Therapeutic Effects of Chinese Medicine Herb Pair, Huzhang ... ·...

Research ArticleTherapeutic Effects of Chinese Medicine Herb PairHuzhang and Guizhi on Monosodium Urate Crystal-InducedGouty Arthritis in Rats Revealed by Anti-InflammatoryAssessments and NMR-Based Metabonomics

Bin Han Huizhu Huang Zhong Li Mengjuan Gong Wan Shi Chunxia ZhuZulian Gu and Zhongjie Zou

School of Traditional Chinese Medicine Guangdong Pharmaceutical University Guangzhou 510006 China

Correspondence should be addressed to Zhongjie Zou zouzhongjie139com

Received 31 October 2015 Accepted 20 January 2016

Academic Editor Antonella Fioravanti

Copyright copy 2016 Bin Han et alThis is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The present study was undertaken to evaluate the therapeutic effects of Huzhang-Guizhi herb pair (HG) firstly included in Hu-Zhang Power documented in Taiping Shenghui Fang on monosodium urate (MSU) crystals-induced gouty arthritis in rats Wefound that pretreatment withHG in rats with gouty arthritis could significantly attenuate the ankle joint swelling and this beneficialantigout effect might be mediated at least in part by inhibiting tumor necrosis factor-alpha (TNF-120572) and interleukin-1 beta (IL-1120573) production in synovial fluid as well as nuclear transcription factor-120581B p65 (NF-120581B p65) protein expression in synovial tissueMoreover metabonomic analysis demonstrated that 5 and 6 potential biomarkers associated with gouty arthritis in plasma andurine respectively which were mainly involved in energy metabolism amino acid metabolism and gut microbe metabolism wereidentified HG could reverse the pathological process of MSU-induced gouty arthritis through regulating the disturbed metabolicpathways These results provided important mechanistic insights into the protective effects of HG against MSU-induced goutyarthritis in rats

1 Introduction

Gout an ancient and common form of inflammatory arthri-tis is a rheumatic disease resulting from deposition of uricacid crystals (monosodium urate MSU) in tissues and fluidswithin the bodyThis process is caused by an overproductionor under excretion of uric acid Certain common medica-tions alcohol and dietary foods are known to be contributoryfactors Acute gout will typically manifest itself as an acutelyred hot and swollen joint with excruciating pain Theseacute gouty flare-ups respond well to treatment with oralanti-inflammatory medicines and may be prevented withmedication and dietary changes Recurrent bouts of acutegout can lead to a degenerative formof chronic arthritis calledgouty arthritis [1]

The goal of anti-inflammatory therapy for goutyarthritis is to eliminate the massive pain swelling and

disability resulted from acute attacks [2] Nonsteroidal anti-inflammatory drugs (NSAIDs) or colchicine is a first-linesystemic treatment according to the current gouty arthritistreatment guidelines [3] However side effects of NSAIDsmay include acute renal failure and gastrointestinal bleedingFurthermore treatment is contraindicated in patients withchronic kidney disease (CKD) [4] As part of complementaryand alternative medicine traditional Chinese medicationand acupuncture have also been used to treat gout whichhave in some circumstances a superior side effect profile toNSAIDs [5ndash7]

Herb pairs (mixture of two herbs) as the basic composi-tion units of Chinese herbal formulas have special clinicalsignificance in traditional Chinese medicine (TCM) andare much simpler than other complex formulas withoutaltering their basic therapeutic features [8] Polygonum cus-pidatum (known as ldquoHu-Zhangrdquo in Chinese) has antiviral

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2016 Article ID 9398435 12 pageshttpdxdoiorg10115520169398435

2 Evidence-Based Complementary and Alternative Medicine

antimicrobial anti-inflammatory neuroprotective and car-dioprotective functions [9] Cassia twig (known as ldquoGui Zhirdquoin Chinese) exhibits antiviral antimicrobial antineoplasticanti-inflammatory antiallergic and analgesic properties [10]The combination of these two herbs known as Huzhang-Guizhi herb pair (HG) (with a ratio of 6 1) is firstly includedin Hu-Zhang Power documented in Taiping Shenghui Fangand our previous study demonstrated the antigout effects ofHG in acute gouty arthritis rat model [11] Meanwhile HGis also used in other formulas such as Lexing-tongfengkewhich has been widely used to treat gouty arthritis inclinics [12] However the biochemical mechanism by whichHG acts in gouty arthritis still remains elusive Our studytherefore aims to elucidate themechanisms bywhichHGactstherapeutically

Metabonomics is a well-established field in systems biol-ogy and has been applied to observe meaningful and relevantbiochemical changes due to disease toxicity nutrition andother variables Metabonomic studies can provide invaluableinformation towards understanding molecular mechanismsand novel insights into the status of dysfunction in biologicalsystems [13] Recently using ultra-performance liquid chro-matography coupled with mass spectrometry (UPLCMS)themetabolic profiles of rats withMSU-induced gouty arthri-tis and the biological effects of acupuncture were investigated[5] As we all know both nuclear magnetic resonance (NMR)spectroscopy and mass spectrometry (MS) (usually with achromatographic separation step) are suitable techniques formetabonomic analysis but have different analytical strengthsand weaknesses and give complementary information [14]To the best of our knowledge metabonomic study of goutyarthritis in rat model based on NMR has not been reported

In this study we monitored intumesce index histopatho-logical changes production of inflammatory cytokines andimmunohistochemistry of nuclear transcription factor-120581Bp65 (NF-120581B p65) to investigate the efficacy and anti-inflammatory mechanism of HG against MSU crystal-induced gout in rats an experimental model for goutyarthritis that is similar to those observed in clinical goutFurthermore a metabonomic method was applied to char-acterize the metabolic profiles associated with MSU-inducedgouty arthritis and observe the protective effects of HG in ratplasma and urine

2 Materials and Methods

21 Chemicals and Reagents Huzhang and Guizhi were pur-chased from Heshuntang (Guangzhou China) and authenti-cated by a specialist in pharmacognosy Distilled water waspurified using a Milli-Q ultrapure water system (MilliporeBedford MA USA) MSU crystals and deuterium oxide(D2O 999) were obtained from Sigma-Aldrich (St Louis

MO USA) Rat tumor necrosis factor-alpha (TNF-120572) andinterleukin-1 beta (IL-1120573) enzyme-linked immunosorbentassay (ELISA) kits were purchased fromAffinity BioReagents(Golden CO USA) NF-120581B p65 antibody was purchasedfrom Santa Cruz Biotechnology Inc (Santa Cruz CA USA)

22 Herbal Extraction A mixture of Huzhang (60 g) andGuizhi (10 g) crushed into small pieces was extracted withwater (1 10 wv) under thermal reflux for 2 h The filtrateswere collected and the residues were then refluxed in water(1 10 wv) for 15 h Two batches of filtrates were combinedand then were concentrated under a vacuum to obtain HGextract at a final concentration of 07 gmL (wv expressed asthe weight of raw materials)

23 AnimalHandling and Sample Collection All experimentswere approved by the local ethics committee and all exper-imental procedures were in compliance with the NationalInstitutes of Health Guide for Care and Use of LaboratoryAnimals

