp 971ndash976 2007
[68] H Li L Yang Y Zhang et al ldquoKaempferol inhibits fibroblastcollagen synthesis proliferation and activation in hypertrophicscar via targeting TGF-beta receptor type Irdquo Biomedicine ampPharmacotherapy = Biomedecine amp Pharmacotherapie vol 83pp 967ndash974 2016
[69] K P Devi D S Malar S F Nabavi et al ldquoKaempferol andinflammation From chemistry to medicinerdquo PharmacologicalResearch vol 99 pp 1ndash10 2015
[70] M Zhou H Ren J Han W Wang Q Zheng and DWang ldquoProtective effects of kaempferol against myocardialischemiareperfusion injury in isolated rat heart via antioxidantactivity and inhibition of glycogen synthase kinase-3120573rdquo Oxida-tive Medicine and Cellular Longevity vol 2015 p 481405 2015
[71] C-F Lee J-S Yang F-J Tsai et al ldquoKaempferol inducesATMp53-mediated death receptor and mitochondrial apop-tosis in human umbilical vein endothelial cellsrdquo InternationalJournal of Oncology vol 48 no 5 pp 2007ndash2014 2016
[72] C Luo H Yang C Tang et al ldquoKaempferol alleviates insulinresistance via hepatic IKKNF-120581B signal in type 2 diabetic ratsrdquoInternational Immunopharmacology vol 28 no 1 pp 744ndash7502015
[73] A B Awad and C S Fink ldquoPhytosterols as anticancer dietarycomponents evidence and mechanism of actionrdquo Journal ofNutrition vol 130 no 9 pp 2127ndash2130 2000
[74] K Farber and H Kettenmann ldquoFunctional role of calciumsignals for microglial functionrdquoGlia vol 54 no 7 pp 656ndash6652006
[75] Y Lu Y Gu X Ding et al ldquoIntracellular Ca2+ homeostasisand JAK1STAT3 pathway are involved in the protective effectof propofol on BV2 microglia against hypoxia-induced inflam-mation and apoptosisrdquo PLoS ONE vol 12 no 5 p e01780982017
[76] K Leroy and J-P Brion ldquoDevelopmental expression and local-ization of glycogen synthase kinase-3120573 in rat brainrdquo Journal ofChemical Neuroanatomy vol 16 no 4 pp 279ndash293 1999
[77] C-M Liu E-M Hur and F-Q Zhou ldquoCoordinating geneexpression and axon assembly to control axon growth Potentialrole ofGSK3 signalingrdquo Frontiers inMolecularNeuroscience vol3 p 3 2012
[78] D A E Cross A A Culbert K A Chalmers L Facci S DSkaper and A D Reith ldquoSelective small-molecule inhibitors ofglycogen synthase kinase-3 activity protect primary neuronesfrom deathrdquo Journal of Neurochemistry vol 77 no 1 pp 94ndash102 2001
[79] V S Ossovskaya and NW Bunnett ldquoProtease-activated recep-tors contribution to physiology and diseaserdquo PhysiologicalReviews vol 84 no 2 pp 579ndash621 2004
[80] M Steinhoff J Buddenkotte V Shpacovitch et al ldquoProteinase-activated receptors transducers of proteinase-mediated signal-ing in inflammation and immune responserdquoEndocrine Reviewsvol 26 no 1 pp 1ndash43 2005
[81] H Krenzlin V Lorenz S Danckwardt O Kempski and BAlessandri ldquoThe importance of thrombin in cerebral injury and
diseaserdquo International Journal of Molecular Sciences vol 17 no1 2016
[82] M J McGinn and J T Povlishock ldquoPathophysiology of Trau-matic Brain InjuryrdquoNeurosurgery Clinics of North America vol27 no 4 pp 397ndash407 2016
[83] T Woodcock and M C Morganti-Kossmann ldquoThe role ofmarkers of inflammation in traumatic brain injuryrdquo Frontiersin Neurology vol 4 article 18 2013
[84] F Tanriverdi H J Schneider G Aimaretti B E Masel F FCasanueva and F Kelestimur ldquoPituitary dysfunction after trau-matic brain injury A clinical and pathophysiological approachrdquoEndocrine Reviews vol 36 no 3 pp 305ndash342 2015
[85] J V Rosenfeld A I Maas P Bragge M C Morganti-Kossmann G T Manley and R L Gruen ldquoEarly managementof severe traumatic brain injuryrdquoThe Lancet vol 380 no 9847pp 1088ndash1098 2012
[86] B M Aertker S Bedi and C S Cox ldquoStrategies for CNS repairfollowing TBIrdquo Experimental Neurology vol 275 no 3 pp 411ndash426 2016
[87] K Adachi Z Mirzadeh M Sakaguchi et al ldquo120573-catenin signal-ing promotes proliferation of progenitor cells in the adultmousesubventricular zonerdquo Stem Cells vol 25 no 11 pp 2827ndash28362007
[88] Y Hirabayashi and Y Gotoh ldquoStage-dependent fate determina-tion of neural precursor cells in mouse forebrainrdquo NeuroscienceResearch vol 51 no 4 pp 331ndash336 2005
[89] S Ding T Y H Wu A Brinker et al ldquoSynthetic smallmolecules that control stem cell faterdquo Proceedings of theNationalAcadamy of Sciences of the United States of America vol 100 no13 pp 7632ndash7637 2003
[90] N Israsena M Hu W Fu L Kan and J A Kessler ldquoThepresence of FGF2 signaling determines whether 120573-cateninexerts effects on proliferation or neuronal differentiation ofneural stem cellsrdquo Developmental Biology vol 268 no 1 pp220ndash231 2004
[91] A Salehi A Jullienne M Baghchechi et al ldquoUp-regulation ofWnt120573-catenin expression is accompanied with vascular repairafter traumatic brain injuryrdquo Journal of Cerebral Blood Flow ampMetabolism vol 38 no 2 pp 274ndash289 2017
[92] L F Reichardt and K J Tomaselli ldquoExtracellular matrixmolecules and their receptors Functions in neural develop-mentrdquoAnnual Review of Neuroscience vol 14 pp 531ndash570 1991
[93] R M Gibson S E Craig L Heenan C Tournier and M JHumphries ldquoActivation of integrin 12057251205731 delays apoptosis ofNtera2 neuronal cellsrdquoMolecularandCellularNeuroscience vol28 no 3 pp 588ndash598 2005
[94] C C Tate A J Garcıa andMC LaPlaca ldquoPlasma fibronectin isneuroprotective following traumatic brain injuryrdquoExperimentalNeurology vol 207 no 1 pp 13ndash22 2007
[95] C Culmsee and M P Mattson ldquop53 in neuronal apoptosisrdquoBiochemical and Biophysical Research Communications vol 331no 3 pp 761ndash777 2005
[96] M S Geddis and V Rehder ldquoThe phosphorylation state ofneuronal processes determines growth cone formation afterneuronal injuryrdquo Journal of Neuroscience Research vol 74 no2 pp 210ndash220 2003
[97] M L Craske M Fivaz N N Batada and T Meyer ldquoSpines andneurite branches function as geometric attractors that enhanceprotein kinase C actionrdquoThe Journal of Cell Biology vol 170 no7 pp 1147ndash1158 2005
20 Evidence-Based Complementary and Alternative Medicine
[98] A Muscella C Vetrugno L G Cossa et al ldquoApoptosis by[Pt(OOrsquo-acac)(gamma-acac)(DMS)] requires PKC-deltamedi-ated p53 activation in malignant pleural mesotheliomardquo PloSone vol 12 no 7 Article ID e0181114 2017
[99] J S Bonini W C Da Silva L R M Bevilaqua J H MedinaI Izquierdo and M Cammarota ldquoOn the participation ofhippocampal PKC in acquisition consolidation and reconsol-idation of spatial memoryrdquo Neuroscience vol 147 no 1 pp 37ndash45 2007
[100] O Zohar R Lavy X Zi et al ldquoPKC activator therapeutic formild traumatic brain injury in micerdquo Neurobiology of Diseasevol 41 no 2 pp 329ndash337 2011
[101] J David Sweatt ldquoTheneuronalMAPkinase cascade a biochem-ical signal integration system subserving synaptic plasticity andmemoryrdquo Journal ofNeurochemistry vol 76 no 1 pp 1ndash10 2001
[102] Y Matsuoka M Picciano B Maleste et al ldquoInflammatoryresponses to amyloidosis in a transgenic mouse model ofAlzheimerrsquos diseaserdquo The American Journal of Pathology vol158 no 4 pp 1345ndash1354 2001
[103] L Esposito L Gan G-Q Yu C Essrich and L MuckeldquoIntracellularly generated amyloid-120573 peptide counteracts theantiapoptotic function of its precursor protein and primesproapoptotic pathways for activation by other insults in neu-roblastoma cellsrdquo Journal of Neurochemistry vol 91 no 6 pp1260ndash1274 2004
[104] D J Loane A Pocivavsek C E-H Moussa et al ldquoAmyloidprecursor protein secretases as therapeutic targets for traumaticbrain injuryrdquo Nature Medicine vol 15 no 4 pp 377ndash379 2009
[105] R Torres B L Firestein H Dong et al ldquoPDZ proteins bindcluster and synaptically colocalize with Eph receptors and theirephrin ligandsrdquo Neuron vol 21 no 6 pp 1453ndash1463 1998
[106] M B Dalva M A Takasu M Z Lin et al ldquoEphB receptorsinteract with NMDA receptors and regulate excitatory synapseformationrdquo Cell vol 103 no 6 pp 945ndash956 2000
[107] J M Basak P B Verghese H Yoon J Kim and D MHoltzman ldquoLow-density lipoprotein receptor represents anapolipoprotein E-independent pathway of A120573 uptake anddegradation by astrocytesrdquoThe Journal of Biological Chemistryvol 287 no 17 pp 13959ndash13971 2012
[108] L Yao X Gu Q Song et al ldquoNanoformulated alpha-mangostinameliorates Alzheimerrsquos disease neuropathology by elevatingLDLR expression and accelerating amyloid-beta clearancerdquoJournal of Controlled Release vol 226 pp 1ndash14 2016
Stem Cells International
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Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
10 Evidence-Based Complementary and Alternative Medicine
Table 4 Top 10 proteins of TBI specific proteins according to 2 centrality indicators
Proteins Degree Proteins BetweennessSRC 164 APP 0071814AKT1 157 ALB 0069343ALB 153 SRC 0058568EGFR 139 EGFR 0052938APP 134 AKT1 0045466GAPDH 131 HTT 0037164HSP90AA1 108 GAPDH 0034549BCL2 106 HSP90AA1 0027596MAPK1 105 CTNNB1 0021759HRAS 105 GNB1 0019492Note the centrality indicators identify the important nodes within the network Higher degree centrality and betweenness centrality indicate greaterimportance
Table 5 Top 10 significantly enriched KEGG pathways in TBI-specific proteins
Pathway ID Pathway description Gene count FDR4010 MAPK signaling pathway 44 299E-235200 Pathways in cancer 43 113E-184722 Neurotrophin signaling pathway 41 101E-344151 PI3K-Akt signaling pathway 40 111E-154510 Focal adhesion 39 292E-225205 Proteoglycans in cancer 39 410E-215010 Alzheimer s disease 34 124E-204015 Rap1 signaling pathway 33 769E-174014 Ras signaling pathway 33 646E-164020 Calcium signaling pathway 31 505E-17
0
001
002
003
004
005
006
007
008
0 20 40 60 80 100 120 140 160 180
Betw
eenn
ess
Degree
APP
ALB SRCEGFR
AKT1HTT
Figure 5 Relationship between degree and betweenness centralityin the TBI-specific protein interaction network
for the 119 potential compounds is shown in Table S4Similarly 5 pharmacologically active ingredients in the JunChen and Zuo-Shi groups including quercetin stigmasterolkaempferol baicalin and beta-sitosterol anchored 33 TBI-specific proteins such as CALM SCN5A F2 ACHE F7ADRA1B NOS3 BCL2 CASP3 and AKT1 11 Jun-specificcompounds targeted 2 specific targets (ALB CTNNB1) while12 Chen-specific compounds anchored 3 unique proteins(PRKCD FN1 and BC3) The Zuo-Shi herbs possessed thelargest number of active compounds (89) and targeted 5
unique proteins including EPHB2 BACE1 LDLR MAPK3and PRSS3 GSK3Bwas the common target between theChenandZuo-Shi drugs KCNMA1 CASP7 andAPPwere targetedby the Jun and Zuo-Shi drugs
The top 10 candidate compounds and targets to treat TBIwere showed in Tables 6 and 7 Formost of active compoundsfrom XFZYD each component hit more than one targetTable 6 demonstrated that quercetin had the highest numberof targets (degree =76) followed by kaempferol (degree=43) beta-sitosterol (degree =35) stigmasterol (degree =19)luteolin (degree=18) baicalein (degree=15) 7-Methoxy-2-methyl isoflavone (degree=12) wogonin (degree=12)nobiletin (degree=11) and naringenin (degree=9) Forinstance quercetin (3310158404101584057-pentahydroxyflavone) is anaturally occurring flavonoid commonly found in fruits andvegetables It regulates multiple biological pathways elicitinginduction of apoptosis as well as inhibiting angiogenesisand proliferation [63 64] Quercetin can attenuate neuronalautophagy and apoptosis in rat traumatic brain injury modelvia activation of PI3KAkt signaling pathway [65] It has alsobeen reported to have a protective ability against oxidativestress and mutagenesis in normal cells [66 67] Kaempferol(3 41015840 5 7 tetrahydroxy flavone) is a yellow-colored flavonoidthat is widely distributed in many botanical families[68] It has been shown to possess a variety of biologicalcharacteristics including effects of anti-inflammatory[69] antioxidative [70] tumor growth inhibition [71] and
Evidence-Based Complementary and Alternative Medicine 11
Mol 22Mol 33
Mol 29Mol 32 Mol 23Mol 20
Mol 97Mol 106
Mol 104Mol 100Mol 94
Mol 63Mol 90
Mol 87
Mol 91Mol 61
Mol 19
Mol 109
Mol 161
Mol 118
Mol 111
Mol 158
Mol 114
Mol 149
Mol 141
Mol 152
Mol 14
CASP3
BCL2
NOS3
SCN5A
F7
ADRA1B
ACHE
F2
Mol 4
Mol 49
Mol 127
APP CASP7
Mol 92
Mol 54
Mol 6
Mol 36
KCNMA1
Mol 27
Mol 13Mol 105Mol 140
Mol 17
Mol 39
Mol 2
Mol 21
Mol 12
Mol 142
Mol 126
Mol 135
Mol 121
Mol 119
Mol 124
Mol 120
Mol 117
Mol 133
Mol 131
Mol 134
PlatycodonGrandiforus
Aurantii Fructus
Licorice
Radix Bupleuri
RehmanniaglutinosaLibosch
AngelicaeSinensis Radix
Mol 8
Mol 73
Mol 75
Mol 77
Mol 80
Mol 7
Mol 82
Mol 76
Mol 74
Mol 60
Mol 46
Mol 150
Mol 151
Mol 112
Mol 115
Mol 11
Mol 116
Mol 113
Mol 110
Mol 1
Mol 9Mol 101Mol 10Mol 93
Mol 103Mol 89 Mol 107
Mol 96PRSS3 LDLRMAPK3 BACE1 EPHB2
Mol 35Mol 81
Mol 86
Mol 85
Mol 84
Mol 88
Mol 83Mol 30
Mol 137
ChuanxiongRhizoma
Mol 3
Mol 57
Mol 139
Achyranthis
Bidentatae
RadixMol 102
CASP8
IGHG1AKT1p53
CHRNA7
Mol 40
SLC6A3 PTPN1
SOD1MAPK1
Mol 38
Mol 95
Carthami Flos
Mol 24
Mol 78 Persicae Semen
Mol 122
Mol 25
Mol 98
TGFB1
GSK3B
PRKCA
Radix Paeoniae Rubra
Mol 52
Mol 42
Mol 62
Mol 138
EGFR
STAT1 PPP3CAPLAT
Stigmasterol
MAP2
Beta-sistosterol Quercetin
Kaempferol
SYNGAP1
CALM
Baicalin
PRKCB
CAV1
CTSD
TNF
DRD1
CDK1
IFNG
FN1BBC3 PRKCD
Mol 132
Mol 160
Mol 58
Mol 67
Mol 69 Mol 43ALB
Mol 64CTNNB1
xiongixioChuanxC nxnxioaamaRhihihizoma
Achyranthisiistntnt
aeeaeBidenBidennntatae
RadixadixR diR di
oniae nianiaiaonRadix PaeoPaePRadix P eeoeonRRRRubra dondonddoodPlatycolatycPlatyycPl ycoyccoPlatPlatycoPlatyccocod
rususrusususruGranG anGranGranGranGrGGranndndiforu
ructusuctuc sructusuctusructusruAurantiirA ntaA ntiitiirantAurantintiurantAur iiii Fru
cecececececececeecLiLLiLLiLiLLLLLLLLiLiLiLLiLLLiLicoric
leuririeuuurieurileurleurieurieurieurieurileRadix BBRadRad Bdix Bdixx R i Bdi BBBadix Radix BBuple
RehmanniaRe mRe m nnRehmanniamReRehmhmannnniamanniaRehmehmanniRR nosasasasaosaosglutilutglutgluulutiglgl titinos
LiboschLibLLibo hschib chLiboschboscLLib chschLibos
icaecaecacaeicaeicAngelAnAngeAngelAnAng lAAngeelielicdix dix dixdixdSinensinensSS nennensSinSSiSinensSinensSine sissis Rad
FloslololFlhamaCartharthhaamami Fl
menmennnnmicae Scae SSem
Figure 6 HB-pC-pT network of XFZYD for treating TBI The triangles with circle backgrounds represent the herbs (HB) the squares andcircles represent the potential compounds (pC) and targets (pT)The red triangles squares and circles represent corresponding HB pC andpT in the Junherbs the same is to aqua representing theChen herbs and periwinkle representing the Zuo-Shi herbsThe claybank squares andcircles represent corresponding pC and pT shared by the Chen and Zuo-Shi herbs the same is to blue representing the overlap between theJun and Zuo-Shi herbs and the green representing the overlap between the Chen and Zuo-Shi herbsThe purple squares and circles representthe corresponding pC and pT shared by the 3 groups of herbs
alleviating insulin resistance in type 2 diabetic rats [72]Beta-sitosterol (BS) is a vegetable-derived compound foundin various plants and is suggested to modulate the immunefunction inflammation and pain levels by controlling theproduction of inflammatory cytokines [73]
For targets analysis CALM possesses the largest degree(degree =84) followed by GSK3B (degree =61) SCN5A(degree = 59) F2 (degree = 43) ACHE (degree=28)F7 (degree=28) ADRA1B (degree=28) NOS3 (degree=20)BCL2 (degree=18) and CASP3 (degree=18) which demon-strated their crucial therapeutic effects for treating TBI Forinstance CALM possess an essential position in calciumsignaling pathway and is related to morphological changesmigration proliferation and secretion of cytokines andreactive oxygen species ofMicroglial cells [74]The activationof CaMKII120572 major isoform of Ca2+calmodulin-dependentprotein kinase (CaMK) in brain is directly associated withthe production of proinflammatory cytokines such as TNF-120572and IL-1120573 [75] GSK-3120573 is a serinethreonine-protein kinasewhich is abundant in the central nervous system (CNS)particularly in neurons [76] It can control gene transcrip-tion axonal transport and cytoskeletal dynamics in growthcones [77] The inhibition of GSK-3120573 attenuates apoptotic
signals and prevents neuronal death [78] There is increasingevidence that prothrombin (F2) and its active derivativethrombin are expressed locally in the central nervous systemBeside the central role in the coagulation cascade the gener-ation of thrombin leads to receptor mediated inflammatoryresponses cell proliferationmodulation cell protection andapoptosis [79 80] The role in brain injury depends upon itsconcentration as higher amounts cause neuroinflammationand apoptosis while lower concentrations might even becytoprotective [81]
Direct tissue damage aswell as hypoxic-ischemic increas-ing anaerobic glycolysis of the brain tissue results in theATP-stores depletion and failure of energy-dependent mem-brane ion pumps especially for voltage-dependent Ca2+ andNa+-channels Accumulated Ca2+ activates lipid peroxidasesproteases and phospholipases and caspases at the sametime increasing the intracellular concentration of free fattyacids and free radicals leading to necrosis or apoptosis ofneurocyte [82] At the same time the activation of residentglial cells microglia and astrocytes and the infiltration ofblood leukocytes secrete various immune mediators elicitedinflammatory responses which subsequently intersect withadjacent pathological cascades including oxidative stress
12 Evidence-Based Complementary and Alternative Medicine
Table 6 Top 10 potential candidate compounds of XFZYD for treating TBI according to 2 centrality indicators
Compound Degree Compound Betweennessquercetin 76 quercetin 01682179kaempferol 43 kaempferol 005501511beta-sitosterol 35 wogonin 005141376Stigmasterol 19 beta-sitosterol 004451294luteolin 18 naringenin 003251738baicalein 15 beta-carotene 002977217-Methoxy-2-methyl isoflavone 12 nobiletin 002787992wogonin 12 7-Methoxy-2-methyl isoflavone 002096439nobiletin 11 luteolin 002090223naringenin 9 baicalein 001453488
Table 7 Top 10 potential targets of XFZYD for treating TBI according to 2 centrality indicators
Targets Degree Targets BetweennessCALM 84 CALM 021452046GSK3B 61 SCN5A 011441591SCN5A 59 GSK3B 009553896F2 43 F2 00689171ACHE 28 BCL2 002651903F7 28 CASP3 002372836ADRA1B 28 F7 002032647NOS3 20 ACHE 001845996BCL2 18 ADRA1B 001704505CASP3 18 NOS3 00166699
excitotoxicity or reparative events including angiogenesisscarring and neurogenesis [83] Function analysis of the 47target proteins regulated by XFZYD was mainly associatedwith core pathophysiology process of TBI (Figure 7) 47 targetproteins were connected with 16 key process related to TBIMost of the targets have one or more links to other biolog-ical process such as apoptosis cell proliferation superoxideanion generation nitric oxide biosynthetic process responseto calcium ion I-kappaB kinaseNF-kappaB signaling andregulation of inflammation The above analysis implied themultifunction character of these target proteins regulated byXFZYD Of these target proteins 25 proteins (53 of the 47)such as TGFB1 EGFR CAV1 MAPK1 PRKCB and AKT1were responsible for regulating the apoptosis process 17 forblood coagulation 14 for cell proliferation and axon genesisand 12 for hypoxia andMAPK cascade We found that TGFB1was the crucial protein because it participated in 9 biologicalprocesses related to TBI such as apoptosis blood coagulationcell proliferation and MAPK cascade followed by EGFR (8)CAV1 (7) MAPK1 (6) PRKCB (6) and AKT1 (6)
Overall these observations strongly support the evidencethat the generated HB-pCminuspT network and pT-F networkhave important roles in treating TBI further validating thedrug targeting approach
Molecule docking was used to further validate the bind-ing mode between candidate compounds and their targetproteins We found that 18 TBI-specific target proteins inter-acted with 91 candidate compounds from XFZYD (Figure 8)
Other 29 target proteins were not discussed for the lackof proper protein crystal structure The detailed moleculedocking results are shown in Table S5The 6 essential proteinsincluding GSK3B AKT1 CDK1 F2 NOS3 and ACHEwere used to elucidate the exact binding mode (Figure 9)Quercetin was located within the binding cavity of AKT1 andCDK1 (Figures 9(a) and 9(b) and S1A B) Four conventionalhydrogen bonds were formed between quercetin and AKT1by interacting with the key amino acids including ILE-290 THR-211 and SER 205 Additionally 120587-120587 interactionsbetween quercetin and TRP-90 were found in the activesite which helped the stabilization of the compound at thebinding site (Figure 9(a) S1A) Figure 9(b) and S1B suggestedthat five conventional hydrogen bonds (LEU-83 ASP-146and LYS-33) and 120587-120587 interaction (PHE-80) were formedbetween quercetin and CDK1 The GSK3B-FA complexes(Figure 9(c) S1C) were stabilized by 6 hydrogen-bondinginteractions between FA and LYS-85 GLU-97 TYR-134 andARG-141 Glyasperin Bmainly bonds to F2 through hydrogenbonds by interacting with the key amino acids includingGLY-193 SER-195 and GLY-219 (Figure 9(d) S1D) andan edge-to-face 120587 minus 120587 interaction was also observed withTYR-228 The GLN-247 GLU-351 and GLY-355 from theactive site pocket of NOS3 participated in the hydrogen-bondformationwith 1-Methoxyphaseollidin (Figure 9(e) S1E)The(-)-Medicocarpin formed a total of 6 hydrogen bonds withSER-293 PHE-295 ARG-296 and TRY-341 in the active siteof ACHE (Figure 9(f) S1F) Besides an edge-to-face 120587 minus 120587
Evidence-Based Complementary and Alternative Medicine 13
KCNMA1
P53
IFNG
PPP3CA
CASP8
SCN5A
I-kappaB kinase NF-kappaB signaling
Regulation of inflammation
Response to calcium ion
TGFB1
TNF
PRKCA
CHRNA7
CAV1
STAT1
MAPK cascadeAKT1
BCL2
APP
EGFR
MAPK1
Angiogenesis
SYNGAP1
MAP2
PTPN1
Axonogenesis
GSK3B
EPHB2
CDK1
CASP7FN1
MAPK3
CTNNB1
F2
Autophagy
Cell proliferation
BACE1
SLC6A3
DRD1
ADRA1BACHE
CALMCASP3
PLAT
Apoptotic process
NOS3
PRKCB
Blood coagulation
Response to hypoxia
Superoxide anion generation
Nitric oxide biosynthetic process
Astrocyte activation
Amyloid-beta regulation
Microglial cell activation
LDLR
PRSS3
F7
PRKCD
SOD1ALB
BBC3
Figure 7 pT-F network of XFZYD for treating TBI 16 biological processes (red square) the 47 target proteins (periwinkle circle) of XFZYDparticipate in for treating TBI
interactionwas also observedwith TYR-337 From the resultshydrogen-bonding and edge-to-face 120587 minus 120587 interactions playkey roles in the proteinminusligand recognition and stabilitywhich may be helpful in determining the inhibitor activitiesAnd the C-T network confirmed the potential therapeuticeffects of the candidate compounds from XFZYD to treatTBI through interacting with the relevant proteins The com-putational analysis further elucidated the accurate moleculemechanisms between active compounds and targets
4 Discussion
Traumatic brain injury (TBI) is a growing public healthproblem worldwide and is a leading cause of death anddisability [84] Although major progress has been made in
understanding the pathophysiology of this injury this hasnot yet led to substantial improvements in outcome by alack of treatments which have proven successful during phaseIII trials for modern medicine [85 86] TCM rooted inthousands of years of history may offer an alternative or acomplementary strategy for the treatment of TBI XFZYDa representative formula in TCM has been used for yearsto treat TBI in China and has been demonstrated to beeffective in clinical practice However its ldquomulticomponentsrdquoand ldquomultitargetsrdquo features make it much difficult to decipherthemolecular mechanisms of XFZYD in the treatment of TBIfrom a systematic perspective if employing routine methods
In the present study a network pharmacology-basedmethod was employed to elucidate the pharmacologicalmechanisms of XFZYD to treat TBI according to the drug
14 Evidence-Based Complementary and Alternative Medicine
Figure 8 C-T network through molecule docking validating 119 potential compounds (green triangles) interacting with 18 potential targets(yellow circles) of XFZYDThe size of the nodes is proportional to the value of degree
combination principle of TCM We first proposed a newmodeling system combining OB and DL screening multipledrug targets prediction and validation network constructionand molecule docking to probe the efficiency of a typicalTCM formula XFZYD for the treatment of TBI The 11herbs from XFZYD possessed 162 bioactive compounds andtargeted 285 proteins There were 5 compounds and 189
target proteins overlapped among the Jun Chen and Zuo-Shigroup Furthermore 47 TBI-specific proteins were targetedby 119 (73) bioactive compounds from XFZYD Similarly 5common compounds and 33 (70) common target proteinsamong the 3 groups of drugs were observed Most of thebioactive ingredients targeted more than one protein The 47target proteins regulated several essential pathophysiological
Evidence-Based Complementary and Alternative Medicine 15
(a) (b) (c)
(d) (e) (f)
Figure 9 Hydrogen-bonding networks within the binding site of the compoundminustarget complexes obtained from molecule docking(a) AKT1-quercetin (b) CDK1-quercetin (c) GSK3B-FA (d) F2-glyasperin B (e) NOS3-1-Methoxyphaseollidin and (f) ACHE-(-)-Medicocarpin The molecules are presented as ball and stick models Active site amino acid residues are represented as lines Dotted bluelines in these pictures represent hydrogen bonds with distance unit of A Other O and N atoms are colored as red and blue respectively
processes of TBI that referred to apoptosis inflammationcell proliferation superoxide anion generation nitric oxidebiosynthetic process response to calcium ion etc The aboveanalysis reveals that the synergistic action mechanisms ofXFZYDmay be (1) bioactive compounds overlapping amongthe different group of herbs (2) specific bioactive com-pounds from different groups of herbs targeting the sameproteins (3) specific bioactive compounds from differentgroups of herbs targeting different proteins which participatein the same pathophysiological process of the disease Toa certain degree the 5 compounds including quercetinstigmasterol kaempferol baicalin and beta-sitosterol playedessential role in XFZYD for TBI treatment MAPK3MAPK1AKT1 PRKCA TNF PRKCB EGFR BCL2 GSK3B CASP3PPP3CA and NOS3 were the main target proteins regulatedby XFZYD in the treatment of TBI
Interestingly Beta-carotene (Mol 43) from Carthami Flos(the Jun herb) specifically regulated 120573-Catenin (CTNNB1)and played critical role for curing TBI The 120573-Catenin is acritical downstream component of the Wnt pathway whichplays essential role in the regulation of mammalian neuraldevelopment [87] In vitro and in vivo studies demonstrate
that the Wnt120573-catenin pathway regulates the proliferationand differentiation of neural progenitor cells [88] Neuronaldifferentiation is induced by overexpression of 120573-cateninor the pharmacological inhibition of GSK3120573 (the phospho-rylating enzyme of 120573-catenin) [89 90] This pathway alsopromotes blood vessel formation during vascular develop-ment as well as the vascular repair process after TBI [91]In addition wogonin (Mol 160) from Achyranthis BidentataeRadix (the Chen herb) specifically targeted fibronectin (FN1)Bcl-2-binding component 3 (BBC3) and Protein KinaseC delta type (PRKCD) FN1 an important component ofthe extracellular matrix (ECM) environment promotes cellmigration neurite outgrowth and synapse formation dur-ing neural development [92] It aggregates in the injuredbrain and plays a neuroprotection role through antiapoptosisand anti-inflammation ways following TBI [93 94] BBC3namely p53 upregulated modulator of apoptosis (PUMA) iscritical for the p53-dependent apoptosis pathway which playsan important role in hippocampal neuronal loss and associ-ated cognitive deficits [95] PRKCD one of PKC isoformsactivates signal transduction pathways involved in neuronalregeneration [96] synaptic transmissionplasticity [97] and
16 Evidence-Based Complementary and Alternative Medicine
activation of apoptosis processes [98] as well as higher brainfunctions such as learning andmemory [99]The activators ofPKCare effective for the treatment of TBI [100] FurthermoreMitogen-activated protein kinase 3 (MAPK3) Beta-secretase1 (BACE1) Ephrin type-B receptor 2 (EphB) and low-densitylipoprotein receptor (LDLR) were specifically targeted bythe ingredients in the Zuo-Shi herbs Naringenin (Mol133) targeted LDLR MAPK3 while euchrenone (Mol 60)anchored BACE1 and nobiletin (Mol 134) targeted EphB2MAPK3 is an essential component of the MAP kinase signaltransduction pathway It has been appreciated recently thatthe ERK12 cascade plays a fundamental role in synapticplasticity and memory [101] BACE1 is responsible for pro-duction of A120573 from amyloid precursor protein (APP) A120573can cause cell death activate inflammatory pathways [102]and prime proapoptotic pathways for activation by otherinsults [103] The blocking of BACE1 can ameliorate motorand cognitive deficits and reduce cell loss after experimentalTBI in mice [104] EphB is localized to synaptic sites inhippocampal neurons [105] The interaction between EphBand NMDA receptors regulates excitatory synapse formation[106] LDLR acts as an important receptor that facilitatesbrain A120573 clearance and inhibits amyloid deposition [107]and then ameliorates Alzheimerrsquos disease neuropathologyafter TBI [108] The analysis above indicates the ldquoJunrdquoldquoChenrdquo and ldquoZuo-Shirdquo herbs from XFZYD trigger theirspecific targets regulation respectively for the therapeuticeffects
