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JournalofChinesePharmaceuticalSciences

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SynthesisofanactivepeptidefromCarapaxTrionycisanditsinhibitory effectontheproliferationofhepaticstellatecells
* ChunLingHu,YinPingTang,YanWenLiu

SchoolofPharmacy,HubeiUniversityofTraditionalChineseMedicine,Wuhan430065,China

Abstract:ThisstudywasaimedtosynthesizeanactivepeptidefromCarapaxTrionycisandtoinvestigateitseffectontheproliferation of hepaticstellate cells (HSCs). An active peptide from CarapaxTrionycis, which was shown to have significant antihepatic fibrosis activity, was synthesized by solid phase method and characterized by MALDITOF MS analysis. The HSCs in log growthphasewastreatedwiththesyntheticpeptideatdifferentconcentrations.Viabilityandapoptosisofhepaticstellatecells weredeterminedbyMTSassayandAnnexinVFITC/PIstaining,respectively.Theactivepeptideshowedstronginhibitionof proliferationandinductionofapoptosisofHSCT6 inaconcentrationdependentmanner.Theresultssuggestthattheactive peptide fromCarapaxTrionyciscouldbesynthesizedefficientlyandhassignificantinhibitoryeffectontheproliferationofHSCT6. Keywords: HepaticstellatecellsSyntheticpeptideCarapaxTrionycisMTSApoptosis CLCnumber:R916 Documentcode:A ArticleID: 10031057(2012)213204

1.Introduction

Liver fibrosis is a woundhealing response to variouschronicliverinjuries,includingalcoholism, persistent viral and helminthic infections, and [1,2] hereditary metal overload .Activation of hepatic stellatecellsplaysacrucialroleinthedevelopment [35] of liver fibrosis . During the activation process, hepatic stellate cells (HSCs) undergo phenotype transformation from vitaminAstoring quiescent cellstomyofibroblastlikeactivatedcells.Activated HSCsareproliferativeandfibrogenic,withaccumu lationofextracellularmatrix(ECM).Thus,waysto eliminate HSCs such as inhibition of proliferation and induction of apoptosis have become important [6] strategiesforthetreatmentofliverfibrosis . Carapax Trionycis, a traditional Chinese medicine, is originated from the shell of Trionyx sinensis Wiegmann.Ithassuchactivitiesasreplenishingyin, restraining yang, softening and resolving hard masses,reducing feverandremoving steam. Clinical experienceindicatedthattraditionalChinesemedicine containingCarapaxTrionycisshowedcurativeeffect [7] when used for the treatment of liver fibrosis .
Receiveddate:20110908. * Correspondingauthor.Tel.:8602788920834 Email:1054681196@qq.com doi:10.5246/jcps.2012.02.016

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Previous studies have demonstratedthatextractsof CarapaxTrionyciswereabletoprotectliveragainst [8] fibrosis in CCl4 animal models . Moreover active peptideswerefirstisolated fromCarapaxTrionycis and shown to have significant antihepatic fibrosis [9,10] activities by our group . However, the natural source of Carapax Trionycis is very limited and extractivecostisveryhigh,whichrestricttheuse oftheseactivepeptides. In this study, we selected one of the active peptidesfromCarapaxTrionyciswhosesequenceis HGRFG(molecularweight:572.3)andsynthesized itbysolidphasemethod.Wedeterminedwhetherthe syntheticpeptideaffectstheproliferationandapoptosis of HSCs by MTS assay and Annexin VFITC/PI staining. Our results may provide experimental support in the search of new antihepatic fibrosis agents.

