Protective Effect of Jianpiyifei II Granule against...

Research ArticleProtective Effect of Jianpiyifei II Granule against ChronicObstructive Pulmonary Disease via NF-120581B Signaling Pathway

Long Fan1 Ruifeng Chen12 Leng Li1 Ziyao Liang1 Xuhua Yu1 Kai Huang1

Shuomiao Yin1 Lei Wu 1 Yuanbin Chen1 Yinji Xu 1 Qi Wang 12 and Lin Lin 1

1The Second Clinical College of Guangzhou University of Chinese Medicine Guangdong Provincial Hospital of Chinese MedicineGuangzhou 510120 China2Institute of Clinical Pharmacology Guangzhou University of Chinese Medicine Guangzhou 510405 China

Correspondence should be addressed to Qi Wang wangqigzucmeducn and Lin Lin drlinlin620163com

Received 29 April 2018 Revised 29 June 2018 Accepted 8 July 2018 Published 5 August 2018

Academic Editor Mark Moss

Copyright copy 2018 Long Fan 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

Jianpiyifei II granule (JPYF II) is an oriental herbal formula used clinically in China to treat chronic obstructive pulmonary disease(COPD) The aim of the present study was to investigate the anti-inflammatory and antioxidative activities of JPYF II in a mousemodel of COPD induced by lipopolysaccharide (LPS) and cigarette smoke (CS) and in RAW2647 cells stimulated with cigarettesmoke extract (CSE) Mice were given LPS via intratracheal instillation on days 1 and 15 and exposed to CS generated from 4cigarettesday for 28 days The mice were treated with 075 15 or 3 gkgd JPYF II by intragastric administration in low middleand high dose groups respectively for two weeks RAW2647 cells were stimulated by CSE and treated with JPYF II at doses of125 25 or 50120583gmL In the mouse model of LPS and CS-induced COPD JPYF II decreased inflammatory cell counts in bronchoalveolar lavage fluid (BALF) in addition to mRNA expression of proinflammatory cytokines and metalloproteinases (MMPs) inlung tissues In addition JPYF II elevated catalase (CAT) and glutathione peroxidase (GSH-Px) activities and reduced the levels ofmalondialdehyde (MDA) and I120581B120572 and p65 phosphorylation and inflammatory cell infiltration in the lung tissues In RAW2647cells stimulated with CSE JPYF II inhibited the mRNA levels of inflammatorymediators and the phosphorylation of I120581B120572 and p65Our results suggest that JPYF II enhanced anti-inflammatory and antioxidative activities in a mouse model of COPD induced byLPS and CS and in RAW2647 cells stimulated with CSE via inhibition of the NF-120581B pathway

1 Introduction

Chronic obstructive pulmonary disease (COPD) describesa collection of chronic inflammatory diseases includingchronic bronchitis and emphysema It is characterized bylimited airflow which arises from an abnormal inflammatoryresponse to inhaled particles and gases in the lungs [1]leading to alveolar and bronchial inflammation in suscep-tible patients Cigarette smoke (CS) contains a complexproinflammatory mixture of chemical components whichcauses chronic inflammation in the airway and lung Only10-20 of smokers develop clinically significant COPDand additional exposure to workplace dust chemicals oroccupational fumes may also result in this disease but CSexposure is deemed to be a major risk factor related to the

onset of COPD [2 3] and almost 90 of COPD deaths aredue to smoking [4]

COPD is characterized by the development of a complexinflammatory response in the lungs due to chronic exposureto irritants such as CS [5] consisting of the infiltration ofinflammatory cells (eg macrophages neutrophils and T-lymphocytes) and increased expression of proinflammatorymediators (eg TNF-120572 IL-6 and IL-1120573) and matrix metal-loproteinases (eg MMP-9 and MMP-12) [6] Furthermorethe inflammatory process can be cyclically amplified asinflammatory cells are attracted from the circulation by thecytokines [7] One important modulator of inflammationNF-120581B governs the generation of TNF-120572 IL-6 and IL-1120573[8] Similarly in the lipopolysaccharide- (LPS-) induced acutelung injury model suppression of the NF-120581B pathway results

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2018 Article ID 4265790 13 pageshttpsdoiorg10115520184265790

2 Evidence-Based Complementary and Alternative Medicine

Table 1 The components of JPYF II

Herbal name Botanical name Family Medicinal part

Radix Astragali Astragalus membranaceus(Fisch) Bunge Leguminosae Root

Rhizoma Cimicifugae Cimicifuga foetida L Ranunculaceae Rhizome

Radix Codonopsis Codonopsis pilosula (Franch)Nannf Campanulaceae Root

Rhizoma AtractylodisMacrocephalae Atractylodes macrocephala koidz Asteraceae Rhizome

Radix Bupleuri Bupleurum chinense DC Umbelliferae RootHerba Cynomorii Cynomorium songaricum Rupr Cynomoriaceae Whole plantFructus Viticis Negundinis Vitex negundo L Verbenaceae FruitSemen Persicae Prunus persica (L) Batsch Rosaceae Seed

in inhibition of lung neutrophil and cytokine generation[9 10] Furthermore recent studies indicate that a numberof MMPs may also contribute to airway remodeling [11 12]It is noteworthy that MMP-9 which is secreted primarilyby macrophages and neutrophils is considered a pivotalexecutor in an inflammatory response In addition it hasbeen shown that emphysema induced by chronicCS exposuredoes not occur in MMP-12minusminus mice [13] indicating thatMMP-12 can have a destructive role in COPD

CS contains more than 1015 free radicals or oxidants andthousands of different chemical components per puff [14ndash17]which elevates the concentration of reactive oxygen species(ROS) produced enzymatically by epithelial and inflamma-tory cells in the lung The production of additional ROSleads to the initiation of an inflammatory response throughthe activation of transcriptional factors such as activatorprotein (AP-1) and NF-120581B in addition to gene expressionof proinflammatory cytokines [17ndash19] Therefore oxidativestress also plays a key role in COPD and affects infiltrationof surfactants to the alveoli mitochondrial respiration andremodeling of the extracellular matrix [17]

CS exposure is recognized as the major factor in thepathogenesis of COPD In addition as an important proin-flammatory glycolipid component of the outer membrane ofGram-negative bacteria LPS is often used in animal modelsof lung diseases including COPD and acute lung injury[20ndash22] Thus a combination of LPS and CS has becomethe preferred in vivo irritant as it shortens the duration ofexposure required forCOPD todevelop into an animalmodeland so enables deeper investigation of its pathological charac-teristics [21 23ndash25] Therefore intratracheal administrationof LPS and exposure to CS were combined to construct amouse model of COPD in the present study Moreover CSactivates innate immune cells such as macrophages whichplay a key role in promoting inflammation and the secretionof inflammatory cytokines It is known that their excessiveactivation has damaging effects and persistence of proin-flammatory activity results in the development of chronicinflammation such as COPD [26 27] Hence exposure ofthemousemacrophage cell lineRAW2647 to cigarette smokeextract (CSE) was also adopted in the present study

Jianpiyifei II granule (JPYF II) an oriental herbalformula consists of eight traditional Chinese medicines(TCMs) as shown in Table 1 including Radix Astragali Rhi-zoma Cimicifugae Radix Codonopsis Rhizoma AtractylodisMacrocephalae Radix Bupleuri Herba Cynomorii FructusViticis Negundinis and Semen Persicae Radix AstragaliRadix Codonopsis and Rhizoma Atractylodis Macrocepha-lae are used in TCM to invigorate Qi [28] and RhizomaCimicifugae can be used to treat a collapse of the mid-dle Qi [29] Modern pharmacological studies suggest thatthey enhance natural immune function Bu-Zhong-Yi-Qi-Tang (BT) a famous herbal formula used in China andJapan whose formulation also includes Radix AstragaliRhizoma Cimicifugae Rhizoma Atractylodis Macrocepha-lae and Radix Bupleuri demonstrates immunomodulationand anti-inflammatory effects [30 31] Furthermore HerbaCynomorii has been used in the treatment of kidney-yangdeficiency which can lead to asthma [32] Fructus ViticisNegundinis has traditionally been used to treat chronicbronchitis and coughs [33] Semen Persicae which regulateslung Qi and removes stasis is traditionally combined withother herbs to dissipate phlegm relieve coughs and treatasthma caused by the upward reversal of lung Qi [34]Hence JPYF II is formulated to treat COPD based onthe traditional uses of the eight medicinal herbs describedabove using the principle of ldquoJun-Chen-Zuo-Shirdquo (ldquoemperor-minister-adjuvant-courierrdquo) Radix Astragali and RhizomaCimicifugae act as ldquoJunrdquo to treat the main disease RadixCodonopsis and Rhizoma Atractylodis Macrocephalae playthe role of ldquoChenrdquo to increase the function of ldquoJunrdquo RadixBupleuri Herba Cynomorii Fructus Viticis Negundinis andSemen Persicae treat the accompanying symptoms as ldquoZuordquo

JPYF II has been prescribed to treat COPD inGuangdongProvincial Hospital of Chinese Medicine for more than adecade In a clinical study performed previously by theauthors it was established that JPYF IIwas able to significantlyincrease walking distance over six minutes and decreaseSt Georgersquos Respiratory Questionnaire (SGRQ) score in atreatment group of 178 patients demonstrating that JPYF IIhad a clear therapeutic effect in stable stage COPD patients[35] In addition our previous studies demonstrated that

Evidence-Based Complementary and Alternative Medicine 3

JPYF II reduced airway resistance and intrathoracic pressureand attenuated ultrastructural damage to lung tissues in ratsexposed to LPS and CS [36] Furthermore JPYF II protectedthe lung tissues of rats from exposure to LPS and CS bydecreasing protein levels of TNF-120572 IL-8 and TGF-1205731 inaddition to the MMP-9TIMP-1 ratio [37] However themechanisms of action of JPYF II or its protective effectsagainst COPD are still not well-understood

Therefore the aim of the present study was to investigatethe anti-inflammatory and antioxidative activities of JPYFII in a mouse model of COPD induced by LPS and CSand in RAW2647 cells stimulated with CSE The resultsdemonstrate that JPYF II reduced pulmonary inflammationvia suppression of the NF-120581Bpathway and excessive oxidativestress which indicated that it was the underlying mechanismof JPYF II in the treatment of COPD and provided atheoretical basis for future research and clinical application