XFZYD is a very famous traditional Chinese formula inpromoting qi circulation and removing blood stasis accord-ing to TCM theory However several limited researches havedemonstrated its efficacy for treating TBI such as anti-inflammatory and synaptic regulation [30 31] which arein accord with our study However previous studies merelypartially deciphered the molecule mechanism of XFZYD fortreating TBI This study reports 119 bioactive compounds inXFZYD that target 47 TBI-specific proteins such as MAPK3MAPK1 AKT1 PRKCA TNF PRKCB and EGFR Theseproteins regulate several crucial pathophysiological processesof TBI such as apoptosis inflammation blood coagulationand axon genesis Our study demonstrates that the therapeu-tic actions of XFZYD refer to ldquomulticompoundsrdquo ldquomultitar-getsrdquo features rather than only the improvement of bloodcirculation With the help of molecule docking methodwe further validate the interactions between bioactive com-pounds and potential targets of XFZYD The hydrogen-bonding and edge-to-face 120587 minus120587 interactions play key roles inthe proteinminusligand recognition and stability This provides avaluable reference for further experimental investigations ofbioactive ingredients and therapeutic targets of XFZYD fortreating TBI
5 Conclusion
Our work successfully illuminates the efficiency of XFZYDfor the treatment of TBI as well as herb combination ruleof TCM formula Network pharmacology with moleculedocking method confirms the ldquomulticompounds multitar-getsrdquo therapeutic actions of XFZYD in the treatment of TBI
The present work may provide valuable evidence for furtherclinical application of XFZYD for treating TBI
Abbreviations
TBI Traumatic brain injuryXFZYD Xuefu Zhuyu decoctionTCM Traditional Chines medicineDL Drug-likenessOB Oral bioavailabilityHB-cC-cT Herb-candidate compound-candidate
targetHB-pC-pT Herb-potential compound-potential targetCALM CalmodulinGSK3B Glycogen synthase kinase-3 betaSCN5A Sodium channel protein type 5 subunit
alphaF2 ProthrombinACHE AcetylcholinesteraseGO Gene OntologyKEGG Kyoto Encyclopedia of Genes and
Genomes
Data Availability
The data used to support the findings of this study areavailable from the corresponding author upon request
Conflicts of Interest
The authors declare that they have no conflicts of interest
Authorsrsquo Contributions
YangWang and Zebing Huang participated in the conceptionand design of the study YuanyuanZhong YangWang ZebingHuang Jiekun Luo Tao Tang and Pengfei Li acquired andanalyzed the data Yuanyuan Zhong Tao Liu and HanjinCui drafted and revised the manuscript The correspondingauthor and all of the authors have read and approved the finalsubmitted manuscript
Acknowledgments
This work was supported by the National Natural ScienceFoundation of China (Nos 81673719 81874409 81603670and 81803948) Project funded byChina Postdoctoral ScienceFoundation (Nos 2016M600639 and 2017T100614)
Supplementary Materials
Table S1 162 bioactive compounds in XFZYD Table S2 targetproteins of XFZYD Table S3 TBI-specific proteins Table S4119 potential compounds of XFZYD for treating TBI TableS5 docking result of 18 target proteins with 91 potential com-pounds Fig S1 the exact bindingmode between active ingre-dients and protein targets obtained from molecule docking(A) AKT1-quercetin (B) CDK1-quercetin (C) GSK3B-FA
Evidence-Based Complementary and Alternative Medicine 17
(D) F2-glyasperin B (E) NOS3-1-Methoxyphaseollidin and(F)ACHE-(-)-Medicocarpin (Supplementary Materials)
References
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[2] J A Langlois W Rutland-Brown and M M Wald ldquoTheepidemiology and impact of traumatic brain injury a briefoverviewrdquo The Journal of Head Trauma Rehabilitation vol 21no 5 pp 375ndash378 2006
[3] H A Alsulaim B J Smart A O Asemota et al ldquoConsciousstatus predictsmortality among patientswith isolated traumaticbrain injury in administrative datardquo The American Journal ofSurgery vol 214 no 2 pp 207ndash210 2017
[4] M Majdan D Plancikova A Maas et al ldquoYears of life lost dueto traumatic brain injury in Europe A cross-sectional analysisof 16 countriesrdquo PLoS Medicine vol 14 no 7 p e1002331 2017
[5] P Cheng P Yin P Ning et al ldquoTrends in traumatic braininjury mortality in China 2006ndash2013 A population-basedlongitudinal studyrdquo PLoS Medicine vol 14 no 7 Article IDe1002332 2017
[6] M V Russo and D B McGavern ldquoInflammatory neuroprotec-tion following traumatic brain injuryrdquo Science vol 353 no 6301pp 783ndash785 2016
[7] WWang H Li J Yu et al ldquoProtective Effects of ChineseHerbalMedicine Rhizoma drynariae in Rats After Traumatic BrainInjury and Identification of Active Compoundrdquo MolecularNeurobiology vol 53 no 7 pp 4809ndash4820 2016
[8] M Das S Mohapatra and S S Mohapatra ldquoNew perspectiveson central and peripheral immune responses to acute traumaticbrain injuryrdquo Journal of Neuroinflammation vol 9 p 236 2012
[9] J W Finnie ldquoNeuroinflammation Beneficial and detrimentaleffects after traumatic brain injuryrdquo Inflammopharmacologyvol 21 no 4 pp 309ndash320 2013
[10] T Cheng W Wang Q Li et al ldquoCerebroprotection of flavanol(minus)-epicatechin after traumatic brain injury viaNrf2-dependentand -independent pathwaysrdquo Free Radical Biology amp Medicinevol 92 pp 15ndash28 2016
[11] W Young ldquoRole of calcium in central nervous system injuriesrdquoJournal of Neurotrauma vol 9 Suppl 1 pp S9ndashS25 1992
[12] R Bullock A Zauner J S Myseros et al ldquoEvidence forProlonged Release of Excitatory Amino Acids in Severe HumanHead Trauma Relationship to Clinical Eventsrdquo Annals of theNew York Academy of Sciences vol 765 no 1 pp 290ndash297 1995
[13] A T Mazzeo A Beat A Singh and M R Bullock ldquoTherole of mitochondrial transition pore and its modulation intraumatic brain injury and delayed neurodegeneration afterTBIrdquo Experimental Neurology vol 218 no 2 pp 363ndash370 2009
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18 Evidence-Based Complementary and Alternative Medicine
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[85] J V Rosenfeld A I Maas P Bragge M C Morganti-Kossmann G T Manley and R L Gruen ldquoEarly managementof severe traumatic brain injuryrdquoThe Lancet vol 380 no 9847pp 1088ndash1098 2012
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[90] N Israsena M Hu W Fu L Kan and J A Kessler ldquoThepresence of FGF2 signaling determines whether 120573-cateninexerts effects on proliferation or neuronal differentiation ofneural stem cellsrdquo Developmental Biology vol 268 no 1 pp220ndash231 2004
[91] A Salehi A Jullienne M Baghchechi et al ldquoUp-regulation ofWnt120573-catenin expression is accompanied with vascular repairafter traumatic brain injuryrdquo Journal of Cerebral Blood Flow ampMetabolism vol 38 no 2 pp 274ndash289 2017
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[95] C Culmsee and M P Mattson ldquop53 in neuronal apoptosisrdquoBiochemical and Biophysical Research Communications vol 331no 3 pp 761ndash777 2005
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20 Evidence-Based Complementary and Alternative Medicine
[98] A Muscella C Vetrugno L G Cossa et al ldquoApoptosis by[Pt(OOrsquo-acac)(gamma-acac)(DMS)] requires PKC-deltamedi-ated p53 activation in malignant pleural mesotheliomardquo PloSone vol 12 no 7 Article ID e0181114 2017
[99] J S Bonini W C Da Silva L R M Bevilaqua J H MedinaI Izquierdo and M Cammarota ldquoOn the participation ofhippocampal PKC in acquisition consolidation and reconsol-idation of spatial memoryrdquo Neuroscience vol 147 no 1 pp 37ndash45 2007
[100] O Zohar R Lavy X Zi et al ldquoPKC activator therapeutic formild traumatic brain injury in micerdquo Neurobiology of Diseasevol 41 no 2 pp 329ndash337 2011
[101] J David Sweatt ldquoTheneuronalMAPkinase cascade a biochem-ical signal integration system subserving synaptic plasticity andmemoryrdquo Journal ofNeurochemistry vol 76 no 1 pp 1ndash10 2001
[102] Y Matsuoka M Picciano B Maleste et al ldquoInflammatoryresponses to amyloidosis in a transgenic mouse model ofAlzheimerrsquos diseaserdquo The American Journal of Pathology vol158 no 4 pp 1345ndash1354 2001
[103] L Esposito L Gan G-Q Yu C Essrich and L MuckeldquoIntracellularly generated amyloid-120573 peptide counteracts theantiapoptotic function of its precursor protein and primesproapoptotic pathways for activation by other insults in neu-roblastoma cellsrdquo Journal of Neurochemistry vol 91 no 6 pp1260ndash1274 2004
[104] D J Loane A Pocivavsek C E-H Moussa et al ldquoAmyloidprecursor protein secretases as therapeutic targets for traumaticbrain injuryrdquo Nature Medicine vol 15 no 4 pp 377ndash379 2009
[105] R Torres B L Firestein H Dong et al ldquoPDZ proteins bindcluster and synaptically colocalize with Eph receptors and theirephrin ligandsrdquo Neuron vol 21 no 6 pp 1453ndash1463 1998
[106] M B Dalva M A Takasu M Z Lin et al ldquoEphB receptorsinteract with NMDA receptors and regulate excitatory synapseformationrdquo Cell vol 103 no 6 pp 945ndash956 2000
[107] J M Basak P B Verghese H Yoon J Kim and D MHoltzman ldquoLow-density lipoprotein receptor represents anapolipoprotein E-independent pathway of A120573 uptake anddegradation by astrocytesrdquoThe Journal of Biological Chemistryvol 287 no 17 pp 13959ndash13971 2012
[108] L Yao X Gu Q Song et al ldquoNanoformulated alpha-mangostinameliorates Alzheimerrsquos disease neuropathology by elevatingLDLR expression and accelerating amyloid-beta clearancerdquoJournal of Controlled Release vol 226 pp 1ndash14 2016
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Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 11
Mol 22Mol 33
Mol 29Mol 32 Mol 23Mol 20
Mol 97Mol 106
Mol 104Mol 100Mol 94
Mol 63Mol 90
Mol 87
Mol 91Mol 61
Mol 19
Mol 109
Mol 161
Mol 118
Mol 111
Mol 158
Mol 114
Mol 149
Mol 141
Mol 152
Mol 14
CASP3
BCL2
NOS3
SCN5A
F7
ADRA1B
ACHE
F2
Mol 4
Mol 49
Mol 127
APP CASP7
Mol 92
Mol 54
Mol 6
Mol 36
KCNMA1
Mol 27
Mol 13Mol 105Mol 140
Mol 17
Mol 39
Mol 2
Mol 21
Mol 12
Mol 142
Mol 126
Mol 135
Mol 121
Mol 119
Mol 124
Mol 120
Mol 117
Mol 133
Mol 131
Mol 134
PlatycodonGrandiforus
Aurantii Fructus
Licorice
Radix Bupleuri
RehmanniaglutinosaLibosch
AngelicaeSinensis Radix
Mol 8
Mol 73
Mol 75
Mol 77
Mol 80
Mol 7
Mol 82
Mol 76
Mol 74
Mol 60
Mol 46
Mol 150
Mol 151
Mol 112
Mol 115
Mol 11
Mol 116
Mol 113
Mol 110
Mol 1
Mol 9Mol 101Mol 10Mol 93
Mol 103Mol 89 Mol 107
Mol 96PRSS3 LDLRMAPK3 BACE1 EPHB2
Mol 35Mol 81
Mol 86
Mol 85
Mol 84
Mol 88
Mol 83Mol 30
Mol 137
ChuanxiongRhizoma
Mol 3
Mol 57
Mol 139
Achyranthis
Bidentatae
RadixMol 102
CASP8
IGHG1AKT1p53
CHRNA7
Mol 40
SLC6A3 PTPN1
SOD1MAPK1
Mol 38
Mol 95
Carthami Flos
Mol 24
Mol 78 Persicae Semen
Mol 122
Mol 25
Mol 98
TGFB1
GSK3B
PRKCA
Radix Paeoniae Rubra
Mol 52
Mol 42
Mol 62
Mol 138
EGFR
STAT1 PPP3CAPLAT
Stigmasterol
MAP2
Beta-sistosterol Quercetin
Kaempferol
SYNGAP1
CALM
Baicalin
PRKCB
CAV1
CTSD
TNF
DRD1
CDK1
IFNG
FN1BBC3 PRKCD
Mol 132
Mol 160
Mol 58
Mol 67
Mol 69 Mol 43ALB
Mol 64CTNNB1
xiongixioChuanxC nxnxioaamaRhihihizoma
Achyranthisiistntnt
aeeaeBidenBidennntatae
RadixadixR diR di
oniae nianiaiaonRadix PaeoPaePRadix P eeoeonRRRRubra dondonddoodPlatycolatycPlatyycPl ycoyccoPlatPlatycoPlatyccocod
rususrusususruGranG anGranGranGranGrGGranndndiforu
ructusuctuc sructusuctusructusruAurantiirA ntaA ntiitiirantAurantintiurantAur iiii Fru
cecececececececeecLiLLiLLiLiLLLLLLLLiLiLiLLiLLLiLicoric
leuririeuuurieurileurleurieurieurieurieurileRadix BBRadRad Bdix Bdixx R i Bdi BBBadix Radix BBuple
RehmanniaRe mRe m nnRehmanniamReRehmhmannnniamanniaRehmehmanniRR nosasasasaosaosglutilutglutgluulutiglgl titinos
LiboschLibLLibo hschib chLiboschboscLLib chschLibos
icaecaecacaeicaeicAngelAnAngeAngelAnAng lAAngeelielicdix dix dixdixdSinensinensSS nennensSinSSiSinensSinensSine sissis Rad
FloslololFlhamaCartharthhaamami Fl
menmennnnmicae Scae SSem
Figure 6 HB-pC-pT network of XFZYD for treating TBI The triangles with circle backgrounds represent the herbs (HB) the squares andcircles represent the potential compounds (pC) and targets (pT)The red triangles squares and circles represent corresponding HB pC andpT in the Junherbs the same is to aqua representing theChen herbs and periwinkle representing the Zuo-Shi herbsThe claybank squares andcircles represent corresponding pC and pT shared by the Chen and Zuo-Shi herbs the same is to blue representing the overlap between theJun and Zuo-Shi herbs and the green representing the overlap between the Chen and Zuo-Shi herbsThe purple squares and circles representthe corresponding pC and pT shared by the 3 groups of herbs
alleviating insulin resistance in type 2 diabetic rats [72]Beta-sitosterol (BS) is a vegetable-derived compound foundin various plants and is suggested to modulate the immunefunction inflammation and pain levels by controlling theproduction of inflammatory cytokines [73]
For targets analysis CALM possesses the largest degree(degree =84) followed by GSK3B (degree =61) SCN5A(degree = 59) F2 (degree = 43) ACHE (degree=28)F7 (degree=28) ADRA1B (degree=28) NOS3 (degree=20)BCL2 (degree=18) and CASP3 (degree=18) which demon-strated their crucial therapeutic effects for treating TBI Forinstance CALM possess an essential position in calciumsignaling pathway and is related to morphological changesmigration proliferation and secretion of cytokines andreactive oxygen species ofMicroglial cells [74]The activationof CaMKII120572 major isoform of Ca2+calmodulin-dependentprotein kinase (CaMK) in brain is directly associated withthe production of proinflammatory cytokines such as TNF-120572and IL-1120573 [75] GSK-3120573 is a serinethreonine-protein kinasewhich is abundant in the central nervous system (CNS)particularly in neurons [76] It can control gene transcrip-tion axonal transport and cytoskeletal dynamics in growthcones [77] The inhibition of GSK-3120573 attenuates apoptotic
signals and prevents neuronal death [78] There is increasingevidence that prothrombin (F2) and its active derivativethrombin are expressed locally in the central nervous systemBeside the central role in the coagulation cascade the gener-ation of thrombin leads to receptor mediated inflammatoryresponses cell proliferationmodulation cell protection andapoptosis [79 80] The role in brain injury depends upon itsconcentration as higher amounts cause neuroinflammationand apoptosis while lower concentrations might even becytoprotective [81]
Direct tissue damage aswell as hypoxic-ischemic increas-ing anaerobic glycolysis of the brain tissue results in theATP-stores depletion and failure of energy-dependent mem-brane ion pumps especially for voltage-dependent Ca2+ andNa+-channels Accumulated Ca2+ activates lipid peroxidasesproteases and phospholipases and caspases at the sametime increasing the intracellular concentration of free fattyacids and free radicals leading to necrosis or apoptosis ofneurocyte [82] At the same time the activation of residentglial cells microglia and astrocytes and the infiltration ofblood leukocytes secrete various immune mediators elicitedinflammatory responses which subsequently intersect withadjacent pathological cascades including oxidative stress
12 Evidence-Based Complementary and Alternative Medicine
Table 6 Top 10 potential candidate compounds of XFZYD for treating TBI according to 2 centrality indicators
Compound Degree Compound Betweennessquercetin 76 quercetin 01682179kaempferol 43 kaempferol 005501511beta-sitosterol 35 wogonin 005141376Stigmasterol 19 beta-sitosterol 004451294luteolin 18 naringenin 003251738baicalein 15 beta-carotene 002977217-Methoxy-2-methyl isoflavone 12 nobiletin 002787992wogonin 12 7-Methoxy-2-methyl isoflavone 002096439nobiletin 11 luteolin 002090223naringenin 9 baicalein 001453488
Table 7 Top 10 potential targets of XFZYD for treating TBI according to 2 centrality indicators
Targets Degree Targets BetweennessCALM 84 CALM 021452046GSK3B 61 SCN5A 011441591SCN5A 59 GSK3B 009553896F2 43 F2 00689171ACHE 28 BCL2 002651903F7 28 CASP3 002372836ADRA1B 28 F7 002032647NOS3 20 ACHE 001845996BCL2 18 ADRA1B 001704505CASP3 18 NOS3 00166699
excitotoxicity or reparative events including angiogenesisscarring and neurogenesis [83] Function analysis of the 47target proteins regulated by XFZYD was mainly associatedwith core pathophysiology process of TBI (Figure 7) 47 targetproteins were connected with 16 key process related to TBIMost of the targets have one or more links to other biolog-ical process such as apoptosis cell proliferation superoxideanion generation nitric oxide biosynthetic process responseto calcium ion I-kappaB kinaseNF-kappaB signaling andregulation of inflammation The above analysis implied themultifunction character of these target proteins regulated byXFZYD Of these target proteins 25 proteins (53 of the 47)such as TGFB1 EGFR CAV1 MAPK1 PRKCB and AKT1were responsible for regulating the apoptosis process 17 forblood coagulation 14 for cell proliferation and axon genesisand 12 for hypoxia andMAPK cascade We found that TGFB1was the crucial protein because it participated in 9 biologicalprocesses related to TBI such as apoptosis blood coagulationcell proliferation and MAPK cascade followed by EGFR (8)CAV1 (7) MAPK1 (6) PRKCB (6) and AKT1 (6)
Overall these observations strongly support the evidencethat the generated HB-pCminuspT network and pT-F networkhave important roles in treating TBI further validating thedrug targeting approach
Molecule docking was used to further validate the bind-ing mode between candidate compounds and their targetproteins We found that 18 TBI-specific target proteins inter-acted with 91 candidate compounds from XFZYD (Figure 8)
Other 29 target proteins were not discussed for the lackof proper protein crystal structure The detailed moleculedocking results are shown in Table S5The 6 essential proteinsincluding GSK3B AKT1 CDK1 F2 NOS3 and ACHEwere used to elucidate the exact binding mode (Figure 9)Quercetin was located within the binding cavity of AKT1 andCDK1 (Figures 9(a) and 9(b) and S1A B) Four conventionalhydrogen bonds were formed between quercetin and AKT1by interacting with the key amino acids including ILE-290 THR-211 and SER 205 Additionally 120587-120587 interactionsbetween quercetin and TRP-90 were found in the activesite which helped the stabilization of the compound at thebinding site (Figure 9(a) S1A) Figure 9(b) and S1B suggestedthat five conventional hydrogen bonds (LEU-83 ASP-146and LYS-33) and 120587-120587 interaction (PHE-80) were formedbetween quercetin and CDK1 The GSK3B-FA complexes(Figure 9(c) S1C) were stabilized by 6 hydrogen-bondinginteractions between FA and LYS-85 GLU-97 TYR-134 andARG-141 Glyasperin Bmainly bonds to F2 through hydrogenbonds by interacting with the key amino acids includingGLY-193 SER-195 and GLY-219 (Figure 9(d) S1D) andan edge-to-face 120587 minus 120587 interaction was also observed withTYR-228 The GLN-247 GLU-351 and GLY-355 from theactive site pocket of NOS3 participated in the hydrogen-bondformationwith 1-Methoxyphaseollidin (Figure 9(e) S1E)The(-)-Medicocarpin formed a total of 6 hydrogen bonds withSER-293 PHE-295 ARG-296 and TRY-341 in the active siteof ACHE (Figure 9(f) S1F) Besides an edge-to-face 120587 minus 120587
Evidence-Based Complementary and Alternative Medicine 13
KCNMA1
P53
IFNG
PPP3CA
CASP8
SCN5A
I-kappaB kinase NF-kappaB signaling
Regulation of inflammation
Response to calcium ion
TGFB1
TNF
PRKCA
CHRNA7
CAV1
STAT1
MAPK cascadeAKT1
BCL2
APP
EGFR
MAPK1
Angiogenesis
SYNGAP1
MAP2
PTPN1
Axonogenesis
GSK3B
EPHB2
CDK1
CASP7FN1
MAPK3
CTNNB1
F2
Autophagy
Cell proliferation
BACE1
SLC6A3
DRD1
ADRA1BACHE
CALMCASP3
PLAT
Apoptotic process
NOS3
PRKCB
Blood coagulation
Response to hypoxia
Superoxide anion generation
Nitric oxide biosynthetic process
Astrocyte activation
Amyloid-beta regulation
Microglial cell activation
LDLR
PRSS3
F7
PRKCD
SOD1ALB
BBC3
Figure 7 pT-F network of XFZYD for treating TBI 16 biological processes (red square) the 47 target proteins (periwinkle circle) of XFZYDparticipate in for treating TBI
interactionwas also observedwith TYR-337 From the resultshydrogen-bonding and edge-to-face 120587 minus 120587 interactions playkey roles in the proteinminusligand recognition and stabilitywhich may be helpful in determining the inhibitor activitiesAnd the C-T network confirmed the potential therapeuticeffects of the candidate compounds from XFZYD to treatTBI through interacting with the relevant proteins The com-putational analysis further elucidated the accurate moleculemechanisms between active compounds and targets
4 Discussion
Traumatic brain injury (TBI) is a growing public healthproblem worldwide and is a leading cause of death anddisability [84] Although major progress has been made in
understanding the pathophysiology of this injury this hasnot yet led to substantial improvements in outcome by alack of treatments which have proven successful during phaseIII trials for modern medicine [85 86] TCM rooted inthousands of years of history may offer an alternative or acomplementary strategy for the treatment of TBI XFZYDa representative formula in TCM has been used for yearsto treat TBI in China and has been demonstrated to beeffective in clinical practice However its ldquomulticomponentsrdquoand ldquomultitargetsrdquo features make it much difficult to decipherthemolecular mechanisms of XFZYD in the treatment of TBIfrom a systematic perspective if employing routine methods
In the present study a network pharmacology-basedmethod was employed to elucidate the pharmacologicalmechanisms of XFZYD to treat TBI according to the drug
14 Evidence-Based Complementary and Alternative Medicine
Figure 8 C-T network through molecule docking validating 119 potential compounds (green triangles) interacting with 18 potential targets(yellow circles) of XFZYDThe size of the nodes is proportional to the value of degree
combination principle of TCM We first proposed a newmodeling system combining OB and DL screening multipledrug targets prediction and validation network constructionand molecule docking to probe the efficiency of a typicalTCM formula XFZYD for the treatment of TBI The 11herbs from XFZYD possessed 162 bioactive compounds andtargeted 285 proteins There were 5 compounds and 189
target proteins overlapped among the Jun Chen and Zuo-Shigroup Furthermore 47 TBI-specific proteins were targetedby 119 (73) bioactive compounds from XFZYD Similarly 5common compounds and 33 (70) common target proteinsamong the 3 groups of drugs were observed Most of thebioactive ingredients targeted more than one protein The 47target proteins regulated several essential pathophysiological
Evidence-Based Complementary and Alternative Medicine 15
(a) (b) (c)
(d) (e) (f)
Figure 9 Hydrogen-bonding networks within the binding site of the compoundminustarget complexes obtained from molecule docking(a) AKT1-quercetin (b) CDK1-quercetin (c) GSK3B-FA (d) F2-glyasperin B (e) NOS3-1-Methoxyphaseollidin and (f) ACHE-(-)-Medicocarpin The molecules are presented as ball and stick models Active site amino acid residues are represented as lines Dotted bluelines in these pictures represent hydrogen bonds with distance unit of A Other O and N atoms are colored as red and blue respectively
processes of TBI that referred to apoptosis inflammationcell proliferation superoxide anion generation nitric oxidebiosynthetic process response to calcium ion etc The aboveanalysis reveals that the synergistic action mechanisms ofXFZYDmay be (1) bioactive compounds overlapping amongthe different group of herbs (2) specific bioactive com-pounds from different groups of herbs targeting the sameproteins (3) specific bioactive compounds from differentgroups of herbs targeting different proteins which participatein the same pathophysiological process of the disease Toa certain degree the 5 compounds including quercetinstigmasterol kaempferol baicalin and beta-sitosterol playedessential role in XFZYD for TBI treatment MAPK3MAPK1AKT1 PRKCA TNF PRKCB EGFR BCL2 GSK3B CASP3PPP3CA and NOS3 were the main target proteins regulatedby XFZYD in the treatment of TBI
Interestingly Beta-carotene (Mol 43) from Carthami Flos(the Jun herb) specifically regulated 120573-Catenin (CTNNB1)and played critical role for curing TBI The 120573-Catenin is acritical downstream component of the Wnt pathway whichplays essential role in the regulation of mammalian neuraldevelopment [87] In vitro and in vivo studies demonstrate
that the Wnt120573-catenin pathway regulates the proliferationand differentiation of neural progenitor cells [88] Neuronaldifferentiation is induced by overexpression of 120573-cateninor the pharmacological inhibition of GSK3120573 (the phospho-rylating enzyme of 120573-catenin) [89 90] This pathway alsopromotes blood vessel formation during vascular develop-ment as well as the vascular repair process after TBI [91]In addition wogonin (Mol 160) from Achyranthis BidentataeRadix (the Chen herb) specifically targeted fibronectin (FN1)Bcl-2-binding component 3 (BBC3) and Protein KinaseC delta type (PRKCD) FN1 an important component ofthe extracellular matrix (ECM) environment promotes cellmigration neurite outgrowth and synapse formation dur-ing neural development [92] It aggregates in the injuredbrain and plays a neuroprotection role through antiapoptosisand anti-inflammation ways following TBI [93 94] BBC3namely p53 upregulated modulator of apoptosis (PUMA) iscritical for the p53-dependent apoptosis pathway which playsan important role in hippocampal neuronal loss and associ-ated cognitive deficits [95] PRKCD one of PKC isoformsactivates signal transduction pathways involved in neuronalregeneration [96] synaptic transmissionplasticity [97] and
16 Evidence-Based Complementary and Alternative Medicine
activation of apoptosis processes [98] as well as higher brainfunctions such as learning andmemory [99]The activators ofPKCare effective for the treatment of TBI [100] FurthermoreMitogen-activated protein kinase 3 (MAPK3) Beta-secretase1 (BACE1) Ephrin type-B receptor 2 (EphB) and low-densitylipoprotein receptor (LDLR) were specifically targeted bythe ingredients in the Zuo-Shi herbs Naringenin (Mol133) targeted LDLR MAPK3 while euchrenone (Mol 60)anchored BACE1 and nobiletin (Mol 134) targeted EphB2MAPK3 is an essential component of the MAP kinase signaltransduction pathway It has been appreciated recently thatthe ERK12 cascade plays a fundamental role in synapticplasticity and memory [101] BACE1 is responsible for pro-duction of A120573 from amyloid precursor protein (APP) A120573can cause cell death activate inflammatory pathways [102]and prime proapoptotic pathways for activation by otherinsults [103] The blocking of BACE1 can ameliorate motorand cognitive deficits and reduce cell loss after experimentalTBI in mice [104] EphB is localized to synaptic sites inhippocampal neurons [105] The interaction between EphBand NMDA receptors regulates excitatory synapse formation[106] LDLR acts as an important receptor that facilitatesbrain A120573 clearance and inhibits amyloid deposition [107]and then ameliorates Alzheimerrsquos disease neuropathologyafter TBI [108] The analysis above indicates the ldquoJunrdquoldquoChenrdquo and ldquoZuo-Shirdquo herbs from XFZYD trigger theirspecific targets regulation respectively for the therapeuticeffects
XFZYD is a very famous traditional Chinese formula inpromoting qi circulation and removing blood stasis accord-ing to TCM theory However several limited researches havedemonstrated its efficacy for treating TBI such as anti-inflammatory and synaptic regulation [30 31] which arein accord with our study However previous studies merelypartially deciphered the molecule mechanism of XFZYD fortreating TBI This study reports 119 bioactive compounds inXFZYD that target 47 TBI-specific proteins such as MAPK3MAPK1 AKT1 PRKCA TNF PRKCB and EGFR Theseproteins regulate several crucial pathophysiological processesof TBI such as apoptosis inflammation blood coagulationand axon genesis Our study demonstrates that the therapeu-tic actions of XFZYD refer to ldquomulticompoundsrdquo ldquomultitar-getsrdquo features rather than only the improvement of bloodcirculation With the help of molecule docking methodwe further validate the interactions between bioactive com-pounds and potential targets of XFZYD The hydrogen-bonding and edge-to-face 120587 minus120587 interactions play key roles inthe proteinminusligand recognition and stability This provides avaluable reference for further experimental investigations ofbioactive ingredients and therapeutic targets of XFZYD fortreating TBI
5 Conclusion
Our work successfully illuminates the efficiency of XFZYDfor the treatment of TBI as well as herb combination ruleof TCM formula Network pharmacology with moleculedocking method confirms the ldquomulticompounds multitar-getsrdquo therapeutic actions of XFZYD in the treatment of TBI
The present work may provide valuable evidence for furtherclinical application of XFZYD for treating TBI
Abbreviations
TBI Traumatic brain injuryXFZYD Xuefu Zhuyu decoctionTCM Traditional Chines medicineDL Drug-likenessOB Oral bioavailabilityHB-cC-cT Herb-candidate compound-candidate