A total of 18 adult male SD rats weighing 180ndash200 gwere commercially obtained from Laboratory Animal Centerof Traditional Chinese Medicine University of GuangzhouChina Rats were maintained under controlled temperature(20 plusmn 2∘C) and lighting (0800ndash2000) conditions with foodandwater available ad libitumAfter 7 days of acclimatizationthe animals were randomly divided into three groups (119899 = 6in each group) (1) control group (2) gouty arthritis modelgroup and (3) HG pretreatment group (HG group) Ratsin the HG pretreatment group were administrated orally bygavage with HG at a dose of 93 gkg of body weight oncedaily for consecutive 7 days Dosing in this study was setaccording to dosages previously detailed in the literature [11]The control andmodel groups received an equivalent volumeof vehicle in the form of sterile saline as the pretreatmentgroup for 7 days Gouty arthritis was induced according toestablished protocol [15] 1 h after the last dosing on day 7MSU crystals (100mgmL suspended in sterile saline) wereinjected in the left ankle joint of each rat in the modelgroup and the HG group in a volume of 100 120583L The controlgroup received a 100 120583L intra-articular injection of singlesterile saline After the injection of MSU urine was collectedcontinuously (24 h) into ice-cooled vessels containing 05mLof 2 sodium azide in metabolism cages Following urinecollection all animals were anesthetized by diethyl etherinhalation and blood samples were acquired via the ratsrsquoorbital venous plexus Heparin was used as an anticoagulantPlasma was separated by centrifugation at 3500 rpm for15min All urine and plasma samples were kept atminus80∘Cuntilthey were defrosted immediately prior to analysis Synoviallavage fluid from each ankle was collected by injecting5mL phosphate buffered saline (PBS) into the joint cavityLavage fluids were then centrifuged at 400 g for 10 minutesand supernatants were stored at minus80∘C before biochemicaldeterminations and synovial tissue was gently separated forimmunohistochemistry examination

24 Measurement of Ankle Edema The development ofarthritis was assessed by measuring the ankle size before andafter induction of gout The circumference of the inflamedankle joint of the animals was measured using a 3mmwide tape measure without elasticity at 24 h after MSUcrystal injection In order to quantify ankle edema the datawere converted to a percentage of changes of ankle size as

Evidence-Based Complementary and Alternative Medicine 3

an intumesce index using the following formula intumesceindex = (measured ankle size minus primary ankle size)primaryankle size

25Measurement of Inflammatory Cytokines in the Ankle JointLavage Fluid The levels of TNF-120572 and IL-1120573 in the anklejoint lavage fluid which could be secreted during the processof acute gouty arthritis induced by MSU were measured byusing commercially available ELISA kits according to themanufacturerrsquos instructions The concentrations of IL-1120573 andTNF-120572 were calculated according to standard curves

26 Histopathological Evaluation and Immunohistochemistryfor NF-120581B p65 Synovial tissue was fixed in 10 formalinsolution dehydrated in ascending grades of alcohol andembedded in paraffin Paraffin sections were then cut to athickness of 5 120583m and stained with hematoxylin and eosin(HampE) for histological evaluation according to standardprocedures The expression of NF-120581B p65 was investigatedby immunohistochemistry analysis of 5120583m paraffin sectionsBriefly sections of synovial tissues from different animalgroups were deparaffinized with xylene and dehydrated ina graded ethanol series Thereafter tissue sections weremicrowaved for 8min twice while immersed in 01M citratebuffer (pH 60) to achieve antigen retrieval After coolingthe sections were washed with PBS three times and endoge-nous peroxidase activity was blocked with 30 H

2O2in

methanol for 25min at room temperature After anotherthree rinses with PBS the tissues were blocked with 3BSA (Sigma St Louis MO USA) for 30min at roomtemperature and incubated with anti-NF-120581B p65 antibody(dilution 1 100) overnight at 4∘C After being washed threetimes in PBS sections were incubated with goat anti-ratsecondary antibody (Dako CA USA) at room temperaturefor 50min Following exposure to 331015840-diaminobenzidinetetrahydrochloride (DAB) sections were counterstainedwithhematoxylin dehydrated with increasing concentration ofethanol and mounted with neutral gum The slides werevisualized under light microscope and the extent of cellimmunopositivity was assessed

27 Sample Preparation and 1HNMR Spectroscopy 400 120583L ofrat urine was diluted with 200 120583L phosphate buffer (02molLNa2HPO4-02molL NaH

2PO4 pH 74) thoroughly mixed in

a vortexmixer and was subsequently centrifuged at 3500 rpmfor 15min at 4∘C to remove sediment 500120583L of supernatantwas transferred into a 5mm NMR tube and 50120583L of D

2O

(containing 005WVTSP-d4) was added 400 120583L of plasma

was transferred into a 5mm NMR tube Finally 50120583L ofphosphate buffer (02molL Na

2HPO4-02molL NaH

2PO4

pH 74) and 50 120583L of D2O were mixed with the plasma The

D2O provided a field-frequency lock solvent for the NMR

spectrometer [16]1H-NMR spectra of the plasma and urine samples were

recorded at 298K on a Bruker AVANCE III 500MHz spec-trometer (BrukerBiospin Rheinstetten Germany) operatingat 50013MHz 1H frequency For plasma samples the water-suppressed Carr-Purcell-Meibom-Gill (CPMG) spin-echo

pulse sequence (RD-90∘-(120591-180∘-120591)119899-ACQ) with a total spin-echo delay (2119899120591) of 100ms was used to attenuate broadsignals from proteins and lipoproteins 128 free inductiondecays (FIDs) were collected into 64 k data points over aspectral width of 10000Hz with a relaxation delay of 3 sand an acquisition time of 328 s For urine samples all 1HNMR spectra were collected using a standard 1D nuclearOverhauser enhancement spectroscopy- (NOESY-) presatu-ration pulse sequence 128 free induction decays (FIDs) werecollected into 64 k data points Spectra were acquired with aspectral width of 10000Hz and an acquisition time of 328 sRelaxation delay was set at 3 s

28 Data Analysis Data were expressed as mean plusmn standarddeviation (SD) Statistical analysis was carried out usingone-way analysis of variance (ANOVA) followed by posthoc Tukeyrsquos test 119875 values less than 005 were consideredsignificant

The obtained spectra of plasma and urine samples wereprocessed with a 03Hz line-broadening factor prior toFourier transformation All the spectra were manually cor-rected for phase and baseline distortions using MestReNovasoftware (Mestrelab Research SL Santiago de CompostelaSpain) The spectra of plasma were referenced internallyto methyl resonance of lactate (120575133) The plasma spectrawere divided and the signal integral was computed in 001intervals across the region 120575050ndash90 The region of 120575468ndash522 was removed to eliminate the effects of imperfect watersaturation The spectra of urine were referenced to thechemical shift of TSP at 1205750The spectra were divided and thesignal integral computed in 001 intervals across the region12057505ndash90 The region of 120575447ndash598 was removed to eliminatethe influence of water and urea Finally all remaining regionsof the spectra were then normalized to the total integratedarea of the spectra to reduce any significant concentrationdifferences

The resultant integral data were imported into SIMCA-P 120 software (Umetrics Umea Sweden) for principalcomponent analysis (PCA) partial least-squares discrimi-nant analysis (PLS-DA) and orthogonal partial least-squaresdiscriminant analysis (OPLS-DA) after mean-centering andpareto scaling a technique that increased the importanceof low abundance ions without significant amplification ofnoise Model parameters for example goodness of fit 1198772and goodness of predication 1198762 were used to evaluatethe quality of these models Metabolites with VIP (variableimportance in the projection) values ge 10 (obtained fromOPLS-DA models) and values of 119875 lt 005 (calculatedfrom independent sample 119905-test orMann-Whitney119880-test) areregarded as potential biomarkers [17]

3 Results

31 Effects of HG on Ankle Edema in MSU Crystal-InducedGouty Arthritis Rats As shown in Figure 1(a) there wasa significant increase in the intumesce index in the goutyarthritis model group as compared to the control group


0

005

01

015

02

025

03

035

04

045

05

Control Model HG

Ank

le in

tum

esce

inde

x

lowast

lowast

(a)

0

20

40

60

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140

160

Control Model HG

TNF-120572

leve

ls (p

gm

L)

lowast

lowast

(b)

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1000

1200

1400

1600

1800

2000

Control Model HG

IL1

-120573le

vels

(pg

mL)

lowast

lowast

(c)