2 Materials and Methods

21 Plant Materials JPYF II is a formulation of RadixAstragali Rhizoma Cimicifugae Radix Codonopsis Rhi-zoma Atractylodis Macrocephalae Radix Bupleuri HerbaCynomorii Fructus Viticis Negundinis and Semen Persicaein a ratio of 31315115151 All were purchased fromGuangdong Provincial Hospital of Chinese Medicine andidentified by their Department of TCMs Additionally spec-imens were deposited in the Second Clinical College ofGuangzhou University of Chinese Medicine (Voucher Nos160717 160718 160719 160720 160721 160722 160723 and160724) The medicinal herbs were powdered and extractedtwice with 10 times their volume of boiling water for 90minutes Each water extract was filtered and dehydratedunder vacuum and then residue was freeze-dried and storedin a refrigerator until required

22 Chromatographic Analysis Chromatographic analysiswas performed using a Thermo Fisher Accela UPLC system(Thermo Fisher Scientific San Jose CA USA) equippedwith a quaternary pump solvent management system anonline degasser a diode-array detector (DAD) a columncompartment and an autosampler using a PhenomenexUPLC Kinetex C18 column (21 times 100mm 17 120583m) The flowrate wasmaintained at 02mLminwith a volume of injectionof 3 120583L The mobile phase consisted of an aqueous solutionof 01 formic acid (A) and acetonitrile (B) with an elutiongradient of 5-25 B from 0 to 5min 25-60 B from 5 to28min 60-90B from28 to 38min and 90B between 38and 42min Detection wavelengths were set at 214 254 and280 nm with a column oven temperature set at 25∘C

23 Mass Conditions Mass spectrometry (MS) was per-formed using a Thermo Fisher Accela LTQ Orbitrap XLhybridmass spectrometer (ThermoFisher Scientific BremenGermany) equipped with an electrospray ionization (ESI)interface The ESI source was set in positive ionization modeMass calibration was performed in accordance with themanufacturerrsquos guidelines using a standard solution mixof Ultramark the tetrapeptide Met-Arg-Phe-Ala (MRFA)

acetate salt sodium taurocholate and sodium dodecyl sul-phate The ionization and tube lens voltages were 42 kVand 86 V respectively Sheath and auxiliary gas (nitrogen)pressures were set at 20 and 5 units respectively Capillarytemperature was set at 350∘C MS acquisition was set with ascan range of 150-1300 mz and a resolving power of 30000for full-scan

24 Reagents Malondialdehyde (MDA) glutathione perox-idase (GSH-Px) and catalase (CAT) kits were purchasedfrom Jiancheng Bioengineering Institute (Nanjing China)I120581B120572 (4812S) p-I120581B120572 (9246S) and p-NF-120581Bp65 (3033S)antibodies were obtained from Cell Signaling Technology(Danvers USA) NF-120581Bp65 (L0413) and 120573-actin (G0213)antibodies were purchased from Santa Cruz Biotechnology(Texas USA)

25 Animals Female Balbc mice (specifically pathogen-free and six to eight weeks old) were purchased from theAnimal Supply Center of Guangdong Academy of MedicalScience All animals were housed under standard conditionsin the Animal Center of Guangdong Provincial Academyof Chinese Medical Sciences at a temperature of 25plusmn1∘Cand 55plusmn5 humidity under a 12 h lightdark cycle withfree access to water and food All experimental procedureswere approved by the Institutional Animal Care and UseCommittee of Guangdong Provincial Academy of ChineseMedical Sciences

26 Experiment Protocols The mouse model of COPD wascreated by intratracheal administration of LPS and CS expo-sure daily Briefly mice were anesthetized with 4 pento-barbital and treated with LPS (02120583g120583L 50120583L) throughintratracheal administration on days 1 and 15 Mice wereexposed to CS generated from 4 cigarettesday in batchesof 10 in an 18 L Perspex chamber (40 cm times 25 cm times 18 cm)for 28 days CS was collected in a 50-mL syringe over 10 smimicking the normal quantity of smoking inhalation andrate of cigarette combustion and delivered four times perday at 11 AM 12 noon 1 PM and 2 PM with 1 cigaretteeach Control mice were exposed to air using a similar pro-cedure Filter-tipped DaQianMen cigarettes (manufacturedby Shanghai Tobacco Group Co Ltd China) emitting 13mgCO 08mg nicotine and 11mg tar per cigarette were used inthis study From the third week of CS exposure mice in thepositive group received 2mgkgd dexamethasone and thosein the treatment groups were treated with 075 15 or 3 gkgdJPYF II by intragastric administration for two weeks Mice inthe control and model groups were intragastrically given anequal volume of saline

27 Lung Homogenate Lungs were harvested rinsed thor-oughly with ice-cold PBS and homogenized with PBS Thehomogenate was centrifuged at 12000 rpm for 15min at 4∘Cand the supernatant was collected into tubes and stored atminus80∘C until required for analysis


28 Broncho Alveolar Lavage Fluid (BALF) Collection Tra-cheostomies were performed and the tracheas were cannu-lated using a 24G blunt needle 05mL ice-cold PBS wasinjected and as much fluid as possible was withdrawn Theprocedure was repeated twice The cells were isolated bycentrifugation fixed and then stained to distinguish theinflammatory cells in the BALF

29 Cell Culture and 3-(45-Dimethylthiazol-2-yl)-25-diphen-yl Tetrazolium Bromide (MTT) Assay RAW2647 cells amouse macrophage cell line were cultured in Dulbeccorsquosmodified Eagle medium (DMEM) containing 100 IUmLpenicillin 100120583gmL streptomycin and 10 fetal bovineserum (FBS) in a humidified atmosphere of 5 CO2 at 37

∘CCells that reached 90 confluence were used for subsequentexperiments An MTT assay was performed to measure cellviability in response to JPYF II RAW2647 cells were treatedwith either JPYF II alone or in combination with CSE for24 hours Then MTT solution (10120583L) was added for 4 hoursat 37∘C Finally 100120583L of dimethyl sulfoxide (DMSO) wasadded to dissolve the resultant formazan crystals Metabolicactivity was determined from optical density at 490 nm usinga microplate reader (PerkinElmer Waltham MA USA)

210 Preparation of CSE CSE was prepared in accordancewith the procedure described by Wirtz and Schmidt [38]Filter-tipped HongShuangXi cigarettes (manufactured byChina Tobacco Guangdong Industrial Co Ltd China) emit-ting 13mg CO 12mg nicotine and 11mg tar per cigarettewere used in this study Briefly a cigarette without filterwas installed on a 50mL syringe and completely combustedwithin 2min Smoke from two cigarettes was dissolved in10mLDMEMwithout serumThe final solution was adjustedto pH 74 and sterilized using a 022120583m filter Absorbanceat 320 nm was measured using a spectrophotometer tostandardize CSE preparation The CSE solution was preparedprior to each experiment using exactly the same procedureand the concentration of the final solution was defined as100 The CSE solution was diluted to the desired concen-trations in accordance with the experiment

211 Antioxidant Detection Concentrations of MDA GSH-Px and CAT in the lung tissues were determined usingappropriate detection kits according to the manufacturerrsquosguidelines Absorbance values weremeasured at 405 412 and532nm respectively

212 Quantitative Real-Time PCR Total RNA was isolatedusing NucleoZOL reagent (MN Duren Germany) fromcultured cells or mouse lung tissues and reverse transcribedto cDNA using a reverse transcription kit (TaKaRa KusatsuJapan) Real-time PCR expression analysis was performedon an ABI 7500 Real-Time PCR System (Applied Biosys-tems Foster City CA USA) using SYBR Premix Ex Taq(TaKaRa Kusatsu Japan) Primers used for PCR were as fol-lows IL-1120573 51015840-GTCCTGTGTAATGAAAGACGGC-31015840 (F)

Figure 1 The total ion chromatogram (TIC) in positive ion (PI)mode of JPYF II

51015840-TGCTTGTGAGGTGCTGATGT-31015840 (R) IL-6 51015840-ACA-AAGCCAGAGTCCTTCAGAG-31015840 (F) 51015840-GAGCATTGG-AAATTGGGGTAGG-31015840 (R) TNF-120572 51015840-ACGGCATGG-ATCTCAAAGAC-31015840 (F) 51015840-GGAGGTTGACTTTCTCCT-GGTA-31015840 (R) MMP-9 51015840-GCCCTGGAACTCACACGA-CA-31015840 (F) 51015840-TTGGAAACTCACACGCCAGAAG-31015840 (R)MMP-12 51015840-ACACTACTGGAGGTATGATGTGAG-31015840 (F)51015840-TGTTTTGGTGACACGACGGA-31015840 (R) 120573-actin 51015840-GCTCCTAGCACCATGAAGATCA-31015840 (F) 51015840-AGGGTG-TAAAACGCAGCTCA-31015840 (R)

213 Western Blot Analysis RAW2647 cells and the homo-genate ofmouse lung tissues were lysed in ice-cold lysis bufferfor 30min Total protein concentration in the lysate wasmea-sured using a bicinchoninic acid (BCA) assay Normalizedquantities of protein were separated using SDS-PAGE andthen transferred to PVDF membranes which were blockedwith 5 skim milk in Tris-buffered saline-Tween 20 (TBST)and incubated overnight at 4∘C with primary antibody Afterbeing washed with TBST the membranes were incubatedwith secondary antibody for 2 h and the protein bandswere visualized using a chemiluminescence detection system(Pierce Rockford IL USA) Scanned images were quantifiedusing Quantity One software (Bio-Rad Hercules CA USA)GAPDH or 120573-actin was used as an internal control

214 Histological Examination Lung tissues were fixed using4 (vv) paraformaldehyde paraffin-embedded cut into4120583m thick slices and then stained with hematoxylin andeosin solution (Sigma-Aldrich CO USA) for histopatholog-ical examination

215 Statistical Analysis Data from all the experiments wereexpressed as means plusmn standard deviation (SD) A one-wayanalysis of variance (ANOVA) with Tukeyrsquos multiple compar-ison test was applied to determine statistical significance P-values lt 005 were considered statistically significant

3 Results

31 Identification of Phytochemicals in JPYF II Using HPLC-ESI-HRMS By comparing the retention times and DAD-spectra of compounds previously reported or standard mate-rials with the experimental compounds the major compo-nents of JPYF II were identified usingHPLC-ESI-HRMS Fig-ure 1 displays the HPLC-ESI-HRMS total ion chromatogram


Table 2 The mass numbers of LCESI-HRMS peaks and the identification results for the compounds of JPYF II

No 119905119877 [M+H]+ Formula Identification Source(min) (mass error ppm)