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2.Materialsandmethods 2.1.Materials The HSCT6 cell line was kindly provided by Prof.HangPin Yao (Zhejiang University, Hangzhou). Thefetalcalfserum(FCS)waspurchasedfromSanli BiologicalCo.,China. TrypsinandHighDMEMwere
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fromGibcolCo.,USA.MTS(3(4,5dimethylthiazol 2yl)5(3carboxymethoxyphenyl)2(4sulfophenyl) 2Htetrazolium)wasfromSigmaChemicalCo.,USA. Annexin VFITC/PI assay kit was supplied by Rose Co.,USA.FmocAAOH,WangresinandTBTUwere obtainedfromGilBiochemicalShanghailimitedCo., China.MALDITOFMSwassuppliedbyShimadzu Co., Japan. Flow cytometry was supplied by BeckmancounterInc.,USA. 2.2. Solidphase synthesis of an active peptide fromCarapaxTrionycis The introduction of solid phase peptide synthesis (SPPS)in1963byBruceMerrifieldopenedthedoor forresearcherstopreparestructurallydiversepeptides. Inthisstudy,theSPPSwascarriedoutonWangresin asthecarrierandNFmocprotectedaminoacid as the starting materials. After condensed with the mix reagents of TBTU/NMM and deprotected with 20%piperidine,thecrudesyntheticpeptideproducts were cleaved from the Wang resin by the cleavage reagents TFA/TIS/H2O. We finally obtained the activepeptideofthesequenceHGRFG. 2.3.CellcultureandMTSassay

2.4.AnnexinVFITC/PIanalysis
2 HSCT6 cellswereculturedin25cm flasksunder humidified 5% CO2 atmosphere at 37 C to 80% confluenceandthentreatedwiththesyntheticpeptide beforeanalysis.Thecontrolgroupwasculturedonly inserummedium.Theexperimentalgroupwastreated withthesyntheticpeptideatdifferentconcentrations (0.5,1.0,2.0and4.0mg/mL)inserummedium. After 24 h, the culture medium was removed and the cells were washed in phosphatebuffered saline(PBS)andresuspended. Subsequently,100L 6 (210 cells/mL)ofthesupernatantwasreactedwith 5 L (1 g/mL) FITCconjugated annexin V and 10L(2g/mL)PI.Thecellswereincubatedatroom temperature for15 min in the darkandanalysed by flow cytometry. Only fluoresceinpositive cells withoutPIstainingwereregardedasapoptoticcells.

HSCT6 cells were maintained in Dulbeccos modifiedEagles medium with10%FCSandincu batedat37Cunder5%CO2 humidifiedatmosphere. HSCs were digested with 0.25% trypsin and adjusted 4 to610 cells/mLwhentheHSCswereinexponential growth phase. The cells were planted into 96well plate at 0.2 mL/well and 5wells/group and treated separately with serum containing the synthetic peptide. After2h,thecellswereadded800pg/mL TGF1 ineachwell. The control group was cultured only in serum medium.ThestimulantgroupwasculturedinTGF1 medium. The experimental group was treated with thesyntheticpeptideatdifferentconcentrations(0.01, 0.05,0.1and0.5mg/mL)inTGF1 medium.After cells were incubated for 72 h, the culture medium was removed, and 20 L MTS/PMS was added in each well for 1 h. OD values at 490 nm were measured.Therateofinhibition(IR)wascalculated asfollowsIR(%)=[(control valueblank)(test value blank)]/(controlvalue blank)100.

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mAU 120 100 80 60

2.5.Statisticalanalysis

Eachexperimentwasrepeatedaminimumofthree times.ResultswereexpressedasmeanSD.Statistical analysis was performed by oneway ANOVA test. TheP valueslowerthan0.05wereconsideredstatisti cally significant.

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3.Results 3.1.Analysisofthesyntheticpeptide The active peptide from Carapax Trionycis was synthesized by classical solid phase method. After purificationbyRPHPLC,thepurityofthesynthetic peptide was over 98%. A typical chromatogram is showninFigure1.
13.926

20 0 20 40

051015202530 t (min)

Figure1.AnHPLCchromatogramofcrudepeptideHGRFG.

Copyright 2012 Journal of Chinese Pharmaceutical Sciences, School of Pharmaceutical Sciences, Peking University

14.616

17.934

20.322 21.658

40

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C.L.Huetal./JournalofChinesePharmaceuticalSciences21(2012)132135

The molecular weight of the synthetic peptide was determined by MALDITOF MS (Fig. 2). The + [M+H] peakwasatm/z573.04,indicatingthesyn theticpeptidehasthesamemolecularweightasthe activepeptideHGRFG(molecularweight:572.3). 3.2. Inhibitory effect of the synthetic peptide on theproliferationofHSCT6 cells Results from MTS assay demonstrated that the synthetic peptide at 0.010.5 mg/mL significantly inhibited HSCT6 proliferation in a concentration dependentmanner(P<0.05,Table1). 3.3.InductionofapoptosisinHSCT6 cells Flow cytometry is a widely used and important method for research and clinical applications. The apoptosis of HSCs was determined using a flow cytometric annexin VFITC/PI assay. The annexin VFITC/PIassayshowedthat24hafterthesynthetic

peptide was administered, the apoptosis rate of cultured HSCT6 cells increased significantly in a concentrationdependent manner. The results of the annexin VFITC/PI assays for the different groups are shown in Figure 3AE, and the changes in the apoptosisrateareshowninFigure3F.