Figure 1 Effects of HG on ankle edema (a) and TNF-120572 (b) and IL-1120573 (c) production in the ankle joint lavage fluid in MSU crystal-inducedgouty arthritis rats lowast119875 lt 005 compared to the gouty arthritis model group

(119875 lt 005) HG could remarkably reduce the intumesce indexin rats with MSU-induced gouty arthritis (119875 lt 005)

32 Effects of HG on TNF-120572 and IL-1120573 Production in the AnkleJoint Lavage Fluid The levels of TNF-120572 and IL-1120573 in the anklejoint lavage fluidwere elevatedmarkedly in the gouty arthritismodel group when compared with the control group (119875 lt005 Figures 1(b) and 1(c)) HG-pretreated rats exhibited asignificant reduction in all tested proinflammatory cytokinesproduction in comparison with those in the gouty arthritismodel group (119875 lt 005)

33 Histopathological Effect of HG on Gouty Arthritis MSUcrystals significantly increased leukocyte infiltration in thesuperficial and deeper layers of the synovium of ankle jointcompared with those in control group and neutrophils weregrossly abundant within the mixed cellular infiltrate in thesynoviumThe synovium became hyperplastic and thickenedby inflammatory cells and formed a pannus that destroyedthe underlying cartilage In contrast pretreatment with HGdecreased leucocytes influx in the synoviumof ratswith goutyarthritis and slight hyperplasia was observed in synovialtissue (Figure 2)

34 Immunohistochemistry for NF-120581B p65 Compared to thecontrol group the positive expression of NF-120581B p65 proteinsin the cytoplasm and the nucleus of synovium increasedsignificantly in the gouty arthritis model group (119875 lt 005)HGcould inhibitNF-120581Bp65 proteins overexpression inducedby MSU crystals (Figure 3)

35 Analysis of 1H-NMR Profiles Typical 1H-NMR spectraobtained for plasma and urine were shown in Figures 4and 5 The metabolite resonances were assigned according

to previous studies [18ndash20] and the Chenomx NMR suite(Chenomx Inc Edmonton AB Canada)

36 The Perturbation of Metabolic Profiles in GoutModel Rats PCA and PLS-DA score plots weredisplayed in Supplementary Material available online athttpdxdoiorg10115520169398435 (Figures 1s and 2s)In order to maximize the separation between experimentalgroups and to focus on metabolic variations significantlycontributing to classifications the OPLS-DA models weresubsequently performed As shown in the OPLS-DA scoreplots (Figures 6(a) and 6(b)) a separation between thecontrol and gouty arthritis model groups was clearly seenindicating that they had completely different metabolicprofilesThemodel parameters were as follows 1198772119883 = 08361198772119884 = 0949 and 1198762 = 0832 for plasma 1198772119883 = 08711198772119884 = 0996 and 1198762 = 0859 for urine In general excellent

models were obtained when values of 1198772119883 1198772119884 and 1198762were above 08 [21] Furthermore to validate the robustnessof these OPLS-DA models 200-iteration permutation testswere performed in the corresponding PLS-DA modelswith the same number of components as the OPLS-DAmodels The validation plots demonstrated that the originalOPLS-DA models were not random and overfitted as bothpermutated 1198762 and 1198772 values were significantly lower thanthe corresponding original values (Figures 7(a) and 7(b)) [16]

Selected according to the VIP values from the OPLS-DAmodels (VIP ge 1) and the 119875 values from univariate statisticalanalysis (119875 lt 005) 5 and 6 endogenous metabolites relatedto gouty arthritis were identified as potential biomarkersin plasma and urine respectively (Figure 8 and Table 1)Compared to the control group 120573-glucose was significantlydownregulated while leucine lysine lactate and glutaminewere significantly upregulated in the plasma of the goutyarthritis model group The gouty arthritis model group


(a) (b)

(c)

Figure 2 Effects of HG on synovial tissue histopathology of rats with gouty arthritis (magnification times200) (a) Control group (b) modelgroup and (c) HG pretreatment group

(a) (b)

(c)

Figure 3 Effects of HG on the activation of NF-120581B p65 in synovial tissue of rats with gouty arthritis (magnification times400) (a) Control group(b) model group and (c) HG pretreatment group


0510152025303540455560

235

120575

(a)

0510152025303540455560

245

120575

(b)

0510152025303540455560

12

3

120575

(c)

Figure 4 Representative 1H NMR spectra of rat plasma Control (a) model (b) and HG pretreatment (c) groups Keys 1 leucine 2 lysine3 lactate 4 glutamine 5 120573-glucose

0510152025303540606570758085

35

6

120575

(a)

0510152025303540606570758085

13

4

120575

(b)

0510152025303540606570758085

26

120575

(c)

Figure 5 Representative 1HNMR spectra of rat urine Control (a) model (b) andHGpretreatment (c) groups Keys 1 pyruvate 2 succinate3 2-oxoglutarate 4 citrate 5 trimethylamine N-oxide 6 hippurate

also showed higher urinary levels of pyruvate succinate 2-oxoglutarate and citrate along with lower levels of trimethy-lamineN-oxide (TMAO) and hippuratewhen comparedwiththe control group

37 Intervention of HG on Metabolic Pattern of Rats withMSU-Induced Gouty Arthritis The OPLS-DA scores plotsconstructed with NMR spectral data from rat plasma andurine samples in the control model and HG pretreatmentgroups showed a tendency recovering to healthy controlgroup in HG pretreatment group as well as an obviousseparation between gouty arthritis model group and HGpretreatment group (Figures 6(c)ndash6(f)) The correspondingvalidation plots (Figures 7(c)ndash7(f)) based on 200 times per-mutation tests demonstrated the robustness of these OPLS-DA models In addition all potential biomarkers associatedwith MSU-induced gouty arthritis in rats were significantlyreversed by HG except trimethylamine N-oxide in urine(Figure 8 and Table 1) The above results which were also inaccordance with those in Figures 1ndash3 indicated that there

was no doubt that pretreatment of HG in rats with goutyarthritis induced substantial and characteristic changes intheir metabolic profiles

4 Discussion

Gouty arthritis is an inflammatory response to MSU micro-crystals that precipitate in joint tissues from supersaturatedbody fluids or are shed from preexisting articular deposits[22] MSU crystals first identified as the aetiological agent ofgouty arthritis in the 18th century can stimulate various typesof cells including monocytes macrophages neutrophils andsynovial cells resulting in a rapid increase in the productionof proinflammatory cytokines and chemokines [7 23 24]The clinical symptoms of inflammatory responses are charac-terized by severe pain edema and erythema in the jointsThepotential forMSU crystal-induced rat joint inflammation as avalid experimental model of gouty arthritis was determinedby testing classic antigout drugs [25 26] This experimentalmodel caused a remarkable accumulation of neutrophils that


minus60

minus40

minus20

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20

40

60

minus50 minus40 minus30 minus20 minus10 0 10 20 30 40 50t[1]

t o[1

]

(a)

minus40

minus20

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minus60 minus40 minus20 0 20 40 60t[1]

t o[1

]

(b)

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minus50

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t o[1

]

(c)

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t o[1

]

(d)

minus30

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20

30


t[2

]

(e)

minus40

minus20

0

20

40

minus100 minus50 0 50 100t[1]

t[2

]

(f)

Figure 6 Metabolic profiles depicted by OPLS-DA score plots of 1H NMR spectral data of rat plasma ((a) (c) and (e)) and urine ((b) (d)and (f)) from control (998771 blue triangle) model (e red dot) and HG pretreatment (X purple diamond) groups

was similar to the situation in humans We used this well-established model to investigate the effects of HG on thearthritis induced by MSU crystals The results demonstratedthat HG significantly attenuated the swell in joint andreduced the intumesce index in rats withMSU-induced goutyarthritis Furthermore histopathological observations alsoshowed that HG decreased leukocyte infiltration (primarilyneutrophils) in the synovium of ankle joint in gouty arthritis

rats These results suggested that HG could protect animalsfromMSU crystal-induced gouty arthritis and joint injury