1 718 47519174 (-136) C20H27NO11 Amygdalinlowast Semen Persicae2 1453 44712805 (-117) C22H22O10 Calycosin-7-O-120573-d-glucosidelowast Radix Astragali3 1511 41423279 (-141) C20H28O8 Lobetyolinlowast Radix Codonopsis4 1760 35713306 (-111) C20H20O6 Vitedoin A Fructus Viticis Negundinis5 1879 35713284 (-119) C20H20O6 Isomer of vitedoin A Fructus Viticis Negundinis

6 1901 53312830 (-125) C25H24O13Calycosin-7-O-120573-d-glycoside-

610158401015840-O-acetate

Radix Astragali

7 2046 43113315 (-118) C22H22O9 Ononinlowast Radix Astragali8 2342 28507553 (-077) C16H12O5 Calycosinlowast Radix Astragali

9 2409 51713391 (-028) C25H24O12Formononetin-7-O-120573-d-glycoside-610158401015840-O-acetate Radix Astragali

10 2854 26908057 (-099) C16H12O4 Formononetinlowast Radix Astragali11 3013 82747821 (-065) C43H70O15 Astragaloside II Radix Astragali12 3032 78147327(002) C42H68O13 Saikosaponin A Radix Bupleuri13 3250 23113776 (-085) C15H18O2 Atractylenolide I Radix Codonopsis

RhizomaAtractylodis

Macrocephalae14 3250 24914832 (-081) C15H20O3 Atractylenolide III Radix Codonopsis

RhizomaAtractylodis

Macrocephalae15 3302 86948914 (-020) C45H72O16 Astragaloside IIsoastragaloside I Radix Astragali16 3328 78147351 (-004) C42H68O13 Saikosaponin D Radix Bupleuri17 3560 23315353 (-033) C15H20O2 Atractylenolide II Radix Codonopsis

RhizomaAtractylodis

MacrocephalaelowastCompared with standard compound

(TIC) With reference to protonated molecular ion massnumber mass difference and retention time compared withthose of standards and literature data a preliminary identi-fication of peaks was performed as listed in Table 2 withproposed compounds

32 Effects of JPYF II on Body Weight of LPS and CS-Exposed Mice Mice exposed to LPS and CS lost weightcomparedwith controlmice (Figure 2) Treatmentwith eitherJPYF II or dexamethasone did not result in body weightreturning to normal However those mice treated with JPYFII did not continue to lose weight unlike those treated withdexamethasone following stimulation with LPS and CS

33 Effects of JPYF II on Inflammatory Cells in BALF Thetotal and differential inflammatory cell numbers in BALFwere quantified to determine the effects of JPYF II oninflammatory cells in LPS and CS-exposed mice As Figure 3demonstrates JPYF II treatment significantly decreased the

total inflammatory cell count in BALF specifically the num-bers of macrophages and neutrophils

34 Effects of JPYF II onmRNAExpression of ProinflammatoryCytokines and MMPs in Lung Tissues As shown in Figure 4LPS and CS-exposed mice demonstrated a notable elevationof the levels of the mRNAof TNF-120572 IL-6 IL-1120573 MMP-9 andMMP-12 in comparison to control mice However JPYF II-treated mice exhibited a significant decrease in TNF-120572 IL-6 IL-1120573 MMP-9 and MMP-12 mRNA levels in lung tissuescompared with LPS and CS-exposed mice

35 Effects of JPYF II on Antioxidant Activity CAT and GSH-Px activities were reduced andMDA levels were increased inthe lung tissues of the LPS and CS-induced mice comparedwith those in control mice However treatment with JPYF IIprevented the decrease in CAT and GSH-Px activities andthe elevation in MDA levels compared with those in LPS andCS-exposed mice The results indicate that JPYF II was able


controlhighdexamethasone

modellow

middle

07

08

09

10

11

12

perc

enta

ge o

f sta

rtin

g w

eigh

t (

)

10 20 300Days

Figure 2 Effects of JPYF II on body weight of mice exposed to LPS and CS Control saline-treated mice model LPS and CS-exposed micelow JPYF II (075 gkgd) + LPS and CS-exposed mice middle JPYF II (15 gkgd) + LPS and CS-exposed mice high JPYF II (3 gkgd) +LPS and CS-exposed mice dexamethasone dexamethasone (2mgkgd) + LPS and CS-exposed mice

to suppress excessive oxidative stress associated with COPD(Figure 5)

36 Expression of p-I120581B120572 and p-p65 in Lung Tissues AsFigure 6 demonstrates phosphorylation of I120581B120572 and NF-120581Bp65 in lung tissues was clearly upregulated in LPS andCS-induced mice which JPYF II treatment significantlyreduced The above results indicate that JPYF II suppressedthe expression of p-I120581B120572 which led to the inhibition of NF-120581B activation

37 JPYF II Decreases Inflammatory Cell Infiltration Caused byExposure to LPS and CS As Figure 7 indicates by compari-son with control mice a clear recruitment of inflammatorycells into the peribronchial and alveolar regions of the lungtissues of LPS and CS-exposed mice was observed Howeverboth JPYF II and dexamethasone-treated mice displayedreduced inflammatory cell infiltration in the bronchial airwaycompared with the LPS and CS-exposed mice

38 Cell Viability of RAW2647 Cells Exposed to CSE andTreated with JPYF II An MTT assay was used to assess theviability of RAW2647 cells exposed to various concentrationsof CSE The results reveal that the metabolism of cells wasinhibited by CSE in a dose- and time-dependent manner(data not shown) A CSE concentration of 15 for 3 h wasselected as the model because of its cytotoxic effects TheRAW2647 cells were then treated with JPYF II (125 25 50100 and 200 120583gmL) alone or in combination with 15 CSEfor 24 h demonstrating that JPYF II displayed no cytotoxiceffects but enhanced the viability of the RAW2647 cells(Figure 8)

39 Effects of JPYF II on the mRNA Expression of Proin-flammatory Cytokines in RAW2647 Cells Stimulated withCSE The ability of JPYF II to suppress the mRNA levels

of CSE-induced inflammatory cytokines (TNF-120572 IL-6 andIL-1120573) was measured Their expression levels were elevatedin RAW2647 cells stimulated with CSE compared withnonstimulated cells whereas the expression of TNF-120572 IL-6 and IL-1120573 mRNA was suppressed in JPYF II-treated cellscompared with CSE-stimulated cells (Figure 9)

310 Phosphorylation of I120581B120572 and p65 in RAW2647 CellsStimulated with CSE As shown in Figure 10 the phospho-rylation of I120581B120572 and p65 was clearly increased in RAW2647cells stimulated with CSE compared with nonstimulated cellswhereas JPYF II significantly suppressed the phosphorylationof I120581B120572 and p65 induced by the stimulation with CSE

4 Discussion

In the present study amousemodel of COPD induced by LPSand CS and RAW2647 cells stimulated with CSE were usedto evaluate the inhibitory effects of JPYF II on COPD In theCOPDmodel JPYF II reduced the total and differential num-bers of inflammatory cells inBALF In addition JPYF II atten-uated inflammatory cell infiltration which was evidenced bydecreases inmRNAexpression of proinflammatory cytokinesandMMPs and the phosphorylation of I120581B120572 and p65 in lungtissues Furthermore JPYF II increased CAT and GSH-Pxactivities whilst decreasing MDA levels indicating that JPYFII could inhibit excessive oxidative stress in the progression ofCOPD In the RAW2647 cells stimulated with CSE JPYF IIclearly suppressed the expression of proinflammatory medi-ators at the mRNA level in addition to the phosphorylationof I120581B120572 and p65 expression

COPD is characterized by reduced airflow and CS isbelieved to be the main risk factor in the progression ofCOPD Exposure to CS elevates inflammatory cell countsleading to constant airway remodeling It also promotesthe production of proinflammatory cytokines and proteases


dexa

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high

mid

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low

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cont

rol

0

1

2

3

4

5To

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in B

ALF

(1 times

5 )

lowastlowastlowast lowastlowastlowast lowastlowast

(a)

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haso

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high

mid

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low

mod

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cont

rol

0

1

2

3

4

5

Tota

l mac

roph

ages

in B

ALF

(1 times

5 )

lowastlowast lowastlowastlowast

(b)

dexa

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haso

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high

mid

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low

mod

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cont

rol

00

05

10

15

Tota

l neu

trop

hils

in B

ALF

(1 times

)

lowastlowastlowast lowastlowastlowast

lowastlowast

(c)

dexa

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haso

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high

mid

dle

low

mod

el

cont

rol

000

005

010

015

Tota

l lym

phoc

ytes

in B

ALF

(1 times

)

(d)

Figure 3 Effects of JPYF II on inflammatory cells in BALF (a) Total cells in BALF (b) total macrophages in BALF (c) total neutrophilsin BALF and (d) total lymphocytes in BALF Control saline-treated mice model LPS and CS-exposed mice low JPYF II (075 gkgd) +LPS and CS-exposed mice middle JPYF II (15 gkgd) + LPS and CS-exposed mice high JPYF II (3 gkgd) + LPS and CS-exposed micedexamethasone dexamethasone (2mgkgd) + LPS and CS-exposed mice Values are presented as means plusmn SD P lt 005 and P lt 0001compared with control mice lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 compared with LPS and CS-exposed mice

which aggravates COPD [39ndash42] Proinflammatory media-tors are associated with exacerbation of the inflammatoryresponse in the development of COPD In addition previousstudies have demonstrated that the concentrations of TNF-120572IL-6 and IL-1120573 increase significantly in human macrophagesexposed to CSE and mice exposed to CS [43 44] Thisstudy demonstrated that treatment with JPYF II significantlysuppressed excessive production of inflammatory cells andproinflammatory cytokines in LPS and CS-exposed miceMoreover JPYF II reduced the mRNA levels of TNF-120572 IL-6 and IL-1120573 in RAW2647 cells stimulated with CSE Theseresults indicate that JPYF II suppressed the inflammatoryresponse associated with COPD

MMPs are proteolytic enzymes which play a major rolein breaking down components of the extracellular matrix

(ECM) [45] Previous studies have demonstrated that a num-ber of MMPs especially MMP-9 may also be associated withairway remodeling [11 12] the migration of inflammatorycells into the lungs and even the destruction of lung tissues[42] Activated MMP-9 has been detected in the phlegmof the majority of COPD patients but absent from healthysubjects in addition to COPD patients possessing enhancedgelatinolytic activity related toMMP-9 in phlegm confirmingthe importance of MMP-9 in COPD [46] Additionallycompared to healthy subjects the levels of MMP-12 in BALFfrom COPD patients were enhanced and the number ofMMP-12-positive macrophages in both BALF and bronchialtissue sections of COPD patients was greater These studiesreveal that MMP-12 also plays an important role in COPD[47] In this study JPYF II treatment significantly suppressed


dexa

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haso

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high

mid

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cont

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00

05

10

15

20

25

TNF-

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rela

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nlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowast

(a)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

00

05

10

15

20

IL-6

mRN

Are

lativ

e exp

ress

ion

lowastlowastlowast


lowastlowastlowast

(b)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

0

1

2

3

4

5

IL-1

mRN

Are

lativ

e exp

ress

ion

lowastlowastlowast


lowast

(c)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

00

05

10

15

20

MM

P-9

mRN

Are

lativ

e exp

ress

ion

lowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowast

(d)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

0

1

2

3

4

5

MM

P-12

mRN

Are

lativ

e exp

ress

ion

lowast lowast

(e)

Figure 4 JPYF II decreased mRNA expression of proinflammatory cytokines and MMPs in lung tissues caused by LPS and CS exposure(a) TNF-120572 (b) IL-6 (c) IL-1120573 (d) MMP-9 and (e) MMP-12 Control saline-treated mice model LPS and CS-exposed mice low JPYF II(075 gkgd) + LPS and CS-exposed mice middle JPYF II (15 gkgd) + LPS and CS-exposed mice high JPYF II (3 gkgd) + LPS and CS-exposed mice dexamethasone dexamethasone (2mgkgd) + LPS and CS-exposed mice Values are presented as means plusmn SD P lt 005 Plt 001 and P lt 0001 compared with control mice lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 compared with LPS and CS-exposed mice

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

0

10

20

30

40

CAT

(Um

g)

lowast


(a)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

0

20

40

60

80

100

GSH

-Px

(Um

g)

lowastlowast

lowastlowast

(b)

de

xam

etha

sone

high

mid

dle

low

mod

el

cont

rol

00

05

10

15

20

25

MD

A (n

mol

mg)

lowastlowast

lowast

lowast

(c)

Figure 5 JPYF II increased the activities of CAT and GSH-Px as well as decreased the level of MDA in lung tissues caused by LPS and CSexposure (a) CAT (b) GSH-Px and (c) MDA Control saline-treated mice model LPS and CS-exposed mice low JPYF II (075 gkgd) +LPS and CS-exposed mice middle JPYF II (15 gkgd) + LPS and CS-exposed mice high JPYF II (3 gkgd) + LPS and CS-exposed micedexamethasone dexamethasone (2mgkgd) + LPS and CS-exposed mice Values are presented as means plusmn SD P lt 0001 compared withcontrol mice lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 compared with LPS and CS-exposed mice


p65

p-p65

IB

p-IB

-actin

Jianpiyifei II(gkgd)

dexamethasone(mgkgd)

CS + LPS

2

(a)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

00

05

10

15

20

Rela

tive i

nten

sity

of p

-p65

00

05

10

15

20

Rela

tive i

nten

sity

of p

-I

B

lowastlowastlowast

lowastlowast

lowastlowastlowast lowastlowast

(b)

Figure 6 JPYF II reduced phosphorylations of I120581B120572 and NF-120581Bp65 in lung tissues caused by LPS and CS exposure (a) Representative figureof protein expression and (b) quantitative analysis of protein expression Control saline-treatedmice model LPS and CS-exposedmice lowJPYF II (075 gkgd) + LPS and CS-exposed mice middle JPYF II (15 gkgd) + LPS and CS-exposed mice high JPYF II (3 gkgd) + LPSand CS-exposed mice dexamethasone dexamethasone (2mgkgd) + LPS and CS-exposed mice Values are presented as means plusmn SD P lt005 and P lt 001 compared with control mice lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 compared with LPS and CS-exposed mice

Peribronchial region

Alveolarregion

control model low middle high dexamethasone

Figure 7 JPYF II inhibited inflammatory cell infiltration caused by LPS and CS exposure Inflammatory infiltration in the peribronchial andalveolar regions was revealed by H amp E staining Control saline-treated mice model LPS and CS-exposed mice low JPYF II (075 gkgd)+ LPS and CS-exposed mice middle JPYF II (15 gkgd) + LPS and CS-exposed mice high JPYF II (3 gkgd) + LPS and CS-exposed micedexamethasone dexamethasone (2mgkgd) + LPS and CS-exposed mice

the elevated levels of MMP-9 and 12 in mice exposed to LPSand CS These results suggest that the ameliorative effect ofJPYF II on COPDmight also be associated with its inhibitoryeffects on MMPs

Excessive ROS produced by epithelial and inflamma-tory cells stimulated by CS creates an oxidantantioxidantimbalance leading to oxidative stress and induces lipid

peroxidation causing serious damage to cellular and subcel-lular organelle membranes and function [48] Antioxidantenzymes can either facilitate antioxidant reactions or directlydecompose ROS [49] GSH-Px and CAT play significant rolesin peroxyl scavenging mechanisms and in maintaining thefunctional integration of cell membranes [50] In additionas the main product of lipid peroxidation MDA is closely


200

gm

L

100

gm

L

50

gm

L

25

gm

L

125

g

mL

cont

rol

0

50

100

150C

ell v

iabi

lity

( o

f con

trol

)

(a)

0

25

5075

100

125

150

Cel

l via

bilit

y (

of c

ontr

ol)

200

gm

L

100

gm

L

50

gm

L

25

gm

L

125

g

mL

cont

rol

mod

el

lowastlowast lowastlowast lowastlowast lowastlowast lowast

(b)

Figure 8 Cell viability was quantified by MTT assay RAW2647 cells were treated with various concentrations of (a) JPYF II or (b) incombination with CSE for 24 h Values are presented as means plusmn SD P lt 0001 compared with control group lowast119875 lt 005 and lowastlowast119875 lt 001compared with LPS and CS-exposed mice

50

gm

L

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

0

5

10

15

20

TNF-

m

RNA

rela

tive e

xpre

ssio

n

lowast

lowastlowastlowastlowast

(a)

50

g

mL

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

0

10

20

30

IL-6

mRN

Are

lativ

e exp

ress

ion


lowastlowastlowast

lowastlowastlowast

(b)

50

gm

L

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

0

10

20

30

IL-1

mRN

Are

lativ

e exp

ress

ion

lowastlowastlowast

lowast

(c)

Figure 9 JPYF II reduced mRNA expression of proinflammatory cytokines in CSE-stimulated RAW2647 cells (a) TNF-120572 (b) IL-6 and (c)IL-1120573 control RAW2647 cells without any treatment model RAW2647 cells induced by CSE 125 120583gmL CSE-stimulated RAW2647 cellstreated with JPYF II (125120583gmL) 25120583gmL CSE-stimulated RAW2647 cells treated with JPYF II (25120583gmL) 50120583gmL CSE-stimulatedRAW2647 cells treated with JPYF II (50 120583gmL) dexamethasone CSE-stimulated RAW2647 cells treated with dexamethasone (50 120583gmL)Values are presented asmeans plusmn SD P lt 0001 compared with control group lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 compared with modelgroup

associated with pulmonary dysfunction and considered abiomarker in the assessment of lipid peroxidation [51] Ourinvestigation indicated that JPYF II administration could sig-nificantly increase CAT and GSH-Px activities and decreaseMDA levels in lung tissues obtained from JPYF II-treatedmice demonstrating the antioxidation properties of JPYF II

NF-120581B is normally present in an inactive form in thecytosol held by its repressor (I120581B) [43] Activation of NF-120581B can be induced by proinflammatory stimuli which causedegradation of I120581B CS is a potential stimulus that can inducethe phosphorylation of NF-120581Bp65 and eventually activateNF-120581B [52] In bronchial biopsies from smokers and in guineapigs exposed to CS NF-120581B has been shown to be significantlyexpressed regulating the production of many inflammatory

cytokines [53] In the present study phosphorylation of I120581B120572and NF-120581Bp65 was meaningfully elevated in mice exposedto LPS and CS and RAW2647 cells stimulated with CSEHowever JPYF II treatment inhibited activation of NF-120581Bindicating that JPYF II suppressed the inflammatory responsecaused by LPS and CS exposure or CSE stimulation probablyassociated with downregulation of the NF-120581B pathway

5 Conclusions

In summary our study demonstrated that JPYF II provided aprotective effect in LPS and CS-induced COPDmice througha reduction of the inflammatory response and oxidativestress In addition JPYF II suppressed the secretion of


p65

p-p65

IB

p-IB

GAPDH

Jianpiyifei II(gmL)

(gmL)dexamethasone

CSE

(a)

50

gm

L

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

50

gm

L

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

lowast lowastlowast



00

05

10

15

20

Rela

tive i

nten

sity

of p

-p65

(fol

d of

cont

rol)

00

05

10

15

20

Rela

tive i

nten

sity

of p

-I

B (f

old

of co

ntro

l)(b)

Figure 10 JPYF II inhibited the phosphorylation of I120581B120572 and NF-120581Bp65 expression in CSE-stimulated RAW2647 cells (a) Representativefigure of protein expression and (b) quantitative analysis of protein expression Control RAW2647 cells without any treatment modelRAW2647 cells induced byCSE dexamethasone CSE-stimulatedRAW2647 cells treatedwith dexamethasone (50120583gmL) 125 120583gmL CSE-stimulated RAW2647 cells treated with JPYF II (125 120583gmL) 25120583gmL CSE-stimulated RAW2647 cells treated with JPYF II (25120583gmL)50120583gmL CSE-stimulated RAW2647 cells treated with JPYF II (50120583gmL) Values are presented as means plusmn SD P lt 005 and P lt 0001compared with control group lowast119875 lt 005 and lowastlowastlowast119875 lt 0001 compared with model group

inflammatory cytokines in RAW2647 cells stimulated withCSEThese effects might be related to suppression of the NF-120581B pathway Hence our study demonstrates that JPYF II hasthe potential to prevent COPD

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

Long Fan Ruifeng Chen and Leng Li contributed equally tothe work

Acknowledgments

This work was supported financially by the National NaturalScience Foundation of China (Grants nos 81573895 81673897

81603554 and 31500285) and the Natural Science Foundationof Guangdong Province (Grant no 2015A030310529)

References

[1] R A Pauwels A S Buist P M A Calverley C R Jenkinsand S S Hurd ldquoGlobal strategy for the diagnosis managementand prevention of chronic obstructive pulmonary diseaseNHLBIWHO Global Initiative for Chronic Obstructive LungDisease (GOLD) workshop summaryrdquo American Journal ofRespiratory and Critical Care Medicine vol 163 no 5 pp 1256ndash1276 2001