Intens. [a.u.] 8000

573.040

6000

4000

2000

352.814 529.043

Table1. InhibitionoftheproliferationofHSCT6 cellsbythesyntheticpeptide(meanSD)

Group 0.01 2.25330.0513 3.85 Syntheticpeptide(mg/mL) 0.05 0.1 * 2.07330.0961 1.84670.0971 12.11 20.94

OD 490 value Inhibitoryrate(%)

*

P<0.05comparedwiththecontrolgroup(n =4).

4 10

4 10

3 10

3 10

FL2H

FL2H

FL2H

2 10

2 10

1 10

1 10

0 10

0 10

0 10

1 10

2 10 FL1H

3 10

4 10

0 10

1 10

2 10 FL1H

3 10

4 10

0 10

1 10

2 10

3 10

4 10

(A)

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(B)
4 10

200400

Figure2.MassspectrumofthesyntheticpeptideHGRFG.

0.5 * 1.24000.0634 47.01

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630.064 729.213

600800

100012001400 m/z

Control

TGF1stimulant 2.80670.0847 20.09

2.34000.0655

(C)

0 10

1 10

2 10 FL1H

3 10

4 10

4 10

(D)

(E)

(F)

90 80 70
** **

3 10

3 10

Apoptosisrate(%)

60 50 40 30 20 10
**

FL2H

FL2H
1 10 2 10 FL1H 3 10 4 10

2 10

1 10

0 10

0 10

0 10

1 10 0 10

2 10

1 10

2 10 FL1H

3 10

4 10

0 Control0.512 4 SyntheticpeptideofCarapaxTrionycis(mg/mL)

Figure 3. Detection of the synthetic peptideinducedapoptosisinHSCT6 by flowcytometry.Panels AE represent the controland synthetic peptidetreatmentsat0.5,1.0,2.0and4.0mg/mL,respectively.F,bargraphshowingtheincreasedHSCT6 cellapoptosisrate(%)24hafterthe syntheticpeptidetreatment.**P<0.05comparedwiththecontrolgroup(n =4).

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4.Discussion Inrecent years,theuseof peptidesastherapeutic agents has gained momentum. Chemical synthesis ofactiveantihepaticfibrosispeptidesfromCarapax Trionycis,whichwereconsideredasleadcompounds in drug development, is an important way in the search of antihepatic fibrosis agents. In this study we synthesized an active peptide from Carapax TrionycisbyFmocsolid phase method. Theresults showedthatthesyntheticpeptidecouldbeobtained bySPPSandwasidenticaltothenaturalpeptide. Hepatic stellate cells play a central role in fibro [11,12] genesisandfibrolysis .Activationandproliferation of HSCs is the key to fibrogenesis while apoptosis [6,13] ofHSCisassociatedwithresolutionoffibrosis . Therefore,inhibitionofHSCsactivationandprolifera tionandinductionofapoptosisofactivatedHSCshave [11,14] been proposed as potential antifibrotic strategies . In this study we found that the synthetic peptide at 0.010.5mg/mLinhibitedHSCT6 proliferationin a concentrationdependent manner by MTS assay AnnexinVFITC/PIassayshowedtheearlyapoptosis rate after treatment with 1.0, 2.0 and 4.0 mg/mL synthetic peptide were significantly higher than control(P<0.05). In summary, our study showed that an active peptidefromCarapaxTrionyciscouldbesynthesized efficiently by solid phase method and the synthetic peptidehassignificantproliferationinhibitionactivity againstHSCs.

Acknowledgments WethankDr.YinPingTangforherexperthelpin preparingthemanuscriptandProfessorYanWenLiu forhisexcellenttechnicalassistance.

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, , *
, 430065 : (HSCT6) , , MALDITOF MS (HSCT6) , HSCT6, MTSHSCT6 Annexin VFITC/PIHSC , , , : MTS
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