TNF-120572 and IL-1120573 are thought to be particularly importantin the pathogenesis of gouty arthritis [27 28] TNF-120572 and IL-1120573 mediate the rapid upregulation of the major endothelialligand for neutrophils E-selectin in response to injection ofMSU crystals into the joint space [27ndash30] Both TNF-120572 andIL-1120573 are powerful stimuli for IL-6 and IL-8 productions


00

05

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00 02 04 06 08 10

R2

Q2

(a)

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R2

Q2

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R2

Q2

(c)

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Q2

(d)

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R2

Q2

(e)

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R2

Q2

(f)

Figure 7 Statistical validation of the OPLS-DA models (Figures 6(a)ndash6(f)) by the corresponding PLS-DA models with the same number ofcomponents

which can respectively account for the pronounced acutephase response [31 32] Here we found that HG couldsignificantly decrease the increased expression of TNF-120572 andIL-1120573 in rats with gouty arthritis

NF-120581B is clearly one of the most important regulatorsof proinflammatory gene expression that can upregulate theexpression of lots of cytokines such as TNF-120572 IL-1120573 and IL-8[33] The activated NF-120581B is reported to form a heterodimer

which usually consists of two proteins p65 and p50 subunitsThe p65 subunit has been demonstrated to exert criticalactivity on the transcription of many inflammatory genesincluding adhesion molecules cytokines and chemokines[34] Moreover cytokines that are stimulated by NF-120581B suchas TNF-120572 and IL-1120573 can also directly activate the NF-120581Bpathway thus establishing a positive autoregulatory loopthat can amplify the inflammatory reaction [35] Activation


0

2

4

6

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Leucine Lysine Glutamine

ControlModelHG

lowast

lowast

lowastlowast

lowast

lowast

(a)

0

50

100

150

200

250

300

350

Lactate

ControlModelHG

120573-Glucose

lowast lowastlowast

lowastlowast

(b)

020406080

100120140160180

ControlModelHG

lowast

lowastlowast lowastlowast

lowastlowastlowastlowast

lowast

lowastlowast

lowast

Pyruvate Succinate 2-Oxoglutarate Citrate TrimethylamineN-oxide

Hippurate

(c)

Figure 8 Bar charts of differentially expressed metabolite markers (mean plusmn SD 119899 = 6) in rat plasma ((a) and (b)) and urine (c) One-wayANOVA was used to determine the significance of difference Compared to model group lowastlowast and lowast represented 119875 lt 001 and 119875 lt 005respectively

of NF-120581B may therefore be a key step in the pathogenesisof gouty arthritis and suppression of NF-120581B is likely tobe effective for the treatment of gouty arthritis In thepresent study immunohistochemistry analysis indicated thatHG suppressed the expression of active NF-120581B p65 in thesynovium

Taken together these findings suggested that the posi-tive feedback loop between NF-120581B and extracellular signalsincluding TNF-120572 and IL-1120573might be interrupted by HG

Changes in a number of metabolites involved in energymetabolism were observed in this work The gouty arthritismodel group showed a drop in glucose and elevated level oflactate in serum together with increased levels of succinate2-oxoglutarate citrate and pyruvate in urine as comparedwith the control group Pyruvate the product of glycolysisrepresents an important junction point in carbohydratecatabolism under aerobic and anaerobic conditions Citrate2-oxoglutarate and succinate are key intermediate productsof tricarboxylic acid (TCA) cycle which involves not only the

glucose aerobic oxidation but also the major pathways for fatand amino acid metabolisms Lactate is the end-product ofglucosemetabolismunder anaerobic conditionsThe elevatedlactate was found to be a common and key biomarker of fourtypes of human arthritis including gout [36] Therefore ourresults demonstrated the acceleration of glycolytic activityinduced by gouty arthritis The downregulation of succinate2-oxoglutarate citrate pyruvate and lactate as well as upreg-ulation of glucose was present in the HG pretreatment groupcompared with those in the model group indicating thatHG was able to efficaciously ameliorate the altered energymetabolism

The metabolite profiling of plasma showed that leucineand lysine increased significantly in model rats indicatinggouty arthritis induced amino acid metabolism disturbanceMeanwhile glutamine the amide of glutamate provides anontoxic storage and transport form of ammonia Thus thealtered level of glutamine in the model group might reflectdisorders of glutamine formation and could also be used as


Table 1 Identification of significantly differential endogenous metabolites associated with gouty arthritis in rat plasma and urine

Metabolite Chemical shift (120575)a VIPb FCc Controld HGd

PlasmaLeucine 096 (t) 16 120 darr

lowastdarrlowast

Lysine 146 (m) 190 (m) 302 (m) 12 134 darrlowast

darrlowast

Lactate 133 (d) 412 (q) 31 115 darrlowast

darrlowast

Glutamine 245 (m) 377 (m) 15 114 darrlowast

darrlowast

120573-Glucose 325 (dd) 465 (d) 45 064 uarrlowastlowast

uarrlowast

UrinePyruvate 238 (s) 13 119 darr

lowastdarrlowast

Succinate 241 (s) 13 126 darrlowast

darrlowast

2-Oxoglutarate 245 (t) 302 (t) 17 130 darrlowast

darrlowast

Citrate 255 (d) 268 (d) 16 121 darrlowast

darrlowast

Trimethylamine N-oxide 328 (s) 21 078 uarrlowast -

Hippurate 398 (d) 756 (t) 764 (t) 784 (d) 24 064 uarrlowastlowast

uarrlowastlowast

aLetters in parentheses indicate the peak multiplicities s singlet d doublet t triplet q quartet m multipletbVIP was obtained from OPLS-DA model (Figure 6)cFold change (FC) was calculated as the ratio of the mean metabolite levels between model and control groups FC with a value gt1 indicates a relatively higherconcentration while a value lt1 means a relatively lower concentration present in model group as compared to the controlsdCompared to model group uarr indicates relative increase in signal while darr indicates relative decrease in signal lowastlowast and lowast represent 119875 lt 001 and 119875 lt 005respectively whereas - denotes no statistically significant difference

an index of disturbance in amino acid metabolism causedby gouty arthritis HG could restore the increased levels ofleucine lysine and glutaminemanifesting its ability to rectifythe disturbance of amino acid metabolism induced by goutyarthritis

Urinary levels of TMAO and hippurate were signifi-cantly decreased in gouty arthritis model rats relative to thehealthy controls Reduced urinary excretion of hippurate wasobserved in patients with acute gout [37] These metabolitesare uniquely produced by bacterial metabolism in the intesti-nal tract indicating that gouty arthritis may be associatedwith variations in intestinal microflora [38] HG effectivelyattenuated the alteration of hippurate reflecting its protectiveaction on gut microbiota metabolism

Although potential biomarkers associated with goutyarthritis were identified and intervention effects of HG wereclearly observed there were limitations in the current studyFirstly owing to the lack of individual herb pretreatmentgroups it was impossible to identify the role of each herbalone It would be better to set five animal groups to comparethe effects of each individual herb and the mixture infuture study Secondly the underlying mechanisms of theobserved changes in metabolic phenotype warrant furtherinvestigation with a goal to verify potential biomarkers inhuman afflicted with gouty arthritis and test newmedicationsfor their efficacyThirdly additional mechanistic informationand metabolite markers may be identified by using MS-based metabonomic techniques which provide complemen-tary information to NMR