[2] D M Mannino and A S Buist ldquoGlobal burden of COPD riskfactors prevalence and future trendsrdquoThe Lancet vol 370 no9589 pp 765ndash773 2007

[3] J Balmes M Becklake P Blanc et al ldquoAmerican ThoracicSociety Statement Occupational contribution to the burden ofairway diseaserdquo American Journal of Respiratory and CriticalCare Medicine vol 167 pp 787ndash797 2003

[4] C A Jimenez-Ruiz S Andreas K E Lewis et al ldquoStatementon smoking cessation in COPD and other pulmonary diseasesand in smokers with comorbidities who find it difficult to quitrdquoEuropean Respiratory Journal vol 46 no 1 pp 61ndash79 2015


[5] T Tsuji K Aoshiba and A Nagai ldquoCigarette smoke inducessenescence in alveolar epithelial cellsrdquo American Journal ofRespiratory Cell and Molecular Biology vol 31 no 6 pp 643ndash649 2004

[6] P J Barnes ldquoNew anti-inflammatory targets for chronicobstructive pulmonary diseaserdquo Nature Reviews Drug Discov-ery vol 12 no 7 pp 543ndash559 2013

[7] P J Barnes ldquoMediators of chronic obstructive pulmonarydiseaserdquo Pharmacological Reviews vol 56 no 4 pp 515ndash5482004

[8] T Chen Y Mou J Tan et al ldquoThe protective effect of CDDO-Me on lipopolysaccharide-induced acute lung injury in micerdquoInternational Immunopharmacology vol 25 no 1 pp 55ndash642015

[9] Z Q Cheng and L Li ldquoGinsenoside Rg3 ameliorateslipopolysaccharide-induced acute lung injury in mice throughinactivating the nuclear factor-120581B (NF-120581B) signaling pathwayrdquoInternational Immunopharmacology vol 34 pp 53ndash59 2016

[10] S Q Yang Z R Yu L Wang et al ldquoThe natural productbergenin ameliorates lipopolysaccharide-induced acute lunginjury by inhibiting NF-kappaB activitionrdquo Journal of Ethno-pharmacology vol 200 pp 147ndash155 2017

[11] F Sampsonas A Kaparianos D Lykouras K Karkouliasand K Spiropoulos ldquoDNA sequence variations of metallopro-teinasesTheir role in asthma and COPDrdquoPostgraduateMedicalJournal vol 83 no 978 pp 244ndash250 2007

[12] K Grzela M Litwiniuk W Zagorska and T Grzela ldquoAirwayRemodeling in Chronic Obstructive Pulmonary Disease andAsthma the Role of Matrix Metalloproteinase-9rdquo ArchivumImmunologiae et Therapia Experimentalis vol 64 no 1 pp 47ndash55 2016

[13] R D Hautamaki D K Kobayashi R M Senior and S DShapiro ldquoRequirement for macrophage elastase for cigarettesmoke-induced emphysema inmicerdquo Science vol 277 no 5334pp 2002ndash2004 1997

[14] D F Church and W A Pryor ldquoFree-radical chemistry ofcigarette smoke and its toxicological implicationsrdquoEnvironmen-tal Health Perspectives vol 64 pp 111ndash126 1985

[15] T Nakayama D F Church and W A Pryor ldquoQuantitativeanalysis of the hydrogen peroxide formed in aqueous cigarettetar extractsrdquo Free Radical Biology amp Medicine vol 7 no 1 pp9ndash15 1989

[16] W A Pryor K Stone C E Cross L Machlin and L PackerldquoOxidants in cigarette smoke radicals hydrogen peroxide per-oxynitrate and peroxynitriterdquoAnnals of the New York Academyof Sciences vol 686 pp 12ndash28 1993

[17] I Rahman ldquoPharmacological antioxidant strategies as thera-peutic interventions for COPDrdquo Biochimica et Biophysica Acta(BBA) - Molecular Basis of Disease vol 1822 no 5 pp 714ndash7282012

[18] R Vlahos and S Bozinovski ldquoGlutathione peroxidase-1 as anovel therapeutic target for COPDrdquo Redox Report vol 18 no4 pp 142ndash149 2013

[19] I Rahman and I M Adcock ldquoOxidative stress and redox reg-ulation of lung inflammation in COPDrdquo European RespiratoryJournal vol 28 no 1 pp 219ndash242 2006

[20] A Kumari N Tyagi D Dash and R Singh ldquoIntranasal Cur-cumin Ameliorates Lipopolysaccharide-Induced Acute LungInjury in Micerdquo Inflammation vol 38 no 3 pp 1103ndash1112 2015

[21] C S Stevenson and M A Birrell ldquoMoving towards a newgeneration of animal models for asthma and COPD with

improved clinical relevancerdquoPharmacologyampTherapeutics vol130 no 2 pp 93ndash105 2011

[22] J Stolk A Rudolphus P Davies et al ldquoInduction of emphy-sema and bronchial mucus cell hyperplasia by intratrachealinstillation of lipopolysaccharide in the hamsterrdquo The Journalof Pathology vol 167 no 3 pp 349ndash356 1992

[23] J G Martin and M Tamaoka ldquoRat models of asthma andchronic obstructive lung diseaserdquo Pulmonary PharmacologyandTherapeutics vol 19 no 6 pp 377ndash385 2006

[24] Y-C Nie H Wu P-B Li et al ldquoCharacteristic comparison ofthree rat models induced by cigarette smoke or combined withLPS to establish a suitable model for study of airway mucushypersecretion in chronic obstructive pulmonary diseaserdquo Pul-monary Pharmacology andTherapeutics vol 25 no 5 pp 349ndash356 2012

[25] E L Hardaker M S Freeman N Dale et al ldquoExposing rodentsto a combination of tobacco smoke and lipopolysaccharideresults in an exaggerated inflammatory response in the lungrdquoBritish Journal of Pharmacology vol 160 no 8 pp 1985ndash19962010

[26] G G Brusselle G F Joos and K R Bracke ldquoNew insights intothe immunology of chronic obstructive pulmonary diseaserdquoThe Lancet vol 378 no 9795 pp 1015ndash1026 2011

[27] A F Valledor M Comalada L F Santamarıa-Babi J Lloberasand A Celada ldquoMacrophage Proinflammatory Activation andDeactivation AQuestion of BalancerdquoAdvances in Immunologyvol 108 pp 1ndash20 2010

[28] Q Tang S Yang J Tong et al ldquoHemostasis and uterinecontraction promoting effect of the extract from drugs inthe Zi-Yin-Tiao-Jing granule a traditional Chinese compoundpreparationrdquo Journal of Ethnopharmacology vol 211 pp 278ndash284 2018

[29] Y Q Guo T Yin X M Wang et al ldquoTraditional usesphytochemistry pharmacology and toxicology of the genusCimicifuga A reviewrdquo Journal of Ethnopharmacology vol 209pp 264ndash282 2017

[30] A Kuroiwa S Liou H Yan A Eshita S Naitoh and ANagayama ldquoEffect of a traditional Japanese herbal medicineHochu-ekki-to (Bu-Zhong-Yi-Qi Tang) on immunity in elderlypersonsrdquo International Immunopharmacology vol 4 no 2 pp317ndash324 2004

[31] S-H Yang and C-L Yu ldquoAntiinflammatory effects of Bu-zhong-yi-qi-tang in patients with perennial allergic rhinitisrdquoJournal of Ethnopharmacology vol 115 no 1 pp 104ndash109 2008

[32] Z H Cui Z Q Guo J H Miao et al ldquoThe genus Cynomoriumin China an ethnopharmacological and phytochemical reviewrdquoJournal of Ethnopharmacology vol 147 no 1 pp 1ndash15 2013

[33] R-U Haq A-U A Shah A-U Khan et al ldquoAntitussive andtoxicological evaluation of Vitex negundordquo Natural ProductResearch (Formerly Natural Product Letters) vol 26 no 5 pp484ndash488 2012

[34] S Y Xi L C Qian H Y Tong et al ldquoToxicity and clinicalreasonable application of Taoren (Semen Persicae) based onancient and modern literature researchrdquo Journal of TraditionalChinese Medicine vol 33 no 2 pp 272ndash279 2013

[35] L Wu L Lin Y J Xu et al ldquoClinical study on 178 cases ofstable stage of chronic obstructive pulmonary disease treatedby Jianpiyifei IIrdquo Journal of Traditional Chinese Medicine vol52 pp 1465ndash1468 2011

[36] L Lin X H Yu Y J Xu M J Zhou L Wu and L N WuldquoEffects of Jianpi Yifei II formula on regulation of oxidantanti-oxidant imbalance and ultrastructure of lung tissues induced by


CSE andLPS in ratsrdquo Journal ofNanjingUniversity of TraditionalChinese Medicine vol 31 pp 39ndash43 2015

[37] L Lin Y J Xu L Wu Z X Chen and X H Yu ldquoInfluenceof Jianpi Yifei II decoction on inflammatory cytokines andmetalloproteases in lung tissues of rats induced by cigarettesmoke and LPSrdquo Journal of Tianjin University of TraditionalChinese Medicine vol 33 pp 342ndash346 2014

[38] H R W Wirtz and M Schmidt ldquoAcute influence of cigarettesmoke on secretion of pulmonary surfactant in rat alveolar typeII cells in culturerdquoEuropeanRespiratory Journal vol 9 no 1 pp24ndash32 1996

[39] A T D C Hoepers M M Menezes and T S FrodeldquoSystematic review of anaemia and inflammatory markers inchronic obstructive pulmonary diseaserdquo Clinical and Experi-mental Pharmacology and Physiology vol 42 no 3 pp 231ndash2392015

[40] S Sethi D A Mahler P Marcus C A Owen B Yawnand S Rennard ldquoInflammation in COPD Implications formanagementrdquoAmerican Journal of Medicine vol 125 no 12 pp1162ndash1170 2012

[41] M F Abd El-Fatah M A Ghazy M S Mostafa M M El-Attar and A Osman ldquoIdentification of MMP-9 as a biomarkerfor detecting progression of chronic obstructive pulmonary dis-easerdquo The International Journal of Biochemistry amp Cell Biologyvol 93 no 6 pp 541ndash547 2015

[42] I K Demedts G G Brusselle K R Bracke K Y Vermaelenand R A Pauwels ldquoMatrix metalloproteinases in asthma andCOPDrdquoCurrent Opinion in Pharmacology vol 5 no 3 pp 257ndash263 2005

[43] R Zhou F Luo H Lei et al ldquoLiujunzi Tang a famous tradi-tional Chinese medicine ameliorates cigarette smoke-inducedmousemodel of COPDrdquo Journal of Ethnopharmacology vol 193pp 643ndash651 2016