targetHB-pC-pT Herb-potential compound-potential targetCALM CalmodulinGSK3B Glycogen synthase kinase-3 betaSCN5A Sodium channel protein type 5 subunit
alphaF2 ProthrombinACHE AcetylcholinesteraseGO Gene OntologyKEGG Kyoto Encyclopedia of Genes and
Genomes
Data Availability
The data used to support the findings of this study areavailable from the corresponding author upon request
Conflicts of Interest
The authors declare that they have no conflicts of interest
Authorsrsquo Contributions
YangWang and Zebing Huang participated in the conceptionand design of the study YuanyuanZhong YangWang ZebingHuang Jiekun Luo Tao Tang and Pengfei Li acquired andanalyzed the data Yuanyuan Zhong Tao Liu and HanjinCui drafted and revised the manuscript The correspondingauthor and all of the authors have read and approved the finalsubmitted manuscript
Acknowledgments
This work was supported by the National Natural ScienceFoundation of China (Nos 81673719 81874409 81603670and 81803948) Project funded byChina Postdoctoral ScienceFoundation (Nos 2016M600639 and 2017T100614)
Supplementary Materials
Table S1 162 bioactive compounds in XFZYD Table S2 targetproteins of XFZYD Table S3 TBI-specific proteins Table S4119 potential compounds of XFZYD for treating TBI TableS5 docking result of 18 target proteins with 91 potential com-pounds Fig S1 the exact bindingmode between active ingre-dients and protein targets obtained from molecule docking(A) AKT1-quercetin (B) CDK1-quercetin (C) GSK3B-FA
Evidence-Based Complementary and Alternative Medicine 17
(D) F2-glyasperin B (E) NOS3-1-Methoxyphaseollidin and(F)ACHE-(-)-Medicocarpin (Supplementary Materials)
References
[1] L Yang Y Chu D Tweedie et al ldquoPost-trauma administrationof the pifithrin-120572 oxygen analog improves histological andfunctional outcomes after experimental traumatic brain injuryrdquoExperimental Neurology vol 269 pp 56ndash66 2015
[2] J A Langlois W Rutland-Brown and M M Wald ldquoTheepidemiology and impact of traumatic brain injury a briefoverviewrdquo The Journal of Head Trauma Rehabilitation vol 21no 5 pp 375ndash378 2006
[3] H A Alsulaim B J Smart A O Asemota et al ldquoConsciousstatus predictsmortality among patientswith isolated traumaticbrain injury in administrative datardquo The American Journal ofSurgery vol 214 no 2 pp 207ndash210 2017
[4] M Majdan D Plancikova A Maas et al ldquoYears of life lost dueto traumatic brain injury in Europe A cross-sectional analysisof 16 countriesrdquo PLoS Medicine vol 14 no 7 p e1002331 2017
[5] P Cheng P Yin P Ning et al ldquoTrends in traumatic braininjury mortality in China 2006ndash2013 A population-basedlongitudinal studyrdquo PLoS Medicine vol 14 no 7 Article IDe1002332 2017
[6] M V Russo and D B McGavern ldquoInflammatory neuroprotec-tion following traumatic brain injuryrdquo Science vol 353 no 6301pp 783ndash785 2016
[7] WWang H Li J Yu et al ldquoProtective Effects of ChineseHerbalMedicine Rhizoma drynariae in Rats After Traumatic BrainInjury and Identification of Active Compoundrdquo MolecularNeurobiology vol 53 no 7 pp 4809ndash4820 2016
[8] M Das S Mohapatra and S S Mohapatra ldquoNew perspectiveson central and peripheral immune responses to acute traumaticbrain injuryrdquo Journal of Neuroinflammation vol 9 p 236 2012
[9] J W Finnie ldquoNeuroinflammation Beneficial and detrimentaleffects after traumatic brain injuryrdquo Inflammopharmacologyvol 21 no 4 pp 309ndash320 2013
[10] T Cheng W Wang Q Li et al ldquoCerebroprotection of flavanol(minus)-epicatechin after traumatic brain injury viaNrf2-dependentand -independent pathwaysrdquo Free Radical Biology amp Medicinevol 92 pp 15ndash28 2016
[11] W Young ldquoRole of calcium in central nervous system injuriesrdquoJournal of Neurotrauma vol 9 Suppl 1 pp S9ndashS25 1992
[12] R Bullock A Zauner J S Myseros et al ldquoEvidence forProlonged Release of Excitatory Amino Acids in Severe HumanHead Trauma Relationship to Clinical Eventsrdquo Annals of theNew York Academy of Sciences vol 765 no 1 pp 290ndash297 1995
[13] A T Mazzeo A Beat A Singh and M R Bullock ldquoTherole of mitochondrial transition pore and its modulation intraumatic brain injury and delayed neurodegeneration afterTBIrdquo Experimental Neurology vol 218 no 2 pp 363ndash370 2009
[14] S Gennai A Monsel Q Hao et al ldquoCell-Based therapy fortraumatic brain injuryrdquo British Journal of Anaesthesia vol 115no 2 pp 203ndash212 2015
[15] J Ghajar ldquoTraumatic brain injuryrdquo The Lancet vol 356 no9233 pp 923ndash929 2000
[16] D J Loane and A I Faden ldquoNeuroprotection for traumaticbrain injury translational challenges and emerging therapeuticstrategiesrdquoTrends in Pharmacological Sciences vol 31 no 12 pp596ndash604 2010
[17] Y Wang X Fan T Tang et al ldquoRhein and rhubarb simi-larly protect the blood-brain barrier after experimental trau-matic brain injury via gp91phox subunit of NADPH oxidaseROSERKMMP-9 signaling pathwayrdquo Scientific Reports vol 6p 37098 2016
[18] D-X Kong X-J Li and H-Y Zhang ldquoWhere is the hopefor drug discovery Let history tell the futurerdquo Drug DiscoveryTherapy vol 14 no 3-4 pp 115ndash119 2009
[19] F Cheung ldquoTCM made in Chinardquo Nature vol 480 no 7378pp S82ndashS83 2011
[20] C Fu Z Xia Y Liu et al ldquoQualitative analysis of majorconstituents from Xue Fu Zhu Yu Decoction using ultra highperformance liquid chromatographywith hybrid ion trap time-of-flight mass spectrometryrdquo Journal of Separation Science vol39 no 17 pp 3457ndash3468 2016
[21] H-J Zhang and Y-Y Cheng ldquoAn HPLCMS method for iden-tifying major constituents in the hypocholesterolemic extractsof Chinese medicine formula lsquoXue-Fu-Zhu-Yu decoctionrsquordquoBiomedical Chromatography vol 20 no 8 pp 821ndash826 2006
[22] L Zhang L Zhu YWang et al ldquoCharacterization and quantifi-cation of major constituents of Xue Fu Zhu Yu by UPLC-DAD-MSMSrdquo Journal of Pharmaceutical and Biomedical Analysisvol 62 pp 203ndash209 2012
[23] X Yang X Xiong G Yang and J Wang ldquoChinese patentmedicine Xuefu Zhuyu capsule for the treatment of unstableangina pectoris a systematic review of randomized controlledtrialsrdquo Complementary Therapies in Medicine vol 22 no 2 pp391ndash399 2014
[24] J Wang X Yang F Chu et al ldquoThe effects of Xuefu Zhuyuand Shengmai on the evolution of syndromes and inflammatorymarkers in patients with unstable angina pectoris after percuta-neous coronary intervention a randomised controlled clinicaltrialrdquoEvidence-BasedComplementary andAlternativeMedicinevol 2013 Article ID 896467 9 pages 2013
[25] Q Zhang H Yu J Qi et al ldquoNatural formulas and the natureof formulas Exploring potential therapeutic targets based ontraditional Chinese herbal formulasrdquo PLoS ONE vol 12 no 2p e0171628 2017
[26] J LeeWHsu T Yen et al ldquoTraditional ChinesemedicineXue-Fu-Zhu-Yu decoction potentiates tissue plasminogen activatoragainst thromboembolic stroke in ratsrdquo Journal of Ethnophar-macology vol 134 no 3 pp 824ndash830 2011
[27] Y-C Shen C-K Lu K-T Liou et al ldquoCommon and uniquemechanisms of Chinese herbal remedies on ischemic strokemice revealed by transcriptome analysesrdquo Journal of Ethnophar-macology vol 173 pp 370ndash382 2015
[28] F Xue-hai G Yan and L Jian-tao ldquoTherapeutic effect of XiefuZhuyu decoction joint brain protein hydrolysat injection intraumatic brain injury and its effect on cerebrospinal fluid ofET-1rdquo Chinese Journal of Biochemical Pharmaceutics no 02 pp55ndash58 2017
[29] L Shuxiang W Jingchun C Jie et al ldquoEffect of Xuefu Zhuyudecoction on the neural functional recovery and living abilityin patients with craniocerebral injuryrdquo Modern Journal ofIntegrated Traditional Chinese andWesternMedicine no 04 pp350ndash352 2015
[30] L Zhong W Ning and W Dong-pi ldquoModel Study of theTherapy of Replenishing Qi and Activating Blood Circulationon Protecting the Brain Impairment Caused by Hypoxic -ischemic Encephalopathy in SD Ratsrdquo Journal of TraditionalChinese Medicine vol 07 pp 1552ndash1555 2011
18 Evidence-Based Complementary and Alternative Medicine
[31] M Sun X-P Zhan C-Y Jin J-Z Shan S Xu and Y-LWang ldquoclinical observation on treatment of post-craniocerebraltraumatic mental disorder by integrative medicinerdquo ChineseJournal of Integrative Medicine vol 14 no 2 pp 137ndash141 2008
[32] Z Xing Z Xia W Peng et al ldquoXuefu Zhuyu decoction atraditional Chinese medicine provides neuroprotection in arat model of traumatic brain injury via an anti-inflammatorypathwayrdquo Scientific Reports vol 6 Article ID 20040 2016
[33] J Zhou T Liu H Cui et al ldquoXuefu zhuyu decoction improvescognitive impairment in experimental traumatic brain injuryvia synaptic regulationrdquo Oncotarget vol 8 no 42 2017
[34] S Li ldquoExploring traditional chinese medicine by a novel thera-peutic concept of network targetrdquo Chinese Journal of IntegrativeMedicine vol 22 no 9 pp 647ndash652 2016
[35] S Li and B Zhang ldquoTraditional Chinese medicine networkpharmacology theory methodology and applicationrdquo ChineseJournal of Natural Medicines vol 11 no 2 pp 110ndash120 2013
[36] A L Hopkins ldquoNetwork pharmacology the next paradigm indrug discoveryrdquoNature Chemical Biology vol 4 no 11 pp 682ndash690 2008
[37] Y Li C Han J Wang et al ldquoInvestigation into the mechanismof Eucommia ulmoides Oliv based on a systems pharmacologyapproachrdquo Journal of Ethnopharmacology vol 151 no 1 pp452ndash460 2014
[38] Y Yao X Zhang Z Wang et al ldquoDeciphering the combinationprinciples of Traditional Chinese Medicine from a systemspharmacology perspective based on Ma-huang DecoctionrdquoJournal of Ethnopharmacology vol 150 no 2 pp 619ndash638 2013
[39] Bo Zhang Xu Wang and Shao Li ldquoAn Integrative Platform ofTCM Network Pharmacology and Its Application on a HerbalFormula Qing-Luo-Yinrdquo Evidence-Based Complementary andAlternativeMedicine vol 2013 Article ID 456747 12 pages 2013
[40] J Ru P Li J Wang et al ldquoTCMSP a database of systemspharmacology for drug discovery from herbal medicinesrdquoJournal of Cheminformatics vol 6 no 1 p 13 2014
[41] J V Turner D J Maddalena and S Agatonovic-KustrinldquoBioavailability Prediction Based on Molecular Structure for aDiverse Series of Drugsrdquo Pharmaceutical Research vol 21 no 1pp 68ndash82 2004
[42] X XuW Zhang CHuang et al ldquoAnovel chemometricmethodfor the prediction of human oral bioavailabilityrdquo InternationalJournal of Molecular Sciences vol 13 no 6 pp 6964ndash6982 2012
[43] A Zhang W Pan J Lv and H Wu ldquoProtective Effect ofAmygdalin on LPS-Induced Acute Lung Injury by InhibitingNF-120581B and NLRP3 Signaling Pathwaysrdquo Inflammation vol 40no 3 pp 745ndash751 2017
[44] X Wei H Liu X Sun et al ldquoHydroxysafflor yellow A protectsrat brains against ischemia-reperfusion injury by antioxidantactionrdquo Neuroscience Letters vol 386 no 1 pp 58ndash62 2005
[45] W PWalters andM A Murcko ldquoPrediction of lsquodrug-likenessrsquordquoAdvanced Drug Delivery Reviews vol 54 no 3 pp 255ndash2712002
[46] Y Yamanishi M Kotera M Kanehisa and S Goto ldquoDrug-target interactionprediction from chemical genomic and phar-macological data in an integrated frameworkrdquo Bioinformaticsvol 26 no 12 pp i246ndashi254 2010
[47] H Yu J Chen X Xu et al ldquoA systematic prediction ofmultiple drug-target interactions from chemical genomic andpharmacological datardquo PLoS ONE vol 7 no 5 Article IDe37608 2012
[48] X Chen Z L Ji and Y Z Chen ldquoTTD therapeutic targetdatabaserdquo Nucleic Acids Research vol 30 no 1 pp 412ndash4152002
[49] A Hamosh A F Scott J S Amberger C A Bocchini andV AMcKusick ldquoOnline Mendelian Inheritance in Man (OMIM) aknowledgebase of human genes and genetic disordersrdquo NucleicAcids Research vol 33 pp D514ndashD517 2005
[50] T S Keshava Prasad RGoel K Kandasamy et al ldquoHuman pro-tein reference databasemdash2009 updaterdquo Nucleic Acids Researchvol 37 no 1 pp D767ndashD772 2009
[51] A Franceschini D Szklarczyk S Frankild et al ldquoSTRING v91protein-protein interaction networks with increased coverageand integrationrdquoNucleic Acids Research vol 41 no 1 pp D808ndashD815 2013
[52] P Shannon A Markiel O Ozier et al ldquoCytoscape a softwareEnvironment for integratedmodels of biomolecular interactionnetworksrdquo Genome Research vol 13 no 11 pp 2498ndash25042003
[53] G Dennis Jr B T Sherman D A Hosack et al ldquoDAVIDDatabase for Annotation Visualization and IntegratedDiscov-eryrdquo Genome Biology vol 4 no 5 p P3 2003
[54] L Yang X Zhao and X Tang ldquoPredicting disease-related pro-teins based on clique backbone in protein-protein interactionnetworkrdquo International Journal of Biological Sciences vol 10 no7 pp 677ndash688 2014
[55] S MThomas and J S Brugge ldquoCellular functions regulated bySRC family kinasesrdquo Annual Review of Cell and DevelopmentalBiology vol 13 pp 513ndash609 1997
[56] D Z Liu F R Sharp K C Van et al ldquoInhibition of Src familykinases protects hippocampal neurons and improves cognitivefunction after traumatic brain injuryrdquo Journal of Neurotraumavol 31 no 14 pp 1268ndash1276 2014
[57] B D Manning and A Toker ldquoAKTPKB Signaling Navigatingthe Networkrdquo Cell vol 169 no 3 pp 381ndash405 2017
[58] Y Kitagishi and S Matsuda ldquoDiets involved in PPAR andPI3KAKTPTEN pathway may contribute to neuroprotectionin a traumatic brain injuryrdquoAlzheimerrsquos ResearchampTherapy vol5 no 5 p 42 2013
[59] D Chen L Bao S-Q Lu and F Xu ldquoSerum albumin andprealbuminpredict the poor outcomeof traumatic brain injuryrdquoPLoS ONE vol 9 no 3 Article ID e93167 2014
[60] J Yang Z Wu N Renier et al ldquoPathological axonal deaththrough aMapk cascade that triggers a local energy deficitrdquoCellvol 160 no 1-2 pp 161ndash176 2015
[61] E J Huang and L F Reichardt ldquoTrk receptors roles in neuronalsignal transductionrdquoAnnual Review of Biochemistry vol 72 pp609ndash642 2003
[62] J W Lee and R Juliano ldquoMitogenic signal transductionby integrin- and growth factor receptor-mediated pathwaysrdquoMolecules and Cells vol 17 no 2 pp 188ndash202 2004
[63] C S Yang J M Landau M T Huang and H L NewmarkldquoInhibition of carcinogenesis by dietary polyphenolic com-poundsrdquo Annual Review of Nutrition vol 21 pp 381ndash406 2001
[64] A Murakami H Ashida and J Terao ldquoMultitargeted cancerprevention by quercetinrdquoCancer Letters vol 269 no 2 pp 315ndash325 2008
[65] G Du Z Zhao Y Chen et al ldquoQuercetin attenuates neuronalautophagy and apoptosis in rat traumatic brain injury modelvia activation of PI3KAkt signaling pathwayrdquo NeurologicalResearch vol 38 no 11 pp 1ndash8 2016
Evidence-Based Complementary and Alternative Medicine 19
[66] Q Cai R O Rahn and R Zhang ldquoDietary flavonoidsquercetin luteolin andgenistein reduce oxidativeDNAdamageand lipid peroxidation and quench free radicalsrdquoCancer Lettersvol 119 no 1 pp 99ndash107 1997
[67] G A Bongiovanni E A Soria and A R Eynard ldquoEffectsof the plant flavonoids silymarin and quercetin on arsenite-induced oxidative stress in CHO-K1 cellsrdquo Food and ChemicalToxicology vol 45 no 6 pp 971ndash976 2007
[68] H Li L Yang Y Zhang et al ldquoKaempferol inhibits fibroblastcollagen synthesis proliferation and activation in hypertrophicscar via targeting TGF-beta receptor type Irdquo Biomedicine ampPharmacotherapy = Biomedecine amp Pharmacotherapie vol 83pp 967ndash974 2016
[69] K P Devi D S Malar S F Nabavi et al ldquoKaempferol andinflammation From chemistry to medicinerdquo PharmacologicalResearch vol 99 pp 1ndash10 2015
[70] M Zhou H Ren J Han W Wang Q Zheng and DWang ldquoProtective effects of kaempferol against myocardialischemiareperfusion injury in isolated rat heart via antioxidantactivity and inhibition of glycogen synthase kinase-3120573rdquo Oxida-tive Medicine and Cellular Longevity vol 2015 p 481405 2015
[71] C-F Lee J-S Yang F-J Tsai et al ldquoKaempferol inducesATMp53-mediated death receptor and mitochondrial apop-tosis in human umbilical vein endothelial cellsrdquo InternationalJournal of Oncology vol 48 no 5 pp 2007ndash2014 2016
[72] C Luo H Yang C Tang et al ldquoKaempferol alleviates insulinresistance via hepatic IKKNF-120581B signal in type 2 diabetic ratsrdquoInternational Immunopharmacology vol 28 no 1 pp 744ndash7502015
[73] A B Awad and C S Fink ldquoPhytosterols as anticancer dietarycomponents evidence and mechanism of actionrdquo Journal ofNutrition vol 130 no 9 pp 2127ndash2130 2000
[74] K Farber and H Kettenmann ldquoFunctional role of calciumsignals for microglial functionrdquoGlia vol 54 no 7 pp 656ndash6652006
[75] Y Lu Y Gu X Ding et al ldquoIntracellular Ca2+ homeostasisand JAK1STAT3 pathway are involved in the protective effectof propofol on BV2 microglia against hypoxia-induced inflam-mation and apoptosisrdquo PLoS ONE vol 12 no 5 p e01780982017
[76] K Leroy and J-P Brion ldquoDevelopmental expression and local-ization of glycogen synthase kinase-3120573 in rat brainrdquo Journal ofChemical Neuroanatomy vol 16 no 4 pp 279ndash293 1999
[77] C-M Liu E-M Hur and F-Q Zhou ldquoCoordinating geneexpression and axon assembly to control axon growth Potentialrole ofGSK3 signalingrdquo Frontiers inMolecularNeuroscience vol3 p 3 2012
[78] D A E Cross A A Culbert K A Chalmers L Facci S DSkaper and A D Reith ldquoSelective small-molecule inhibitors ofglycogen synthase kinase-3 activity protect primary neuronesfrom deathrdquo Journal of Neurochemistry vol 77 no 1 pp 94ndash102 2001
[79] V S Ossovskaya and NW Bunnett ldquoProtease-activated recep-tors contribution to physiology and diseaserdquo PhysiologicalReviews vol 84 no 2 pp 579ndash621 2004
[80] M Steinhoff J Buddenkotte V Shpacovitch et al ldquoProteinase-activated receptors transducers of proteinase-mediated signal-ing in inflammation and immune responserdquoEndocrine Reviewsvol 26 no 1 pp 1ndash43 2005
[81] H Krenzlin V Lorenz S Danckwardt O Kempski and BAlessandri ldquoThe importance of thrombin in cerebral injury and
diseaserdquo International Journal of Molecular Sciences vol 17 no1 2016
[82] M J McGinn and J T Povlishock ldquoPathophysiology of Trau-matic Brain InjuryrdquoNeurosurgery Clinics of North America vol27 no 4 pp 397ndash407 2016
[83] T Woodcock and M C Morganti-Kossmann ldquoThe role ofmarkers of inflammation in traumatic brain injuryrdquo Frontiersin Neurology vol 4 article 18 2013
[84] F Tanriverdi H J Schneider G Aimaretti B E Masel F FCasanueva and F Kelestimur ldquoPituitary dysfunction after trau-matic brain injury A clinical and pathophysiological approachrdquoEndocrine Reviews vol 36 no 3 pp 305ndash342 2015
[85] J V Rosenfeld A I Maas P Bragge M C Morganti-Kossmann G T Manley and R L Gruen ldquoEarly managementof severe traumatic brain injuryrdquoThe Lancet vol 380 no 9847pp 1088ndash1098 2012
[86] B M Aertker S Bedi and C S Cox ldquoStrategies for CNS repairfollowing TBIrdquo Experimental Neurology vol 275 no 3 pp 411ndash426 2016
[87] K Adachi Z Mirzadeh M Sakaguchi et al ldquo120573-catenin signal-ing promotes proliferation of progenitor cells in the adultmousesubventricular zonerdquo Stem Cells vol 25 no 11 pp 2827ndash28362007
[88] Y Hirabayashi and Y Gotoh ldquoStage-dependent fate determina-tion of neural precursor cells in mouse forebrainrdquo NeuroscienceResearch vol 51 no 4 pp 331ndash336 2005
[89] S Ding T Y H Wu A Brinker et al ldquoSynthetic smallmolecules that control stem cell faterdquo Proceedings of theNationalAcadamy of Sciences of the United States of America vol 100 no13 pp 7632ndash7637 2003
[90] N Israsena M Hu W Fu L Kan and J A Kessler ldquoThepresence of FGF2 signaling determines whether 120573-cateninexerts effects on proliferation or neuronal differentiation ofneural stem cellsrdquo Developmental Biology vol 268 no 1 pp220ndash231 2004
[91] A Salehi A Jullienne M Baghchechi et al ldquoUp-regulation ofWnt120573-catenin expression is accompanied with vascular repairafter traumatic brain injuryrdquo Journal of Cerebral Blood Flow ampMetabolism vol 38 no 2 pp 274ndash289 2017
[92] L F Reichardt and K J Tomaselli ldquoExtracellular matrixmolecules and their receptors Functions in neural develop-mentrdquoAnnual Review of Neuroscience vol 14 pp 531ndash570 1991
[93] R M Gibson S E Craig L Heenan C Tournier and M JHumphries ldquoActivation of integrin 12057251205731 delays apoptosis ofNtera2 neuronal cellsrdquoMolecularandCellularNeuroscience vol28 no 3 pp 588ndash598 2005
[94] C C Tate A J Garcıa andMC LaPlaca ldquoPlasma fibronectin isneuroprotective following traumatic brain injuryrdquoExperimentalNeurology vol 207 no 1 pp 13ndash22 2007
[95] C Culmsee and M P Mattson ldquop53 in neuronal apoptosisrdquoBiochemical and Biophysical Research Communications vol 331no 3 pp 761ndash777 2005
[96] M S Geddis and V Rehder ldquoThe phosphorylation state ofneuronal processes determines growth cone formation afterneuronal injuryrdquo Journal of Neuroscience Research vol 74 no2 pp 210ndash220 2003
[97] M L Craske M Fivaz N N Batada and T Meyer ldquoSpines andneurite branches function as geometric attractors that enhanceprotein kinase C actionrdquoThe Journal of Cell Biology vol 170 no7 pp 1147ndash1158 2005
20 Evidence-Based Complementary and Alternative Medicine
[98] A Muscella C Vetrugno L G Cossa et al ldquoApoptosis by[Pt(OOrsquo-acac)(gamma-acac)(DMS)] requires PKC-deltamedi-ated p53 activation in malignant pleural mesotheliomardquo PloSone vol 12 no 7 Article ID e0181114 2017
[99] J S Bonini W C Da Silva L R M Bevilaqua J H MedinaI Izquierdo and M Cammarota ldquoOn the participation ofhippocampal PKC in acquisition consolidation and reconsol-idation of spatial memoryrdquo Neuroscience vol 147 no 1 pp 37ndash45 2007
[100] O Zohar R Lavy X Zi et al ldquoPKC activator therapeutic formild traumatic brain injury in micerdquo Neurobiology of Diseasevol 41 no 2 pp 329ndash337 2011
[101] J David Sweatt ldquoTheneuronalMAPkinase cascade a biochem-ical signal integration system subserving synaptic plasticity andmemoryrdquo Journal ofNeurochemistry vol 76 no 1 pp 1ndash10 2001
[102] Y Matsuoka M Picciano B Maleste et al ldquoInflammatoryresponses to amyloidosis in a transgenic mouse model ofAlzheimerrsquos diseaserdquo The American Journal of Pathology vol158 no 4 pp 1345ndash1354 2001
[103] L Esposito L Gan G-Q Yu C Essrich and L MuckeldquoIntracellularly generated amyloid-120573 peptide counteracts theantiapoptotic function of its precursor protein and primesproapoptotic pathways for activation by other insults in neu-roblastoma cellsrdquo Journal of Neurochemistry vol 91 no 6 pp1260ndash1274 2004
[104] D J Loane A Pocivavsek C E-H Moussa et al ldquoAmyloidprecursor protein secretases as therapeutic targets for traumaticbrain injuryrdquo Nature Medicine vol 15 no 4 pp 377ndash379 2009
[105] R Torres B L Firestein H Dong et al ldquoPDZ proteins bindcluster and synaptically colocalize with Eph receptors and theirephrin ligandsrdquo Neuron vol 21 no 6 pp 1453ndash1463 1998
[106] M B Dalva M A Takasu M Z Lin et al ldquoEphB receptorsinteract with NMDA receptors and regulate excitatory synapseformationrdquo Cell vol 103 no 6 pp 945ndash956 2000
[107] J M Basak P B Verghese H Yoon J Kim and D MHoltzman ldquoLow-density lipoprotein receptor represents anapolipoprotein E-independent pathway of A120573 uptake anddegradation by astrocytesrdquoThe Journal of Biological Chemistryvol 287 no 17 pp 13959ndash13971 2012
[108] L Yao X Gu Q Song et al ldquoNanoformulated alpha-mangostinameliorates Alzheimerrsquos disease neuropathology by elevatingLDLR expression and accelerating amyloid-beta clearancerdquoJournal of Controlled Release vol 226 pp 1ndash14 2016
Stem Cells International
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Evidence-Based Complementary andAlternative Medicine
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Submit your manuscripts atwwwhindawicom
12 Evidence-Based Complementary and Alternative Medicine
Table 6 Top 10 potential candidate compounds of XFZYD for treating TBI according to 2 centrality indicators
Compound Degree Compound Betweennessquercetin 76 quercetin 01682179kaempferol 43 kaempferol 005501511beta-sitosterol 35 wogonin 005141376Stigmasterol 19 beta-sitosterol 004451294luteolin 18 naringenin 003251738baicalein 15 beta-carotene 002977217-Methoxy-2-methyl isoflavone 12 nobiletin 002787992wogonin 12 7-Methoxy-2-methyl isoflavone 002096439nobiletin 11 luteolin 002090223naringenin 9 baicalein 001453488
Table 7 Top 10 potential targets of XFZYD for treating TBI according to 2 centrality indicators
Targets Degree Targets BetweennessCALM 84 CALM 021452046GSK3B 61 SCN5A 011441591SCN5A 59 GSK3B 009553896F2 43 F2 00689171ACHE 28 BCL2 002651903F7 28 CASP3 002372836ADRA1B 28 F7 002032647NOS3 20 ACHE 001845996BCL2 18 ADRA1B 001704505CASP3 18 NOS3 00166699
excitotoxicity or reparative events including angiogenesisscarring and neurogenesis [83] Function analysis of the 47target proteins regulated by XFZYD was mainly associatedwith core pathophysiology process of TBI (Figure 7) 47 targetproteins were connected with 16 key process related to TBIMost of the targets have one or more links to other biolog-ical process such as apoptosis cell proliferation superoxideanion generation nitric oxide biosynthetic process responseto calcium ion I-kappaB kinaseNF-kappaB signaling andregulation of inflammation The above analysis implied themultifunction character of these target proteins regulated byXFZYD Of these target proteins 25 proteins (53 of the 47)such as TGFB1 EGFR CAV1 MAPK1 PRKCB and AKT1were responsible for regulating the apoptosis process 17 forblood coagulation 14 for cell proliferation and axon genesisand 12 for hypoxia andMAPK cascade We found that TGFB1was the crucial protein because it participated in 9 biologicalprocesses related to TBI such as apoptosis blood coagulationcell proliferation and MAPK cascade followed by EGFR (8)CAV1 (7) MAPK1 (6) PRKCB (6) and AKT1 (6)
Overall these observations strongly support the evidencethat the generated HB-pCminuspT network and pT-F networkhave important roles in treating TBI further validating thedrug targeting approach
Molecule docking was used to further validate the bind-ing mode between candidate compounds and their targetproteins We found that 18 TBI-specific target proteins inter-acted with 91 candidate compounds from XFZYD (Figure 8)
Other 29 target proteins were not discussed for the lackof proper protein crystal structure The detailed moleculedocking results are shown in Table S5The 6 essential proteinsincluding GSK3B AKT1 CDK1 F2 NOS3 and ACHEwere used to elucidate the exact binding mode (Figure 9)Quercetin was located within the binding cavity of AKT1 andCDK1 (Figures 9(a) and 9(b) and S1A B) Four conventionalhydrogen bonds were formed between quercetin and AKT1by interacting with the key amino acids including ILE-290 THR-211 and SER 205 Additionally 120587-120587 interactionsbetween quercetin and TRP-90 were found in the activesite which helped the stabilization of the compound at thebinding site (Figure 9(a) S1A) Figure 9(b) and S1B suggestedthat five conventional hydrogen bonds (LEU-83 ASP-146and LYS-33) and 120587-120587 interaction (PHE-80) were formedbetween quercetin and CDK1 The GSK3B-FA complexes(Figure 9(c) S1C) were stabilized by 6 hydrogen-bondinginteractions between FA and LYS-85 GLU-97 TYR-134 andARG-141 Glyasperin Bmainly bonds to F2 through hydrogenbonds by interacting with the key amino acids includingGLY-193 SER-195 and GLY-219 (Figure 9(d) S1D) andan edge-to-face 120587 minus 120587 interaction was also observed withTYR-228 The GLN-247 GLU-351 and GLY-355 from theactive site pocket of NOS3 participated in the hydrogen-bondformationwith 1-Methoxyphaseollidin (Figure 9(e) S1E)The(-)-Medicocarpin formed a total of 6 hydrogen bonds withSER-293 PHE-295 ARG-296 and TRY-341 in the active siteof ACHE (Figure 9(f) S1F) Besides an edge-to-face 120587 minus 120587
Evidence-Based Complementary and Alternative Medicine 13
KCNMA1
P53
IFNG
PPP3CA
CASP8
SCN5A
I-kappaB kinase NF-kappaB signaling
Regulation of inflammation
Response to calcium ion
TGFB1
TNF
PRKCA
CHRNA7
CAV1
STAT1
MAPK cascadeAKT1
BCL2
APP
EGFR
MAPK1
Angiogenesis
SYNGAP1
MAP2
PTPN1
Axonogenesis
GSK3B
EPHB2
CDK1
CASP7FN1
MAPK3
CTNNB1
F2
Autophagy
Cell proliferation
BACE1
SLC6A3
DRD1
ADRA1BACHE
CALMCASP3
PLAT
Apoptotic process
NOS3
PRKCB
Blood coagulation
Response to hypoxia
Superoxide anion generation
Nitric oxide biosynthetic process
Astrocyte activation
Amyloid-beta regulation
Microglial cell activation
LDLR
PRSS3
F7
PRKCD
SOD1ALB
BBC3
Figure 7 pT-F network of XFZYD for treating TBI 16 biological processes (red square) the 47 target proteins (periwinkle circle) of XFZYDparticipate in for treating TBI
interactionwas also observedwith TYR-337 From the resultshydrogen-bonding and edge-to-face 120587 minus 120587 interactions playkey roles in the proteinminusligand recognition and stabilitywhich may be helpful in determining the inhibitor activitiesAnd the C-T network confirmed the potential therapeuticeffects of the candidate compounds from XFZYD to treatTBI through interacting with the relevant proteins The com-putational analysis further elucidated the accurate moleculemechanisms between active compounds and targets
4 Discussion
Traumatic brain injury (TBI) is a growing public healthproblem worldwide and is a leading cause of death anddisability [84] Although major progress has been made in