5 Conclusions

In summary antigout effects ofHGwere observed in rats withMSU-induced gouty arthritis as evidenced by inhibition of

joint swelling and suppression of inflammatory cell infiltra-tionThis beneficial antigout effectmight bemediated at leastin part by inhibiting TNF-120572 IL-1120573 and NF-120581B p65 proteinexpression in synovial fluid and synovial tissue In addition1H NMR-based metabonomic approach was firstly appliedin this study to reveal that the pathophysiological process ofMSU-induced gouty arthritiswas closely related to disruptionof several metabolic pathways including energy metabolismamino acid metabolism and gut microbe metabolism andHG possessed protective effects against these metabolicdysfunctionsThese results offered new insights into in-depthunderstanding of the pathogenesis of gouty arthritis as wellas discovery of targets for clinical diagnosis and treatmentand also provided further evidence for the potential use ofHG as an antigout agent

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

This work was supported by National Natural Science Foun-dation of China (nos 81173194 81403075) and Science andTechnology Planning Project of Guangdong Province China(no 2012B060300031)

References

[1] T Neogi ldquoClinical practice GoutrdquoThe New England Journal ofMedicine vol 364 no 5 pp 443ndash452 2011

[2] J F Baker and H R Schumacher ldquoUpdate on gout andhyperuricemiardquo International Journal of Clinical Practice vol64 no 3 pp 371ndash377 2010


[3] B N Cronstein and P Sunkureddi ldquoMechanistic aspects ofinflammation and clinical management of inflammation inacute gouty arthritisrdquo Journal of Clinical Rheumatology vol 19no 1 pp 19ndash29 2013

[4] D C Brater ldquoAnti-inflammatory agents and renal functionrdquoSeminars inArthritis andRheumatism vol 32 no 3 supplement1 pp 33ndash42 2002

[5] S-L Wen Y-J Liu H-L Yin et al ldquoEffect of acupuncture onrats with acute gouty arthritis inflammation a metabonomicmethod for profiling of both urine and plasma metabolicperturbationrdquoAmerican Journal of ChineseMedicine vol 39 no2 pp 287ndash300 2011

[6] Y Y Kou Y F Li M Xu W Li M Yang and R Li ldquoEffectsof RuPeng15 Powder (RPP15) on monosodium urate crystal-induced gouty arthritis in ratsrdquo Evidence-Based Complementaryand Alternative Medicine vol 2015 Article ID 527019 7 pages2015

[7] Y Ma L-L Zhou H-Y Yan and M Liu ldquoEffects of extractsfrom Paederia scandens (LOUR) MERRILL (Rubiaceae) onMSUcrystal-induced rats gouty arthritisrdquoTheAmerican Journalof Chinese Medicine vol 37 no 4 pp 669ndash683 2009

[8] L Liu J-A Duan Y Tang et al ldquoTaoren-Honghua herb pairand its main components promoting blood circulation throughinfluencing on hemorheology plasma coagulation and plateletaggregationrdquo Journal of Ethnopharmacology vol 139 no 2 pp381ndash387 2012

[9] H Zhang C Li S-T Kwok Q-W Zhang and S-W ChanldquoA review of the pharmacological effects of the dried rootof Polygonum cuspidatum (Hu Zhang) and its constituentsrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 208349 13 pages 2013

[10] Y Xu S Su T Wang J Duan and Z Wang ldquoA review of thepharmacological effects of chemical constituents of Cassia twig(Guizhi)rdquo Zhong Yao Cai vol 36 no 4 pp 674ndash678 2013

[11] YWang B Han Z Jia et al ldquoStudies on the intervention effectsof different ratios of cold-heat herb pair in Tongfengke capsulein Rat Gout Modelrdquo Zhongguo Shi Yan Fang Ji Xue Za Zhi vol18 no 15 pp 185ndash189 2012

[12] B Han L Qiu and Z Li ldquoClinical observations of the anti-gouteffects of Lexing-tongfengkerdquo Xin Zhong Yi vol 41 no 7 pp60ndash61 2009

[13] M D Reily and A A Tymiak ldquoMetabolomics in the pharma-ceutical industryrdquo Drug Discovery Today Technologies vol 13pp 25ndash31 2015

[14] J C Lindon and J K Nicholson ldquoSpectroscopic and statisticaltechniques for information recovery in metabonomics andmetabolomicsrdquo Annual Review of Analytical Chemistry vol 1no 1 pp 45ndash69 2008

[15] Y Jiang X-Y You K-L Fu and W-L Yin ldquoEffects of extractfrom Mangifera indica leaf on monosodium urate crystal-induced gouty arthritis in ratsrdquo Evidence-Based Complementaryand Alternative Medicine vol 2012 Article ID 967573 6 pages2012

[16] Z J Zou Z H Liu M J Gong B Han S M Wang andS W Liang ldquoIntervention effects of puerarin on blood stasisin rats revealed by a 1H NMR-based metabonomic approachrdquoPhytomedicine vol 22 no 3 pp 333ndash343 2015

[17] Z Zou B Han M Gong S Wang and S Liang ldquoNMR-basedmetabonomic studies on stomach heat and cold syndromes andintervention effects of the corresponding formulasrdquo Evidence-Based Complementary and Alternative Medicine vol 2014Article ID 528396 11 pages 2014

[18] J C Lindon J K Nicholson and J R Everett ldquoNMR spec-troscopy of biofluidsrdquo Annual Reports on NMR Spectroscopyvol 38 pp 1ndash88 1999

[19] J K Nicholson P J D Foxall M Spraul R D Farrant andJ C Lindon ldquo750MHz 1H and 1H-13C NMR spectroscopy ofhuman blood plasmardquo Analytical Chemistry vol 67 no 5 pp793ndash811 1995

[20] L Wei P Liao H Wu et al ldquoMetabolic profiling studies on thetoxicological effects of realgar in rats by 1HNMR spectroscopyrdquoToxicology and Applied Pharmacology vol 234 no 3 pp 314ndash325 2009

[21] J Xuan G Pan Y Qiu et al ldquoMetabolomic profiling to identifypotential serum biomarkers for schizophrenia and risperidoneactionrdquo Journal of Proteome Research vol 10 no 12 pp 5433ndash5443 2011

[22] H R Schumacher Jr ldquoCrystal-induced arthritis an overviewrdquoThe American Journal of Medicine vol 100 no 2 pp 46Sndash52S1996

[23] R Terkeltaub C Zachariae D Santoro J Martin P Peveri andK Matsushima ldquoMonocyte-derived neutrophil chemotacticfactorinterleukin-8 is a potential mediator of crystal-inducedinflammationrdquo Arthritis amp Rheumatism vol 34 no 7 pp 894ndash903 1991

[24] P-A Guerne R Terkeltaub B Zuraw and M Lotz ldquoInflam-matory microcrystals stimulate interleukin-6 production andsecretion by humanmonocytes and synoviocytesrdquoArthritis andRheumatism vol 32 no 11 pp 1443ndash1452 1989

[25] S J Getting H C Christian R J Flower and M PerrettildquoActivation of melanocortin type 3 receptor as a molecularmechanism for adrenocorticotropic hormone efficacy in goutyarthritisrdquo Arthritis and Rheumatism vol 46 no 10 pp 2765ndash2775 2002

[26] S J Getting R J Flower L Parente et al ldquoMolecular determi-nants ofmonosodiumurate crystal-inducedmurine peritonitisa role for endogenous mast cells and a distinct requirementfor endothelial-derived selectinsrdquo Journal of Pharmacology andExperimental Therapeutics vol 283 no 1 pp 123ndash130 1997

[27] F S DiGiovine S EMalawista GNuki andGWDuff ldquoInter-leukin 1 (IL 1) as a mediator of crystal arthritis Stimulation of Tcell and synovial fibroblastmitogenesis by urate crystal-inducedIL 1rdquoThe Journal of Immunology vol 138 no 10 pp 3213ndash32181987