[44] E Mortaz I M Adcock F L M Ricciardolo et alldquoAnti-inflammatory Eeffects of Lactobacillus rahmnosus andbifidobacterium breve on cigarette smoke activated humanmacrophagesrdquo PLoS ONE vol 10 no 8 Article ID e01364552015

[45] W C Parks C L Wilson and Y S Lopez-Boado ldquoMatrixmetalloproteinases as modulators of inflammation and innateimmunityrdquo Nature Reviews Immunology vol 4 no 8 pp 617ndash629 2004

[46] D Cataldo C Munaut A Noel et al ldquoMMP-2- and MMP-9-linked gelatinolytic activity in the sputum from patients withasthma and chronic obstructive pulmonary diseaserdquo Interna-tional Archives of Allergy and Immunology vol 123 no 3 pp259ndash267 2000

[47] S Molet C Belleguic H Lena et al ldquoIncrease in macrophageelastase (MMP-12) in lungs from patients with chronic obstruc-tive pulmonary diseaserdquo Inflammation Research vol 54 no 1pp 31ndash36 2005

[48] S Carnesecchi J-C Pache and C Barazzone-Argiroffo ldquoNOXenzymes potential target for the treatment of acute lung injuryrdquoCellular and Molecular Life Sciences vol 69 no 14 pp 2373ndash2385 2012

[49] M Christofidou-Solomidou and V R Muzykantov ldquoAntioxi-dant strategies in respiratory medicinerdquo Treatments in Respira-tory Medicine vol 5 no 1 pp 47ndash78 2006

[50] S Singh S K Verma S Kumar et al ldquoEvaluation of OxidativeStress and Antioxidant Status in Chronic Obstructive Pul-monary Diseaserdquo Scandinavian Journal of Immunology vol 85no 2 pp 130ndash137 2017

[51] J Wang C-H Ma and S-M Wang ldquoEffects of acteoside onlipopolysaccharide-induced inflammation in acute lung injuryvia regulation of NF-kappaB pathway in vivo and in vitrordquoToxicology and Applied Pharmacology vol 285 no 2 pp 128ndash135 2015

[52] N-R Shin J-WKo S-H Park et al ldquoProtective effect ofHwan-gRyunHaeDok-Tang water extract against chronic obstructivepulmonary disease induced by cigarette smoke and lipopolysac-charide in a mouse modelrdquo Journal of Ethnopharmacology vol200 pp 60ndash65 2017

[53] W Wang X G Li and J Xu ldquoExposure to cigarette smokedownregulates 1205732-adrenergic receptor expression and upregu-lates inflammation in alveolar macrophagesrdquo Inhalation Toxi-cology vol 27 no 10 pp 488ndash494 2015

Stem Cells International

Hindawiwwwhindawicom Volume 2018


MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

OphthalmologyJournal of


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Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom


Table 1 The components of JPYF II

Herbal name Botanical name Family Medicinal part

Radix Astragali Astragalus membranaceus(Fisch) Bunge Leguminosae Root

Rhizoma Cimicifugae Cimicifuga foetida L Ranunculaceae Rhizome

Radix Codonopsis Codonopsis pilosula (Franch)Nannf Campanulaceae Root

Rhizoma AtractylodisMacrocephalae Atractylodes macrocephala koidz Asteraceae Rhizome

Radix Bupleuri Bupleurum chinense DC Umbelliferae RootHerba Cynomorii Cynomorium songaricum Rupr Cynomoriaceae Whole plantFructus Viticis Negundinis Vitex negundo L Verbenaceae FruitSemen Persicae Prunus persica (L) Batsch Rosaceae Seed

in inhibition of lung neutrophil and cytokine generation[9 10] Furthermore recent studies indicate that a numberof MMPs may also contribute to airway remodeling [11 12]It is noteworthy that MMP-9 which is secreted primarilyby macrophages and neutrophils is considered a pivotalexecutor in an inflammatory response In addition it hasbeen shown that emphysema induced by chronicCS exposuredoes not occur in MMP-12minusminus mice [13] indicating thatMMP-12 can have a destructive role in COPD

CS contains more than 1015 free radicals or oxidants andthousands of different chemical components per puff [14ndash17]which elevates the concentration of reactive oxygen species(ROS) produced enzymatically by epithelial and inflamma-tory cells in the lung The production of additional ROSleads to the initiation of an inflammatory response throughthe activation of transcriptional factors such as activatorprotein (AP-1) and NF-120581B in addition to gene expressionof proinflammatory cytokines [17ndash19] Therefore oxidativestress also plays a key role in COPD and affects infiltrationof surfactants to the alveoli mitochondrial respiration andremodeling of the extracellular matrix [17]

CS exposure is recognized as the major factor in thepathogenesis of COPD In addition as an important proin-flammatory glycolipid component of the outer membrane ofGram-negative bacteria LPS is often used in animal modelsof lung diseases including COPD and acute lung injury[20ndash22] Thus a combination of LPS and CS has becomethe preferred in vivo irritant as it shortens the duration ofexposure required forCOPD todevelop into an animalmodeland so enables deeper investigation of its pathological charac-teristics [21 23ndash25] Therefore intratracheal administrationof LPS and exposure to CS were combined to construct amouse model of COPD in the present study Moreover CSactivates innate immune cells such as macrophages whichplay a key role in promoting inflammation and the secretionof inflammatory cytokines It is known that their excessiveactivation has damaging effects and persistence of proin-flammatory activity results in the development of chronicinflammation such as COPD [26 27] Hence exposure ofthemousemacrophage cell lineRAW2647 to cigarette smokeextract (CSE) was also adopted in the present study

Jianpiyifei II granule (JPYF II) an oriental herbalformula consists of eight traditional Chinese medicines(TCMs) as shown in Table 1 including Radix Astragali Rhi-zoma Cimicifugae Radix Codonopsis Rhizoma AtractylodisMacrocephalae Radix Bupleuri Herba Cynomorii FructusViticis Negundinis and Semen Persicae Radix AstragaliRadix Codonopsis and Rhizoma Atractylodis Macrocepha-lae are used in TCM to invigorate Qi [28] and RhizomaCimicifugae can be used to treat a collapse of the mid-dle Qi [29] Modern pharmacological studies suggest thatthey enhance natural immune function Bu-Zhong-Yi-Qi-Tang (BT) a famous herbal formula used in China andJapan whose formulation also includes Radix AstragaliRhizoma Cimicifugae Rhizoma Atractylodis Macrocepha-lae and Radix Bupleuri demonstrates immunomodulationand anti-inflammatory effects [30 31] Furthermore HerbaCynomorii has been used in the treatment of kidney-yangdeficiency which can lead to asthma [32] Fructus ViticisNegundinis has traditionally been used to treat chronicbronchitis and coughs [33] Semen Persicae which regulateslung Qi and removes stasis is traditionally combined withother herbs to dissipate phlegm relieve coughs and treatasthma caused by the upward reversal of lung Qi [34]Hence JPYF II is formulated to treat COPD based onthe traditional uses of the eight medicinal herbs describedabove using the principle of ldquoJun-Chen-Zuo-Shirdquo (ldquoemperor-minister-adjuvant-courierrdquo) Radix Astragali and RhizomaCimicifugae act as ldquoJunrdquo to treat the main disease RadixCodonopsis and Rhizoma Atractylodis Macrocephalae playthe role of ldquoChenrdquo to increase the function of ldquoJunrdquo RadixBupleuri Herba Cynomorii Fructus Viticis Negundinis andSemen Persicae treat the accompanying symptoms as ldquoZuordquo

JPYF II has been prescribed to treat COPD inGuangdongProvincial Hospital of Chinese Medicine for more than adecade In a clinical study performed previously by theauthors it was established that JPYF IIwas able to significantlyincrease walking distance over six minutes and decreaseSt Georgersquos Respiratory Questionnaire (SGRQ) score in atreatment group of 178 patients demonstrating that JPYF IIhad a clear therapeutic effect in stable stage COPD patients[35] In addition our previous studies demonstrated that

























3 Results







610158401015840-O-acetate

Radix Astragali




RhizomaAtractylodis


RhizomaAtractylodis


RhizomaAtractylodis










modellow

middle

07

08

09

10

11

12

perc

enta

ge o

f sta

rtin

g w

eigh

t (

)

10 20 300Days









4 Discussion




dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

0

1

2

3

4

5To

tal c

ells

in B

ALF

(1 times

5 )


(a)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

0

1

2

3

4

5

Tota

l mac

roph

ages

in B

ALF

(1 times

5 )


(b)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

00

05

10

15

Tota

l neu

trop

hils

in B

ALF

(1 times

)


lowastlowast

(c)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

000

005

010

015

Tota

l lym

phoc

ytes

in B

ALF

(1 times

)

(d)






dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

00

05

10

15

20

25

TNF-

m

RNA

rela

tive e

xpre

ssio

nlowastlowastlowast


lowastlowast

(a)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

00

05

10

15

20

IL-6

mRN

Are

lativ

e exp

ress

ion

lowastlowastlowast


lowastlowastlowast

(b)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

0

1

2

3

4

5

IL-1

mRN

Are

lativ

e exp

ress

ion

lowastlowastlowast


lowast

(c)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

00

05

10

15

20

MM

P-9

mRN

Are

lativ

e exp

ress

ion


lowastlowastlowast

(d)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

0

1

2

3

4

5

MM

P-12

mRN

Are

lativ

e exp

ress

ion

lowast lowast

(e)


dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

0

10

20

30

40

CAT

(Um

g)

lowast


(a)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

0

20

40

60

80

100

GSH

-Px

(Um

g)

lowastlowast

lowastlowast

(b)

de

xam

etha

sone

high

mid

dle

low

mod

el

cont

rol

00

05

10

15

20

25

MD

A (n

mol

mg)

lowastlowast

lowast

lowast

(c)



p65

p-p65

IB

p-IB

-actin



CS + LPS

2

(a)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

00

05

10

15

20

Rela

tive i

nten

sity

of p

-p65

00

05

10

15

20

Rela

tive i

nten

sity

of p

-I

B

lowastlowastlowast

lowastlowast


(b)



Alveolarregion







200

gm

L

100

gm

L

50

gm

L

25

gm

L

125

g

mL

cont

rol

0

50

100

150C

ell v

iabi

lity

( o

f con

trol

)

(a)

0

25

5075

100

125

150

Cel

l via

bilit

y (

of c

ontr

ol)