understanding the pathophysiology of this injury this hasnot yet led to substantial improvements in outcome by alack of treatments which have proven successful during phaseIII trials for modern medicine [85 86] TCM rooted inthousands of years of history may offer an alternative or acomplementary strategy for the treatment of TBI XFZYDa representative formula in TCM has been used for yearsto treat TBI in China and has been demonstrated to beeffective in clinical practice However its ldquomulticomponentsrdquoand ldquomultitargetsrdquo features make it much difficult to decipherthemolecular mechanisms of XFZYD in the treatment of TBIfrom a systematic perspective if employing routine methods
In the present study a network pharmacology-basedmethod was employed to elucidate the pharmacologicalmechanisms of XFZYD to treat TBI according to the drug
14 Evidence-Based Complementary and Alternative Medicine
Figure 8 C-T network through molecule docking validating 119 potential compounds (green triangles) interacting with 18 potential targets(yellow circles) of XFZYDThe size of the nodes is proportional to the value of degree
combination principle of TCM We first proposed a newmodeling system combining OB and DL screening multipledrug targets prediction and validation network constructionand molecule docking to probe the efficiency of a typicalTCM formula XFZYD for the treatment of TBI The 11herbs from XFZYD possessed 162 bioactive compounds andtargeted 285 proteins There were 5 compounds and 189
target proteins overlapped among the Jun Chen and Zuo-Shigroup Furthermore 47 TBI-specific proteins were targetedby 119 (73) bioactive compounds from XFZYD Similarly 5common compounds and 33 (70) common target proteinsamong the 3 groups of drugs were observed Most of thebioactive ingredients targeted more than one protein The 47target proteins regulated several essential pathophysiological
Evidence-Based Complementary and Alternative Medicine 15
(a) (b) (c)
(d) (e) (f)
Figure 9 Hydrogen-bonding networks within the binding site of the compoundminustarget complexes obtained from molecule docking(a) AKT1-quercetin (b) CDK1-quercetin (c) GSK3B-FA (d) F2-glyasperin B (e) NOS3-1-Methoxyphaseollidin and (f) ACHE-(-)-Medicocarpin The molecules are presented as ball and stick models Active site amino acid residues are represented as lines Dotted bluelines in these pictures represent hydrogen bonds with distance unit of A Other O and N atoms are colored as red and blue respectively
processes of TBI that referred to apoptosis inflammationcell proliferation superoxide anion generation nitric oxidebiosynthetic process response to calcium ion etc The aboveanalysis reveals that the synergistic action mechanisms ofXFZYDmay be (1) bioactive compounds overlapping amongthe different group of herbs (2) specific bioactive com-pounds from different groups of herbs targeting the sameproteins (3) specific bioactive compounds from differentgroups of herbs targeting different proteins which participatein the same pathophysiological process of the disease Toa certain degree the 5 compounds including quercetinstigmasterol kaempferol baicalin and beta-sitosterol playedessential role in XFZYD for TBI treatment MAPK3MAPK1AKT1 PRKCA TNF PRKCB EGFR BCL2 GSK3B CASP3PPP3CA and NOS3 were the main target proteins regulatedby XFZYD in the treatment of TBI
Interestingly Beta-carotene (Mol 43) from Carthami Flos(the Jun herb) specifically regulated 120573-Catenin (CTNNB1)and played critical role for curing TBI The 120573-Catenin is acritical downstream component of the Wnt pathway whichplays essential role in the regulation of mammalian neuraldevelopment [87] In vitro and in vivo studies demonstrate
that the Wnt120573-catenin pathway regulates the proliferationand differentiation of neural progenitor cells [88] Neuronaldifferentiation is induced by overexpression of 120573-cateninor the pharmacological inhibition of GSK3120573 (the phospho-rylating enzyme of 120573-catenin) [89 90] This pathway alsopromotes blood vessel formation during vascular develop-ment as well as the vascular repair process after TBI [91]In addition wogonin (Mol 160) from Achyranthis BidentataeRadix (the Chen herb) specifically targeted fibronectin (FN1)Bcl-2-binding component 3 (BBC3) and Protein KinaseC delta type (PRKCD) FN1 an important component ofthe extracellular matrix (ECM) environment promotes cellmigration neurite outgrowth and synapse formation dur-ing neural development [92] It aggregates in the injuredbrain and plays a neuroprotection role through antiapoptosisand anti-inflammation ways following TBI [93 94] BBC3namely p53 upregulated modulator of apoptosis (PUMA) iscritical for the p53-dependent apoptosis pathway which playsan important role in hippocampal neuronal loss and associ-ated cognitive deficits [95] PRKCD one of PKC isoformsactivates signal transduction pathways involved in neuronalregeneration [96] synaptic transmissionplasticity [97] and
16 Evidence-Based Complementary and Alternative Medicine
activation of apoptosis processes [98] as well as higher brainfunctions such as learning andmemory [99]The activators ofPKCare effective for the treatment of TBI [100] FurthermoreMitogen-activated protein kinase 3 (MAPK3) Beta-secretase1 (BACE1) Ephrin type-B receptor 2 (EphB) and low-densitylipoprotein receptor (LDLR) were specifically targeted bythe ingredients in the Zuo-Shi herbs Naringenin (Mol133) targeted LDLR MAPK3 while euchrenone (Mol 60)anchored BACE1 and nobiletin (Mol 134) targeted EphB2MAPK3 is an essential component of the MAP kinase signaltransduction pathway It has been appreciated recently thatthe ERK12 cascade plays a fundamental role in synapticplasticity and memory [101] BACE1 is responsible for pro-duction of A120573 from amyloid precursor protein (APP) A120573can cause cell death activate inflammatory pathways [102]and prime proapoptotic pathways for activation by otherinsults [103] The blocking of BACE1 can ameliorate motorand cognitive deficits and reduce cell loss after experimentalTBI in mice [104] EphB is localized to synaptic sites inhippocampal neurons [105] The interaction between EphBand NMDA receptors regulates excitatory synapse formation[106] LDLR acts as an important receptor that facilitatesbrain A120573 clearance and inhibits amyloid deposition [107]and then ameliorates Alzheimerrsquos disease neuropathologyafter TBI [108] The analysis above indicates the ldquoJunrdquoldquoChenrdquo and ldquoZuo-Shirdquo herbs from XFZYD trigger theirspecific targets regulation respectively for the therapeuticeffects
XFZYD is a very famous traditional Chinese formula inpromoting qi circulation and removing blood stasis accord-ing to TCM theory However several limited researches havedemonstrated its efficacy for treating TBI such as anti-inflammatory and synaptic regulation [30 31] which arein accord with our study However previous studies merelypartially deciphered the molecule mechanism of XFZYD fortreating TBI This study reports 119 bioactive compounds inXFZYD that target 47 TBI-specific proteins such as MAPK3MAPK1 AKT1 PRKCA TNF PRKCB and EGFR Theseproteins regulate several crucial pathophysiological processesof TBI such as apoptosis inflammation blood coagulationand axon genesis Our study demonstrates that the therapeu-tic actions of XFZYD refer to ldquomulticompoundsrdquo ldquomultitar-getsrdquo features rather than only the improvement of bloodcirculation With the help of molecule docking methodwe further validate the interactions between bioactive com-pounds and potential targets of XFZYD The hydrogen-bonding and edge-to-face 120587 minus120587 interactions play key roles inthe proteinminusligand recognition and stability This provides avaluable reference for further experimental investigations ofbioactive ingredients and therapeutic targets of XFZYD fortreating TBI
5 Conclusion
Our work successfully illuminates the efficiency of XFZYDfor the treatment of TBI as well as herb combination ruleof TCM formula Network pharmacology with moleculedocking method confirms the ldquomulticompounds multitar-getsrdquo therapeutic actions of XFZYD in the treatment of TBI
The present work may provide valuable evidence for furtherclinical application of XFZYD for treating TBI
Abbreviations
TBI Traumatic brain injuryXFZYD Xuefu Zhuyu decoctionTCM Traditional Chines medicineDL Drug-likenessOB Oral bioavailabilityHB-cC-cT Herb-candidate compound-candidate
targetHB-pC-pT Herb-potential compound-potential targetCALM CalmodulinGSK3B Glycogen synthase kinase-3 betaSCN5A Sodium channel protein type 5 subunit
alphaF2 ProthrombinACHE AcetylcholinesteraseGO Gene OntologyKEGG Kyoto Encyclopedia of Genes and
Genomes
Data Availability
The data used to support the findings of this study areavailable from the corresponding author upon request
Conflicts of Interest
The authors declare that they have no conflicts of interest
Authorsrsquo Contributions
YangWang and Zebing Huang participated in the conceptionand design of the study YuanyuanZhong YangWang ZebingHuang Jiekun Luo Tao Tang and Pengfei Li acquired andanalyzed the data Yuanyuan Zhong Tao Liu and HanjinCui drafted and revised the manuscript The correspondingauthor and all of the authors have read and approved the finalsubmitted manuscript
Acknowledgments
This work was supported by the National Natural ScienceFoundation of China (Nos 81673719 81874409 81603670and 81803948) Project funded byChina Postdoctoral ScienceFoundation (Nos 2016M600639 and 2017T100614)
Supplementary Materials
Table S1 162 bioactive compounds in XFZYD Table S2 targetproteins of XFZYD Table S3 TBI-specific proteins Table S4119 potential compounds of XFZYD for treating TBI TableS5 docking result of 18 target proteins with 91 potential com-pounds Fig S1 the exact bindingmode between active ingre-dients and protein targets obtained from molecule docking(A) AKT1-quercetin (B) CDK1-quercetin (C) GSK3B-FA
Evidence-Based Complementary and Alternative Medicine 17
(D) F2-glyasperin B (E) NOS3-1-Methoxyphaseollidin and(F)ACHE-(-)-Medicocarpin (Supplementary Materials)
References
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[2] J A Langlois W Rutland-Brown and M M Wald ldquoTheepidemiology and impact of traumatic brain injury a briefoverviewrdquo The Journal of Head Trauma Rehabilitation vol 21no 5 pp 375ndash378 2006
[3] H A Alsulaim B J Smart A O Asemota et al ldquoConsciousstatus predictsmortality among patientswith isolated traumaticbrain injury in administrative datardquo The American Journal ofSurgery vol 214 no 2 pp 207ndash210 2017
[4] M Majdan D Plancikova A Maas et al ldquoYears of life lost dueto traumatic brain injury in Europe A cross-sectional analysisof 16 countriesrdquo PLoS Medicine vol 14 no 7 p e1002331 2017
[5] P Cheng P Yin P Ning et al ldquoTrends in traumatic braininjury mortality in China 2006ndash2013 A population-basedlongitudinal studyrdquo PLoS Medicine vol 14 no 7 Article IDe1002332 2017
[6] M V Russo and D B McGavern ldquoInflammatory neuroprotec-tion following traumatic brain injuryrdquo Science vol 353 no 6301pp 783ndash785 2016
[7] WWang H Li J Yu et al ldquoProtective Effects of ChineseHerbalMedicine Rhizoma drynariae in Rats After Traumatic BrainInjury and Identification of Active Compoundrdquo MolecularNeurobiology vol 53 no 7 pp 4809ndash4820 2016
[8] M Das S Mohapatra and S S Mohapatra ldquoNew perspectiveson central and peripheral immune responses to acute traumaticbrain injuryrdquo Journal of Neuroinflammation vol 9 p 236 2012
[9] J W Finnie ldquoNeuroinflammation Beneficial and detrimentaleffects after traumatic brain injuryrdquo Inflammopharmacologyvol 21 no 4 pp 309ndash320 2013
[10] T Cheng W Wang Q Li et al ldquoCerebroprotection of flavanol(minus)-epicatechin after traumatic brain injury viaNrf2-dependentand -independent pathwaysrdquo Free Radical Biology amp Medicinevol 92 pp 15ndash28 2016
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[12] R Bullock A Zauner J S Myseros et al ldquoEvidence forProlonged Release of Excitatory Amino Acids in Severe HumanHead Trauma Relationship to Clinical Eventsrdquo Annals of theNew York Academy of Sciences vol 765 no 1 pp 290ndash297 1995
[13] A T Mazzeo A Beat A Singh and M R Bullock ldquoTherole of mitochondrial transition pore and its modulation intraumatic brain injury and delayed neurodegeneration afterTBIrdquo Experimental Neurology vol 218 no 2 pp 363ndash370 2009
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18 Evidence-Based Complementary and Alternative Medicine
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[80] M Steinhoff J Buddenkotte V Shpacovitch et al ldquoProteinase-activated receptors transducers of proteinase-mediated signal-ing in inflammation and immune responserdquoEndocrine Reviewsvol 26 no 1 pp 1ndash43 2005
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[82] M J McGinn and J T Povlishock ldquoPathophysiology of Trau-matic Brain InjuryrdquoNeurosurgery Clinics of North America vol27 no 4 pp 397ndash407 2016
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[85] J V Rosenfeld A I Maas P Bragge M C Morganti-Kossmann G T Manley and R L Gruen ldquoEarly managementof severe traumatic brain injuryrdquoThe Lancet vol 380 no 9847pp 1088ndash1098 2012
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[89] S Ding T Y H Wu A Brinker et al ldquoSynthetic smallmolecules that control stem cell faterdquo Proceedings of theNationalAcadamy of Sciences of the United States of America vol 100 no13 pp 7632ndash7637 2003
[90] N Israsena M Hu W Fu L Kan and J A Kessler ldquoThepresence of FGF2 signaling determines whether 120573-cateninexerts effects on proliferation or neuronal differentiation ofneural stem cellsrdquo Developmental Biology vol 268 no 1 pp220ndash231 2004
[91] A Salehi A Jullienne M Baghchechi et al ldquoUp-regulation ofWnt120573-catenin expression is accompanied with vascular repairafter traumatic brain injuryrdquo Journal of Cerebral Blood Flow ampMetabolism vol 38 no 2 pp 274ndash289 2017
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20 Evidence-Based Complementary and Alternative Medicine
[98] A Muscella C Vetrugno L G Cossa et al ldquoApoptosis by[Pt(OOrsquo-acac)(gamma-acac)(DMS)] requires PKC-deltamedi-ated p53 activation in malignant pleural mesotheliomardquo PloSone vol 12 no 7 Article ID e0181114 2017
[99] J S Bonini W C Da Silva L R M Bevilaqua J H MedinaI Izquierdo and M Cammarota ldquoOn the participation ofhippocampal PKC in acquisition consolidation and reconsol-idation of spatial memoryrdquo Neuroscience vol 147 no 1 pp 37ndash45 2007
[100] O Zohar R Lavy X Zi et al ldquoPKC activator therapeutic formild traumatic brain injury in micerdquo Neurobiology of Diseasevol 41 no 2 pp 329ndash337 2011
[101] J David Sweatt ldquoTheneuronalMAPkinase cascade a biochem-ical signal integration system subserving synaptic plasticity andmemoryrdquo Journal ofNeurochemistry vol 76 no 1 pp 1ndash10 2001
[102] Y Matsuoka M Picciano B Maleste et al ldquoInflammatoryresponses to amyloidosis in a transgenic mouse model ofAlzheimerrsquos diseaserdquo The American Journal of Pathology vol158 no 4 pp 1345ndash1354 2001
[103] L Esposito L Gan G-Q Yu C Essrich and L MuckeldquoIntracellularly generated amyloid-120573 peptide counteracts theantiapoptotic function of its precursor protein and primesproapoptotic pathways for activation by other insults in neu-roblastoma cellsrdquo Journal of Neurochemistry vol 91 no 6 pp1260ndash1274 2004
[104] D J Loane A Pocivavsek C E-H Moussa et al ldquoAmyloidprecursor protein secretases as therapeutic targets for traumaticbrain injuryrdquo Nature Medicine vol 15 no 4 pp 377ndash379 2009
[105] R Torres B L Firestein H Dong et al ldquoPDZ proteins bindcluster and synaptically colocalize with Eph receptors and theirephrin ligandsrdquo Neuron vol 21 no 6 pp 1453ndash1463 1998
[106] M B Dalva M A Takasu M Z Lin et al ldquoEphB receptorsinteract with NMDA receptors and regulate excitatory synapseformationrdquo Cell vol 103 no 6 pp 945ndash956 2000
[107] J M Basak P B Verghese H Yoon J Kim and D MHoltzman ldquoLow-density lipoprotein receptor represents anapolipoprotein E-independent pathway of A120573 uptake anddegradation by astrocytesrdquoThe Journal of Biological Chemistryvol 287 no 17 pp 13959ndash13971 2012
[108] L Yao X Gu Q Song et al ldquoNanoformulated alpha-mangostinameliorates Alzheimerrsquos disease neuropathology by elevatingLDLR expression and accelerating amyloid-beta clearancerdquoJournal of Controlled Release vol 226 pp 1ndash14 2016
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Evidence-Based Complementary and Alternative Medicine 13
KCNMA1
P53
IFNG
PPP3CA
CASP8
SCN5A
I-kappaB kinase NF-kappaB signaling
Regulation of inflammation
Response to calcium ion
TGFB1
TNF
PRKCA
CHRNA7
CAV1
STAT1
MAPK cascadeAKT1
BCL2
APP
EGFR
MAPK1
Angiogenesis
SYNGAP1
MAP2
PTPN1
Axonogenesis
GSK3B
EPHB2
CDK1
CASP7FN1
MAPK3
CTNNB1
F2
Autophagy
Cell proliferation
BACE1
SLC6A3
DRD1
ADRA1BACHE
CALMCASP3
PLAT
Apoptotic process
NOS3
PRKCB
Blood coagulation
Response to hypoxia
Superoxide anion generation
Nitric oxide biosynthetic process
Astrocyte activation
Amyloid-beta regulation
Microglial cell activation
LDLR
PRSS3
F7
PRKCD
SOD1ALB
BBC3
Figure 7 pT-F network of XFZYD for treating TBI 16 biological processes (red square) the 47 target proteins (periwinkle circle) of XFZYDparticipate in for treating TBI
interactionwas also observedwith TYR-337 From the resultshydrogen-bonding and edge-to-face 120587 minus 120587 interactions playkey roles in the proteinminusligand recognition and stabilitywhich may be helpful in determining the inhibitor activitiesAnd the C-T network confirmed the potential therapeuticeffects of the candidate compounds from XFZYD to treatTBI through interacting with the relevant proteins The com-putational analysis further elucidated the accurate moleculemechanisms between active compounds and targets
4 Discussion
Traumatic brain injury (TBI) is a growing public healthproblem worldwide and is a leading cause of death anddisability [84] Although major progress has been made in
understanding the pathophysiology of this injury this hasnot yet led to substantial improvements in outcome by alack of treatments which have proven successful during phaseIII trials for modern medicine [85 86] TCM rooted inthousands of years of history may offer an alternative or acomplementary strategy for the treatment of TBI XFZYDa representative formula in TCM has been used for yearsto treat TBI in China and has been demonstrated to beeffective in clinical practice However its ldquomulticomponentsrdquoand ldquomultitargetsrdquo features make it much difficult to decipherthemolecular mechanisms of XFZYD in the treatment of TBIfrom a systematic perspective if employing routine methods
In the present study a network pharmacology-basedmethod was employed to elucidate the pharmacologicalmechanisms of XFZYD to treat TBI according to the drug
14 Evidence-Based Complementary and Alternative Medicine
Figure 8 C-T network through molecule docking validating 119 potential compounds (green triangles) interacting with 18 potential targets(yellow circles) of XFZYDThe size of the nodes is proportional to the value of degree
combination principle of TCM We first proposed a newmodeling system combining OB and DL screening multipledrug targets prediction and validation network constructionand molecule docking to probe the efficiency of a typicalTCM formula XFZYD for the treatment of TBI The 11herbs from XFZYD possessed 162 bioactive compounds andtargeted 285 proteins There were 5 compounds and 189
target proteins overlapped among the Jun Chen and Zuo-Shigroup Furthermore 47 TBI-specific proteins were targetedby 119 (73) bioactive compounds from XFZYD Similarly 5common compounds and 33 (70) common target proteinsamong the 3 groups of drugs were observed Most of thebioactive ingredients targeted more than one protein The 47target proteins regulated several essential pathophysiological
Evidence-Based Complementary and Alternative Medicine 15
(a) (b) (c)
(d) (e) (f)
Figure 9 Hydrogen-bonding networks within the binding site of the compoundminustarget complexes obtained from molecule docking(a) AKT1-quercetin (b) CDK1-quercetin (c) GSK3B-FA (d) F2-glyasperin B (e) NOS3-1-Methoxyphaseollidin and (f) ACHE-(-)-Medicocarpin The molecules are presented as ball and stick models Active site amino acid residues are represented as lines Dotted bluelines in these pictures represent hydrogen bonds with distance unit of A Other O and N atoms are colored as red and blue respectively
processes of TBI that referred to apoptosis inflammationcell proliferation superoxide anion generation nitric oxidebiosynthetic process response to calcium ion etc The aboveanalysis reveals that the synergistic action mechanisms ofXFZYDmay be (1) bioactive compounds overlapping amongthe different group of herbs (2) specific bioactive com-pounds from different groups of herbs targeting the sameproteins (3) specific bioactive compounds from differentgroups of herbs targeting different proteins which participatein the same pathophysiological process of the disease Toa certain degree the 5 compounds including quercetinstigmasterol kaempferol baicalin and beta-sitosterol playedessential role in XFZYD for TBI treatment MAPK3MAPK1AKT1 PRKCA TNF PRKCB EGFR BCL2 GSK3B CASP3PPP3CA and NOS3 were the main target proteins regulatedby XFZYD in the treatment of TBI
Interestingly Beta-carotene (Mol 43) from Carthami Flos(the Jun herb) specifically regulated 120573-Catenin (CTNNB1)and played critical role for curing TBI The 120573-Catenin is acritical downstream component of the Wnt pathway whichplays essential role in the regulation of mammalian neuraldevelopment [87] In vitro and in vivo studies demonstrate
that the Wnt120573-catenin pathway regulates the proliferationand differentiation of neural progenitor cells [88] Neuronaldifferentiation is induced by overexpression of 120573-cateninor the pharmacological inhibition of GSK3120573 (the phospho-rylating enzyme of 120573-catenin) [89 90] This pathway alsopromotes blood vessel formation during vascular develop-ment as well as the vascular repair process after TBI [91]In addition wogonin (Mol 160) from Achyranthis BidentataeRadix (the Chen herb) specifically targeted fibronectin (FN1)Bcl-2-binding component 3 (BBC3) and Protein KinaseC delta type (PRKCD) FN1 an important component ofthe extracellular matrix (ECM) environment promotes cellmigration neurite outgrowth and synapse formation dur-ing neural development [92] It aggregates in the injuredbrain and plays a neuroprotection role through antiapoptosisand anti-inflammation ways following TBI [93 94] BBC3namely p53 upregulated modulator of apoptosis (PUMA) iscritical for the p53-dependent apoptosis pathway which playsan important role in hippocampal neuronal loss and associ-ated cognitive deficits [95] PRKCD one of PKC isoformsactivates signal transduction pathways involved in neuronalregeneration [96] synaptic transmissionplasticity [97] and
16 Evidence-Based Complementary and Alternative Medicine
activation of apoptosis processes [98] as well as higher brainfunctions such as learning andmemory [99]The activators ofPKCare effective for the treatment of TBI [100] FurthermoreMitogen-activated protein kinase 3 (MAPK3) Beta-secretase1 (BACE1) Ephrin type-B receptor 2 (EphB) and low-densitylipoprotein receptor (LDLR) were specifically targeted bythe ingredients in the Zuo-Shi herbs Naringenin (Mol133) targeted LDLR MAPK3 while euchrenone (Mol 60)anchored BACE1 and nobiletin (Mol 134) targeted EphB2MAPK3 is an essential component of the MAP kinase signaltransduction pathway It has been appreciated recently thatthe ERK12 cascade plays a fundamental role in synapticplasticity and memory [101] BACE1 is responsible for pro-duction of A120573 from amyloid precursor protein (APP) A120573can cause cell death activate inflammatory pathways [102]and prime proapoptotic pathways for activation by otherinsults [103] The blocking of BACE1 can ameliorate motorand cognitive deficits and reduce cell loss after experimentalTBI in mice [104] EphB is localized to synaptic sites inhippocampal neurons [105] The interaction between EphBand NMDA receptors regulates excitatory synapse formation[106] LDLR acts as an important receptor that facilitatesbrain A120573 clearance and inhibits amyloid deposition [107]and then ameliorates Alzheimerrsquos disease neuropathologyafter TBI [108] The analysis above indicates the ldquoJunrdquoldquoChenrdquo and ldquoZuo-Shirdquo herbs from XFZYD trigger theirspecific targets regulation respectively for the therapeuticeffects
XFZYD is a very famous traditional Chinese formula inpromoting qi circulation and removing blood stasis accord-ing to TCM theory However several limited researches havedemonstrated its efficacy for treating TBI such as anti-inflammatory and synaptic regulation [30 31] which arein accord with our study However previous studies merelypartially deciphered the molecule mechanism of XFZYD fortreating TBI This study reports 119 bioactive compounds inXFZYD that target 47 TBI-specific proteins such as MAPK3MAPK1 AKT1 PRKCA TNF PRKCB and EGFR Theseproteins regulate several crucial pathophysiological processesof TBI such as apoptosis inflammation blood coagulationand axon genesis Our study demonstrates that the therapeu-tic actions of XFZYD refer to ldquomulticompoundsrdquo ldquomultitar-getsrdquo features rather than only the improvement of bloodcirculation With the help of molecule docking methodwe further validate the interactions between bioactive com-pounds and potential targets of XFZYD The hydrogen-bonding and edge-to-face 120587 minus120587 interactions play key roles inthe proteinminusligand recognition and stability This provides avaluable reference for further experimental investigations ofbioactive ingredients and therapeutic targets of XFZYD fortreating TBI
5 Conclusion
Our work successfully illuminates the efficiency of XFZYDfor the treatment of TBI as well as herb combination ruleof TCM formula Network pharmacology with moleculedocking method confirms the ldquomulticompounds multitar-getsrdquo therapeutic actions of XFZYD in the treatment of TBI
The present work may provide valuable evidence for furtherclinical application of XFZYD for treating TBI
Abbreviations
TBI Traumatic brain injuryXFZYD Xuefu Zhuyu decoctionTCM Traditional Chines medicineDL Drug-likenessOB Oral bioavailabilityHB-cC-cT Herb-candidate compound-candidate
targetHB-pC-pT Herb-potential compound-potential targetCALM CalmodulinGSK3B Glycogen synthase kinase-3 betaSCN5A Sodium channel protein type 5 subunit
alphaF2 ProthrombinACHE AcetylcholinesteraseGO Gene OntologyKEGG Kyoto Encyclopedia of Genes and
Genomes
Data Availability
The data used to support the findings of this study areavailable from the corresponding author upon request
Conflicts of Interest
The authors declare that they have no conflicts of interest
Authorsrsquo Contributions
YangWang and Zebing Huang participated in the conceptionand design of the study YuanyuanZhong YangWang ZebingHuang Jiekun Luo Tao Tang and Pengfei Li acquired andanalyzed the data Yuanyuan Zhong Tao Liu and HanjinCui drafted and revised the manuscript The correspondingauthor and all of the authors have read and approved the finalsubmitted manuscript
Acknowledgments
This work was supported by the National Natural ScienceFoundation of China (Nos 81673719 81874409 81603670and 81803948) Project funded byChina Postdoctoral ScienceFoundation (Nos 2016M600639 and 2017T100614)
Supplementary Materials
Table S1 162 bioactive compounds in XFZYD Table S2 targetproteins of XFZYD Table S3 TBI-specific proteins Table S4119 potential compounds of XFZYD for treating TBI TableS5 docking result of 18 target proteins with 91 potential com-pounds Fig S1 the exact bindingmode between active ingre-dients and protein targets obtained from molecule docking(A) AKT1-quercetin (B) CDK1-quercetin (C) GSK3B-FA
Evidence-Based Complementary and Alternative Medicine 17
(D) F2-glyasperin B (E) NOS3-1-Methoxyphaseollidin and(F)ACHE-(-)-Medicocarpin (Supplementary Materials)
References
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[2] J A Langlois W Rutland-Brown and M M Wald ldquoTheepidemiology and impact of traumatic brain injury a briefoverviewrdquo The Journal of Head Trauma Rehabilitation vol 21no 5 pp 375ndash378 2006
[3] H A Alsulaim B J Smart A O Asemota et al ldquoConsciousstatus predictsmortality among patientswith isolated traumaticbrain injury in administrative datardquo The American Journal ofSurgery vol 214 no 2 pp 207ndash210 2017
[4] M Majdan D Plancikova A Maas et al ldquoYears of life lost dueto traumatic brain injury in Europe A cross-sectional analysisof 16 countriesrdquo PLoS Medicine vol 14 no 7 p e1002331 2017
[5] P Cheng P Yin P Ning et al ldquoTrends in traumatic braininjury mortality in China 2006ndash2013 A population-basedlongitudinal studyrdquo PLoS Medicine vol 14 no 7 Article IDe1002332 2017
[6] M V Russo and D B McGavern ldquoInflammatory neuroprotec-tion following traumatic brain injuryrdquo Science vol 353 no 6301pp 783ndash785 2016
[7] WWang H Li J Yu et al ldquoProtective Effects of ChineseHerbalMedicine Rhizoma drynariae in Rats After Traumatic BrainInjury and Identification of Active Compoundrdquo MolecularNeurobiology vol 53 no 7 pp 4809ndash4820 2016