[28] F S di Giovine S E Malawista E Thornton and G WDuff ldquoUrate crystals stimulate production of tumor necrosisfactor alpha from human blood monocytes and synovial cellsCytokinemRNAandprotein kinetics and cellular distributionrdquoJournal of Clinical Investigation vol 87 no 4 pp 1375ndash1381 1991

[29] P T Chapman F Jamar A A Harrison R M Binns A MPeters and D O Haskard ldquoNoninvasive imaging of E-selectinexpression by activated endothelium in urate crystal-inducedarthritisrdquo Arthritis amp Rheumatism vol 37 no 12 pp 1752ndash17561994

[30] P T Chapman F Jamar A A Harrison et al ldquoCharacterizationof E-selectin expression leucocyte traffic and clinical sequelaein urate crystal-induced inflammation an insight into goutrdquoBritish Journal of Rheumatology vol 35 no 4 pp 323ndash334 1996

[31] P Defilippi P Poupart J Tavernier W Fiers and J ContentldquoInduction and regulation of mRNA encoding 26-kDa proteinin human cell lines treated with recombinant human tumornecrosis factorrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 84 no 13 pp 4557ndash45611987


[32] K Matsushima K Morishita T Yoshimura et al ldquoMolecularcloning of a human monocyte-derived neutrophil chemotacticfactor (MDNCF) and the induction ofMDNCFmRNAby inter-leukin 1 and tumor necrosis factorrdquoThe Journal of ExperimentalMedicine vol 167 no 6 pp 1883ndash1893 1988

[33] P P Tak and G S Firestein ldquoNF-120581B a key role in inflammatorydiseasesrdquoThe Journal of Clinical Investigation vol 107 no 1 pp7ndash11 2001

[34] Y Si Y Zhang J Zhao et al ldquoNiacin inhibits vascularinflammation via downregulating nuclear transcription factor-b signaling pathwayrdquo Mediators of Inflammation vol 2014Article ID 263786 12 pages 2014

[35] M Grilli J J-S Chiu and M J Lenardo ldquoNF-120581B and Relparticipants in a multiform transcriptional regulatory systemrdquoInternational Review of Cytology vol 143 pp 1ndash62 1993

[36] M Jiang T Chen H Feng et al ldquoSerum metabolic signaturesof four types of human arthritisrdquo Journal of Proteome Researchvol 12 no 8 pp 3769ndash3779 2013

[37] Y Liu X Sun D Di J Quan J Zhang and X YangldquoA metabolic profiling analysis of symptomatic gout inhuman serum and urine using high performance liquidchromatography-diode array detector techniquerdquo ClinicaChimica Acta vol 412 no 23-24 pp 2132ndash2140 2011

[38] P Zheng Y Wang L Chen et al ldquoIdentification and validationof urinary metabolite biomarkers for major depressive disor-derrdquo Molecular and Cellular Proteomics vol 12 no 1 pp 207ndash214 2013


antimicrobial anti-inflammatory neuroprotective and car-dioprotective functions [9] Cassia twig (known as ldquoGui Zhirdquoin Chinese) exhibits antiviral antimicrobial antineoplasticanti-inflammatory antiallergic and analgesic properties [10]The combination of these two herbs known as Huzhang-Guizhi herb pair (HG) (with a ratio of 6 1) is firstly includedin Hu-Zhang Power documented in Taiping Shenghui Fangand our previous study demonstrated the antigout effects ofHG in acute gouty arthritis rat model [11] Meanwhile HGis also used in other formulas such as Lexing-tongfengkewhich has been widely used to treat gouty arthritis inclinics [12] However the biochemical mechanism by whichHG acts in gouty arthritis still remains elusive Our studytherefore aims to elucidate themechanisms bywhichHGactstherapeutically

Metabonomics is a well-established field in systems biol-ogy and has been applied to observe meaningful and relevantbiochemical changes due to disease toxicity nutrition andother variables Metabonomic studies can provide invaluableinformation towards understanding molecular mechanismsand novel insights into the status of dysfunction in biologicalsystems [13] Recently using ultra-performance liquid chro-matography coupled with mass spectrometry (UPLCMS)themetabolic profiles of rats withMSU-induced gouty arthri-tis and the biological effects of acupuncture were investigated[5] As we all know both nuclear magnetic resonance (NMR)spectroscopy and mass spectrometry (MS) (usually with achromatographic separation step) are suitable techniques formetabonomic analysis but have different analytical strengthsand weaknesses and give complementary information [14]To the best of our knowledge metabonomic study of goutyarthritis in rat model based on NMR has not been reported

In this study we monitored intumesce index histopatho-logical changes production of inflammatory cytokines andimmunohistochemistry of nuclear transcription factor-120581Bp65 (NF-120581B p65) to investigate the efficacy and anti-inflammatory mechanism of HG against MSU crystal-induced gout in rats an experimental model for goutyarthritis that is similar to those observed in clinical goutFurthermore a metabonomic method was applied to char-acterize the metabolic profiles associated with MSU-inducedgouty arthritis and observe the protective effects of HG in ratplasma and urine

2 Materials and Methods

21 Chemicals and Reagents Huzhang and Guizhi were pur-chased from Heshuntang (Guangzhou China) and authenti-cated by a specialist in pharmacognosy Distilled water waspurified using a Milli-Q ultrapure water system (MilliporeBedford MA USA) MSU crystals and deuterium oxide(D2O 999) were obtained from Sigma-Aldrich (St Louis

MO USA) Rat tumor necrosis factor-alpha (TNF-120572) andinterleukin-1 beta (IL-1120573) enzyme-linked immunosorbentassay (ELISA) kits were purchased fromAffinity BioReagents(Golden CO USA) NF-120581B p65 antibody was purchasedfrom Santa Cruz Biotechnology Inc (Santa Cruz CA USA)

22 Herbal Extraction A mixture of Huzhang (60 g) andGuizhi (10 g) crushed into small pieces was extracted withwater (1 10 wv) under thermal reflux for 2 h The filtrateswere collected and the residues were then refluxed in water(1 10 wv) for 15 h Two batches of filtrates were combinedand then were concentrated under a vacuum to obtain HGextract at a final concentration of 07 gmL (wv expressed asthe weight of raw materials)

23 AnimalHandling and Sample Collection All experimentswere approved by the local ethics committee and all exper-imental procedures were in compliance with the NationalInstitutes of Health Guide for Care and Use of LaboratoryAnimals

A total of 18 adult male SD rats weighing 180ndash200 gwere commercially obtained from Laboratory Animal Centerof Traditional Chinese Medicine University of GuangzhouChina Rats were maintained under controlled temperature(20 plusmn 2∘C) and lighting (0800ndash2000) conditions with foodandwater available ad libitumAfter 7 days of acclimatizationthe animals were randomly divided into three groups (119899 = 6in each group) (1) control group (2) gouty arthritis modelgroup and (3) HG pretreatment group (HG group) Ratsin the HG pretreatment group were administrated orally bygavage with HG at a dose of 93 gkg of body weight oncedaily for consecutive 7 days Dosing in this study was setaccording to dosages previously detailed in the literature [11]The control andmodel groups received an equivalent volumeof vehicle in the form of sterile saline as the pretreatmentgroup for 7 days Gouty arthritis was induced according toestablished protocol [15] 1 h after the last dosing on day 7MSU crystals (100mgmL suspended in sterile saline) wereinjected in the left ankle joint of each rat in the modelgroup and the HG group in a volume of 100 120583L The controlgroup received a 100 120583L intra-articular injection of singlesterile saline After the injection of MSU urine was collectedcontinuously (24 h) into ice-cooled vessels containing 05mLof 2 sodium azide in metabolism cages Following urinecollection all animals were anesthetized by diethyl etherinhalation and blood samples were acquired via the ratsrsquoorbital venous plexus Heparin was used as an anticoagulantPlasma was separated by centrifugation at 3500 rpm for15min All urine and plasma samples were kept atminus80∘Cuntilthey were defrosted immediately prior to analysis Synoviallavage fluid from each ankle was collected by injecting5mL phosphate buffered saline (PBS) into the joint cavityLavage fluids were then centrifuged at 400 g for 10 minutesand supernatants were stored at minus80∘C before biochemicaldeterminations and synovial tissue was gently separated forimmunohistochemistry examination