200

gm

L

100

gm

L

50

gm

L

25

gm

L

125

g

mL

cont

rol

mod

el


(b)


50

gm

L

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

0

5

10

15

20

TNF-

m

RNA

rela

tive e

xpre

ssio

n

lowast


(a)

50

g

mL

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

0

10

20

30

IL-6

mRN

Are

lativ

e exp

ress

ion


lowastlowastlowast

lowastlowastlowast

(b)

50

gm

L

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

0

10

20

30

IL-1

mRN

Are

lativ

e exp

ress

ion

lowastlowastlowast

lowast

(c)





5 Conclusions



p65

p-p65

IB

p-IB

GAPDH

Jianpiyifei II(gmL)

(gmL)dexamethasone

CSE

(a)

50

gm

L

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

50

gm

L

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

lowast lowastlowast



00

05

10

15

20

Rela

tive i

nten

sity

of p

-p65

(fol

d of

cont

rol)

00

05

10

15

20

Rela

tive i

nten

sity

of p

-I

B (f

old

of co

ntro

l)(b)



Data Availability






Acknowledgments



References






























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of











































3 Results







610158401015840-O-acetate

Radix Astragali




RhizomaAtractylodis


RhizomaAtractylodis


RhizomaAtractylodis










modellow

middle

07

08

09

10

11

12

perc

enta

ge o

f sta

rtin

g w

eigh

t (

)

10 20 300Days









4 Discussion




dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

0

1

2

3

4

5To

tal c

ells

in B

ALF

(1 times

5 )


(a)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

0

1

2

3

4

5

Tota

l mac

roph

ages

in B

ALF

(1 times

5 )


(b)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

00

05

10

15

Tota

l neu

trop

hils

in B

ALF

(1 times

)


lowastlowast

(c)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

000

005

010

015

Tota

l lym

phoc

ytes

in B

ALF

(1 times

)

(d)






dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

00

05

10

15

20

25

TNF-

m

RNA

rela

tive e

xpre

ssio

nlowastlowastlowast


lowastlowast

(a)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

00

05

10

15

20

IL-6

mRN

Are

lativ

e exp

ress

ion

lowastlowastlowast


lowastlowastlowast

(b)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

0

1

2

3

4

5

IL-1

mRN

Are

lativ

e exp

ress

ion

lowastlowastlowast


lowast

(c)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

00

05

10

15

20

MM

P-9

mRN

Are

lativ

e exp

ress

ion


lowastlowastlowast

(d)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

0

1

2

3

4

5

MM

P-12

mRN

Are

lativ

e exp

ress

ion

lowast lowast

(e)


dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

0

10

20

30

40

CAT

(Um

g)

lowast


(a)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

0

20

40

60

80

100

GSH

-Px

(Um

g)

lowastlowast

lowastlowast

(b)

de

xam

etha

sone

high

mid

dle

low

mod

el

cont

rol

00

05

10

15

20

25

MD

A (n

mol

mg)

lowastlowast

lowast

lowast

(c)



p65

p-p65

IB

p-IB

-actin



CS + LPS

2

(a)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

00

05

10

15

20

Rela

tive i

nten

sity

of p

-p65

00

05

10

15

20

Rela

tive i

nten

sity

of p

-I

B

lowastlowastlowast

lowastlowast


(b)



Alveolarregion







200

gm

L

100

gm

L

50

gm

L

25

gm

L

125

g

mL

cont

rol

0

50

100

150C

ell v

iabi

lity

( o

f con

trol

)

(a)

0

25

5075

100

125

150

Cel

l via

bilit

y (

of c

ontr

ol)

200

gm

L

100

gm

L

50

gm

L

25

gm

L

125

g

mL

cont

rol

mod

el


(b)


50

gm

L

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

0

5

10

15

20

TNF-

m

RNA

rela

tive e

xpre

ssio

n

lowast


(a)

50

g

mL

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

0

10

20

30

IL-6

mRN

Are

lativ

e exp

ress

ion


lowastlowastlowast

lowastlowastlowast

(b)

50

gm

L

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

0

10

20

30

IL-1

mRN

Are

lativ

e exp

ress

ion

lowastlowastlowast

lowast

(c)





5 Conclusions



p65

p-p65

IB

p-IB

GAPDH

Jianpiyifei II(gmL)

(gmL)dexamethasone

CSE

(a)

50

gm

L

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

50

gm

L

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

lowast lowastlowast



00

05

10

15

20

Rela

tive i

nten

sity

of p

-p65

(fol

d of

cont

rol)

00

05

10

15

20

Rela

tive i

nten

sity

of p

-I

B (f

old

of co

ntro

l)(b)



Data Availability






Acknowledgments



References






























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of
























610158401015840-O-acetate

Radix Astragali




RhizomaAtractylodis


RhizomaAtractylodis


RhizomaAtractylodis










modellow

middle

07

08

09

10

11

12

perc

enta

ge o

f sta

rtin

g w

eigh

t (

)

10 20 300Days









4 Discussion




dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

0

1

2

3

4

5To

tal c

ells

in B

ALF

(1 times

5 )


(a)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

0

1

2

3

4

5

Tota

l mac

roph

ages

in B

ALF

(1 times

5 )


(b)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

00

05

10

15

Tota

l neu

trop

hils

in B

ALF

(1 times

)


lowastlowast

(c)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

000

005

010

015

Tota

l lym

phoc

ytes

in B

ALF

(1 times

)

(d)






dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

00

05

10

15

20

25

TNF-

m

RNA

rela

tive e

xpre

ssio

nlowastlowastlowast


lowastlowast

(a)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

00

05

10

15

20

IL-6

mRN

Are

lativ

e exp

ress

ion

lowastlowastlowast


lowastlowastlowast

(b)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

0

1

2

3

4

5

IL-1

mRN

Are

lativ

e exp

ress

ion

lowastlowastlowast


lowast

(c)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

00

05

10

15

20

MM

P-9

mRN

Are

lativ

e exp

ress

ion


lowastlowastlowast

(d)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

0

1

2

3

4

5

MM

P-12

mRN

Are

lativ

e exp

ress

ion

lowast lowast

(e)


dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

0

10

20

30

40

CAT

(Um

g)

lowast


(a)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

0

20

40

60

80

100

GSH

-Px

(Um

g)

lowastlowast

lowastlowast

(b)

de

xam

etha

sone

high

mid

dle

low

mod

el

cont

rol

00

05

10

15

20

25

MD

A (n

mol

mg)

lowastlowast

lowast

lowast

(c)



p65

p-p65

IB

p-IB

-actin



CS + LPS

2

(a)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

00

05

10

15

20

Rela

tive i

nten

sity

of p

-p65

00

05

10

15

20

Rela

tive i

nten

sity

of p

-I

B

lowastlowastlowast

lowastlowast


(b)



Alveolarregion







200

gm

L

100

gm

L

50

gm

L

25

gm

L

125

g

mL

cont

rol

0

50

100

150C

ell v

iabi

lity

( o

f con

trol

)

(a)

0

25

5075

100

125

150

Cel

l via

bilit

y (

of c

ontr

ol)

200

gm

L

100

gm

L

50

gm

L

25

gm

L

125

g

mL

cont

rol

mod

el


(b)


50

gm

L

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

0

5

10

15

20

TNF-

m

RNA

rela

tive e

xpre

ssio

n

lowast


(a)

50

g

mL

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

0

10

20

30

IL-6

mRN

Are

lativ

e exp

ress

ion


lowastlowastlowast

lowastlowastlowast

(b)

50

gm

L

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

0

10

20

30

IL-1

mRN

Are

lativ

e exp

ress

ion

lowastlowastlowast

lowast

(c)





5 Conclusions



p65

p-p65

IB

p-IB

GAPDH

Jianpiyifei II(gmL)

(gmL)dexamethasone

CSE

(a)

50

gm

L

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

50

gm

L

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

lowast lowastlowast



00

05

10

15

20

Rela

tive i

nten

sity

of p

-p65

(fol

d of

cont

rol)

00

05

10

15

20

Rela

tive i

nten

sity

of p

-I

B (f

old

of co

ntro

l)(b)



Data Availability






Acknowledgments



References






























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















modellow

middle

07

08

09

10

11

12

perc

enta

ge o

f sta

rtin

g w

eigh

t (

)

10 20 300Days









4 Discussion




dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

0

1

2

3

4

5To

tal c

ells

in B

ALF

(1 times

5 )


(a)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

0

1

2

3

4

5

Tota

l mac

roph

ages

in B

ALF

(1 times

5 )


(b)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

00

05

10

15

Tota

l neu

trop

hils

in B

ALF

(1 times

)


lowastlowast

(c)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

000

005

010

015

Tota

l lym

phoc

ytes

in B

ALF

(1 times

)

(d)






dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

00

05

10

15

20

25

TNF-

m

RNA

rela

tive e

xpre

ssio

nlowastlowastlowast


lowastlowast

(a)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

00

05

10

15

20

IL-6

mRN

Are

lativ

e exp

ress

ion

lowastlowastlowast


lowastlowastlowast

(b)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

0

1

2

3

4

5

IL-1

mRN

Are

lativ

e exp

ress

ion

lowastlowastlowast


lowast

(c)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

00

05

10

15

20

MM

P-9

mRN

Are

lativ

e exp

ress

ion


lowastlowastlowast

(d)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

0

1

2

3

4

5

MM

P-12

mRN

Are

lativ

e exp

ress

ion

lowast lowast

(e)


dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

0

10

20

30

40

CAT

(Um

g)

lowast


(a)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

0

20

40

60

80

100

GSH

-Px

(Um

g)

lowastlowast

lowastlowast

(b)

de

xam

etha

sone

high

mid

dle

low

mod

el

cont

rol

00

05

10

15

20

25

MD

A (n

mol

mg)

lowastlowast

lowast

lowast

(c)



p65

p-p65

IB

p-IB

-actin



CS + LPS

2

(a)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

00

05

10

15

20

Rela

tive i

nten

sity

of p

-p65

00

05

10

15

20

Rela

tive i

nten

sity

of p

-I

B

lowastlowastlowast

lowastlowast


(b)



Alveolarregion







200

gm

L

100

gm

L

50

gm

L

25

gm

L

125

g

mL

cont

rol

0

50

100

150C

ell v

iabi

lity

( o

f con

trol

)

(a)

0

25

5075

100

125

150

Cel

l via

bilit

y (

of c

ontr

ol)

200

gm

L

100

gm

L

50

gm

L

25

gm

L

125

g

mL

cont

rol

mod

el


(b)


50

gm

L

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

0

5

10

15

20

TNF-

m

RNA

rela

tive e

xpre

ssio

n

lowast


(a)