[8] M Das S Mohapatra and S S Mohapatra ldquoNew perspectiveson central and peripheral immune responses to acute traumaticbrain injuryrdquo Journal of Neuroinflammation vol 9 p 236 2012
[9] J W Finnie ldquoNeuroinflammation Beneficial and detrimentaleffects after traumatic brain injuryrdquo Inflammopharmacologyvol 21 no 4 pp 309ndash320 2013
[10] T Cheng W Wang Q Li et al ldquoCerebroprotection of flavanol(minus)-epicatechin after traumatic brain injury viaNrf2-dependentand -independent pathwaysrdquo Free Radical Biology amp Medicinevol 92 pp 15ndash28 2016
[11] W Young ldquoRole of calcium in central nervous system injuriesrdquoJournal of Neurotrauma vol 9 Suppl 1 pp S9ndashS25 1992
[12] R Bullock A Zauner J S Myseros et al ldquoEvidence forProlonged Release of Excitatory Amino Acids in Severe HumanHead Trauma Relationship to Clinical Eventsrdquo Annals of theNew York Academy of Sciences vol 765 no 1 pp 290ndash297 1995
[13] A T Mazzeo A Beat A Singh and M R Bullock ldquoTherole of mitochondrial transition pore and its modulation intraumatic brain injury and delayed neurodegeneration afterTBIrdquo Experimental Neurology vol 218 no 2 pp 363ndash370 2009
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[19] F Cheung ldquoTCM made in Chinardquo Nature vol 480 no 7378pp S82ndashS83 2011
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18 Evidence-Based Complementary and Alternative Medicine
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[88] Y Hirabayashi and Y Gotoh ldquoStage-dependent fate determina-tion of neural precursor cells in mouse forebrainrdquo NeuroscienceResearch vol 51 no 4 pp 331ndash336 2005
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[90] N Israsena M Hu W Fu L Kan and J A Kessler ldquoThepresence of FGF2 signaling determines whether 120573-cateninexerts effects on proliferation or neuronal differentiation ofneural stem cellsrdquo Developmental Biology vol 268 no 1 pp220ndash231 2004
[91] A Salehi A Jullienne M Baghchechi et al ldquoUp-regulation ofWnt120573-catenin expression is accompanied with vascular repairafter traumatic brain injuryrdquo Journal of Cerebral Blood Flow ampMetabolism vol 38 no 2 pp 274ndash289 2017
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[95] C Culmsee and M P Mattson ldquop53 in neuronal apoptosisrdquoBiochemical and Biophysical Research Communications vol 331no 3 pp 761ndash777 2005
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20 Evidence-Based Complementary and Alternative Medicine
[98] A Muscella C Vetrugno L G Cossa et al ldquoApoptosis by[Pt(OOrsquo-acac)(gamma-acac)(DMS)] requires PKC-deltamedi-ated p53 activation in malignant pleural mesotheliomardquo PloSone vol 12 no 7 Article ID e0181114 2017
[99] J S Bonini W C Da Silva L R M Bevilaqua J H MedinaI Izquierdo and M Cammarota ldquoOn the participation ofhippocampal PKC in acquisition consolidation and reconsol-idation of spatial memoryrdquo Neuroscience vol 147 no 1 pp 37ndash45 2007
[100] O Zohar R Lavy X Zi et al ldquoPKC activator therapeutic formild traumatic brain injury in micerdquo Neurobiology of Diseasevol 41 no 2 pp 329ndash337 2011
[101] J David Sweatt ldquoTheneuronalMAPkinase cascade a biochem-ical signal integration system subserving synaptic plasticity andmemoryrdquo Journal ofNeurochemistry vol 76 no 1 pp 1ndash10 2001
[102] Y Matsuoka M Picciano B Maleste et al ldquoInflammatoryresponses to amyloidosis in a transgenic mouse model ofAlzheimerrsquos diseaserdquo The American Journal of Pathology vol158 no 4 pp 1345ndash1354 2001
[103] L Esposito L Gan G-Q Yu C Essrich and L MuckeldquoIntracellularly generated amyloid-120573 peptide counteracts theantiapoptotic function of its precursor protein and primesproapoptotic pathways for activation by other insults in neu-roblastoma cellsrdquo Journal of Neurochemistry vol 91 no 6 pp1260ndash1274 2004
[104] D J Loane A Pocivavsek C E-H Moussa et al ldquoAmyloidprecursor protein secretases as therapeutic targets for traumaticbrain injuryrdquo Nature Medicine vol 15 no 4 pp 377ndash379 2009
[105] R Torres B L Firestein H Dong et al ldquoPDZ proteins bindcluster and synaptically colocalize with Eph receptors and theirephrin ligandsrdquo Neuron vol 21 no 6 pp 1453ndash1463 1998
[106] M B Dalva M A Takasu M Z Lin et al ldquoEphB receptorsinteract with NMDA receptors and regulate excitatory synapseformationrdquo Cell vol 103 no 6 pp 945ndash956 2000
[107] J M Basak P B Verghese H Yoon J Kim and D MHoltzman ldquoLow-density lipoprotein receptor represents anapolipoprotein E-independent pathway of A120573 uptake anddegradation by astrocytesrdquoThe Journal of Biological Chemistryvol 287 no 17 pp 13959ndash13971 2012
[108] L Yao X Gu Q Song et al ldquoNanoformulated alpha-mangostinameliorates Alzheimerrsquos disease neuropathology by elevatingLDLR expression and accelerating amyloid-beta clearancerdquoJournal of Controlled Release vol 226 pp 1ndash14 2016
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Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
14 Evidence-Based Complementary and Alternative Medicine
Figure 8 C-T network through molecule docking validating 119 potential compounds (green triangles) interacting with 18 potential targets(yellow circles) of XFZYDThe size of the nodes is proportional to the value of degree
combination principle of TCM We first proposed a newmodeling system combining OB and DL screening multipledrug targets prediction and validation network constructionand molecule docking to probe the efficiency of a typicalTCM formula XFZYD for the treatment of TBI The 11herbs from XFZYD possessed 162 bioactive compounds andtargeted 285 proteins There were 5 compounds and 189
target proteins overlapped among the Jun Chen and Zuo-Shigroup Furthermore 47 TBI-specific proteins were targetedby 119 (73) bioactive compounds from XFZYD Similarly 5common compounds and 33 (70) common target proteinsamong the 3 groups of drugs were observed Most of thebioactive ingredients targeted more than one protein The 47target proteins regulated several essential pathophysiological
Evidence-Based Complementary and Alternative Medicine 15
(a) (b) (c)
(d) (e) (f)
Figure 9 Hydrogen-bonding networks within the binding site of the compoundminustarget complexes obtained from molecule docking(a) AKT1-quercetin (b) CDK1-quercetin (c) GSK3B-FA (d) F2-glyasperin B (e) NOS3-1-Methoxyphaseollidin and (f) ACHE-(-)-Medicocarpin The molecules are presented as ball and stick models Active site amino acid residues are represented as lines Dotted bluelines in these pictures represent hydrogen bonds with distance unit of A Other O and N atoms are colored as red and blue respectively
processes of TBI that referred to apoptosis inflammationcell proliferation superoxide anion generation nitric oxidebiosynthetic process response to calcium ion etc The aboveanalysis reveals that the synergistic action mechanisms ofXFZYDmay be (1) bioactive compounds overlapping amongthe different group of herbs (2) specific bioactive com-pounds from different groups of herbs targeting the sameproteins (3) specific bioactive compounds from differentgroups of herbs targeting different proteins which participatein the same pathophysiological process of the disease Toa certain degree the 5 compounds including quercetinstigmasterol kaempferol baicalin and beta-sitosterol playedessential role in XFZYD for TBI treatment MAPK3MAPK1AKT1 PRKCA TNF PRKCB EGFR BCL2 GSK3B CASP3PPP3CA and NOS3 were the main target proteins regulatedby XFZYD in the treatment of TBI
Interestingly Beta-carotene (Mol 43) from Carthami Flos(the Jun herb) specifically regulated 120573-Catenin (CTNNB1)and played critical role for curing TBI The 120573-Catenin is acritical downstream component of the Wnt pathway whichplays essential role in the regulation of mammalian neuraldevelopment [87] In vitro and in vivo studies demonstrate
that the Wnt120573-catenin pathway regulates the proliferationand differentiation of neural progenitor cells [88] Neuronaldifferentiation is induced by overexpression of 120573-cateninor the pharmacological inhibition of GSK3120573 (the phospho-rylating enzyme of 120573-catenin) [89 90] This pathway alsopromotes blood vessel formation during vascular develop-ment as well as the vascular repair process after TBI [91]In addition wogonin (Mol 160) from Achyranthis BidentataeRadix (the Chen herb) specifically targeted fibronectin (FN1)Bcl-2-binding component 3 (BBC3) and Protein KinaseC delta type (PRKCD) FN1 an important component ofthe extracellular matrix (ECM) environment promotes cellmigration neurite outgrowth and synapse formation dur-ing neural development [92] It aggregates in the injuredbrain and plays a neuroprotection role through antiapoptosisand anti-inflammation ways following TBI [93 94] BBC3namely p53 upregulated modulator of apoptosis (PUMA) iscritical for the p53-dependent apoptosis pathway which playsan important role in hippocampal neuronal loss and associ-ated cognitive deficits [95] PRKCD one of PKC isoformsactivates signal transduction pathways involved in neuronalregeneration [96] synaptic transmissionplasticity [97] and
16 Evidence-Based Complementary and Alternative Medicine
activation of apoptosis processes [98] as well as higher brainfunctions such as learning andmemory [99]The activators ofPKCare effective for the treatment of TBI [100] FurthermoreMitogen-activated protein kinase 3 (MAPK3) Beta-secretase1 (BACE1) Ephrin type-B receptor 2 (EphB) and low-densitylipoprotein receptor (LDLR) were specifically targeted bythe ingredients in the Zuo-Shi herbs Naringenin (Mol133) targeted LDLR MAPK3 while euchrenone (Mol 60)anchored BACE1 and nobiletin (Mol 134) targeted EphB2MAPK3 is an essential component of the MAP kinase signaltransduction pathway It has been appreciated recently thatthe ERK12 cascade plays a fundamental role in synapticplasticity and memory [101] BACE1 is responsible for pro-duction of A120573 from amyloid precursor protein (APP) A120573can cause cell death activate inflammatory pathways [102]and prime proapoptotic pathways for activation by otherinsults [103] The blocking of BACE1 can ameliorate motorand cognitive deficits and reduce cell loss after experimentalTBI in mice [104] EphB is localized to synaptic sites inhippocampal neurons [105] The interaction between EphBand NMDA receptors regulates excitatory synapse formation[106] LDLR acts as an important receptor that facilitatesbrain A120573 clearance and inhibits amyloid deposition [107]and then ameliorates Alzheimerrsquos disease neuropathologyafter TBI [108] The analysis above indicates the ldquoJunrdquoldquoChenrdquo and ldquoZuo-Shirdquo herbs from XFZYD trigger theirspecific targets regulation respectively for the therapeuticeffects
XFZYD is a very famous traditional Chinese formula inpromoting qi circulation and removing blood stasis accord-ing to TCM theory However several limited researches havedemonstrated its efficacy for treating TBI such as anti-inflammatory and synaptic regulation [30 31] which arein accord with our study However previous studies merelypartially deciphered the molecule mechanism of XFZYD fortreating TBI This study reports 119 bioactive compounds inXFZYD that target 47 TBI-specific proteins such as MAPK3MAPK1 AKT1 PRKCA TNF PRKCB and EGFR Theseproteins regulate several crucial pathophysiological processesof TBI such as apoptosis inflammation blood coagulationand axon genesis Our study demonstrates that the therapeu-tic actions of XFZYD refer to ldquomulticompoundsrdquo ldquomultitar-getsrdquo features rather than only the improvement of bloodcirculation With the help of molecule docking methodwe further validate the interactions between bioactive com-pounds and potential targets of XFZYD The hydrogen-bonding and edge-to-face 120587 minus120587 interactions play key roles inthe proteinminusligand recognition and stability This provides avaluable reference for further experimental investigations ofbioactive ingredients and therapeutic targets of XFZYD fortreating TBI
5 Conclusion
Our work successfully illuminates the efficiency of XFZYDfor the treatment of TBI as well as herb combination ruleof TCM formula Network pharmacology with moleculedocking method confirms the ldquomulticompounds multitar-getsrdquo therapeutic actions of XFZYD in the treatment of TBI
The present work may provide valuable evidence for furtherclinical application of XFZYD for treating TBI
Abbreviations
TBI Traumatic brain injuryXFZYD Xuefu Zhuyu decoctionTCM Traditional Chines medicineDL Drug-likenessOB Oral bioavailabilityHB-cC-cT Herb-candidate compound-candidate
targetHB-pC-pT Herb-potential compound-potential targetCALM CalmodulinGSK3B Glycogen synthase kinase-3 betaSCN5A Sodium channel protein type 5 subunit
alphaF2 ProthrombinACHE AcetylcholinesteraseGO Gene OntologyKEGG Kyoto Encyclopedia of Genes and
Genomes
Data Availability
The data used to support the findings of this study areavailable from the corresponding author upon request
Conflicts of Interest
The authors declare that they have no conflicts of interest
Authorsrsquo Contributions
YangWang and Zebing Huang participated in the conceptionand design of the study YuanyuanZhong YangWang ZebingHuang Jiekun Luo Tao Tang and Pengfei Li acquired andanalyzed the data Yuanyuan Zhong Tao Liu and HanjinCui drafted and revised the manuscript The correspondingauthor and all of the authors have read and approved the finalsubmitted manuscript
Acknowledgments
This work was supported by the National Natural ScienceFoundation of China (Nos 81673719 81874409 81603670and 81803948) Project funded byChina Postdoctoral ScienceFoundation (Nos 2016M600639 and 2017T100614)
Supplementary Materials
Table S1 162 bioactive compounds in XFZYD Table S2 targetproteins of XFZYD Table S3 TBI-specific proteins Table S4119 potential compounds of XFZYD for treating TBI TableS5 docking result of 18 target proteins with 91 potential com-pounds Fig S1 the exact bindingmode between active ingre-dients and protein targets obtained from molecule docking(A) AKT1-quercetin (B) CDK1-quercetin (C) GSK3B-FA
Evidence-Based Complementary and Alternative Medicine 17
(D) F2-glyasperin B (E) NOS3-1-Methoxyphaseollidin and(F)ACHE-(-)-Medicocarpin (Supplementary Materials)
References
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[2] J A Langlois W Rutland-Brown and M M Wald ldquoTheepidemiology and impact of traumatic brain injury a briefoverviewrdquo The Journal of Head Trauma Rehabilitation vol 21no 5 pp 375ndash378 2006
[3] H A Alsulaim B J Smart A O Asemota et al ldquoConsciousstatus predictsmortality among patientswith isolated traumaticbrain injury in administrative datardquo The American Journal ofSurgery vol 214 no 2 pp 207ndash210 2017
[4] M Majdan D Plancikova A Maas et al ldquoYears of life lost dueto traumatic brain injury in Europe A cross-sectional analysisof 16 countriesrdquo PLoS Medicine vol 14 no 7 p e1002331 2017
[5] P Cheng P Yin P Ning et al ldquoTrends in traumatic braininjury mortality in China 2006ndash2013 A population-basedlongitudinal studyrdquo PLoS Medicine vol 14 no 7 Article IDe1002332 2017
[6] M V Russo and D B McGavern ldquoInflammatory neuroprotec-tion following traumatic brain injuryrdquo Science vol 353 no 6301pp 783ndash785 2016
[7] WWang H Li J Yu et al ldquoProtective Effects of ChineseHerbalMedicine Rhizoma drynariae in Rats After Traumatic BrainInjury and Identification of Active Compoundrdquo MolecularNeurobiology vol 53 no 7 pp 4809ndash4820 2016
[8] M Das S Mohapatra and S S Mohapatra ldquoNew perspectiveson central and peripheral immune responses to acute traumaticbrain injuryrdquo Journal of Neuroinflammation vol 9 p 236 2012
[9] J W Finnie ldquoNeuroinflammation Beneficial and detrimentaleffects after traumatic brain injuryrdquo Inflammopharmacologyvol 21 no 4 pp 309ndash320 2013
[10] T Cheng W Wang Q Li et al ldquoCerebroprotection of flavanol(minus)-epicatechin after traumatic brain injury viaNrf2-dependentand -independent pathwaysrdquo Free Radical Biology amp Medicinevol 92 pp 15ndash28 2016
[11] W Young ldquoRole of calcium in central nervous system injuriesrdquoJournal of Neurotrauma vol 9 Suppl 1 pp S9ndashS25 1992
[12] R Bullock A Zauner J S Myseros et al ldquoEvidence forProlonged Release of Excitatory Amino Acids in Severe HumanHead Trauma Relationship to Clinical Eventsrdquo Annals of theNew York Academy of Sciences vol 765 no 1 pp 290ndash297 1995
[13] A T Mazzeo A Beat A Singh and M R Bullock ldquoTherole of mitochondrial transition pore and its modulation intraumatic brain injury and delayed neurodegeneration afterTBIrdquo Experimental Neurology vol 218 no 2 pp 363ndash370 2009
[14] S Gennai A Monsel Q Hao et al ldquoCell-Based therapy fortraumatic brain injuryrdquo British Journal of Anaesthesia vol 115no 2 pp 203ndash212 2015
[15] J Ghajar ldquoTraumatic brain injuryrdquo The Lancet vol 356 no9233 pp 923ndash929 2000
[16] D J Loane and A I Faden ldquoNeuroprotection for traumaticbrain injury translational challenges and emerging therapeuticstrategiesrdquoTrends in Pharmacological Sciences vol 31 no 12 pp596ndash604 2010
[17] Y Wang X Fan T Tang et al ldquoRhein and rhubarb simi-larly protect the blood-brain barrier after experimental trau-matic brain injury via gp91phox subunit of NADPH oxidaseROSERKMMP-9 signaling pathwayrdquo Scientific Reports vol 6p 37098 2016
[18] D-X Kong X-J Li and H-Y Zhang ldquoWhere is the hopefor drug discovery Let history tell the futurerdquo Drug DiscoveryTherapy vol 14 no 3-4 pp 115ndash119 2009
[19] F Cheung ldquoTCM made in Chinardquo Nature vol 480 no 7378pp S82ndashS83 2011
[20] C Fu Z Xia Y Liu et al ldquoQualitative analysis of majorconstituents from Xue Fu Zhu Yu Decoction using ultra highperformance liquid chromatographywith hybrid ion trap time-of-flight mass spectrometryrdquo Journal of Separation Science vol39 no 17 pp 3457ndash3468 2016
[21] H-J Zhang and Y-Y Cheng ldquoAn HPLCMS method for iden-tifying major constituents in the hypocholesterolemic extractsof Chinese medicine formula lsquoXue-Fu-Zhu-Yu decoctionrsquordquoBiomedical Chromatography vol 20 no 8 pp 821ndash826 2006
[22] L Zhang L Zhu YWang et al ldquoCharacterization and quantifi-cation of major constituents of Xue Fu Zhu Yu by UPLC-DAD-MSMSrdquo Journal of Pharmaceutical and Biomedical Analysisvol 62 pp 203ndash209 2012
[23] X Yang X Xiong G Yang and J Wang ldquoChinese patentmedicine Xuefu Zhuyu capsule for the treatment of unstableangina pectoris a systematic review of randomized controlledtrialsrdquo Complementary Therapies in Medicine vol 22 no 2 pp391ndash399 2014
[24] J Wang X Yang F Chu et al ldquoThe effects of Xuefu Zhuyuand Shengmai on the evolution of syndromes and inflammatorymarkers in patients with unstable angina pectoris after percuta-neous coronary intervention a randomised controlled clinicaltrialrdquoEvidence-BasedComplementary andAlternativeMedicinevol 2013 Article ID 896467 9 pages 2013
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18 Evidence-Based Complementary and Alternative Medicine
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[41] J V Turner D J Maddalena and S Agatonovic-KustrinldquoBioavailability Prediction Based on Molecular Structure for aDiverse Series of Drugsrdquo Pharmaceutical Research vol 21 no 1pp 68ndash82 2004
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[89] S Ding T Y H Wu A Brinker et al ldquoSynthetic smallmolecules that control stem cell faterdquo Proceedings of theNationalAcadamy of Sciences of the United States of America vol 100 no13 pp 7632ndash7637 2003
[90] N Israsena M Hu W Fu L Kan and J A Kessler ldquoThepresence of FGF2 signaling determines whether 120573-cateninexerts effects on proliferation or neuronal differentiation ofneural stem cellsrdquo Developmental Biology vol 268 no 1 pp220ndash231 2004
[91] A Salehi A Jullienne M Baghchechi et al ldquoUp-regulation ofWnt120573-catenin expression is accompanied with vascular repairafter traumatic brain injuryrdquo Journal of Cerebral Blood Flow ampMetabolism vol 38 no 2 pp 274ndash289 2017
[92] L F Reichardt and K J Tomaselli ldquoExtracellular matrixmolecules and their receptors Functions in neural develop-mentrdquoAnnual Review of Neuroscience vol 14 pp 531ndash570 1991
[93] R M Gibson S E Craig L Heenan C Tournier and M JHumphries ldquoActivation of integrin 12057251205731 delays apoptosis ofNtera2 neuronal cellsrdquoMolecularandCellularNeuroscience vol28 no 3 pp 588ndash598 2005
[94] C C Tate A J Garcıa andMC LaPlaca ldquoPlasma fibronectin isneuroprotective following traumatic brain injuryrdquoExperimentalNeurology vol 207 no 1 pp 13ndash22 2007
[95] C Culmsee and M P Mattson ldquop53 in neuronal apoptosisrdquoBiochemical and Biophysical Research Communications vol 331no 3 pp 761ndash777 2005
[96] M S Geddis and V Rehder ldquoThe phosphorylation state ofneuronal processes determines growth cone formation afterneuronal injuryrdquo Journal of Neuroscience Research vol 74 no2 pp 210ndash220 2003
[97] M L Craske M Fivaz N N Batada and T Meyer ldquoSpines andneurite branches function as geometric attractors that enhanceprotein kinase C actionrdquoThe Journal of Cell Biology vol 170 no7 pp 1147ndash1158 2005
20 Evidence-Based Complementary and Alternative Medicine
[98] A Muscella C Vetrugno L G Cossa et al ldquoApoptosis by[Pt(OOrsquo-acac)(gamma-acac)(DMS)] requires PKC-deltamedi-ated p53 activation in malignant pleural mesotheliomardquo PloSone vol 12 no 7 Article ID e0181114 2017
[99] J S Bonini W C Da Silva L R M Bevilaqua J H MedinaI Izquierdo and M Cammarota ldquoOn the participation ofhippocampal PKC in acquisition consolidation and reconsol-idation of spatial memoryrdquo Neuroscience vol 147 no 1 pp 37ndash45 2007
[100] O Zohar R Lavy X Zi et al ldquoPKC activator therapeutic formild traumatic brain injury in micerdquo Neurobiology of Diseasevol 41 no 2 pp 329ndash337 2011
[101] J David Sweatt ldquoTheneuronalMAPkinase cascade a biochem-ical signal integration system subserving synaptic plasticity andmemoryrdquo Journal ofNeurochemistry vol 76 no 1 pp 1ndash10 2001
[102] Y Matsuoka M Picciano B Maleste et al ldquoInflammatoryresponses to amyloidosis in a transgenic mouse model ofAlzheimerrsquos diseaserdquo The American Journal of Pathology vol158 no 4 pp 1345ndash1354 2001
[103] L Esposito L Gan G-Q Yu C Essrich and L MuckeldquoIntracellularly generated amyloid-120573 peptide counteracts theantiapoptotic function of its precursor protein and primesproapoptotic pathways for activation by other insults in neu-roblastoma cellsrdquo Journal of Neurochemistry vol 91 no 6 pp1260ndash1274 2004
[104] D J Loane A Pocivavsek C E-H Moussa et al ldquoAmyloidprecursor protein secretases as therapeutic targets for traumaticbrain injuryrdquo Nature Medicine vol 15 no 4 pp 377ndash379 2009
[105] R Torres B L Firestein H Dong et al ldquoPDZ proteins bindcluster and synaptically colocalize with Eph receptors and theirephrin ligandsrdquo Neuron vol 21 no 6 pp 1453ndash1463 1998
[106] M B Dalva M A Takasu M Z Lin et al ldquoEphB receptorsinteract with NMDA receptors and regulate excitatory synapseformationrdquo Cell vol 103 no 6 pp 945ndash956 2000
[107] J M Basak P B Verghese H Yoon J Kim and D MHoltzman ldquoLow-density lipoprotein receptor represents anapolipoprotein E-independent pathway of A120573 uptake anddegradation by astrocytesrdquoThe Journal of Biological Chemistryvol 287 no 17 pp 13959ndash13971 2012
[108] L Yao X Gu Q Song et al ldquoNanoformulated alpha-mangostinameliorates Alzheimerrsquos disease neuropathology by elevatingLDLR expression and accelerating amyloid-beta clearancerdquoJournal of Controlled Release vol 226 pp 1ndash14 2016
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Evidence-Based Complementary andAlternative Medicine
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Submit your manuscripts atwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 15
(a) (b) (c)
(d) (e) (f)
Figure 9 Hydrogen-bonding networks within the binding site of the compoundminustarget complexes obtained from molecule docking(a) AKT1-quercetin (b) CDK1-quercetin (c) GSK3B-FA (d) F2-glyasperin B (e) NOS3-1-Methoxyphaseollidin and (f) ACHE-(-)-Medicocarpin The molecules are presented as ball and stick models Active site amino acid residues are represented as lines Dotted bluelines in these pictures represent hydrogen bonds with distance unit of A Other O and N atoms are colored as red and blue respectively
processes of TBI that referred to apoptosis inflammationcell proliferation superoxide anion generation nitric oxidebiosynthetic process response to calcium ion etc The aboveanalysis reveals that the synergistic action mechanisms ofXFZYDmay be (1) bioactive compounds overlapping amongthe different group of herbs (2) specific bioactive com-pounds from different groups of herbs targeting the sameproteins (3) specific bioactive compounds from differentgroups of herbs targeting different proteins which participatein the same pathophysiological process of the disease Toa certain degree the 5 compounds including quercetinstigmasterol kaempferol baicalin and beta-sitosterol playedessential role in XFZYD for TBI treatment MAPK3MAPK1AKT1 PRKCA TNF PRKCB EGFR BCL2 GSK3B CASP3PPP3CA and NOS3 were the main target proteins regulatedby XFZYD in the treatment of TBI
Interestingly Beta-carotene (Mol 43) from Carthami Flos(the Jun herb) specifically regulated 120573-Catenin (CTNNB1)and played critical role for curing TBI The 120573-Catenin is acritical downstream component of the Wnt pathway whichplays essential role in the regulation of mammalian neuraldevelopment [87] In vitro and in vivo studies demonstrate
that the Wnt120573-catenin pathway regulates the proliferationand differentiation of neural progenitor cells [88] Neuronaldifferentiation is induced by overexpression of 120573-cateninor the pharmacological inhibition of GSK3120573 (the phospho-rylating enzyme of 120573-catenin) [89 90] This pathway alsopromotes blood vessel formation during vascular develop-ment as well as the vascular repair process after TBI [91]In addition wogonin (Mol 160) from Achyranthis BidentataeRadix (the Chen herb) specifically targeted fibronectin (FN1)Bcl-2-binding component 3 (BBC3) and Protein KinaseC delta type (PRKCD) FN1 an important component ofthe extracellular matrix (ECM) environment promotes cellmigration neurite outgrowth and synapse formation dur-ing neural development [92] It aggregates in the injuredbrain and plays a neuroprotection role through antiapoptosisand anti-inflammation ways following TBI [93 94] BBC3namely p53 upregulated modulator of apoptosis (PUMA) iscritical for the p53-dependent apoptosis pathway which playsan important role in hippocampal neuronal loss and associ-ated cognitive deficits [95] PRKCD one of PKC isoformsactivates signal transduction pathways involved in neuronalregeneration [96] synaptic transmissionplasticity [97] and
16 Evidence-Based Complementary and Alternative Medicine
activation of apoptosis processes [98] as well as higher brainfunctions such as learning andmemory [99]The activators ofPKCare effective for the treatment of TBI [100] FurthermoreMitogen-activated protein kinase 3 (MAPK3) Beta-secretase1 (BACE1) Ephrin type-B receptor 2 (EphB) and low-densitylipoprotein receptor (LDLR) were specifically targeted bythe ingredients in the Zuo-Shi herbs Naringenin (Mol133) targeted LDLR MAPK3 while euchrenone (Mol 60)anchored BACE1 and nobiletin (Mol 134) targeted EphB2MAPK3 is an essential component of the MAP kinase signaltransduction pathway It has been appreciated recently thatthe ERK12 cascade plays a fundamental role in synapticplasticity and memory [101] BACE1 is responsible for pro-duction of A120573 from amyloid precursor protein (APP) A120573can cause cell death activate inflammatory pathways [102]and prime proapoptotic pathways for activation by otherinsults [103] The blocking of BACE1 can ameliorate motorand cognitive deficits and reduce cell loss after experimentalTBI in mice [104] EphB is localized to synaptic sites inhippocampal neurons [105] The interaction between EphBand NMDA receptors regulates excitatory synapse formation[106] LDLR acts as an important receptor that facilitatesbrain A120573 clearance and inhibits amyloid deposition [107]and then ameliorates Alzheimerrsquos disease neuropathologyafter TBI [108] The analysis above indicates the ldquoJunrdquoldquoChenrdquo and ldquoZuo-Shirdquo herbs from XFZYD trigger theirspecific targets regulation respectively for the therapeuticeffects
XFZYD is a very famous traditional Chinese formula inpromoting qi circulation and removing blood stasis accord-ing to TCM theory However several limited researches havedemonstrated its efficacy for treating TBI such as anti-inflammatory and synaptic regulation [30 31] which arein accord with our study However previous studies merelypartially deciphered the molecule mechanism of XFZYD fortreating TBI This study reports 119 bioactive compounds inXFZYD that target 47 TBI-specific proteins such as MAPK3MAPK1 AKT1 PRKCA TNF PRKCB and EGFR Theseproteins regulate several crucial pathophysiological processesof TBI such as apoptosis inflammation blood coagulationand axon genesis Our study demonstrates that the therapeu-tic actions of XFZYD refer to ldquomulticompoundsrdquo ldquomultitar-getsrdquo features rather than only the improvement of bloodcirculation With the help of molecule docking methodwe further validate the interactions between bioactive com-pounds and potential targets of XFZYD The hydrogen-bonding and edge-to-face 120587 minus120587 interactions play key roles inthe proteinminusligand recognition and stability This provides avaluable reference for further experimental investigations ofbioactive ingredients and therapeutic targets of XFZYD fortreating TBI