24 Measurement of Ankle Edema The development ofarthritis was assessed by measuring the ankle size before andafter induction of gout The circumference of the inflamedankle joint of the animals was measured using a 3mmwide tape measure without elasticity at 24 h after MSUcrystal injection In order to quantify ankle edema the datawere converted to a percentage of changes of ankle size as





2O2in





2O



2HPO4-02molL NaH

2PO4









3 Results



0

005

01

015

02

025

03

035

04

045

05

Control Model HG

Ank

le in

tum

esce

inde

x

lowast

lowast

(a)

0

20

40

60

80

100

120

140

160

Control Model HG

TNF-120572

leve

ls (p

gm

L)

lowast

lowast

(b)

0

200

400

600

800

1000

1200

1400

1600

1800

2000

Control Model HG

IL1

-120573le

vels

(pg

mL)

lowast

lowast

(c)












(a) (b)

(c)


(a) (b)

(c)



0510152025303540455560

235

120575

(a)

0510152025303540455560

245

120575

(b)

0510152025303540455560

12

3

120575

(c)


0510152025303540606570758085

35

6

120575

(a)

0510152025303540606570758085

13

4

120575

(b)

0510152025303540606570758085

26

120575

(c)





4 Discussion



minus60

minus40

minus20

0

20

40

60


t o[1

]

(a)

minus40

minus20

0

20

40


t o[1

]

(b)

minus100

minus50

0

50

100


t o[1

]

(c)

minus100

minus50

0

50

100

minus100 minus50 0 50 100t[1]

t o[1

]

(d)

minus30

minus20

minus10

0

10

20

30


t[2

]

(e)

minus40

minus20

0

20

40

minus100 minus50 0 50 100t[1]

t[2

]

(f)






00

05

10

00 02 04 06 08 10

R2

Q2

(a)

00

05

10

00 02 04 06 08 10

R2

Q2

(b)

00

05

10

00 02 04 06 08 10

R2

Q2

(c)

00

05

10

00 02 04 06 08 10

R2

Q2

(d)

minus05

00

05

10

00 02 04 06 08 10

R2

Q2

(e)

00

05

10

00 02 04 06 08 10

R2

Q2

(f)






0

2

4

6

8

10

12

14

16

18


ControlModelHG

lowast

lowast

lowastlowast

lowast

lowast

(a)

0

50

100

150

200

250

300

350

Lactate

ControlModelHG

120573-Glucose

lowast lowastlowast

lowastlowast

(b)

020406080

100120140160180

ControlModelHG

lowast



lowast

lowastlowast

lowast


Hippurate

(c)











lowastdarrlowast


darrlowast


darrlowast


darrlowast


uarrlowast


lowastdarrlowast


darrlowast


darrlowast


darrlowast



uarrlowastlowast





5 Conclusions





Acknowledgments


References













































2O2in





2O



2HPO4-02molL NaH

2PO4









3 Results



0

005

01

015

02

025

03

035

04

045

05

Control Model HG

Ank

le in

tum

esce

inde

x

lowast

lowast

(a)

0

20

40

60

80

100

120

140

160

Control Model HG

TNF-120572

leve

ls (p

gm

L)

lowast

lowast

(b)

0

200

400

600

800

1000

1200

1400

1600

1800

2000

Control Model HG

IL1

-120573le

vels

(pg

mL)

lowast

lowast

(c)












(a) (b)

(c)


(a) (b)

(c)



0510152025303540455560

235

120575

(a)

0510152025303540455560

245

120575

(b)

0510152025303540455560

12

3

120575

(c)


0510152025303540606570758085

35

6

120575

(a)

0510152025303540606570758085

13

4

120575

(b)

0510152025303540606570758085

26

120575

(c)





4 Discussion



minus60

minus40

minus20

0

20

40

60


t o[1

]

(a)

minus40

minus20

0

20

40


t o[1

]

(b)

minus100

minus50

0

50

100


t o[1

]

(c)

minus100

minus50

0

50

100

minus100 minus50 0 50 100t[1]

t o[1

]

(d)

minus30

minus20

minus10

0

10

20

30


t[2

]

(e)

minus40

minus20

0

20

40

minus100 minus50 0 50 100t[1]

t[2

]

(f)






00

05

10

00 02 04 06 08 10

R2

Q2

(a)

00

05

10

00 02 04 06 08 10

R2

Q2

(b)

00

05

10

00 02 04 06 08 10

R2

Q2

(c)

00

05

10

00 02 04 06 08 10

R2

Q2

(d)

minus05

00

05

10

00 02 04 06 08 10

R2

Q2

(e)

00

05

10

00 02 04 06 08 10

R2

Q2

(f)






0

2

4

6

8

10

12

14

16

18


ControlModelHG

lowast

lowast

lowastlowast

lowast

lowast

(a)

0

50

100

150

200

250

300

350

Lactate

ControlModelHG

120573-Glucose

lowast lowastlowast

lowastlowast

(b)

020406080

100120140160180

ControlModelHG

lowast



lowast

lowastlowast

lowast


Hippurate

(c)











lowastdarrlowast


darrlowast


darrlowast


darrlowast


uarrlowast


lowastdarrlowast


darrlowast


darrlowast


darrlowast



uarrlowastlowast





5 Conclusions





Acknowledgments


References










































0

005

01

015

02

025

03

035

04

045

05

Control Model HG

Ank

le in

tum

esce

inde

x

lowast

lowast

(a)

0

20

40

60

80

100

120

140

160

Control Model HG

TNF-120572

leve

ls (p

gm

L)

lowast

lowast

(b)

0

200

400

600

800

1000

1200

1400

1600

1800

2000

Control Model HG

IL1

-120573le

vels

(pg

mL)

lowast

lowast

(c)












(a) (b)

(c)


(a) (b)

(c)



0510152025303540455560

235

120575

(a)

0510152025303540455560

245

120575

(b)

0510152025303540455560

12

3

120575

(c)


0510152025303540606570758085

35

6

120575

(a)

0510152025303540606570758085

13

4

120575

(b)

0510152025303540606570758085

26

120575

(c)





4 Discussion



minus60

minus40

minus20

0

20

40

60


t o[1

]

(a)

minus40

minus20

0

20

40


t o[1

]

(b)

minus100

minus50

0

50

100


t o[1

]

(c)

minus100

minus50

0

50

100

minus100 minus50 0 50 100t[1]

t o[1

]

(d)

minus30

minus20

minus10

0

10

20

30


t[2

]

(e)

minus40

minus20

0

20

40

minus100 minus50 0 50 100t[1]

t[2

]

(f)






00

05

10

00 02 04 06 08 10

R2

Q2

(a)

00

05

10

00 02 04 06 08 10

R2

Q2

(b)

00

05

10

00 02 04 06 08 10

R2

Q2

(c)

00

05

10

00 02 04 06 08 10

R2

Q2

(d)

minus05

00

05

10

00 02 04 06 08 10

R2

Q2

(e)

00

05

10

00 02 04 06 08 10

R2

Q2

(f)






0

2

4

6

8

10

12

14

16

18


ControlModelHG

lowast

lowast

lowastlowast

lowast

lowast

(a)

0

50

100

150

200

250

300

350

Lactate

ControlModelHG

120573-Glucose

lowast lowastlowast

lowastlowast

(b)