50

g

mL

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

0

10

20

30

IL-6

mRN

Are

lativ

e exp

ress

ion


lowastlowastlowast

lowastlowastlowast

(b)

50

gm

L

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

0

10

20

30

IL-1

mRN

Are

lativ

e exp

ress

ion

lowastlowastlowast

lowast

(c)





5 Conclusions



p65

p-p65

IB

p-IB

GAPDH

Jianpiyifei II(gmL)

(gmL)dexamethasone

CSE

(a)

50

gm

L

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

50

gm

L

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

lowast lowastlowast



00

05

10

15

20

Rela

tive i

nten

sity

of p

-p65

(fol

d of

cont

rol)

00

05

10

15

20

Rela

tive i

nten

sity

of p

-I

B (f

old

of co

ntro

l)(b)



Data Availability






Acknowledgments



References






























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

0

1

2

3

4

5To

tal c

ells

in B

ALF

(1 times

5 )


(a)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

0

1

2

3

4

5

Tota

l mac

roph

ages

in B

ALF

(1 times

5 )


(b)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

00

05

10

15

Tota

l neu

trop

hils

in B

ALF

(1 times

)


lowastlowast

(c)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

000

005

010

015

Tota

l lym

phoc

ytes

in B

ALF

(1 times

)

(d)






dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

00

05

10

15

20

25

TNF-

m

RNA

rela

tive e

xpre

ssio

nlowastlowastlowast


lowastlowast

(a)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

00

05

10

15

20

IL-6

mRN

Are

lativ

e exp

ress

ion

lowastlowastlowast


lowastlowastlowast

(b)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

0

1

2

3

4

5

IL-1

mRN

Are

lativ

e exp

ress

ion

lowastlowastlowast


lowast

(c)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

00

05

10

15

20

MM

P-9

mRN

Are

lativ

e exp

ress

ion


lowastlowastlowast

(d)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

0

1

2

3

4

5

MM

P-12

mRN

Are

lativ

e exp

ress

ion

lowast lowast

(e)


dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

0

10

20

30

40

CAT

(Um

g)

lowast


(a)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

0

20

40

60

80

100

GSH

-Px

(Um

g)

lowastlowast

lowastlowast

(b)

de

xam

etha

sone

high

mid

dle

low

mod

el

cont

rol

00

05

10

15

20

25

MD

A (n

mol

mg)

lowastlowast

lowast

lowast

(c)



p65

p-p65

IB

p-IB

-actin



CS + LPS

2

(a)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

00

05

10

15

20

Rela

tive i

nten

sity

of p

-p65

00

05

10

15

20

Rela

tive i

nten

sity

of p

-I

B

lowastlowastlowast

lowastlowast


(b)



Alveolarregion







200

gm

L

100

gm

L

50

gm

L

25

gm

L

125

g

mL

cont

rol

0

50

100

150C

ell v

iabi

lity

( o

f con

trol

)

(a)

0

25

5075

100

125

150

Cel

l via

bilit

y (

of c

ontr

ol)

200

gm

L

100

gm

L

50

gm

L

25

gm

L

125

g

mL

cont

rol

mod

el


(b)


50

gm

L

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

0

5

10

15

20

TNF-

m

RNA

rela

tive e

xpre

ssio

n

lowast


(a)

50

g

mL

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

0

10

20

30

IL-6

mRN

Are

lativ

e exp

ress

ion


lowastlowastlowast

lowastlowastlowast

(b)

50

gm

L

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

0

10

20

30

IL-1

mRN

Are

lativ

e exp

ress

ion

lowastlowastlowast

lowast

(c)





5 Conclusions



p65

p-p65

IB

p-IB

GAPDH

Jianpiyifei II(gmL)

(gmL)dexamethasone

CSE

(a)

50

gm

L

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

50

gm

L

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

lowast lowastlowast



00

05

10

15

20

Rela

tive i

nten

sity

of p

-p65

(fol

d of

cont

rol)

00

05

10

15

20

Rela

tive i

nten

sity

of p

-I

B (f

old

of co

ntro

l)(b)



Data Availability






Acknowledgments



References






























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

00

05

10

15

20

25

TNF-

m

RNA

rela

tive e

xpre

ssio

nlowastlowastlowast


lowastlowast

(a)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

00

05

10

15

20

IL-6

mRN

Are

lativ

e exp

ress

ion

lowastlowastlowast


lowastlowastlowast

(b)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

0

1

2

3

4

5

IL-1

mRN

Are

lativ

e exp

ress

ion

lowastlowastlowast


lowast

(c)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

00

05

10

15

20

MM

P-9

mRN

Are

lativ

e exp

ress

ion


lowastlowastlowast

(d)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

0

1

2

3

4

5

MM

P-12

mRN

Are

lativ

e exp

ress

ion

lowast lowast

(e)


dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

0

10

20

30

40

CAT

(Um

g)

lowast


(a)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

0

20

40

60

80

100

GSH

-Px

(Um

g)

lowastlowast

lowastlowast

(b)

de

xam

etha

sone

high

mid

dle

low

mod

el

cont

rol

00

05

10

15

20

25

MD

A (n

mol

mg)

lowastlowast

lowast

lowast

(c)



p65

p-p65

IB

p-IB

-actin



CS + LPS

2

(a)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

00

05

10

15

20

Rela

tive i

nten

sity

of p

-p65

00

05

10

15

20

Rela

tive i

nten

sity

of p

-I

B

lowastlowastlowast

lowastlowast


(b)



Alveolarregion







200

gm

L

100

gm

L

50

gm

L

25

gm

L

125

g

mL

cont

rol

0

50

100

150C

ell v

iabi

lity

( o

f con

trol

)

(a)

0

25

5075

100

125

150

Cel

l via

bilit

y (

of c

ontr

ol)

200

gm

L

100

gm

L

50

gm

L

25

gm

L

125

g

mL

cont

rol

mod

el


(b)


50

gm

L

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

0

5

10

15

20

TNF-

m

RNA

rela

tive e

xpre

ssio

n

lowast


(a)

50

g

mL

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

0

10

20

30

IL-6

mRN

Are

lativ

e exp

ress

ion


lowastlowastlowast

lowastlowastlowast

(b)

50

gm

L

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

0

10

20

30

IL-1

mRN

Are

lativ

e exp

ress

ion

lowastlowastlowast

lowast

(c)





5 Conclusions



p65

p-p65

IB

p-IB

GAPDH

Jianpiyifei II(gmL)

(gmL)dexamethasone

CSE

(a)

50

gm

L

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

50

gm

L

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

lowast lowastlowast



00

05

10

15

20

Rela

tive i

nten

sity

of p

-p65

(fol

d of

cont

rol)

00

05

10

15

20

Rela

tive i

nten

sity

of p

-I

B (f

old

of co

ntro

l)(b)



Data Availability






Acknowledgments



References






























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















p65

p-p65

IB

p-IB

-actin



CS + LPS

2

(a)

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

dexa

met

haso

ne

high

mid

dle

low

mod

el

cont

rol

00

05

10

15

20

Rela

tive i

nten

sity

of p

-p65

00

05

10

15

20

Rela

tive i

nten

sity

of p

-I

B

lowastlowastlowast

lowastlowast


(b)



Alveolarregion







200

gm

L

100

gm

L

50

gm

L

25

gm

L

125

g

mL

cont

rol

0

50

100

150C

ell v

iabi

lity

( o

f con

trol

)

(a)

0

25

5075

100

125

150

Cel

l via

bilit

y (

of c

ontr

ol)

200

gm

L

100

gm

L

50

gm

L

25

gm

L

125

g

mL

cont

rol

mod

el


(b)


50

gm

L

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

0

5

10

15

20

TNF-

m

RNA

rela

tive e

xpre

ssio

n

lowast


(a)

50

g

mL

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

0

10

20

30

IL-6

mRN

Are

lativ

e exp

ress

ion


lowastlowastlowast

lowastlowastlowast

(b)

50

gm

L

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

0

10

20

30

IL-1

mRN

Are

lativ

e exp

ress

ion

lowastlowastlowast

lowast

(c)





5 Conclusions



p65

p-p65

IB

p-IB

GAPDH

Jianpiyifei II(gmL)

(gmL)dexamethasone

CSE

(a)

50

gm

L

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

50

gm

L

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

lowast lowastlowast



00

05

10

15

20

Rela

tive i

nten

sity

of p

-p65

(fol

d of

cont

rol)

00

05

10

15

20

Rela

tive i

nten

sity

of p

-I

B (f

old

of co

ntro

l)(b)



Data Availability






Acknowledgments



References






























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















200

gm

L

100

gm

L

50

gm

L

25

gm

L

125

g

mL

cont

rol

0

50

100

150C

ell v

iabi

lity

( o

f con

trol

)

(a)

0

25

5075

100

125

150

Cel

l via

bilit

y (

of c

ontr

ol)

200

gm

L

100

gm

L

50

gm

L

25

gm

L

125

g

mL

cont

rol

mod

el


(b)


50

gm

L

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

0

5

10

15

20

TNF-

m

RNA

rela

tive e

xpre

ssio

n

lowast


(a)

50

g

mL

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

0

10

20

30

IL-6

mRN

Are

lativ

e exp

ress

ion


lowastlowastlowast

lowastlowastlowast

(b)

50

gm

L

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

0

10

20

30

IL-1

mRN

Are

lativ

e exp

ress

ion

lowastlowastlowast

lowast

(c)





5 Conclusions



p65

p-p65

IB

p-IB

GAPDH

Jianpiyifei II(gmL)

(gmL)dexamethasone

CSE

(a)

50

gm

L

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

50

gm

L

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

lowast lowastlowast



00

05

10

15

20

Rela

tive i

nten

sity

of p

-p65

(fol

d of

cont

rol)

00

05

10

15

20

Rela

tive i

nten

sity

of p

-I

B (f

old

of co

ntro

l)(b)



Data Availability






Acknowledgments



References






























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















p65

p-p65

IB

p-IB

GAPDH

Jianpiyifei II(gmL)

(gmL)dexamethasone

CSE

(a)

50

gm

L

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

50

gm

L

25

gm

L

125

g

mL

cont

rol

dexa

met

haso

ne

mod

el

lowast lowastlowast



00

05

10

15

20

Rela

tive i

nten

sity

of p

-p65

(fol

d of

cont

rol)

00

05

10

15

20

Rela

tive i

nten

sity

of p

-I

B (f

old

of co

ntro

l)(b)



Data Availability






Acknowledgments



References






























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of












































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















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