5 Conclusion
Our work successfully illuminates the efficiency of XFZYDfor the treatment of TBI as well as herb combination ruleof TCM formula Network pharmacology with moleculedocking method confirms the ldquomulticompounds multitar-getsrdquo therapeutic actions of XFZYD in the treatment of TBI
The present work may provide valuable evidence for furtherclinical application of XFZYD for treating TBI
Abbreviations
TBI Traumatic brain injuryXFZYD Xuefu Zhuyu decoctionTCM Traditional Chines medicineDL Drug-likenessOB Oral bioavailabilityHB-cC-cT Herb-candidate compound-candidate
targetHB-pC-pT Herb-potential compound-potential targetCALM CalmodulinGSK3B Glycogen synthase kinase-3 betaSCN5A Sodium channel protein type 5 subunit
alphaF2 ProthrombinACHE AcetylcholinesteraseGO Gene OntologyKEGG Kyoto Encyclopedia of Genes and
Genomes
Data Availability
The data used to support the findings of this study areavailable from the corresponding author upon request
Conflicts of Interest
The authors declare that they have no conflicts of interest
Authorsrsquo Contributions
YangWang and Zebing Huang participated in the conceptionand design of the study YuanyuanZhong YangWang ZebingHuang Jiekun Luo Tao Tang and Pengfei Li acquired andanalyzed the data Yuanyuan Zhong Tao Liu and HanjinCui drafted and revised the manuscript The correspondingauthor and all of the authors have read and approved the finalsubmitted manuscript
Acknowledgments
This work was supported by the National Natural ScienceFoundation of China (Nos 81673719 81874409 81603670and 81803948) Project funded byChina Postdoctoral ScienceFoundation (Nos 2016M600639 and 2017T100614)
Supplementary Materials
Table S1 162 bioactive compounds in XFZYD Table S2 targetproteins of XFZYD Table S3 TBI-specific proteins Table S4119 potential compounds of XFZYD for treating TBI TableS5 docking result of 18 target proteins with 91 potential com-pounds Fig S1 the exact bindingmode between active ingre-dients and protein targets obtained from molecule docking(A) AKT1-quercetin (B) CDK1-quercetin (C) GSK3B-FA
Evidence-Based Complementary and Alternative Medicine 17
(D) F2-glyasperin B (E) NOS3-1-Methoxyphaseollidin and(F)ACHE-(-)-Medicocarpin (Supplementary Materials)
References
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[2] J A Langlois W Rutland-Brown and M M Wald ldquoTheepidemiology and impact of traumatic brain injury a briefoverviewrdquo The Journal of Head Trauma Rehabilitation vol 21no 5 pp 375ndash378 2006
[3] H A Alsulaim B J Smart A O Asemota et al ldquoConsciousstatus predictsmortality among patientswith isolated traumaticbrain injury in administrative datardquo The American Journal ofSurgery vol 214 no 2 pp 207ndash210 2017
[4] M Majdan D Plancikova A Maas et al ldquoYears of life lost dueto traumatic brain injury in Europe A cross-sectional analysisof 16 countriesrdquo PLoS Medicine vol 14 no 7 p e1002331 2017
[5] P Cheng P Yin P Ning et al ldquoTrends in traumatic braininjury mortality in China 2006ndash2013 A population-basedlongitudinal studyrdquo PLoS Medicine vol 14 no 7 Article IDe1002332 2017
[6] M V Russo and D B McGavern ldquoInflammatory neuroprotec-tion following traumatic brain injuryrdquo Science vol 353 no 6301pp 783ndash785 2016
[7] WWang H Li J Yu et al ldquoProtective Effects of ChineseHerbalMedicine Rhizoma drynariae in Rats After Traumatic BrainInjury and Identification of Active Compoundrdquo MolecularNeurobiology vol 53 no 7 pp 4809ndash4820 2016
[8] M Das S Mohapatra and S S Mohapatra ldquoNew perspectiveson central and peripheral immune responses to acute traumaticbrain injuryrdquo Journal of Neuroinflammation vol 9 p 236 2012
[9] J W Finnie ldquoNeuroinflammation Beneficial and detrimentaleffects after traumatic brain injuryrdquo Inflammopharmacologyvol 21 no 4 pp 309ndash320 2013
[10] T Cheng W Wang Q Li et al ldquoCerebroprotection of flavanol(minus)-epicatechin after traumatic brain injury viaNrf2-dependentand -independent pathwaysrdquo Free Radical Biology amp Medicinevol 92 pp 15ndash28 2016
[11] W Young ldquoRole of calcium in central nervous system injuriesrdquoJournal of Neurotrauma vol 9 Suppl 1 pp S9ndashS25 1992
[12] R Bullock A Zauner J S Myseros et al ldquoEvidence forProlonged Release of Excitatory Amino Acids in Severe HumanHead Trauma Relationship to Clinical Eventsrdquo Annals of theNew York Academy of Sciences vol 765 no 1 pp 290ndash297 1995
[13] A T Mazzeo A Beat A Singh and M R Bullock ldquoTherole of mitochondrial transition pore and its modulation intraumatic brain injury and delayed neurodegeneration afterTBIrdquo Experimental Neurology vol 218 no 2 pp 363ndash370 2009
[14] S Gennai A Monsel Q Hao et al ldquoCell-Based therapy fortraumatic brain injuryrdquo British Journal of Anaesthesia vol 115no 2 pp 203ndash212 2015
[15] J Ghajar ldquoTraumatic brain injuryrdquo The Lancet vol 356 no9233 pp 923ndash929 2000
[16] D J Loane and A I Faden ldquoNeuroprotection for traumaticbrain injury translational challenges and emerging therapeuticstrategiesrdquoTrends in Pharmacological Sciences vol 31 no 12 pp596ndash604 2010
[17] Y Wang X Fan T Tang et al ldquoRhein and rhubarb simi-larly protect the blood-brain barrier after experimental trau-matic brain injury via gp91phox subunit of NADPH oxidaseROSERKMMP-9 signaling pathwayrdquo Scientific Reports vol 6p 37098 2016
[18] D-X Kong X-J Li and H-Y Zhang ldquoWhere is the hopefor drug discovery Let history tell the futurerdquo Drug DiscoveryTherapy vol 14 no 3-4 pp 115ndash119 2009
[19] F Cheung ldquoTCM made in Chinardquo Nature vol 480 no 7378pp S82ndashS83 2011
[20] C Fu Z Xia Y Liu et al ldquoQualitative analysis of majorconstituents from Xue Fu Zhu Yu Decoction using ultra highperformance liquid chromatographywith hybrid ion trap time-of-flight mass spectrometryrdquo Journal of Separation Science vol39 no 17 pp 3457ndash3468 2016
[21] H-J Zhang and Y-Y Cheng ldquoAn HPLCMS method for iden-tifying major constituents in the hypocholesterolemic extractsof Chinese medicine formula lsquoXue-Fu-Zhu-Yu decoctionrsquordquoBiomedical Chromatography vol 20 no 8 pp 821ndash826 2006
[22] L Zhang L Zhu YWang et al ldquoCharacterization and quantifi-cation of major constituents of Xue Fu Zhu Yu by UPLC-DAD-MSMSrdquo Journal of Pharmaceutical and Biomedical Analysisvol 62 pp 203ndash209 2012
[23] X Yang X Xiong G Yang and J Wang ldquoChinese patentmedicine Xuefu Zhuyu capsule for the treatment of unstableangina pectoris a systematic review of randomized controlledtrialsrdquo Complementary Therapies in Medicine vol 22 no 2 pp391ndash399 2014
[24] J Wang X Yang F Chu et al ldquoThe effects of Xuefu Zhuyuand Shengmai on the evolution of syndromes and inflammatorymarkers in patients with unstable angina pectoris after percuta-neous coronary intervention a randomised controlled clinicaltrialrdquoEvidence-BasedComplementary andAlternativeMedicinevol 2013 Article ID 896467 9 pages 2013
[25] Q Zhang H Yu J Qi et al ldquoNatural formulas and the natureof formulas Exploring potential therapeutic targets based ontraditional Chinese herbal formulasrdquo PLoS ONE vol 12 no 2p e0171628 2017
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18 Evidence-Based Complementary and Alternative Medicine
[31] M Sun X-P Zhan C-Y Jin J-Z Shan S Xu and Y-LWang ldquoclinical observation on treatment of post-craniocerebraltraumatic mental disorder by integrative medicinerdquo ChineseJournal of Integrative Medicine vol 14 no 2 pp 137ndash141 2008
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[39] Bo Zhang Xu Wang and Shao Li ldquoAn Integrative Platform ofTCM Network Pharmacology and Its Application on a HerbalFormula Qing-Luo-Yinrdquo Evidence-Based Complementary andAlternativeMedicine vol 2013 Article ID 456747 12 pages 2013
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[41] J V Turner D J Maddalena and S Agatonovic-KustrinldquoBioavailability Prediction Based on Molecular Structure for aDiverse Series of Drugsrdquo Pharmaceutical Research vol 21 no 1pp 68ndash82 2004
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diseaserdquo International Journal of Molecular Sciences vol 17 no1 2016
[82] M J McGinn and J T Povlishock ldquoPathophysiology of Trau-matic Brain InjuryrdquoNeurosurgery Clinics of North America vol27 no 4 pp 397ndash407 2016
[83] T Woodcock and M C Morganti-Kossmann ldquoThe role ofmarkers of inflammation in traumatic brain injuryrdquo Frontiersin Neurology vol 4 article 18 2013
[84] F Tanriverdi H J Schneider G Aimaretti B E Masel F FCasanueva and F Kelestimur ldquoPituitary dysfunction after trau-matic brain injury A clinical and pathophysiological approachrdquoEndocrine Reviews vol 36 no 3 pp 305ndash342 2015
[85] J V Rosenfeld A I Maas P Bragge M C Morganti-Kossmann G T Manley and R L Gruen ldquoEarly managementof severe traumatic brain injuryrdquoThe Lancet vol 380 no 9847pp 1088ndash1098 2012
[86] B M Aertker S Bedi and C S Cox ldquoStrategies for CNS repairfollowing TBIrdquo Experimental Neurology vol 275 no 3 pp 411ndash426 2016
[87] K Adachi Z Mirzadeh M Sakaguchi et al ldquo120573-catenin signal-ing promotes proliferation of progenitor cells in the adultmousesubventricular zonerdquo Stem Cells vol 25 no 11 pp 2827ndash28362007
[88] Y Hirabayashi and Y Gotoh ldquoStage-dependent fate determina-tion of neural precursor cells in mouse forebrainrdquo NeuroscienceResearch vol 51 no 4 pp 331ndash336 2005
[89] S Ding T Y H Wu A Brinker et al ldquoSynthetic smallmolecules that control stem cell faterdquo Proceedings of theNationalAcadamy of Sciences of the United States of America vol 100 no13 pp 7632ndash7637 2003
[90] N Israsena M Hu W Fu L Kan and J A Kessler ldquoThepresence of FGF2 signaling determines whether 120573-cateninexerts effects on proliferation or neuronal differentiation ofneural stem cellsrdquo Developmental Biology vol 268 no 1 pp220ndash231 2004
[91] A Salehi A Jullienne M Baghchechi et al ldquoUp-regulation ofWnt120573-catenin expression is accompanied with vascular repairafter traumatic brain injuryrdquo Journal of Cerebral Blood Flow ampMetabolism vol 38 no 2 pp 274ndash289 2017
[92] L F Reichardt and K J Tomaselli ldquoExtracellular matrixmolecules and their receptors Functions in neural develop-mentrdquoAnnual Review of Neuroscience vol 14 pp 531ndash570 1991
[93] R M Gibson S E Craig L Heenan C Tournier and M JHumphries ldquoActivation of integrin 12057251205731 delays apoptosis ofNtera2 neuronal cellsrdquoMolecularandCellularNeuroscience vol28 no 3 pp 588ndash598 2005
[94] C C Tate A J Garcıa andMC LaPlaca ldquoPlasma fibronectin isneuroprotective following traumatic brain injuryrdquoExperimentalNeurology vol 207 no 1 pp 13ndash22 2007
[95] C Culmsee and M P Mattson ldquop53 in neuronal apoptosisrdquoBiochemical and Biophysical Research Communications vol 331no 3 pp 761ndash777 2005
[96] M S Geddis and V Rehder ldquoThe phosphorylation state ofneuronal processes determines growth cone formation afterneuronal injuryrdquo Journal of Neuroscience Research vol 74 no2 pp 210ndash220 2003
[97] M L Craske M Fivaz N N Batada and T Meyer ldquoSpines andneurite branches function as geometric attractors that enhanceprotein kinase C actionrdquoThe Journal of Cell Biology vol 170 no7 pp 1147ndash1158 2005
20 Evidence-Based Complementary and Alternative Medicine
[98] A Muscella C Vetrugno L G Cossa et al ldquoApoptosis by[Pt(OOrsquo-acac)(gamma-acac)(DMS)] requires PKC-deltamedi-ated p53 activation in malignant pleural mesotheliomardquo PloSone vol 12 no 7 Article ID e0181114 2017
[99] J S Bonini W C Da Silva L R M Bevilaqua J H MedinaI Izquierdo and M Cammarota ldquoOn the participation ofhippocampal PKC in acquisition consolidation and reconsol-idation of spatial memoryrdquo Neuroscience vol 147 no 1 pp 37ndash45 2007
[100] O Zohar R Lavy X Zi et al ldquoPKC activator therapeutic formild traumatic brain injury in micerdquo Neurobiology of Diseasevol 41 no 2 pp 329ndash337 2011
[101] J David Sweatt ldquoTheneuronalMAPkinase cascade a biochem-ical signal integration system subserving synaptic plasticity andmemoryrdquo Journal ofNeurochemistry vol 76 no 1 pp 1ndash10 2001
[102] Y Matsuoka M Picciano B Maleste et al ldquoInflammatoryresponses to amyloidosis in a transgenic mouse model ofAlzheimerrsquos diseaserdquo The American Journal of Pathology vol158 no 4 pp 1345ndash1354 2001
[103] L Esposito L Gan G-Q Yu C Essrich and L MuckeldquoIntracellularly generated amyloid-120573 peptide counteracts theantiapoptotic function of its precursor protein and primesproapoptotic pathways for activation by other insults in neu-roblastoma cellsrdquo Journal of Neurochemistry vol 91 no 6 pp1260ndash1274 2004
[104] D J Loane A Pocivavsek C E-H Moussa et al ldquoAmyloidprecursor protein secretases as therapeutic targets for traumaticbrain injuryrdquo Nature Medicine vol 15 no 4 pp 377ndash379 2009
[105] R Torres B L Firestein H Dong et al ldquoPDZ proteins bindcluster and synaptically colocalize with Eph receptors and theirephrin ligandsrdquo Neuron vol 21 no 6 pp 1453ndash1463 1998
[106] M B Dalva M A Takasu M Z Lin et al ldquoEphB receptorsinteract with NMDA receptors and regulate excitatory synapseformationrdquo Cell vol 103 no 6 pp 945ndash956 2000
[107] J M Basak P B Verghese H Yoon J Kim and D MHoltzman ldquoLow-density lipoprotein receptor represents anapolipoprotein E-independent pathway of A120573 uptake anddegradation by astrocytesrdquoThe Journal of Biological Chemistryvol 287 no 17 pp 13959ndash13971 2012
[108] L Yao X Gu Q Song et al ldquoNanoformulated alpha-mangostinameliorates Alzheimerrsquos disease neuropathology by elevatingLDLR expression and accelerating amyloid-beta clearancerdquoJournal of Controlled Release vol 226 pp 1ndash14 2016
Stem Cells International
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Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
16 Evidence-Based Complementary and Alternative Medicine
activation of apoptosis processes [98] as well as higher brainfunctions such as learning andmemory [99]The activators ofPKCare effective for the treatment of TBI [100] FurthermoreMitogen-activated protein kinase 3 (MAPK3) Beta-secretase1 (BACE1) Ephrin type-B receptor 2 (EphB) and low-densitylipoprotein receptor (LDLR) were specifically targeted bythe ingredients in the Zuo-Shi herbs Naringenin (Mol133) targeted LDLR MAPK3 while euchrenone (Mol 60)anchored BACE1 and nobiletin (Mol 134) targeted EphB2MAPK3 is an essential component of the MAP kinase signaltransduction pathway It has been appreciated recently thatthe ERK12 cascade plays a fundamental role in synapticplasticity and memory [101] BACE1 is responsible for pro-duction of A120573 from amyloid precursor protein (APP) A120573can cause cell death activate inflammatory pathways [102]and prime proapoptotic pathways for activation by otherinsults [103] The blocking of BACE1 can ameliorate motorand cognitive deficits and reduce cell loss after experimentalTBI in mice [104] EphB is localized to synaptic sites inhippocampal neurons [105] The interaction between EphBand NMDA receptors regulates excitatory synapse formation[106] LDLR acts as an important receptor that facilitatesbrain A120573 clearance and inhibits amyloid deposition [107]and then ameliorates Alzheimerrsquos disease neuropathologyafter TBI [108] The analysis above indicates the ldquoJunrdquoldquoChenrdquo and ldquoZuo-Shirdquo herbs from XFZYD trigger theirspecific targets regulation respectively for the therapeuticeffects
XFZYD is a very famous traditional Chinese formula inpromoting qi circulation and removing blood stasis accord-ing to TCM theory However several limited researches havedemonstrated its efficacy for treating TBI such as anti-inflammatory and synaptic regulation [30 31] which arein accord with our study However previous studies merelypartially deciphered the molecule mechanism of XFZYD fortreating TBI This study reports 119 bioactive compounds inXFZYD that target 47 TBI-specific proteins such as MAPK3MAPK1 AKT1 PRKCA TNF PRKCB and EGFR Theseproteins regulate several crucial pathophysiological processesof TBI such as apoptosis inflammation blood coagulationand axon genesis Our study demonstrates that the therapeu-tic actions of XFZYD refer to ldquomulticompoundsrdquo ldquomultitar-getsrdquo features rather than only the improvement of bloodcirculation With the help of molecule docking methodwe further validate the interactions between bioactive com-pounds and potential targets of XFZYD The hydrogen-bonding and edge-to-face 120587 minus120587 interactions play key roles inthe proteinminusligand recognition and stability This provides avaluable reference for further experimental investigations ofbioactive ingredients and therapeutic targets of XFZYD fortreating TBI
5 Conclusion
Our work successfully illuminates the efficiency of XFZYDfor the treatment of TBI as well as herb combination ruleof TCM formula Network pharmacology with moleculedocking method confirms the ldquomulticompounds multitar-getsrdquo therapeutic actions of XFZYD in the treatment of TBI
The present work may provide valuable evidence for furtherclinical application of XFZYD for treating TBI
Abbreviations
TBI Traumatic brain injuryXFZYD Xuefu Zhuyu decoctionTCM Traditional Chines medicineDL Drug-likenessOB Oral bioavailabilityHB-cC-cT Herb-candidate compound-candidate
targetHB-pC-pT Herb-potential compound-potential targetCALM CalmodulinGSK3B Glycogen synthase kinase-3 betaSCN5A Sodium channel protein type 5 subunit
alphaF2 ProthrombinACHE AcetylcholinesteraseGO Gene OntologyKEGG Kyoto Encyclopedia of Genes and
Genomes
Data Availability
The data used to support the findings of this study areavailable from the corresponding author upon request
Conflicts of Interest
The authors declare that they have no conflicts of interest
Authorsrsquo Contributions
YangWang and Zebing Huang participated in the conceptionand design of the study YuanyuanZhong YangWang ZebingHuang Jiekun Luo Tao Tang and Pengfei Li acquired andanalyzed the data Yuanyuan Zhong Tao Liu and HanjinCui drafted and revised the manuscript The correspondingauthor and all of the authors have read and approved the finalsubmitted manuscript
Acknowledgments
This work was supported by the National Natural ScienceFoundation of China (Nos 81673719 81874409 81603670and 81803948) Project funded byChina Postdoctoral ScienceFoundation (Nos 2016M600639 and 2017T100614)
Supplementary Materials
Table S1 162 bioactive compounds in XFZYD Table S2 targetproteins of XFZYD Table S3 TBI-specific proteins Table S4119 potential compounds of XFZYD for treating TBI TableS5 docking result of 18 target proteins with 91 potential com-pounds Fig S1 the exact bindingmode between active ingre-dients and protein targets obtained from molecule docking(A) AKT1-quercetin (B) CDK1-quercetin (C) GSK3B-FA
Evidence-Based Complementary and Alternative Medicine 17
(D) F2-glyasperin B (E) NOS3-1-Methoxyphaseollidin and(F)ACHE-(-)-Medicocarpin (Supplementary Materials)
References
[1] L Yang Y Chu D Tweedie et al ldquoPost-trauma administrationof the pifithrin-120572 oxygen analog improves histological andfunctional outcomes after experimental traumatic brain injuryrdquoExperimental Neurology vol 269 pp 56ndash66 2015
[2] J A Langlois W Rutland-Brown and M M Wald ldquoTheepidemiology and impact of traumatic brain injury a briefoverviewrdquo The Journal of Head Trauma Rehabilitation vol 21no 5 pp 375ndash378 2006
[3] H A Alsulaim B J Smart A O Asemota et al ldquoConsciousstatus predictsmortality among patientswith isolated traumaticbrain injury in administrative datardquo The American Journal ofSurgery vol 214 no 2 pp 207ndash210 2017
[4] M Majdan D Plancikova A Maas et al ldquoYears of life lost dueto traumatic brain injury in Europe A cross-sectional analysisof 16 countriesrdquo PLoS Medicine vol 14 no 7 p e1002331 2017
[5] P Cheng P Yin P Ning et al ldquoTrends in traumatic braininjury mortality in China 2006ndash2013 A population-basedlongitudinal studyrdquo PLoS Medicine vol 14 no 7 Article IDe1002332 2017
[6] M V Russo and D B McGavern ldquoInflammatory neuroprotec-tion following traumatic brain injuryrdquo Science vol 353 no 6301pp 783ndash785 2016
[7] WWang H Li J Yu et al ldquoProtective Effects of ChineseHerbalMedicine Rhizoma drynariae in Rats After Traumatic BrainInjury and Identification of Active Compoundrdquo MolecularNeurobiology vol 53 no 7 pp 4809ndash4820 2016
[8] M Das S Mohapatra and S S Mohapatra ldquoNew perspectiveson central and peripheral immune responses to acute traumaticbrain injuryrdquo Journal of Neuroinflammation vol 9 p 236 2012
[9] J W Finnie ldquoNeuroinflammation Beneficial and detrimentaleffects after traumatic brain injuryrdquo Inflammopharmacologyvol 21 no 4 pp 309ndash320 2013
[10] T Cheng W Wang Q Li et al ldquoCerebroprotection of flavanol(minus)-epicatechin after traumatic brain injury viaNrf2-dependentand -independent pathwaysrdquo Free Radical Biology amp Medicinevol 92 pp 15ndash28 2016
[11] W Young ldquoRole of calcium in central nervous system injuriesrdquoJournal of Neurotrauma vol 9 Suppl 1 pp S9ndashS25 1992
[12] R Bullock A Zauner J S Myseros et al ldquoEvidence forProlonged Release of Excitatory Amino Acids in Severe HumanHead Trauma Relationship to Clinical Eventsrdquo Annals of theNew York Academy of Sciences vol 765 no 1 pp 290ndash297 1995
[13] A T Mazzeo A Beat A Singh and M R Bullock ldquoTherole of mitochondrial transition pore and its modulation intraumatic brain injury and delayed neurodegeneration afterTBIrdquo Experimental Neurology vol 218 no 2 pp 363ndash370 2009
[14] S Gennai A Monsel Q Hao et al ldquoCell-Based therapy fortraumatic brain injuryrdquo British Journal of Anaesthesia vol 115no 2 pp 203ndash212 2015
[15] J Ghajar ldquoTraumatic brain injuryrdquo The Lancet vol 356 no9233 pp 923ndash929 2000
[16] D J Loane and A I Faden ldquoNeuroprotection for traumaticbrain injury translational challenges and emerging therapeuticstrategiesrdquoTrends in Pharmacological Sciences vol 31 no 12 pp596ndash604 2010
[17] Y Wang X Fan T Tang et al ldquoRhein and rhubarb simi-larly protect the blood-brain barrier after experimental trau-matic brain injury via gp91phox subunit of NADPH oxidaseROSERKMMP-9 signaling pathwayrdquo Scientific Reports vol 6p 37098 2016
[18] D-X Kong X-J Li and H-Y Zhang ldquoWhere is the hopefor drug discovery Let history tell the futurerdquo Drug DiscoveryTherapy vol 14 no 3-4 pp 115ndash119 2009
[19] F Cheung ldquoTCM made in Chinardquo Nature vol 480 no 7378pp S82ndashS83 2011
[20] C Fu Z Xia Y Liu et al ldquoQualitative analysis of majorconstituents from Xue Fu Zhu Yu Decoction using ultra highperformance liquid chromatographywith hybrid ion trap time-of-flight mass spectrometryrdquo Journal of Separation Science vol39 no 17 pp 3457ndash3468 2016
[21] H-J Zhang and Y-Y Cheng ldquoAn HPLCMS method for iden-tifying major constituents in the hypocholesterolemic extractsof Chinese medicine formula lsquoXue-Fu-Zhu-Yu decoctionrsquordquoBiomedical Chromatography vol 20 no 8 pp 821ndash826 2006
[22] L Zhang L Zhu YWang et al ldquoCharacterization and quantifi-cation of major constituents of Xue Fu Zhu Yu by UPLC-DAD-MSMSrdquo Journal of Pharmaceutical and Biomedical Analysisvol 62 pp 203ndash209 2012
[23] X Yang X Xiong G Yang and J Wang ldquoChinese patentmedicine Xuefu Zhuyu capsule for the treatment of unstableangina pectoris a systematic review of randomized controlledtrialsrdquo Complementary Therapies in Medicine vol 22 no 2 pp391ndash399 2014
[24] J Wang X Yang F Chu et al ldquoThe effects of Xuefu Zhuyuand Shengmai on the evolution of syndromes and inflammatorymarkers in patients with unstable angina pectoris after percuta-neous coronary intervention a randomised controlled clinicaltrialrdquoEvidence-BasedComplementary andAlternativeMedicinevol 2013 Article ID 896467 9 pages 2013
[25] Q Zhang H Yu J Qi et al ldquoNatural formulas and the natureof formulas Exploring potential therapeutic targets based ontraditional Chinese herbal formulasrdquo PLoS ONE vol 12 no 2p e0171628 2017
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[30] L Zhong W Ning and W Dong-pi ldquoModel Study of theTherapy of Replenishing Qi and Activating Blood Circulationon Protecting the Brain Impairment Caused by Hypoxic -ischemic Encephalopathy in SD Ratsrdquo Journal of TraditionalChinese Medicine vol 07 pp 1552ndash1555 2011
18 Evidence-Based Complementary and Alternative Medicine
[31] M Sun X-P Zhan C-Y Jin J-Z Shan S Xu and Y-LWang ldquoclinical observation on treatment of post-craniocerebraltraumatic mental disorder by integrative medicinerdquo ChineseJournal of Integrative Medicine vol 14 no 2 pp 137ndash141 2008
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[33] J Zhou T Liu H Cui et al ldquoXuefu zhuyu decoction improvescognitive impairment in experimental traumatic brain injuryvia synaptic regulationrdquo Oncotarget vol 8 no 42 2017
[34] S Li ldquoExploring traditional chinese medicine by a novel thera-peutic concept of network targetrdquo Chinese Journal of IntegrativeMedicine vol 22 no 9 pp 647ndash652 2016
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[37] Y Li C Han J Wang et al ldquoInvestigation into the mechanismof Eucommia ulmoides Oliv based on a systems pharmacologyapproachrdquo Journal of Ethnopharmacology vol 151 no 1 pp452ndash460 2014
[38] Y Yao X Zhang Z Wang et al ldquoDeciphering the combinationprinciples of Traditional Chinese Medicine from a systemspharmacology perspective based on Ma-huang DecoctionrdquoJournal of Ethnopharmacology vol 150 no 2 pp 619ndash638 2013
[39] Bo Zhang Xu Wang and Shao Li ldquoAn Integrative Platform ofTCM Network Pharmacology and Its Application on a HerbalFormula Qing-Luo-Yinrdquo Evidence-Based Complementary andAlternativeMedicine vol 2013 Article ID 456747 12 pages 2013
[40] J Ru P Li J Wang et al ldquoTCMSP a database of systemspharmacology for drug discovery from herbal medicinesrdquoJournal of Cheminformatics vol 6 no 1 p 13 2014
[41] J V Turner D J Maddalena and S Agatonovic-KustrinldquoBioavailability Prediction Based on Molecular Structure for aDiverse Series of Drugsrdquo Pharmaceutical Research vol 21 no 1pp 68ndash82 2004
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[43] A Zhang W Pan J Lv and H Wu ldquoProtective Effect ofAmygdalin on LPS-Induced Acute Lung Injury by InhibitingNF-120581B and NLRP3 Signaling Pathwaysrdquo Inflammation vol 40no 3 pp 745ndash751 2017
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[48] X Chen Z L Ji and Y Z Chen ldquoTTD therapeutic targetdatabaserdquo Nucleic Acids Research vol 30 no 1 pp 412ndash4152002
[49] A Hamosh A F Scott J S Amberger C A Bocchini andV AMcKusick ldquoOnline Mendelian Inheritance in Man (OMIM) aknowledgebase of human genes and genetic disordersrdquo NucleicAcids Research vol 33 pp D514ndashD517 2005
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[53] G Dennis Jr B T Sherman D A Hosack et al ldquoDAVIDDatabase for Annotation Visualization and IntegratedDiscov-eryrdquo Genome Biology vol 4 no 5 p P3 2003
[54] L Yang X Zhao and X Tang ldquoPredicting disease-related pro-teins based on clique backbone in protein-protein interactionnetworkrdquo International Journal of Biological Sciences vol 10 no7 pp 677ndash688 2014
[55] S MThomas and J S Brugge ldquoCellular functions regulated bySRC family kinasesrdquo Annual Review of Cell and DevelopmentalBiology vol 13 pp 513ndash609 1997
[56] D Z Liu F R Sharp K C Van et al ldquoInhibition of Src familykinases protects hippocampal neurons and improves cognitivefunction after traumatic brain injuryrdquo Journal of Neurotraumavol 31 no 14 pp 1268ndash1276 2014
[57] B D Manning and A Toker ldquoAKTPKB Signaling Navigatingthe Networkrdquo Cell vol 169 no 3 pp 381ndash405 2017
[58] Y Kitagishi and S Matsuda ldquoDiets involved in PPAR andPI3KAKTPTEN pathway may contribute to neuroprotectionin a traumatic brain injuryrdquoAlzheimerrsquos ResearchampTherapy vol5 no 5 p 42 2013
[59] D Chen L Bao S-Q Lu and F Xu ldquoSerum albumin andprealbuminpredict the poor outcomeof traumatic brain injuryrdquoPLoS ONE vol 9 no 3 Article ID e93167 2014
[60] J Yang Z Wu N Renier et al ldquoPathological axonal deaththrough aMapk cascade that triggers a local energy deficitrdquoCellvol 160 no 1-2 pp 161ndash176 2015
[61] E J Huang and L F Reichardt ldquoTrk receptors roles in neuronalsignal transductionrdquoAnnual Review of Biochemistry vol 72 pp609ndash642 2003
[62] J W Lee and R Juliano ldquoMitogenic signal transductionby integrin- and growth factor receptor-mediated pathwaysrdquoMolecules and Cells vol 17 no 2 pp 188ndash202 2004
[63] C S Yang J M Landau M T Huang and H L NewmarkldquoInhibition of carcinogenesis by dietary polyphenolic com-poundsrdquo Annual Review of Nutrition vol 21 pp 381ndash406 2001