020406080

100120140160180

ControlModelHG

lowast



lowast

lowastlowast

lowast


Hippurate

(c)











lowastdarrlowast


darrlowast


darrlowast


darrlowast


uarrlowast


lowastdarrlowast


darrlowast


darrlowast


darrlowast



uarrlowastlowast





5 Conclusions





Acknowledgments


References










































(a) (b)

(c)


(a) (b)

(c)



0510152025303540455560

235

120575

(a)

0510152025303540455560

245

120575

(b)

0510152025303540455560

12

3

120575

(c)


0510152025303540606570758085

35

6

120575

(a)

0510152025303540606570758085

13

4

120575

(b)

0510152025303540606570758085

26

120575

(c)





4 Discussion



minus60

minus40

minus20

0

20

40

60


t o[1

]

(a)

minus40

minus20

0

20

40


t o[1

]

(b)

minus100

minus50

0

50

100


t o[1

]

(c)

minus100

minus50

0

50

100

minus100 minus50 0 50 100t[1]

t o[1

]

(d)

minus30

minus20

minus10

0

10

20

30


t[2

]

(e)

minus40

minus20

0

20

40

minus100 minus50 0 50 100t[1]

t[2

]

(f)






00

05

10

00 02 04 06 08 10

R2

Q2

(a)

00

05

10

00 02 04 06 08 10

R2

Q2

(b)

00

05

10

00 02 04 06 08 10

R2

Q2

(c)

00

05

10

00 02 04 06 08 10

R2

Q2

(d)

minus05

00

05

10

00 02 04 06 08 10

R2

Q2

(e)

00

05

10

00 02 04 06 08 10

R2

Q2

(f)






0

2

4

6

8

10

12

14

16

18


ControlModelHG

lowast

lowast

lowastlowast

lowast

lowast

(a)

0

50

100

150

200

250

300

350

Lactate

ControlModelHG

120573-Glucose

lowast lowastlowast

lowastlowast

(b)

020406080

100120140160180

ControlModelHG

lowast



lowast

lowastlowast

lowast


Hippurate

(c)











lowastdarrlowast


darrlowast


darrlowast


darrlowast


uarrlowast


lowastdarrlowast


darrlowast


darrlowast


darrlowast



uarrlowastlowast





5 Conclusions





Acknowledgments


References










































0510152025303540455560

235

120575

(a)

0510152025303540455560

245

120575

(b)

0510152025303540455560

12

3

120575

(c)


0510152025303540606570758085

35

6

120575

(a)

0510152025303540606570758085

13

4

120575

(b)

0510152025303540606570758085

26

120575

(c)





4 Discussion



minus60

minus40

minus20

0

20

40

60


t o[1

]

(a)

minus40

minus20

0

20

40


t o[1

]

(b)

minus100

minus50

0

50

100


t o[1

]

(c)

minus100

minus50

0

50

100

minus100 minus50 0 50 100t[1]

t o[1

]

(d)

minus30

minus20

minus10

0

10

20

30


t[2

]

(e)

minus40

minus20

0

20

40

minus100 minus50 0 50 100t[1]

t[2

]

(f)






00

05

10

00 02 04 06 08 10

R2

Q2

(a)

00

05

10

00 02 04 06 08 10

R2

Q2

(b)

00

05

10

00 02 04 06 08 10

R2

Q2

(c)

00

05

10

00 02 04 06 08 10

R2

Q2

(d)

minus05

00

05

10

00 02 04 06 08 10

R2

Q2

(e)

00

05

10

00 02 04 06 08 10

R2

Q2

(f)






0

2

4

6

8

10

12

14

16

18


ControlModelHG

lowast

lowast

lowastlowast

lowast

lowast

(a)

0

50

100

150

200

250

300

350

Lactate

ControlModelHG

120573-Glucose

lowast lowastlowast

lowastlowast

(b)

020406080

100120140160180

ControlModelHG

lowast



lowast

lowastlowast

lowast


Hippurate

(c)











lowastdarrlowast


darrlowast


darrlowast


darrlowast


uarrlowast


lowastdarrlowast


darrlowast


darrlowast


darrlowast



uarrlowastlowast





5 Conclusions





Acknowledgments


References










































minus60

minus40

minus20

0

20

40

60


t o[1

]

(a)

minus40

minus20

0

20

40


t o[1

]

(b)

minus100

minus50

0

50

100


t o[1

]

(c)

minus100

minus50

0

50

100

minus100 minus50 0 50 100t[1]

t o[1

]

(d)

minus30

minus20

minus10

0

10

20

30


t[2

]

(e)

minus40

minus20

0

20

40

minus100 minus50 0 50 100t[1]

t[2

]

(f)






00

05

10

00 02 04 06 08 10

R2

Q2

(a)

00

05

10

00 02 04 06 08 10

R2

Q2

(b)

00

05

10

00 02 04 06 08 10

R2

Q2

(c)

00

05

10

00 02 04 06 08 10

R2

Q2

(d)

minus05

00

05

10

00 02 04 06 08 10

R2

Q2

(e)

00

05

10

00 02 04 06 08 10

R2

Q2

(f)






0

2

4

6

8

10

12

14

16

18


ControlModelHG

lowast

lowast

lowastlowast

lowast

lowast

(a)

0

50

100

150

200

250

300

350

Lactate

ControlModelHG

120573-Glucose

lowast lowastlowast

lowastlowast

(b)

020406080

100120140160180

ControlModelHG

lowast



lowast

lowastlowast

lowast


Hippurate

(c)











lowastdarrlowast


darrlowast


darrlowast


darrlowast


uarrlowast


lowastdarrlowast


darrlowast


darrlowast


darrlowast



uarrlowastlowast





5 Conclusions





Acknowledgments


References










































00

05

10

00 02 04 06 08 10

R2

Q2

(a)

00

05

10

00 02 04 06 08 10

R2

Q2

(b)

00

05

10

00 02 04 06 08 10

R2

Q2

(c)

00

05

10

00 02 04 06 08 10

R2

Q2

(d)

minus05

00

05

10

00 02 04 06 08 10

R2

Q2

(e)

00

05

10

00 02 04 06 08 10

R2

Q2

(f)






0

2

4

6

8

10

12

14

16

18


ControlModelHG

lowast

lowast

lowastlowast

lowast

lowast

(a)

0

50

100

150

200

250

300

350

Lactate

ControlModelHG

120573-Glucose

lowast lowastlowast

lowastlowast

(b)

020406080

100120140160180

ControlModelHG

lowast



lowast

lowastlowast

lowast


Hippurate

(c)











lowastdarrlowast


darrlowast


darrlowast


darrlowast


uarrlowast


lowastdarrlowast


darrlowast


darrlowast


darrlowast



uarrlowastlowast





5 Conclusions





Acknowledgments


References










































0

2

4

6

8

10

12

14

16

18


ControlModelHG

lowast

lowast

lowastlowast

lowast

lowast

(a)

0

50

100

150

200

250

300

350

Lactate

ControlModelHG

120573-Glucose

lowast lowastlowast

lowastlowast

(b)

020406080

100120140160180

ControlModelHG

lowast



lowast

lowastlowast

lowast


Hippurate

(c)











lowastdarrlowast


darrlowast


darrlowast


darrlowast


uarrlowast


lowastdarrlowast


darrlowast


darrlowast


darrlowast



uarrlowastlowast





5 Conclusions





Acknowledgments


References













































lowastdarrlowast


darrlowast


darrlowast


darrlowast


uarrlowast


lowastdarrlowast


darrlowast


darrlowast


darrlowast



uarrlowastlowast





5 Conclusions





Acknowledgments


References









































Therapeutic Effects of Chinese Medicine Herb Pair, Huzhang ... ·...

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