[64] A Murakami H Ashida and J Terao ldquoMultitargeted cancerprevention by quercetinrdquoCancer Letters vol 269 no 2 pp 315ndash325 2008
[65] G Du Z Zhao Y Chen et al ldquoQuercetin attenuates neuronalautophagy and apoptosis in rat traumatic brain injury modelvia activation of PI3KAkt signaling pathwayrdquo NeurologicalResearch vol 38 no 11 pp 1ndash8 2016
Evidence-Based Complementary and Alternative Medicine 19
[66] Q Cai R O Rahn and R Zhang ldquoDietary flavonoidsquercetin luteolin andgenistein reduce oxidativeDNAdamageand lipid peroxidation and quench free radicalsrdquoCancer Lettersvol 119 no 1 pp 99ndash107 1997
[67] G A Bongiovanni E A Soria and A R Eynard ldquoEffectsof the plant flavonoids silymarin and quercetin on arsenite-induced oxidative stress in CHO-K1 cellsrdquo Food and ChemicalToxicology vol 45 no 6 pp 971ndash976 2007
[68] H Li L Yang Y Zhang et al ldquoKaempferol inhibits fibroblastcollagen synthesis proliferation and activation in hypertrophicscar via targeting TGF-beta receptor type Irdquo Biomedicine ampPharmacotherapy = Biomedecine amp Pharmacotherapie vol 83pp 967ndash974 2016
[69] K P Devi D S Malar S F Nabavi et al ldquoKaempferol andinflammation From chemistry to medicinerdquo PharmacologicalResearch vol 99 pp 1ndash10 2015
[70] M Zhou H Ren J Han W Wang Q Zheng and DWang ldquoProtective effects of kaempferol against myocardialischemiareperfusion injury in isolated rat heart via antioxidantactivity and inhibition of glycogen synthase kinase-3120573rdquo Oxida-tive Medicine and Cellular Longevity vol 2015 p 481405 2015
[71] C-F Lee J-S Yang F-J Tsai et al ldquoKaempferol inducesATMp53-mediated death receptor and mitochondrial apop-tosis in human umbilical vein endothelial cellsrdquo InternationalJournal of Oncology vol 48 no 5 pp 2007ndash2014 2016
[72] C Luo H Yang C Tang et al ldquoKaempferol alleviates insulinresistance via hepatic IKKNF-120581B signal in type 2 diabetic ratsrdquoInternational Immunopharmacology vol 28 no 1 pp 744ndash7502015
[73] A B Awad and C S Fink ldquoPhytosterols as anticancer dietarycomponents evidence and mechanism of actionrdquo Journal ofNutrition vol 130 no 9 pp 2127ndash2130 2000
[74] K Farber and H Kettenmann ldquoFunctional role of calciumsignals for microglial functionrdquoGlia vol 54 no 7 pp 656ndash6652006
[75] Y Lu Y Gu X Ding et al ldquoIntracellular Ca2+ homeostasisand JAK1STAT3 pathway are involved in the protective effectof propofol on BV2 microglia against hypoxia-induced inflam-mation and apoptosisrdquo PLoS ONE vol 12 no 5 p e01780982017
[76] K Leroy and J-P Brion ldquoDevelopmental expression and local-ization of glycogen synthase kinase-3120573 in rat brainrdquo Journal ofChemical Neuroanatomy vol 16 no 4 pp 279ndash293 1999
[77] C-M Liu E-M Hur and F-Q Zhou ldquoCoordinating geneexpression and axon assembly to control axon growth Potentialrole ofGSK3 signalingrdquo Frontiers inMolecularNeuroscience vol3 p 3 2012
[78] D A E Cross A A Culbert K A Chalmers L Facci S DSkaper and A D Reith ldquoSelective small-molecule inhibitors ofglycogen synthase kinase-3 activity protect primary neuronesfrom deathrdquo Journal of Neurochemistry vol 77 no 1 pp 94ndash102 2001
[79] V S Ossovskaya and NW Bunnett ldquoProtease-activated recep-tors contribution to physiology and diseaserdquo PhysiologicalReviews vol 84 no 2 pp 579ndash621 2004
[80] M Steinhoff J Buddenkotte V Shpacovitch et al ldquoProteinase-activated receptors transducers of proteinase-mediated signal-ing in inflammation and immune responserdquoEndocrine Reviewsvol 26 no 1 pp 1ndash43 2005
[81] H Krenzlin V Lorenz S Danckwardt O Kempski and BAlessandri ldquoThe importance of thrombin in cerebral injury and
diseaserdquo International Journal of Molecular Sciences vol 17 no1 2016
[82] M J McGinn and J T Povlishock ldquoPathophysiology of Trau-matic Brain InjuryrdquoNeurosurgery Clinics of North America vol27 no 4 pp 397ndash407 2016
[83] T Woodcock and M C Morganti-Kossmann ldquoThe role ofmarkers of inflammation in traumatic brain injuryrdquo Frontiersin Neurology vol 4 article 18 2013
[84] F Tanriverdi H J Schneider G Aimaretti B E Masel F FCasanueva and F Kelestimur ldquoPituitary dysfunction after trau-matic brain injury A clinical and pathophysiological approachrdquoEndocrine Reviews vol 36 no 3 pp 305ndash342 2015
[85] J V Rosenfeld A I Maas P Bragge M C Morganti-Kossmann G T Manley and R L Gruen ldquoEarly managementof severe traumatic brain injuryrdquoThe Lancet vol 380 no 9847pp 1088ndash1098 2012
[86] B M Aertker S Bedi and C S Cox ldquoStrategies for CNS repairfollowing TBIrdquo Experimental Neurology vol 275 no 3 pp 411ndash426 2016
[87] K Adachi Z Mirzadeh M Sakaguchi et al ldquo120573-catenin signal-ing promotes proliferation of progenitor cells in the adultmousesubventricular zonerdquo Stem Cells vol 25 no 11 pp 2827ndash28362007
[88] Y Hirabayashi and Y Gotoh ldquoStage-dependent fate determina-tion of neural precursor cells in mouse forebrainrdquo NeuroscienceResearch vol 51 no 4 pp 331ndash336 2005
[89] S Ding T Y H Wu A Brinker et al ldquoSynthetic smallmolecules that control stem cell faterdquo Proceedings of theNationalAcadamy of Sciences of the United States of America vol 100 no13 pp 7632ndash7637 2003
[90] N Israsena M Hu W Fu L Kan and J A Kessler ldquoThepresence of FGF2 signaling determines whether 120573-cateninexerts effects on proliferation or neuronal differentiation ofneural stem cellsrdquo Developmental Biology vol 268 no 1 pp220ndash231 2004
[91] A Salehi A Jullienne M Baghchechi et al ldquoUp-regulation ofWnt120573-catenin expression is accompanied with vascular repairafter traumatic brain injuryrdquo Journal of Cerebral Blood Flow ampMetabolism vol 38 no 2 pp 274ndash289 2017
[92] L F Reichardt and K J Tomaselli ldquoExtracellular matrixmolecules and their receptors Functions in neural develop-mentrdquoAnnual Review of Neuroscience vol 14 pp 531ndash570 1991
[93] R M Gibson S E Craig L Heenan C Tournier and M JHumphries ldquoActivation of integrin 12057251205731 delays apoptosis ofNtera2 neuronal cellsrdquoMolecularandCellularNeuroscience vol28 no 3 pp 588ndash598 2005
[94] C C Tate A J Garcıa andMC LaPlaca ldquoPlasma fibronectin isneuroprotective following traumatic brain injuryrdquoExperimentalNeurology vol 207 no 1 pp 13ndash22 2007
[95] C Culmsee and M P Mattson ldquop53 in neuronal apoptosisrdquoBiochemical and Biophysical Research Communications vol 331no 3 pp 761ndash777 2005
[96] M S Geddis and V Rehder ldquoThe phosphorylation state ofneuronal processes determines growth cone formation afterneuronal injuryrdquo Journal of Neuroscience Research vol 74 no2 pp 210ndash220 2003
[97] M L Craske M Fivaz N N Batada and T Meyer ldquoSpines andneurite branches function as geometric attractors that enhanceprotein kinase C actionrdquoThe Journal of Cell Biology vol 170 no7 pp 1147ndash1158 2005
20 Evidence-Based Complementary and Alternative Medicine
[98] A Muscella C Vetrugno L G Cossa et al ldquoApoptosis by[Pt(OOrsquo-acac)(gamma-acac)(DMS)] requires PKC-deltamedi-ated p53 activation in malignant pleural mesotheliomardquo PloSone vol 12 no 7 Article ID e0181114 2017
[99] J S Bonini W C Da Silva L R M Bevilaqua J H MedinaI Izquierdo and M Cammarota ldquoOn the participation ofhippocampal PKC in acquisition consolidation and reconsol-idation of spatial memoryrdquo Neuroscience vol 147 no 1 pp 37ndash45 2007
[100] O Zohar R Lavy X Zi et al ldquoPKC activator therapeutic formild traumatic brain injury in micerdquo Neurobiology of Diseasevol 41 no 2 pp 329ndash337 2011
[101] J David Sweatt ldquoTheneuronalMAPkinase cascade a biochem-ical signal integration system subserving synaptic plasticity andmemoryrdquo Journal ofNeurochemistry vol 76 no 1 pp 1ndash10 2001
[102] Y Matsuoka M Picciano B Maleste et al ldquoInflammatoryresponses to amyloidosis in a transgenic mouse model ofAlzheimerrsquos diseaserdquo The American Journal of Pathology vol158 no 4 pp 1345ndash1354 2001
[103] L Esposito L Gan G-Q Yu C Essrich and L MuckeldquoIntracellularly generated amyloid-120573 peptide counteracts theantiapoptotic function of its precursor protein and primesproapoptotic pathways for activation by other insults in neu-roblastoma cellsrdquo Journal of Neurochemistry vol 91 no 6 pp1260ndash1274 2004
[104] D J Loane A Pocivavsek C E-H Moussa et al ldquoAmyloidprecursor protein secretases as therapeutic targets for traumaticbrain injuryrdquo Nature Medicine vol 15 no 4 pp 377ndash379 2009
[105] R Torres B L Firestein H Dong et al ldquoPDZ proteins bindcluster and synaptically colocalize with Eph receptors and theirephrin ligandsrdquo Neuron vol 21 no 6 pp 1453ndash1463 1998
[106] M B Dalva M A Takasu M Z Lin et al ldquoEphB receptorsinteract with NMDA receptors and regulate excitatory synapseformationrdquo Cell vol 103 no 6 pp 945ndash956 2000
[107] J M Basak P B Verghese H Yoon J Kim and D MHoltzman ldquoLow-density lipoprotein receptor represents anapolipoprotein E-independent pathway of A120573 uptake anddegradation by astrocytesrdquoThe Journal of Biological Chemistryvol 287 no 17 pp 13959ndash13971 2012
[108] L Yao X Gu Q Song et al ldquoNanoformulated alpha-mangostinameliorates Alzheimerrsquos disease neuropathology by elevatingLDLR expression and accelerating amyloid-beta clearancerdquoJournal of Controlled Release vol 226 pp 1ndash14 2016
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 17
(D) F2-glyasperin B (E) NOS3-1-Methoxyphaseollidin and(F)ACHE-(-)-Medicocarpin (Supplementary Materials)
References
[1] L Yang Y Chu D Tweedie et al ldquoPost-trauma administrationof the pifithrin-120572 oxygen analog improves histological andfunctional outcomes after experimental traumatic brain injuryrdquoExperimental Neurology vol 269 pp 56ndash66 2015
[2] J A Langlois W Rutland-Brown and M M Wald ldquoTheepidemiology and impact of traumatic brain injury a briefoverviewrdquo The Journal of Head Trauma Rehabilitation vol 21no 5 pp 375ndash378 2006
[3] H A Alsulaim B J Smart A O Asemota et al ldquoConsciousstatus predictsmortality among patientswith isolated traumaticbrain injury in administrative datardquo The American Journal ofSurgery vol 214 no 2 pp 207ndash210 2017
[4] M Majdan D Plancikova A Maas et al ldquoYears of life lost dueto traumatic brain injury in Europe A cross-sectional analysisof 16 countriesrdquo PLoS Medicine vol 14 no 7 p e1002331 2017
[5] P Cheng P Yin P Ning et al ldquoTrends in traumatic braininjury mortality in China 2006ndash2013 A population-basedlongitudinal studyrdquo PLoS Medicine vol 14 no 7 Article IDe1002332 2017
[6] M V Russo and D B McGavern ldquoInflammatory neuroprotec-tion following traumatic brain injuryrdquo Science vol 353 no 6301pp 783ndash785 2016
[7] WWang H Li J Yu et al ldquoProtective Effects of ChineseHerbalMedicine Rhizoma drynariae in Rats After Traumatic BrainInjury and Identification of Active Compoundrdquo MolecularNeurobiology vol 53 no 7 pp 4809ndash4820 2016
[8] M Das S Mohapatra and S S Mohapatra ldquoNew perspectiveson central and peripheral immune responses to acute traumaticbrain injuryrdquo Journal of Neuroinflammation vol 9 p 236 2012
[9] J W Finnie ldquoNeuroinflammation Beneficial and detrimentaleffects after traumatic brain injuryrdquo Inflammopharmacologyvol 21 no 4 pp 309ndash320 2013
[10] T Cheng W Wang Q Li et al ldquoCerebroprotection of flavanol(minus)-epicatechin after traumatic brain injury viaNrf2-dependentand -independent pathwaysrdquo Free Radical Biology amp Medicinevol 92 pp 15ndash28 2016
[11] W Young ldquoRole of calcium in central nervous system injuriesrdquoJournal of Neurotrauma vol 9 Suppl 1 pp S9ndashS25 1992
[12] R Bullock A Zauner J S Myseros et al ldquoEvidence forProlonged Release of Excitatory Amino Acids in Severe HumanHead Trauma Relationship to Clinical Eventsrdquo Annals of theNew York Academy of Sciences vol 765 no 1 pp 290ndash297 1995
[13] A T Mazzeo A Beat A Singh and M R Bullock ldquoTherole of mitochondrial transition pore and its modulation intraumatic brain injury and delayed neurodegeneration afterTBIrdquo Experimental Neurology vol 218 no 2 pp 363ndash370 2009
[14] S Gennai A Monsel Q Hao et al ldquoCell-Based therapy fortraumatic brain injuryrdquo British Journal of Anaesthesia vol 115no 2 pp 203ndash212 2015
[15] J Ghajar ldquoTraumatic brain injuryrdquo The Lancet vol 356 no9233 pp 923ndash929 2000
[16] D J Loane and A I Faden ldquoNeuroprotection for traumaticbrain injury translational challenges and emerging therapeuticstrategiesrdquoTrends in Pharmacological Sciences vol 31 no 12 pp596ndash604 2010
[17] Y Wang X Fan T Tang et al ldquoRhein and rhubarb simi-larly protect the blood-brain barrier after experimental trau-matic brain injury via gp91phox subunit of NADPH oxidaseROSERKMMP-9 signaling pathwayrdquo Scientific Reports vol 6p 37098 2016
[18] D-X Kong X-J Li and H-Y Zhang ldquoWhere is the hopefor drug discovery Let history tell the futurerdquo Drug DiscoveryTherapy vol 14 no 3-4 pp 115ndash119 2009
[19] F Cheung ldquoTCM made in Chinardquo Nature vol 480 no 7378pp S82ndashS83 2011
[20] C Fu Z Xia Y Liu et al ldquoQualitative analysis of majorconstituents from Xue Fu Zhu Yu Decoction using ultra highperformance liquid chromatographywith hybrid ion trap time-of-flight mass spectrometryrdquo Journal of Separation Science vol39 no 17 pp 3457ndash3468 2016
[21] H-J Zhang and Y-Y Cheng ldquoAn HPLCMS method for iden-tifying major constituents in the hypocholesterolemic extractsof Chinese medicine formula lsquoXue-Fu-Zhu-Yu decoctionrsquordquoBiomedical Chromatography vol 20 no 8 pp 821ndash826 2006
[22] L Zhang L Zhu YWang et al ldquoCharacterization and quantifi-cation of major constituents of Xue Fu Zhu Yu by UPLC-DAD-MSMSrdquo Journal of Pharmaceutical and Biomedical Analysisvol 62 pp 203ndash209 2012
[23] X Yang X Xiong G Yang and J Wang ldquoChinese patentmedicine Xuefu Zhuyu capsule for the treatment of unstableangina pectoris a systematic review of randomized controlledtrialsrdquo Complementary Therapies in Medicine vol 22 no 2 pp391ndash399 2014
[24] J Wang X Yang F Chu et al ldquoThe effects of Xuefu Zhuyuand Shengmai on the evolution of syndromes and inflammatorymarkers in patients with unstable angina pectoris after percuta-neous coronary intervention a randomised controlled clinicaltrialrdquoEvidence-BasedComplementary andAlternativeMedicinevol 2013 Article ID 896467 9 pages 2013
[25] Q Zhang H Yu J Qi et al ldquoNatural formulas and the natureof formulas Exploring potential therapeutic targets based ontraditional Chinese herbal formulasrdquo PLoS ONE vol 12 no 2p e0171628 2017
[26] J LeeWHsu T Yen et al ldquoTraditional ChinesemedicineXue-Fu-Zhu-Yu decoction potentiates tissue plasminogen activatoragainst thromboembolic stroke in ratsrdquo Journal of Ethnophar-macology vol 134 no 3 pp 824ndash830 2011
[27] Y-C Shen C-K Lu K-T Liou et al ldquoCommon and uniquemechanisms of Chinese herbal remedies on ischemic strokemice revealed by transcriptome analysesrdquo Journal of Ethnophar-macology vol 173 pp 370ndash382 2015
[28] F Xue-hai G Yan and L Jian-tao ldquoTherapeutic effect of XiefuZhuyu decoction joint brain protein hydrolysat injection intraumatic brain injury and its effect on cerebrospinal fluid ofET-1rdquo Chinese Journal of Biochemical Pharmaceutics no 02 pp55ndash58 2017
[29] L Shuxiang W Jingchun C Jie et al ldquoEffect of Xuefu Zhuyudecoction on the neural functional recovery and living abilityin patients with craniocerebral injuryrdquo Modern Journal ofIntegrated Traditional Chinese andWesternMedicine no 04 pp350ndash352 2015
[30] L Zhong W Ning and W Dong-pi ldquoModel Study of theTherapy of Replenishing Qi and Activating Blood Circulationon Protecting the Brain Impairment Caused by Hypoxic -ischemic Encephalopathy in SD Ratsrdquo Journal of TraditionalChinese Medicine vol 07 pp 1552ndash1555 2011
18 Evidence-Based Complementary and Alternative Medicine
[31] M Sun X-P Zhan C-Y Jin J-Z Shan S Xu and Y-LWang ldquoclinical observation on treatment of post-craniocerebraltraumatic mental disorder by integrative medicinerdquo ChineseJournal of Integrative Medicine vol 14 no 2 pp 137ndash141 2008
[32] Z Xing Z Xia W Peng et al ldquoXuefu Zhuyu decoction atraditional Chinese medicine provides neuroprotection in arat model of traumatic brain injury via an anti-inflammatorypathwayrdquo Scientific Reports vol 6 Article ID 20040 2016
[33] J Zhou T Liu H Cui et al ldquoXuefu zhuyu decoction improvescognitive impairment in experimental traumatic brain injuryvia synaptic regulationrdquo Oncotarget vol 8 no 42 2017
[34] S Li ldquoExploring traditional chinese medicine by a novel thera-peutic concept of network targetrdquo Chinese Journal of IntegrativeMedicine vol 22 no 9 pp 647ndash652 2016
[35] S Li and B Zhang ldquoTraditional Chinese medicine networkpharmacology theory methodology and applicationrdquo ChineseJournal of Natural Medicines vol 11 no 2 pp 110ndash120 2013
[36] A L Hopkins ldquoNetwork pharmacology the next paradigm indrug discoveryrdquoNature Chemical Biology vol 4 no 11 pp 682ndash690 2008
[37] Y Li C Han J Wang et al ldquoInvestigation into the mechanismof Eucommia ulmoides Oliv based on a systems pharmacologyapproachrdquo Journal of Ethnopharmacology vol 151 no 1 pp452ndash460 2014
[38] Y Yao X Zhang Z Wang et al ldquoDeciphering the combinationprinciples of Traditional Chinese Medicine from a systemspharmacology perspective based on Ma-huang DecoctionrdquoJournal of Ethnopharmacology vol 150 no 2 pp 619ndash638 2013
[39] Bo Zhang Xu Wang and Shao Li ldquoAn Integrative Platform ofTCM Network Pharmacology and Its Application on a HerbalFormula Qing-Luo-Yinrdquo Evidence-Based Complementary andAlternativeMedicine vol 2013 Article ID 456747 12 pages 2013
[40] J Ru P Li J Wang et al ldquoTCMSP a database of systemspharmacology for drug discovery from herbal medicinesrdquoJournal of Cheminformatics vol 6 no 1 p 13 2014
[41] J V Turner D J Maddalena and S Agatonovic-KustrinldquoBioavailability Prediction Based on Molecular Structure for aDiverse Series of Drugsrdquo Pharmaceutical Research vol 21 no 1pp 68ndash82 2004
[42] X XuW Zhang CHuang et al ldquoAnovel chemometricmethodfor the prediction of human oral bioavailabilityrdquo InternationalJournal of Molecular Sciences vol 13 no 6 pp 6964ndash6982 2012
[43] A Zhang W Pan J Lv and H Wu ldquoProtective Effect ofAmygdalin on LPS-Induced Acute Lung Injury by InhibitingNF-120581B and NLRP3 Signaling Pathwaysrdquo Inflammation vol 40no 3 pp 745ndash751 2017
[44] X Wei H Liu X Sun et al ldquoHydroxysafflor yellow A protectsrat brains against ischemia-reperfusion injury by antioxidantactionrdquo Neuroscience Letters vol 386 no 1 pp 58ndash62 2005
[45] W PWalters andM A Murcko ldquoPrediction of lsquodrug-likenessrsquordquoAdvanced Drug Delivery Reviews vol 54 no 3 pp 255ndash2712002
[46] Y Yamanishi M Kotera M Kanehisa and S Goto ldquoDrug-target interactionprediction from chemical genomic and phar-macological data in an integrated frameworkrdquo Bioinformaticsvol 26 no 12 pp i246ndashi254 2010
[47] H Yu J Chen X Xu et al ldquoA systematic prediction ofmultiple drug-target interactions from chemical genomic andpharmacological datardquo PLoS ONE vol 7 no 5 Article IDe37608 2012
[48] X Chen Z L Ji and Y Z Chen ldquoTTD therapeutic targetdatabaserdquo Nucleic Acids Research vol 30 no 1 pp 412ndash4152002
[49] A Hamosh A F Scott J S Amberger C A Bocchini andV AMcKusick ldquoOnline Mendelian Inheritance in Man (OMIM) aknowledgebase of human genes and genetic disordersrdquo NucleicAcids Research vol 33 pp D514ndashD517 2005
[50] T S Keshava Prasad RGoel K Kandasamy et al ldquoHuman pro-tein reference databasemdash2009 updaterdquo Nucleic Acids Researchvol 37 no 1 pp D767ndashD772 2009
[51] A Franceschini D Szklarczyk S Frankild et al ldquoSTRING v91protein-protein interaction networks with increased coverageand integrationrdquoNucleic Acids Research vol 41 no 1 pp D808ndashD815 2013
[52] P Shannon A Markiel O Ozier et al ldquoCytoscape a softwareEnvironment for integratedmodels of biomolecular interactionnetworksrdquo Genome Research vol 13 no 11 pp 2498ndash25042003
[53] G Dennis Jr B T Sherman D A Hosack et al ldquoDAVIDDatabase for Annotation Visualization and IntegratedDiscov-eryrdquo Genome Biology vol 4 no 5 p P3 2003
[54] L Yang X Zhao and X Tang ldquoPredicting disease-related pro-teins based on clique backbone in protein-protein interactionnetworkrdquo International Journal of Biological Sciences vol 10 no7 pp 677ndash688 2014
[55] S MThomas and J S Brugge ldquoCellular functions regulated bySRC family kinasesrdquo Annual Review of Cell and DevelopmentalBiology vol 13 pp 513ndash609 1997
[56] D Z Liu F R Sharp K C Van et al ldquoInhibition of Src familykinases protects hippocampal neurons and improves cognitivefunction after traumatic brain injuryrdquo Journal of Neurotraumavol 31 no 14 pp 1268ndash1276 2014
[57] B D Manning and A Toker ldquoAKTPKB Signaling Navigatingthe Networkrdquo Cell vol 169 no 3 pp 381ndash405 2017
[58] Y Kitagishi and S Matsuda ldquoDiets involved in PPAR andPI3KAKTPTEN pathway may contribute to neuroprotectionin a traumatic brain injuryrdquoAlzheimerrsquos ResearchampTherapy vol5 no 5 p 42 2013
[59] D Chen L Bao S-Q Lu and F Xu ldquoSerum albumin andprealbuminpredict the poor outcomeof traumatic brain injuryrdquoPLoS ONE vol 9 no 3 Article ID e93167 2014
[60] J Yang Z Wu N Renier et al ldquoPathological axonal deaththrough aMapk cascade that triggers a local energy deficitrdquoCellvol 160 no 1-2 pp 161ndash176 2015
[61] E J Huang and L F Reichardt ldquoTrk receptors roles in neuronalsignal transductionrdquoAnnual Review of Biochemistry vol 72 pp609ndash642 2003
[62] J W Lee and R Juliano ldquoMitogenic signal transductionby integrin- and growth factor receptor-mediated pathwaysrdquoMolecules and Cells vol 17 no 2 pp 188ndash202 2004
[63] C S Yang J M Landau M T Huang and H L NewmarkldquoInhibition of carcinogenesis by dietary polyphenolic com-poundsrdquo Annual Review of Nutrition vol 21 pp 381ndash406 2001
[64] A Murakami H Ashida and J Terao ldquoMultitargeted cancerprevention by quercetinrdquoCancer Letters vol 269 no 2 pp 315ndash325 2008
[65] G Du Z Zhao Y Chen et al ldquoQuercetin attenuates neuronalautophagy and apoptosis in rat traumatic brain injury modelvia activation of PI3KAkt signaling pathwayrdquo NeurologicalResearch vol 38 no 11 pp 1ndash8 2016
Evidence-Based Complementary and Alternative Medicine 19
[66] Q Cai R O Rahn and R Zhang ldquoDietary flavonoidsquercetin luteolin andgenistein reduce oxidativeDNAdamageand lipid peroxidation and quench free radicalsrdquoCancer Lettersvol 119 no 1 pp 99ndash107 1997
[67] G A Bongiovanni E A Soria and A R Eynard ldquoEffectsof the plant flavonoids silymarin and quercetin on arsenite-induced oxidative stress in CHO-K1 cellsrdquo Food and ChemicalToxicology vol 45 no 6 pp 971ndash976 2007
[68] H Li L Yang Y Zhang et al ldquoKaempferol inhibits fibroblastcollagen synthesis proliferation and activation in hypertrophicscar via targeting TGF-beta receptor type Irdquo Biomedicine ampPharmacotherapy = Biomedecine amp Pharmacotherapie vol 83pp 967ndash974 2016
[69] K P Devi D S Malar S F Nabavi et al ldquoKaempferol andinflammation From chemistry to medicinerdquo PharmacologicalResearch vol 99 pp 1ndash10 2015
[70] M Zhou H Ren J Han W Wang Q Zheng and DWang ldquoProtective effects of kaempferol against myocardialischemiareperfusion injury in isolated rat heart via antioxidantactivity and inhibition of glycogen synthase kinase-3120573rdquo Oxida-tive Medicine and Cellular Longevity vol 2015 p 481405 2015
[71] C-F Lee J-S Yang F-J Tsai et al ldquoKaempferol inducesATMp53-mediated death receptor and mitochondrial apop-tosis in human umbilical vein endothelial cellsrdquo InternationalJournal of Oncology vol 48 no 5 pp 2007ndash2014 2016
[72] C Luo H Yang C Tang et al ldquoKaempferol alleviates insulinresistance via hepatic IKKNF-120581B signal in type 2 diabetic ratsrdquoInternational Immunopharmacology vol 28 no 1 pp 744ndash7502015
[73] A B Awad and C S Fink ldquoPhytosterols as anticancer dietarycomponents evidence and mechanism of actionrdquo Journal ofNutrition vol 130 no 9 pp 2127ndash2130 2000
[74] K Farber and H Kettenmann ldquoFunctional role of calciumsignals for microglial functionrdquoGlia vol 54 no 7 pp 656ndash6652006
[75] Y Lu Y Gu X Ding et al ldquoIntracellular Ca2+ homeostasisand JAK1STAT3 pathway are involved in the protective effectof propofol on BV2 microglia against hypoxia-induced inflam-mation and apoptosisrdquo PLoS ONE vol 12 no 5 p e01780982017
[76] K Leroy and J-P Brion ldquoDevelopmental expression and local-ization of glycogen synthase kinase-3120573 in rat brainrdquo Journal ofChemical Neuroanatomy vol 16 no 4 pp 279ndash293 1999
[77] C-M Liu E-M Hur and F-Q Zhou ldquoCoordinating geneexpression and axon assembly to control axon growth Potentialrole ofGSK3 signalingrdquo Frontiers inMolecularNeuroscience vol3 p 3 2012
[78] D A E Cross A A Culbert K A Chalmers L Facci S DSkaper and A D Reith ldquoSelective small-molecule inhibitors ofglycogen synthase kinase-3 activity protect primary neuronesfrom deathrdquo Journal of Neurochemistry vol 77 no 1 pp 94ndash102 2001
[79] V S Ossovskaya and NW Bunnett ldquoProtease-activated recep-tors contribution to physiology and diseaserdquo PhysiologicalReviews vol 84 no 2 pp 579ndash621 2004
[80] M Steinhoff J Buddenkotte V Shpacovitch et al ldquoProteinase-activated receptors transducers of proteinase-mediated signal-ing in inflammation and immune responserdquoEndocrine Reviewsvol 26 no 1 pp 1ndash43 2005
[81] H Krenzlin V Lorenz S Danckwardt O Kempski and BAlessandri ldquoThe importance of thrombin in cerebral injury and
diseaserdquo International Journal of Molecular Sciences vol 17 no1 2016
[82] M J McGinn and J T Povlishock ldquoPathophysiology of Trau-matic Brain InjuryrdquoNeurosurgery Clinics of North America vol27 no 4 pp 397ndash407 2016
[83] T Woodcock and M C Morganti-Kossmann ldquoThe role ofmarkers of inflammation in traumatic brain injuryrdquo Frontiersin Neurology vol 4 article 18 2013
[84] F Tanriverdi H J Schneider G Aimaretti B E Masel F FCasanueva and F Kelestimur ldquoPituitary dysfunction after trau-matic brain injury A clinical and pathophysiological approachrdquoEndocrine Reviews vol 36 no 3 pp 305ndash342 2015
[85] J V Rosenfeld A I Maas P Bragge M C Morganti-Kossmann G T Manley and R L Gruen ldquoEarly managementof severe traumatic brain injuryrdquoThe Lancet vol 380 no 9847pp 1088ndash1098 2012
[86] B M Aertker S Bedi and C S Cox ldquoStrategies for CNS repairfollowing TBIrdquo Experimental Neurology vol 275 no 3 pp 411ndash426 2016
[87] K Adachi Z Mirzadeh M Sakaguchi et al ldquo120573-catenin signal-ing promotes proliferation of progenitor cells in the adultmousesubventricular zonerdquo Stem Cells vol 25 no 11 pp 2827ndash28362007
[88] Y Hirabayashi and Y Gotoh ldquoStage-dependent fate determina-tion of neural precursor cells in mouse forebrainrdquo NeuroscienceResearch vol 51 no 4 pp 331ndash336 2005
[89] S Ding T Y H Wu A Brinker et al ldquoSynthetic smallmolecules that control stem cell faterdquo Proceedings of theNationalAcadamy of Sciences of the United States of America vol 100 no13 pp 7632ndash7637 2003
[90] N Israsena M Hu W Fu L Kan and J A Kessler ldquoThepresence of FGF2 signaling determines whether 120573-cateninexerts effects on proliferation or neuronal differentiation ofneural stem cellsrdquo Developmental Biology vol 268 no 1 pp220ndash231 2004
[91] A Salehi A Jullienne M Baghchechi et al ldquoUp-regulation ofWnt120573-catenin expression is accompanied with vascular repairafter traumatic brain injuryrdquo Journal of Cerebral Blood Flow ampMetabolism vol 38 no 2 pp 274ndash289 2017
[92] L F Reichardt and K J Tomaselli ldquoExtracellular matrixmolecules and their receptors Functions in neural develop-mentrdquoAnnual Review of Neuroscience vol 14 pp 531ndash570 1991
[93] R M Gibson S E Craig L Heenan C Tournier and M JHumphries ldquoActivation of integrin 12057251205731 delays apoptosis ofNtera2 neuronal cellsrdquoMolecularandCellularNeuroscience vol28 no 3 pp 588ndash598 2005
[94] C C Tate A J Garcıa andMC LaPlaca ldquoPlasma fibronectin isneuroprotective following traumatic brain injuryrdquoExperimentalNeurology vol 207 no 1 pp 13ndash22 2007
[95] C Culmsee and M P Mattson ldquop53 in neuronal apoptosisrdquoBiochemical and Biophysical Research Communications vol 331no 3 pp 761ndash777 2005
[96] M S Geddis and V Rehder ldquoThe phosphorylation state ofneuronal processes determines growth cone formation afterneuronal injuryrdquo Journal of Neuroscience Research vol 74 no2 pp 210ndash220 2003
[97] M L Craske M Fivaz N N Batada and T Meyer ldquoSpines andneurite branches function as geometric attractors that enhanceprotein kinase C actionrdquoThe Journal of Cell Biology vol 170 no7 pp 1147ndash1158 2005
20 Evidence-Based Complementary and Alternative Medicine
[98] A Muscella C Vetrugno L G Cossa et al ldquoApoptosis by[Pt(OOrsquo-acac)(gamma-acac)(DMS)] requires PKC-deltamedi-ated p53 activation in malignant pleural mesotheliomardquo PloSone vol 12 no 7 Article ID e0181114 2017
[99] J S Bonini W C Da Silva L R M Bevilaqua J H MedinaI Izquierdo and M Cammarota ldquoOn the participation ofhippocampal PKC in acquisition consolidation and reconsol-idation of spatial memoryrdquo Neuroscience vol 147 no 1 pp 37ndash45 2007
[100] O Zohar R Lavy X Zi et al ldquoPKC activator therapeutic formild traumatic brain injury in micerdquo Neurobiology of Diseasevol 41 no 2 pp 329ndash337 2011
[101] J David Sweatt ldquoTheneuronalMAPkinase cascade a biochem-ical signal integration system subserving synaptic plasticity andmemoryrdquo Journal ofNeurochemistry vol 76 no 1 pp 1ndash10 2001
[102] Y Matsuoka M Picciano B Maleste et al ldquoInflammatoryresponses to amyloidosis in a transgenic mouse model ofAlzheimerrsquos diseaserdquo The American Journal of Pathology vol158 no 4 pp 1345ndash1354 2001
[103] L Esposito L Gan G-Q Yu C Essrich and L MuckeldquoIntracellularly generated amyloid-120573 peptide counteracts theantiapoptotic function of its precursor protein and primesproapoptotic pathways for activation by other insults in neu-roblastoma cellsrdquo Journal of Neurochemistry vol 91 no 6 pp1260ndash1274 2004
[104] D J Loane A Pocivavsek C E-H Moussa et al ldquoAmyloidprecursor protein secretases as therapeutic targets for traumaticbrain injuryrdquo Nature Medicine vol 15 no 4 pp 377ndash379 2009
[105] R Torres B L Firestein H Dong et al ldquoPDZ proteins bindcluster and synaptically colocalize with Eph receptors and theirephrin ligandsrdquo Neuron vol 21 no 6 pp 1453ndash1463 1998
[106] M B Dalva M A Takasu M Z Lin et al ldquoEphB receptorsinteract with NMDA receptors and regulate excitatory synapseformationrdquo Cell vol 103 no 6 pp 945ndash956 2000
[107] J M Basak P B Verghese H Yoon J Kim and D MHoltzman ldquoLow-density lipoprotein receptor represents anapolipoprotein E-independent pathway of A120573 uptake anddegradation by astrocytesrdquoThe Journal of Biological Chemistryvol 287 no 17 pp 13959ndash13971 2012
[108] L Yao X Gu Q Song et al ldquoNanoformulated alpha-mangostinameliorates Alzheimerrsquos disease neuropathology by elevatingLDLR expression and accelerating amyloid-beta clearancerdquoJournal of Controlled Release vol 226 pp 1ndash14 2016
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
18 Evidence-Based Complementary and Alternative Medicine
[31] M Sun X-P Zhan C-Y Jin J-Z Shan S Xu and Y-LWang ldquoclinical observation on treatment of post-craniocerebraltraumatic mental disorder by integrative medicinerdquo ChineseJournal of Integrative Medicine vol 14 no 2 pp 137ndash141 2008
[32] Z Xing Z Xia W Peng et al ldquoXuefu Zhuyu decoction atraditional Chinese medicine provides neuroprotection in arat model of traumatic brain injury via an anti-inflammatorypathwayrdquo Scientific Reports vol 6 Article ID 20040 2016
[33] J Zhou T Liu H Cui et al ldquoXuefu zhuyu decoction improvescognitive impairment in experimental traumatic brain injuryvia synaptic regulationrdquo Oncotarget vol 8 no 42 2017
[34] S Li ldquoExploring traditional chinese medicine by a novel thera-peutic concept of network targetrdquo Chinese Journal of IntegrativeMedicine vol 22 no 9 pp 647ndash652 2016
[35] S Li and B Zhang ldquoTraditional Chinese medicine networkpharmacology theory methodology and applicationrdquo ChineseJournal of Natural Medicines vol 11 no 2 pp 110ndash120 2013
[36] A L Hopkins ldquoNetwork pharmacology the next paradigm indrug discoveryrdquoNature Chemical Biology vol 4 no 11 pp 682ndash690 2008
[37] Y Li C Han J Wang et al ldquoInvestigation into the mechanismof Eucommia ulmoides Oliv based on a systems pharmacologyapproachrdquo Journal of Ethnopharmacology vol 151 no 1 pp452ndash460 2014
[38] Y Yao X Zhang Z Wang et al ldquoDeciphering the combinationprinciples of Traditional Chinese Medicine from a systemspharmacology perspective based on Ma-huang DecoctionrdquoJournal of Ethnopharmacology vol 150 no 2 pp 619ndash638 2013
[39] Bo Zhang Xu Wang and Shao Li ldquoAn Integrative Platform ofTCM Network Pharmacology and Its Application on a HerbalFormula Qing-Luo-Yinrdquo Evidence-Based Complementary andAlternativeMedicine vol 2013 Article ID 456747 12 pages 2013
[40] J Ru P Li J Wang et al ldquoTCMSP a database of systemspharmacology for drug discovery from herbal medicinesrdquoJournal of Cheminformatics vol 6 no 1 p 13 2014
[41] J V Turner D J Maddalena and S Agatonovic-KustrinldquoBioavailability Prediction Based on Molecular Structure for aDiverse Series of Drugsrdquo Pharmaceutical Research vol 21 no 1pp 68ndash82 2004
[42] X XuW Zhang CHuang et al ldquoAnovel chemometricmethodfor the prediction of human oral bioavailabilityrdquo InternationalJournal of Molecular Sciences vol 13 no 6 pp 6964ndash6982 2012
[43] A Zhang W Pan J Lv and H Wu ldquoProtective Effect ofAmygdalin on LPS-Induced Acute Lung Injury by InhibitingNF-120581B and NLRP3 Signaling Pathwaysrdquo Inflammation vol 40no 3 pp 745ndash751 2017
[44] X Wei H Liu X Sun et al ldquoHydroxysafflor yellow A protectsrat brains against ischemia-reperfusion injury by antioxidantactionrdquo Neuroscience Letters vol 386 no 1 pp 58ndash62 2005
[45] W PWalters andM A Murcko ldquoPrediction of lsquodrug-likenessrsquordquoAdvanced Drug Delivery Reviews vol 54 no 3 pp 255ndash2712002
[46] Y Yamanishi M Kotera M Kanehisa and S Goto ldquoDrug-target interactionprediction from chemical genomic and phar-macological data in an integrated frameworkrdquo Bioinformaticsvol 26 no 12 pp i246ndashi254 2010
[47] H Yu J Chen X Xu et al ldquoA systematic prediction ofmultiple drug-target interactions from chemical genomic andpharmacological datardquo PLoS ONE vol 7 no 5 Article IDe37608 2012
[48] X Chen Z L Ji and Y Z Chen ldquoTTD therapeutic targetdatabaserdquo Nucleic Acids Research vol 30 no 1 pp 412ndash4152002
[49] A Hamosh A F Scott J S Amberger C A Bocchini andV AMcKusick ldquoOnline Mendelian Inheritance in Man (OMIM) aknowledgebase of human genes and genetic disordersrdquo NucleicAcids Research vol 33 pp D514ndashD517 2005
[50] T S Keshava Prasad RGoel K Kandasamy et al ldquoHuman pro-tein reference databasemdash2009 updaterdquo Nucleic Acids Researchvol 37 no 1 pp D767ndashD772 2009
[51] A Franceschini D Szklarczyk S Frankild et al ldquoSTRING v91protein-protein interaction networks with increased coverageand integrationrdquoNucleic Acids Research vol 41 no 1 pp D808ndashD815 2013
[52] P Shannon A Markiel O Ozier et al ldquoCytoscape a softwareEnvironment for integratedmodels of biomolecular interactionnetworksrdquo Genome Research vol 13 no 11 pp 2498ndash25042003
[53] G Dennis Jr B T Sherman D A Hosack et al ldquoDAVIDDatabase for Annotation Visualization and IntegratedDiscov-eryrdquo Genome Biology vol 4 no 5 p P3 2003
[54] L Yang X Zhao and X Tang ldquoPredicting disease-related pro-teins based on clique backbone in protein-protein interactionnetworkrdquo International Journal of Biological Sciences vol 10 no7 pp 677ndash688 2014
[55] S MThomas and J S Brugge ldquoCellular functions regulated bySRC family kinasesrdquo Annual Review of Cell and DevelopmentalBiology vol 13 pp 513ndash609 1997
[56] D Z Liu F R Sharp K C Van et al ldquoInhibition of Src familykinases protects hippocampal neurons and improves cognitivefunction after traumatic brain injuryrdquo Journal of Neurotraumavol 31 no 14 pp 1268ndash1276 2014
[57] B D Manning and A Toker ldquoAKTPKB Signaling Navigatingthe Networkrdquo Cell vol 169 no 3 pp 381ndash405 2017
[58] Y Kitagishi and S Matsuda ldquoDiets involved in PPAR andPI3KAKTPTEN pathway may contribute to neuroprotectionin a traumatic brain injuryrdquoAlzheimerrsquos ResearchampTherapy vol5 no 5 p 42 2013
[59] D Chen L Bao S-Q Lu and F Xu ldquoSerum albumin andprealbuminpredict the poor outcomeof traumatic brain injuryrdquoPLoS ONE vol 9 no 3 Article ID e93167 2014
[60] J Yang Z Wu N Renier et al ldquoPathological axonal deaththrough aMapk cascade that triggers a local energy deficitrdquoCellvol 160 no 1-2 pp 161ndash176 2015
[61] E J Huang and L F Reichardt ldquoTrk receptors roles in neuronalsignal transductionrdquoAnnual Review of Biochemistry vol 72 pp609ndash642 2003
[62] J W Lee and R Juliano ldquoMitogenic signal transductionby integrin- and growth factor receptor-mediated pathwaysrdquoMolecules and Cells vol 17 no 2 pp 188ndash202 2004
[63] C S Yang J M Landau M T Huang and H L NewmarkldquoInhibition of carcinogenesis by dietary polyphenolic com-poundsrdquo Annual Review of Nutrition vol 21 pp 381ndash406 2001
[64] A Murakami H Ashida and J Terao ldquoMultitargeted cancerprevention by quercetinrdquoCancer Letters vol 269 no 2 pp 315ndash325 2008
[65] G Du Z Zhao Y Chen et al ldquoQuercetin attenuates neuronalautophagy and apoptosis in rat traumatic brain injury modelvia activation of PI3KAkt signaling pathwayrdquo NeurologicalResearch vol 38 no 11 pp 1ndash8 2016
Evidence-Based Complementary and Alternative Medicine 19
[66] Q Cai R O Rahn and R Zhang ldquoDietary flavonoidsquercetin luteolin andgenistein reduce oxidativeDNAdamageand lipid peroxidation and quench free radicalsrdquoCancer Lettersvol 119 no 1 pp 99ndash107 1997
[67] G A Bongiovanni E A Soria and A R Eynard ldquoEffectsof the plant flavonoids silymarin and quercetin on arsenite-induced oxidative stress in CHO-K1 cellsrdquo Food and ChemicalToxicology vol 45 no 6 pp 971ndash976 2007
[68] H Li L Yang Y Zhang et al ldquoKaempferol inhibits fibroblastcollagen synthesis proliferation and activation in hypertrophicscar via targeting TGF-beta receptor type Irdquo Biomedicine ampPharmacotherapy = Biomedecine amp Pharmacotherapie vol 83pp 967ndash974 2016
[69] K P Devi D S Malar S F Nabavi et al ldquoKaempferol andinflammation From chemistry to medicinerdquo PharmacologicalResearch vol 99 pp 1ndash10 2015
[70] M Zhou H Ren J Han W Wang Q Zheng and DWang ldquoProtective effects of kaempferol against myocardialischemiareperfusion injury in isolated rat heart via antioxidantactivity and inhibition of glycogen synthase kinase-3120573rdquo Oxida-tive Medicine and Cellular Longevity vol 2015 p 481405 2015
[71] C-F Lee J-S Yang F-J Tsai et al ldquoKaempferol inducesATMp53-mediated death receptor and mitochondrial apop-tosis in human umbilical vein endothelial cellsrdquo InternationalJournal of Oncology vol 48 no 5 pp 2007ndash2014 2016
[72] C Luo H Yang C Tang et al ldquoKaempferol alleviates insulinresistance via hepatic IKKNF-120581B signal in type 2 diabetic ratsrdquoInternational Immunopharmacology vol 28 no 1 pp 744ndash7502015
[73] A B Awad and C S Fink ldquoPhytosterols as anticancer dietarycomponents evidence and mechanism of actionrdquo Journal ofNutrition vol 130 no 9 pp 2127ndash2130 2000
[74] K Farber and H Kettenmann ldquoFunctional role of calciumsignals for microglial functionrdquoGlia vol 54 no 7 pp 656ndash6652006
[75] Y Lu Y Gu X Ding et al ldquoIntracellular Ca2+ homeostasisand JAK1STAT3 pathway are involved in the protective effectof propofol on BV2 microglia against hypoxia-induced inflam-mation and apoptosisrdquo PLoS ONE vol 12 no 5 p e01780982017
[76] K Leroy and J-P Brion ldquoDevelopmental expression and local-ization of glycogen synthase kinase-3120573 in rat brainrdquo Journal ofChemical Neuroanatomy vol 16 no 4 pp 279ndash293 1999
[77] C-M Liu E-M Hur and F-Q Zhou ldquoCoordinating geneexpression and axon assembly to control axon growth Potentialrole ofGSK3 signalingrdquo Frontiers inMolecularNeuroscience vol3 p 3 2012
[78] D A E Cross A A Culbert K A Chalmers L Facci S DSkaper and A D Reith ldquoSelective small-molecule inhibitors ofglycogen synthase kinase-3 activity protect primary neuronesfrom deathrdquo Journal of Neurochemistry vol 77 no 1 pp 94ndash102 2001
[79] V S Ossovskaya and NW Bunnett ldquoProtease-activated recep-tors contribution to physiology and diseaserdquo PhysiologicalReviews vol 84 no 2 pp 579ndash621 2004
[80] M Steinhoff J Buddenkotte V Shpacovitch et al ldquoProteinase-activated receptors transducers of proteinase-mediated signal-ing in inflammation and immune responserdquoEndocrine Reviewsvol 26 no 1 pp 1ndash43 2005
[81] H Krenzlin V Lorenz S Danckwardt O Kempski and BAlessandri ldquoThe importance of thrombin in cerebral injury and
diseaserdquo International Journal of Molecular Sciences vol 17 no1 2016
[82] M J McGinn and J T Povlishock ldquoPathophysiology of Trau-matic Brain InjuryrdquoNeurosurgery Clinics of North America vol27 no 4 pp 397ndash407 2016
[83] T Woodcock and M C Morganti-Kossmann ldquoThe role ofmarkers of inflammation in traumatic brain injuryrdquo Frontiersin Neurology vol 4 article 18 2013
[84] F Tanriverdi H J Schneider G Aimaretti B E Masel F FCasanueva and F Kelestimur ldquoPituitary dysfunction after trau-matic brain injury A clinical and pathophysiological approachrdquoEndocrine Reviews vol 36 no 3 pp 305ndash342 2015
[85] J V Rosenfeld A I Maas P Bragge M C Morganti-Kossmann G T Manley and R L Gruen ldquoEarly managementof severe traumatic brain injuryrdquoThe Lancet vol 380 no 9847pp 1088ndash1098 2012
[86] B M Aertker S Bedi and C S Cox ldquoStrategies for CNS repairfollowing TBIrdquo Experimental Neurology vol 275 no 3 pp 411ndash426 2016
[87] K Adachi Z Mirzadeh M Sakaguchi et al ldquo120573-catenin signal-ing promotes proliferation of progenitor cells in the adultmousesubventricular zonerdquo Stem Cells vol 25 no 11 pp 2827ndash28362007
[88] Y Hirabayashi and Y Gotoh ldquoStage-dependent fate determina-tion of neural precursor cells in mouse forebrainrdquo NeuroscienceResearch vol 51 no 4 pp 331ndash336 2005
[89] S Ding T Y H Wu A Brinker et al ldquoSynthetic smallmolecules that control stem cell faterdquo Proceedings of theNationalAcadamy of Sciences of the United States of America vol 100 no13 pp 7632ndash7637 2003
[90] N Israsena M Hu W Fu L Kan and J A Kessler ldquoThepresence of FGF2 signaling determines whether 120573-cateninexerts effects on proliferation or neuronal differentiation ofneural stem cellsrdquo Developmental Biology vol 268 no 1 pp220ndash231 2004
[91] A Salehi A Jullienne M Baghchechi et al ldquoUp-regulation ofWnt120573-catenin expression is accompanied with vascular repairafter traumatic brain injuryrdquo Journal of Cerebral Blood Flow ampMetabolism vol 38 no 2 pp 274ndash289 2017
[92] L F Reichardt and K J Tomaselli ldquoExtracellular matrixmolecules and their receptors Functions in neural develop-mentrdquoAnnual Review of Neuroscience vol 14 pp 531ndash570 1991
[93] R M Gibson S E Craig L Heenan C Tournier and M JHumphries ldquoActivation of integrin 12057251205731 delays apoptosis ofNtera2 neuronal cellsrdquoMolecularandCellularNeuroscience vol28 no 3 pp 588ndash598 2005
[94] C C Tate A J Garcıa andMC LaPlaca ldquoPlasma fibronectin isneuroprotective following traumatic brain injuryrdquoExperimentalNeurology vol 207 no 1 pp 13ndash22 2007
[95] C Culmsee and M P Mattson ldquop53 in neuronal apoptosisrdquoBiochemical and Biophysical Research Communications vol 331no 3 pp 761ndash777 2005
[96] M S Geddis and V Rehder ldquoThe phosphorylation state ofneuronal processes determines growth cone formation afterneuronal injuryrdquo Journal of Neuroscience Research vol 74 no2 pp 210ndash220 2003
[97] M L Craske M Fivaz N N Batada and T Meyer ldquoSpines andneurite branches function as geometric attractors that enhanceprotein kinase C actionrdquoThe Journal of Cell Biology vol 170 no7 pp 1147ndash1158 2005
20 Evidence-Based Complementary and Alternative Medicine
[98] A Muscella C Vetrugno L G Cossa et al ldquoApoptosis by[Pt(OOrsquo-acac)(gamma-acac)(DMS)] requires PKC-deltamedi-ated p53 activation in malignant pleural mesotheliomardquo PloSone vol 12 no 7 Article ID e0181114 2017
[99] J S Bonini W C Da Silva L R M Bevilaqua J H MedinaI Izquierdo and M Cammarota ldquoOn the participation ofhippocampal PKC in acquisition consolidation and reconsol-idation of spatial memoryrdquo Neuroscience vol 147 no 1 pp 37ndash45 2007
[100] O Zohar R Lavy X Zi et al ldquoPKC activator therapeutic formild traumatic brain injury in micerdquo Neurobiology of Diseasevol 41 no 2 pp 329ndash337 2011
[101] J David Sweatt ldquoTheneuronalMAPkinase cascade a biochem-ical signal integration system subserving synaptic plasticity andmemoryrdquo Journal ofNeurochemistry vol 76 no 1 pp 1ndash10 2001
[102] Y Matsuoka M Picciano B Maleste et al ldquoInflammatoryresponses to amyloidosis in a transgenic mouse model ofAlzheimerrsquos diseaserdquo The American Journal of Pathology vol158 no 4 pp 1345ndash1354 2001
[103] L Esposito L Gan G-Q Yu C Essrich and L MuckeldquoIntracellularly generated amyloid-120573 peptide counteracts theantiapoptotic function of its precursor protein and primesproapoptotic pathways for activation by other insults in neu-roblastoma cellsrdquo Journal of Neurochemistry vol 91 no 6 pp1260ndash1274 2004
[104] D J Loane A Pocivavsek C E-H Moussa et al ldquoAmyloidprecursor protein secretases as therapeutic targets for traumaticbrain injuryrdquo Nature Medicine vol 15 no 4 pp 377ndash379 2009
[105] R Torres B L Firestein H Dong et al ldquoPDZ proteins bindcluster and synaptically colocalize with Eph receptors and theirephrin ligandsrdquo Neuron vol 21 no 6 pp 1453ndash1463 1998
[106] M B Dalva M A Takasu M Z Lin et al ldquoEphB receptorsinteract with NMDA receptors and regulate excitatory synapseformationrdquo Cell vol 103 no 6 pp 945ndash956 2000
[107] J M Basak P B Verghese H Yoon J Kim and D MHoltzman ldquoLow-density lipoprotein receptor represents anapolipoprotein E-independent pathway of A120573 uptake anddegradation by astrocytesrdquoThe Journal of Biological Chemistryvol 287 no 17 pp 13959ndash13971 2012
[108] L Yao X Gu Q Song et al ldquoNanoformulated alpha-mangostinameliorates Alzheimerrsquos disease neuropathology by elevatingLDLR expression and accelerating amyloid-beta clearancerdquoJournal of Controlled Release vol 226 pp 1ndash14 2016
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 19
[66] Q Cai R O Rahn and R Zhang ldquoDietary flavonoidsquercetin luteolin andgenistein reduce oxidativeDNAdamageand lipid peroxidation and quench free radicalsrdquoCancer Lettersvol 119 no 1 pp 99ndash107 1997
[67] G A Bongiovanni E A Soria and A R Eynard ldquoEffectsof the plant flavonoids silymarin and quercetin on arsenite-induced oxidative stress in CHO-K1 cellsrdquo Food and ChemicalToxicology vol 45 no 6 pp 971ndash976 2007
[68] H Li L Yang Y Zhang et al ldquoKaempferol inhibits fibroblastcollagen synthesis proliferation and activation in hypertrophicscar via targeting TGF-beta receptor type Irdquo Biomedicine ampPharmacotherapy = Biomedecine amp Pharmacotherapie vol 83pp 967ndash974 2016
[69] K P Devi D S Malar S F Nabavi et al ldquoKaempferol andinflammation From chemistry to medicinerdquo PharmacologicalResearch vol 99 pp 1ndash10 2015
[70] M Zhou H Ren J Han W Wang Q Zheng and DWang ldquoProtective effects of kaempferol against myocardialischemiareperfusion injury in isolated rat heart via antioxidantactivity and inhibition of glycogen synthase kinase-3120573rdquo Oxida-tive Medicine and Cellular Longevity vol 2015 p 481405 2015
[71] C-F Lee J-S Yang F-J Tsai et al ldquoKaempferol inducesATMp53-mediated death receptor and mitochondrial apop-tosis in human umbilical vein endothelial cellsrdquo InternationalJournal of Oncology vol 48 no 5 pp 2007ndash2014 2016
[72] C Luo H Yang C Tang et al ldquoKaempferol alleviates insulinresistance via hepatic IKKNF-120581B signal in type 2 diabetic ratsrdquoInternational Immunopharmacology vol 28 no 1 pp 744ndash7502015
[73] A B Awad and C S Fink ldquoPhytosterols as anticancer dietarycomponents evidence and mechanism of actionrdquo Journal ofNutrition vol 130 no 9 pp 2127ndash2130 2000
[74] K Farber and H Kettenmann ldquoFunctional role of calciumsignals for microglial functionrdquoGlia vol 54 no 7 pp 656ndash6652006
[75] Y Lu Y Gu X Ding et al ldquoIntracellular Ca2+ homeostasisand JAK1STAT3 pathway are involved in the protective effectof propofol on BV2 microglia against hypoxia-induced inflam-mation and apoptosisrdquo PLoS ONE vol 12 no 5 p e01780982017
[76] K Leroy and J-P Brion ldquoDevelopmental expression and local-ization of glycogen synthase kinase-3120573 in rat brainrdquo Journal ofChemical Neuroanatomy vol 16 no 4 pp 279ndash293 1999
[77] C-M Liu E-M Hur and F-Q Zhou ldquoCoordinating geneexpression and axon assembly to control axon growth Potentialrole ofGSK3 signalingrdquo Frontiers inMolecularNeuroscience vol3 p 3 2012
[78] D A E Cross A A Culbert K A Chalmers L Facci S DSkaper and A D Reith ldquoSelective small-molecule inhibitors ofglycogen synthase kinase-3 activity protect primary neuronesfrom deathrdquo Journal of Neurochemistry vol 77 no 1 pp 94ndash102 2001
[79] V S Ossovskaya and NW Bunnett ldquoProtease-activated recep-tors contribution to physiology and diseaserdquo PhysiologicalReviews vol 84 no 2 pp 579ndash621 2004
[80] M Steinhoff J Buddenkotte V Shpacovitch et al ldquoProteinase-activated receptors transducers of proteinase-mediated signal-ing in inflammation and immune responserdquoEndocrine Reviewsvol 26 no 1 pp 1ndash43 2005
[81] H Krenzlin V Lorenz S Danckwardt O Kempski and BAlessandri ldquoThe importance of thrombin in cerebral injury and
diseaserdquo International Journal of Molecular Sciences vol 17 no1 2016
[82] M J McGinn and J T Povlishock ldquoPathophysiology of Trau-matic Brain InjuryrdquoNeurosurgery Clinics of North America vol27 no 4 pp 397ndash407 2016
[83] T Woodcock and M C Morganti-Kossmann ldquoThe role ofmarkers of inflammation in traumatic brain injuryrdquo Frontiersin Neurology vol 4 article 18 2013
[84] F Tanriverdi H J Schneider G Aimaretti B E Masel F FCasanueva and F Kelestimur ldquoPituitary dysfunction after trau-matic brain injury A clinical and pathophysiological approachrdquoEndocrine Reviews vol 36 no 3 pp 305ndash342 2015
[85] J V Rosenfeld A I Maas P Bragge M C Morganti-Kossmann G T Manley and R L Gruen ldquoEarly managementof severe traumatic brain injuryrdquoThe Lancet vol 380 no 9847pp 1088ndash1098 2012
[86] B M Aertker S Bedi and C S Cox ldquoStrategies for CNS repairfollowing TBIrdquo Experimental Neurology vol 275 no 3 pp 411ndash426 2016
[87] K Adachi Z Mirzadeh M Sakaguchi et al ldquo120573-catenin signal-ing promotes proliferation of progenitor cells in the adultmousesubventricular zonerdquo Stem Cells vol 25 no 11 pp 2827ndash28362007
[88] Y Hirabayashi and Y Gotoh ldquoStage-dependent fate determina-tion of neural precursor cells in mouse forebrainrdquo NeuroscienceResearch vol 51 no 4 pp 331ndash336 2005
[89] S Ding T Y H Wu A Brinker et al ldquoSynthetic smallmolecules that control stem cell faterdquo Proceedings of theNationalAcadamy of Sciences of the United States of America vol 100 no13 pp 7632ndash7637 2003
[90] N Israsena M Hu W Fu L Kan and J A Kessler ldquoThepresence of FGF2 signaling determines whether 120573-cateninexerts effects on proliferation or neuronal differentiation ofneural stem cellsrdquo Developmental Biology vol 268 no 1 pp220ndash231 2004
[91] A Salehi A Jullienne M Baghchechi et al ldquoUp-regulation ofWnt120573-catenin expression is accompanied with vascular repairafter traumatic brain injuryrdquo Journal of Cerebral Blood Flow ampMetabolism vol 38 no 2 pp 274ndash289 2017
[92] L F Reichardt and K J Tomaselli ldquoExtracellular matrixmolecules and their receptors Functions in neural develop-mentrdquoAnnual Review of Neuroscience vol 14 pp 531ndash570 1991
[93] R M Gibson S E Craig L Heenan C Tournier and M JHumphries ldquoActivation of integrin 12057251205731 delays apoptosis ofNtera2 neuronal cellsrdquoMolecularandCellularNeuroscience vol28 no 3 pp 588ndash598 2005
[94] C C Tate A J Garcıa andMC LaPlaca ldquoPlasma fibronectin isneuroprotective following traumatic brain injuryrdquoExperimentalNeurology vol 207 no 1 pp 13ndash22 2007
[95] C Culmsee and M P Mattson ldquop53 in neuronal apoptosisrdquoBiochemical and Biophysical Research Communications vol 331no 3 pp 761ndash777 2005
[96] M S Geddis and V Rehder ldquoThe phosphorylation state ofneuronal processes determines growth cone formation afterneuronal injuryrdquo Journal of Neuroscience Research vol 74 no2 pp 210ndash220 2003
[97] M L Craske M Fivaz N N Batada and T Meyer ldquoSpines andneurite branches function as geometric attractors that enhanceprotein kinase C actionrdquoThe Journal of Cell Biology vol 170 no7 pp 1147ndash1158 2005
20 Evidence-Based Complementary and Alternative Medicine
[98] A Muscella C Vetrugno L G Cossa et al ldquoApoptosis by[Pt(OOrsquo-acac)(gamma-acac)(DMS)] requires PKC-deltamedi-ated p53 activation in malignant pleural mesotheliomardquo PloSone vol 12 no 7 Article ID e0181114 2017
[99] J S Bonini W C Da Silva L R M Bevilaqua J H MedinaI Izquierdo and M Cammarota ldquoOn the participation ofhippocampal PKC in acquisition consolidation and reconsol-idation of spatial memoryrdquo Neuroscience vol 147 no 1 pp 37ndash45 2007
[100] O Zohar R Lavy X Zi et al ldquoPKC activator therapeutic formild traumatic brain injury in micerdquo Neurobiology of Diseasevol 41 no 2 pp 329ndash337 2011
[101] J David Sweatt ldquoTheneuronalMAPkinase cascade a biochem-ical signal integration system subserving synaptic plasticity andmemoryrdquo Journal ofNeurochemistry vol 76 no 1 pp 1ndash10 2001
[102] Y Matsuoka M Picciano B Maleste et al ldquoInflammatoryresponses to amyloidosis in a transgenic mouse model ofAlzheimerrsquos diseaserdquo The American Journal of Pathology vol158 no 4 pp 1345ndash1354 2001
[103] L Esposito L Gan G-Q Yu C Essrich and L MuckeldquoIntracellularly generated amyloid-120573 peptide counteracts theantiapoptotic function of its precursor protein and primesproapoptotic pathways for activation by other insults in neu-roblastoma cellsrdquo Journal of Neurochemistry vol 91 no 6 pp1260ndash1274 2004
[104] D J Loane A Pocivavsek C E-H Moussa et al ldquoAmyloidprecursor protein secretases as therapeutic targets for traumaticbrain injuryrdquo Nature Medicine vol 15 no 4 pp 377ndash379 2009
[105] R Torres B L Firestein H Dong et al ldquoPDZ proteins bindcluster and synaptically colocalize with Eph receptors and theirephrin ligandsrdquo Neuron vol 21 no 6 pp 1453ndash1463 1998
[106] M B Dalva M A Takasu M Z Lin et al ldquoEphB receptorsinteract with NMDA receptors and regulate excitatory synapseformationrdquo Cell vol 103 no 6 pp 945ndash956 2000
[107] J M Basak P B Verghese H Yoon J Kim and D MHoltzman ldquoLow-density lipoprotein receptor represents anapolipoprotein E-independent pathway of A120573 uptake anddegradation by astrocytesrdquoThe Journal of Biological Chemistryvol 287 no 17 pp 13959ndash13971 2012
[108] L Yao X Gu Q Song et al ldquoNanoformulated alpha-mangostinameliorates Alzheimerrsquos disease neuropathology by elevatingLDLR expression and accelerating amyloid-beta clearancerdquoJournal of Controlled Release vol 226 pp 1ndash14 2016
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
20 Evidence-Based Complementary and Alternative Medicine
[98] A Muscella C Vetrugno L G Cossa et al ldquoApoptosis by[Pt(OOrsquo-acac)(gamma-acac)(DMS)] requires PKC-deltamedi-ated p53 activation in malignant pleural mesotheliomardquo PloSone vol 12 no 7 Article ID e0181114 2017
[99] J S Bonini W C Da Silva L R M Bevilaqua J H MedinaI Izquierdo and M Cammarota ldquoOn the participation ofhippocampal PKC in acquisition consolidation and reconsol-idation of spatial memoryrdquo Neuroscience vol 147 no 1 pp 37ndash45 2007
[100] O Zohar R Lavy X Zi et al ldquoPKC activator therapeutic formild traumatic brain injury in micerdquo Neurobiology of Diseasevol 41 no 2 pp 329ndash337 2011
[101] J David Sweatt ldquoTheneuronalMAPkinase cascade a biochem-ical signal integration system subserving synaptic plasticity andmemoryrdquo Journal ofNeurochemistry vol 76 no 1 pp 1ndash10 2001
[102] Y Matsuoka M Picciano B Maleste et al ldquoInflammatoryresponses to amyloidosis in a transgenic mouse model ofAlzheimerrsquos diseaserdquo The American Journal of Pathology vol158 no 4 pp 1345ndash1354 2001
[103] L Esposito L Gan G-Q Yu C Essrich and L MuckeldquoIntracellularly generated amyloid-120573 peptide counteracts theantiapoptotic function of its precursor protein and primesproapoptotic pathways for activation by other insults in neu-roblastoma cellsrdquo Journal of Neurochemistry vol 91 no 6 pp1260ndash1274 2004
[104] D J Loane A Pocivavsek C E-H Moussa et al ldquoAmyloidprecursor protein secretases as therapeutic targets for traumaticbrain injuryrdquo Nature Medicine vol 15 no 4 pp 377ndash379 2009
[105] R Torres B L Firestein H Dong et al ldquoPDZ proteins bindcluster and synaptically colocalize with Eph receptors and theirephrin ligandsrdquo Neuron vol 21 no 6 pp 1453ndash1463 1998
[106] M B Dalva M A Takasu M Z Lin et al ldquoEphB receptorsinteract with NMDA receptors and regulate excitatory synapseformationrdquo Cell vol 103 no 6 pp 945ndash956 2000
[107] J M Basak P B Verghese H Yoon J Kim and D MHoltzman ldquoLow-density lipoprotein receptor represents anapolipoprotein E-independent pathway of A120573 uptake anddegradation by astrocytesrdquoThe Journal of Biological Chemistryvol 287 no 17 pp 13959ndash13971 2012
[108] L Yao X Gu Q Song et al ldquoNanoformulated alpha-mangostinameliorates Alzheimerrsquos disease neuropathology by elevatingLDLR expression and accelerating amyloid-beta clearancerdquoJournal of Controlled Release vol 226 pp 1ndash14 2016
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
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EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom