JournalofEthnopharmacology150(2013)181–186ContentslistsavailableatScienceDirectJournalofEthnopharmacologyjournalhomepage:www.elsevier.com/locate/jepTheanti-osteoporoticeffectofvelvetantlerpolypeptidesfromCervuselaphusLinnaeusinovariectomizedratsLian-ZhuZhanga,b,nn,Ji-LeXina,Xi-PingZhanga,QinFuc,YangZhanga,n,Qiu-LiZhouaaDepartmentofBiopharmacy,SchoolofPharmaceuticalSciences,JilinUniversity,No.1163XinminStreet,Changchun130021,ChinaAffiliatedHospitaltoChangchunUniversityofChineseMedicine,Changchun130021,ChinacTumorHospitalofJilinProvince,Changchun130021,ChinabarticleinfoArticlehistory:Received23May2013Receivedinrevisedform23July2013Accepted12August2013Availableonline27August2013Keywords:VelvetantlerpolypeptideCartilagecellOsteoblast-likecellOvariectomyOsteoporosisabstractEthnopharmacologicalrelevance:Thedeervelvetantleriswellknownforitstraditionalmedicinalvalue,andiswidelyusedintheclinic.ItisrecordedintheCompendiumofMateriaMedicathatthedeervelvetantlerreplenishesvitalessenceandstrengthensthebone.Aimofthestudy:Thegoalofthisstudywastoinvestigatetheanti-osteoporoticeffectoftotalvelvetantlerpolypeptidesfromCervuselaphusLinnaeus(TVAPL)onovariectomizedrats(OVX),andtheirpossiblemechanismoftheaction.Materialsandmethods:Wistarratsweredividedintofivegroups:sham-operatedgroup,OVXgroup,andOVXratstreatedwith20,40,or60mk/kgTVAPLfor12weeks.Calciumandphosphoruslevels,boneweightcoefficient(BWC),bonemineraldensity(BMD),andbonemineralcontent(BMC)wereevaluated.TheMTTassaywasusedtomeasuretheactivitiesofinterleukin-1(IL-1)andinterleukin-6(IL-6).Inaddition,cartilagecellsandosteoblast-likecellswereexposedtoTVAPL,naturalvelvetantlerpolypep-tides(nVAP),andsyntheticvelvetantlerpolypeptides(sVAP),todeterminetheireffectsoncellproliferationusingthetritiatedthymidineincorporationassay.Finally,theenzyme-linkedimmunosor-bentassaywasusedtodeterminetheeffectsofnVAPandsVAPoncytokinesrelatedtobonemetabolism.Results:TheadministrationofTVAPLfor12weekssignificantlyreversedosteoporosisinOVXrats,therebyimprovingtheBWC,BMD,BMC,andbonemicroarchitecture.IL-1andIL-6weresignificantlyactivatedintheOVXgroup,andtheiractivationwasinhibitedbyTVAPL.Inaddition,nVAPandsVAPpromotedtheproliferationofcartilageandosteoblast-likecells(po0.01orpo0.001),andinhibitedthesecretionofIL-1αfromTHP-1monocyticcellsinvitro.Conclusion:TheseresultssuggestthatTVAPLareeffectiveinpreventingbonelossinOVXrats.TheeffectofTVAPLonosteoporosisisduetoinhibitionofIL-1andIL-6bynVAP,andpromotionofmitosis.sVAPhassimilarbioactivityasnVAP.Thus,bothTVAPLandsVAPmaybepotentialtherapeuticagentsforthetreatmentofpostmenopausalosteoporosis.&2013ElsevierIrelandLtd.Allrightsreserved.1.IntroductionOsteoporosisischaracterizedbysystemicbonemassreductionandbonemicrostructuredamage,whichcausesbonefragilityandahighriskoffracture.Therearemanyfactorsthatcauseosteoporosis,ofwhichestrogendeficiencyinpostmenopausalwomenisparticu-larlyimportant.Therefore,itisofgreatimportancetodevelopatherapeuticagentthatpreventspostmenopausalosteoporosis.ItisrecordedinHuangDiNeiJing,anAncientChineseMedicalBook,thatthekidneygovernsthebones,aconceptthathasalsoCorrespondingauthor.Tel.:þ8613844165956;fax:þ83186177218.Correspondingauthorat:DepartmentofBiopharmacy,SchoolofPharmaceuticalSciences,JilinUniversity,No.1163XinminStreet,ChaoyangDistrict,Changchun130021,China,Tel.:þ8613844165956;fax:þ83186177218.E-mailaddress:zyang@jlu.edu.cn(Y.Zhang).nnnbeendemonstratedinclinicalstudies(Guoetal.,1995;Wangetal.,2002).Therefore,healthykidneycontributestostrongbones;whenthekidneyisweak,osteoporosiscanoccur.Kidneydefi-ciencyisverycommoninmenopausalfemales.Thevelvetantler(VA)anditsextractsarecommonlyusedinthetreatmentofkidneydeficiencyrelatedtobonedisease(Zhaoetal.,2003;ZhaoandZhao,2006;Lietal.,2007).AccordingtotheChinesePharmacopoeia,antlersofboththeCervuselaphusLinnaeus(CEL)andCervusnipponTemminch(CNT)areusedasVAs.TheCNTantlerhasbeenmoreacceptedbecauseoftradition;however,thebreedingofCNTrequiresaspecialenvironment,whichlimitstheamountofantlerproductionfromthisspecies.Ontheotherhand,CELareeasilybred,andarewidelyculturedallovertheworld,resultinginhighVAproduction.Consequently,CELhavebeenusedtostudythecomponentsandefficacyofVAextracts,andtodeterminetheactivecompoundsinVA.0378-8741/$-seefrontmatter&2013ElsevierIrelandLtd.Allrightsreserved.http://dx.doi.org/10.1016/j.jep.2013.08.029182L.-Z.Zhangetal./JournalofEthnopharmacology150(2013)181–186Manyreports,includingpreviousstudiesfromourlaboratory,haveshownthattotalvelvetantlerpolypeptidesfromCervusnipponTemminchantler(TVAPT)promotestheproliferationofrabbitcostalcartilagecells,humanfetalarticularcartilagecells,andtheosteoblast-likecellsofchickenembryoskullboneinvitro(Guoetal.,1998;Zhouetal.,1999).TVAPThasalsobeenshowntoacceleratethehealingofdistalradialfractures(Zhouetal.,1999)andwoundsinrats(Zhaetal.,2012).Inaddition,manystudieshavedemonstratedthatVAextractsareeffectiveintreatingbonediseases(Leeetal.,2011).Together,thesestudiessuggestthattotalantlerpolypeptides(TVAP)arethemainactivecomponentsofVA.InordertodetermineiftheCELantlerhasthesameefficacyastheCNTantler,weobtainedtotalvelvetantlerpolypeptidesfromCervuselaphusLinnaeus(TVAPL)throughthesamemethodusedtoextractTVAPT.Inaddition,weisolatedthenaturalpolypeptide(nTVAP)andanalyzeditsprimarystructure,whichhasbeenreportedtobeanewpolypeptidefromtheCELantler(Wengetal.,2001a,2002).Moreover,wechemicallysynthesizedthesyntheticvelvetantlerpolypeptide(sVAP)accordingtotheprimarystructureofnVAP.TVAPLhasbeenshowntorepairthemechanicalinjuryofratskin(Zhouetal.,2001),andtorestoretheimbalanceofbonereconstructioninducedbyretinoicacid(Duanetal.,2007).TVAPL,nVAP,andsVAPsignificantlyincreasedDNAsynthesisinratepidermalcellsandmousefibroblastsinvitro(Wengetal.,2001b).Inthisstudy,theovariectomised(OVX)ratmodelofosteo-porosiswasusedtodeterminetheeffectsofTVAPLonboneloss,thebonemetabolicbalance,andboneformation.Inaddition,theeffectsofnVAPandsVAPonmitosisandcytokinesecretionwereinvestigatedasapossiblemechanismofTVAPL.2.Materialsandmethods2.1.MaterialsTVAPLandnTVAPwereisolatedbytheResearchCenterofNewDrugsattheChangchunCollegeofTraditionalChineseMedicine.TVAPLisalightyellowpowder(proteincontent56%)thatiseasilydissolvedinwater.nVAPisanivory-whiteandwater-solublemono-peptideisolatedfromTVAPL,withamolecularweightof3216Daand99.4%purity.sVAP,apeptidewithamolecularweightof3200Daand98%purity,wassynthesizedbyMeiLian(Xi'an)BiotechnologyCo.,Ltd.,accordingtotheprimarystructureofnVAP,andhassamephysicochemicalpropertyasnVAP.TheTHP-1humanmonocyticcelllinewaspurchasedfromtheJilinProvinceTumorHospital.Tritiatedthymidine([3H]TdR)wasobtainedfromtheAtomicEnergyInstituteoftheChineseAcademyofScience.Collagenase,trypsin,andpancreatinwerepurchasedfromSigma.2.2.AnimalgroupingandtreatmentsAllexperimentalprocedureswereconductedinaccordancewithinstitutionalguidelinesforthecareanduseoflaboratoryanimals,andalleffortsweremadetominimizeanimalsuffering.TheprotocolswereapprovedbytheAnimalCare&WelfareCommitteeofJilinUniversity.Theanimalsinthisstudyincluded50Wistarrats,2whiterabbits,50chicks,and10BALB/Cmice.ProtocolsforculturinghumanfetalarticularcartilagewereapprovedbytheHumanCare&WelfareCommitteeoftheSecondClinicalHospitalofJilinUniversity;thiscartilagewasfromtwoinducedabortionsat6–7months.Fifty3-month-oldfemalewistarratsweighing250–300gwereusedinthisstudy(DepartmentofExperimentalAnimals,NormanBethuneMedicalUniversity).TheAnimalCareandWelfareCom-mitteeofJilinUniversityapprovedtheuseofanimalsinthisstudy(50rats).Theratswerehousedinstainlesssteelcages,andallowedfreeaccesstofoodandwater.Thetemperaturewaskeptat22721C,andthehumiditywas55710%witha12hdark/lightcycle.Fortyratswereintraperitoneallyinjectedwith2%sodiumpentobarbital(40mg/kg),andreceivedabilateralovariectomy(OVX)(Leietal.,2001)understerileconditions.Theshamgroupof10ratshadashamoperation.Thefifthdayaftersurgery,theOVXratswererandomlyseparatedintofourgroups(10ratsineachgroup):OVXgroup,OVXþTVAPL(60mg/kg),OVXþTVAPL(40mg/kg),andOVXþTVAPL(20mg/kg).TheadministrationofTVAPLwasperformedtheseventhdayaftertheovariectomy.TVAPLweresubcutaneouslyinjectedonceevery2daysfor12weeks;theshamgroupandthemodelgroup(OVXgroupwithoutdrug)wereinjectedwithphysiologicalsalineinthesameway.After12weeks,ratsweresacrificedbydecapitation.Thespleenswereobtainedundersterileconditionsformea-surementofinterleukin-1(IL-1)andIL-6activities.Theleftfemursoftheratsineachgroupweredriedtoaconstantweightbybaking,andwerethenusedtomeasurecalciumandphosphoruslevels(atomicabsorptionspectrometers,Perkin-Elmer)afteraciddigestion.Thebonemineraldensity(BMD)andbonemineralcontent(BMC)oftherightone-thirdofthedistalfemursweremeasured(dualenergyX-rayabsorptiometry,DEVA2000).Thelefttibialspecimensweredissectedandfixedovernightin10%formalin.Thebonesweredecalcified,dehydratedin70%(vol/vol)ethanol,embeddedinparaffin,andcutinto5mmsagittalsections.Theslidesweredried,deparaffinized,andstainedwithhaematoxylinandeosin(H&E)toassessmorphology.2.3.MeasurementofIL-1andIL-6activitiesTheratspleensweregentlyteasedtoobtainmononuclearcells.Thecellsweredilutedto2Â106cells/mLwithRPMI10culturemediumcontaining10%fetalbovineserum(FBS),and10mg/Llipopolysaccharide(LPS).Thenthecellswereplatedinto24-welldishesandincubatedwith5%CO2at371Cfor48h(Tokiwa,Japan).ThesupernatantofspleniccellswascollectedfordetectionofIL-1andIL-6,whichwerestoredatÀ201C(Juetal.,2000).Asuspensionofmouse(10BALB/Cmice,male,18–20g,purchasedfromtheDepartmentofExperimentalAnimals,NormanBethuneMedicalUniversity)thymocyteswasobtainedaccordingtostan-dardmethods(Wangetal.,2006),andthecellconcentrationwasadjustedto1.5Â106cells/mL.Thethymocytecellsuspension(90μL)andthesupernatantofsplenicculture(10μL)wereplatedinto96-welldishes.ThesampleswereanalyzedwiththeMTTmethod.Theopticaldensityat492nmwasmeasuredusingamicroplatereader(Bio-Rad,USA),andrepresentedIL-1activity(Kimbleetal.,1994;Pfeilschifteretal.,19).IL-6activitywasmeasuredinproliferating7TD1cells,whichwereobtainedfromtheDepartmentofImmunology(JilinUni-versity,China)andculturedat371C,5%CO2with10%NBS/RPMI10.The7TD1celllineisanexclusivelyIL-6-dependentcellline(Kimbleetal.,1994;Kimbleetal.,1997)thatcanbeusedtodeterminetheIL-6activity.Thecellconcentrationwasadjustedto8Â104cells/mL,afterwhichcellswereplatedin96-welldishes,andculturedat371C,5%CO2for48h.ThesampleswereanalyzedwiththeMTTmethod.TheODvaluesofthewellsrepresentedIL-6activity.2.4.[3H]TdRincorporationassayCartilagecellswereisolatedaccordingtoapreviouslypub-lishedprotocolfromKatoetal.(Katoetal.,1983).Briefly,cellsweretakenfromthecostalcartilageofrabbitandhumanfetalarticularcartilage(inducedabortion,6–7monthsold),andL.-Z.Zhangetal./JournalofEthnopharmacology150(2013)181–186183osteoblast-likecells(Songetal.,1987)wereobtainedfromtheskullboneof18-day-oldchickenembryos.Thecellswereincu-batedinMinimumEssentialMedium(MEM)with10%FBS,andplatedinto96-welldishesatadensityof1Â105cells/mL,100μLperwell,andculturedat5%CO2,371Cfor6d.Subsequently,theculturemediumwasreplacedwithMEMwithoutFBS.Thecellswereincubatedwith100μLTVAP,nVAP,andsVAPatdifferentconcentrationsfor24h.Tenmicrolitersof[3H]TdR(37kBq/well)wasadded3hbeforeterminatingtheincubation(Puzasetal.,1981;Mohanetal.,1986).Thecpmvalueof[3H]TdRwascounted(Wallac1409DSAliquidscintillationcounter,Perkin-Elmer)torepresentDNAsynthesis.2.5.IL-1αandtumornecrosisfactor(TNF)measurementTHP-1cellsweresuspendedinRPMImedium(containing10%FBS)atadensityof0.32Â106cells/mL,andthenplatedin96-welldishes(100μL/well).nVAPandsVAP(ataconcentrationof1,10,100mg/L),dilutedinRPMImedium,wereaddedtothewells.After30min,differentconcentrationsofLPS(10,100mg/L)wereadded,andthefinalvolumeineachwellwas200μL.Thesamplesunderwentthreefreeze-thawcyclesaftera24-hincubation,andwerecollectedina1.5mLeppendorftube,followedbycentrifuga-tionfor10minat12,000Âgat41C.ThesupernatantwascollectedtoevaluatelevelsofIL-1αandTNFusingtheenzyme-linkedimmunosorbentassay(ELISA)(Auwerx,1991).2.6.StatisticalanalysisSPSSversion11.5wasusedtoanalyzethedata.Resultswereexpressedasmean7standarddeviation.DifferencesamonggroupswereevaluatedwithStudent'st-test.pvalueslessthan0.05wereconsideredstatisticallysignificant.3.Results3.1.InhibitionofbonelossbyTVAPLOVXratshadsignificantlydecreasedBWC,BMC,BMDandbonecalciumlevels(Fig.1,po0.01orpo0.001).However,administrationof20,40,and60mg/kgTVAPLsignificantlyincreasedthosemarkers(Fig.1,po0.05).BonelossintheOVXratwasinhibitedbyTVAPLtreatmentaswell.Histologicalresultsshowedthatcomparedtotheshamgroup,tibialtrabecularbonevolumeandcorticalthicknesswerenotablydecreasedinthemodelgroup,andthearrangementofthetrabecularbonewasrelativelysparse,likeisolatedislands,whileTVAPLtreatments(40and60mg/kg)significantlyincreasedtrabe-cularbonevolumeandcorticalthickness,andalsopromotedtrabe-cularnetworkformationwithrestoredspatialstructure(Fig.2).ThesedataindicatethatTVAPLinhibitsbonelossandregulatesthebalancebetweenboneresorptionandboneremodelinginthehighturnoverosteoporosismodelofOVXrats.Fig.1.EffectsofTVAPLonbonelossinvivoafteradministrationfor12weeks:(A)BWC,(B)BMC,and(C)BMD.npo0.05,nnpo0.01,andnnnpo0.001,versusOVXgroup.184L.-Z.Zhangetal./JournalofEthnopharmacology150(2013)181–186Fig.2.TheeffectofTVAPLonOVXrattibias(H&E100Â):(A)intheshamoperationgroup,thenumberandthethicknessofthetrabeculaewereingoodcondition,(B)intheOVXgroup,thenumberoftrabeculaesignificantlydecreased,andthetrabeculaeweresparselyarranged,likeseparatedislands,(C)intheTVAPL(20mg/kg)group,thenumberoftrabeculaewasslightlymorethaninthemodelgroup,andthearchitecturewasinbadcondition,(D)intheTVAPL(40mg/kg)group,thenumberoftrabeculaeincreasedtosomeextent,andthetrabeculaewereconnected,and(E)intheTVAPL(60mg/kg)group,thenumberandthethicknessoftrabeculaenotablyincreased,andformedanalmostperfectnetwork.Fig.3.EffectsofTVAPLonIL-1andIL-6inOVXrats:(A)IL-1activityinmousethymocyteswasmeasuredwiththeMTTassay,(B)IL-6activitydetectedthrough7TD1cell(anIL-6-dependentcellline)usingtheMTTmethod.npo0.05,andnnpo0.01,comparedtotheOVXgroup.3.2.IL-1andIL-6activityInthemodelgroup,IL-1andIL-6activityinthespleencellsclearlyincreased(po0.01,po0.05),butwasinhibitedafteradministrationofTVAPL(po0.05,Fig.3).TheresultssuggestthatinhibitionofbonelossbyTVAPLcanbeattributedtotheinactiva-tionofIL-1andIL-6.3.3.Proliferationofcartilageandosteoblast-likecellsTVAPL(50–200mg/L)andnVAP(5–50mg/L)significantlyinducedtheincorporationof[3H]TdRintoDNAinhumanfetalarticularcartilageandosteoblast-likecells(Table1).ThisindicatesthatnVAPisanactivecomponentinTVAPLthatstimulatescellproliferation,andsVAPhassimilaractivityasnVAPinpromotingmitosis.3.4.CytokinesinTHP-1cellnVAPandsVAPinhibitedLPS-induced(LPSdoses:10mg/Land100mg/L)IL-1αsecretioninTHP-1cells(Table2).nVAPandsVAPbilaterallyregulatedtheTNFconcentrationinTHP-1cell:for100mg/LLPSstimuli,nVAPandsVAPinhibitedTNFproduction,butincreasedTNFsecretionataLPSconcentrationof10mg/L.L.-Z.Zhangetal./JournalofEthnopharmacology150(2013)181–186185Table1Incorporationof[3H]TdRintotheDNAofchondrocytesandosteoblast-likecellsinvitro.ColumnArepresentsrabbitcostalchondrocytes,columnBrepresentschondrocytesofhumanfetalarticularcartilage,andcolumnCrepresentsosteoblastsofchickembryocalvaria.GroupingDose(mg/L)[3H]TdR/BqperwellABCControl70785772175725TVAPL50108729a8571799713100144740a103728a125727a200169733b165749b138720bnVAP5165735b131746a11172310178734b122733a138724a20206736b152731b156719b40222727b177759b177730b50251756b200752b168728bsVAP5127740a108727a10272210130735a112723a10771820159737b134738a114719a40196740b178749b177725b50210752b2117b190742bapo0.05Comparedtothecontrol.bpo0.01Comparedtothecontrol.Table2EffectsofnVAPandsVAPoncytokineproductioninTHP-1cellline(x7s).GroupDose(mg/L)IL-1αTNFLPS100mg/LLPS10mg/LLPS100mg/LLPS10mg/LControl1.0570.110.5270.0812.073.17.071.7nVAP1001.0070.230.4270.035.671.4a4.870.9100.2770.04b0.1770.05b4.571.1a8.072.310.1470.01b0.3670.02a4.271.3a12.071.1asVAP1000.9870.210.5070.076.271.4a5.171.0100.4670.15b0.2570.01b5.371.5a7.871.810.2270.11b0.3370.04a4.972.1a11.372.0aapo0.05Comparedtothecontrol.bpo0.01Comparedtothecontrol.4.DiscussionThereisadynamicbalancebetweenresorptionandremodelinginnormalbonemetabolism.Oncethisbalanceisdisrupted(e.g.,boneresorptionismorethanboneremodeling),osteoporosisoccurs.Thebalanceisinfluencedbymanyfactors,especiallythelevelofestrogen(Roush,2011);ovariectomizedratshavedramaticallyreducedestrogenlevels.Theinhibitionofboneresorptionbyestrogenisreduced,andthedynamicbalanceofbonemetabolisminclinestowardsboneresorption,leadingtoosteoporosis(Lelovasetal.,2008).Inthisstudy,TVAPLenhancedtheBWC,BMC,BMD,Ca2þandphosphorouslevelsintheboneoftheOVXrat,andeffectivelyinhibitedosteoporosis.DocumentsaboutbonemorphologyindicatethatinOVXratswithsevereosteoporosis,bonelossismainlymanifestedasasignificantreductionofthevolumeoftrabecularbones,accompaniedbydamageofnettrabecularstructure.TVAPLincreasedthenumberandthethicknessofthetrabeculae,restoredthetrabecularnetwork,andthereforealleviatedbonefragility.Atthesametime,bonestrengthwasimproved,andbonelosswasreversedintheOVXrat.OurpreviousworkdemonstratedthatTVAPLcouldcorrectthenegativebalanceofbonereconstruction,andincreasebonemassinratswithosteoporosisinducedbyretinoicacid(Duanetal.,2007).Retinoicacidmainlyactivatesosteoclasts,andenhancesboneresorp-tionwithoutinhibitingtheactivityofosteoblastsorhavinganobviouseffectonboneremodelingandbonematrixmineralization(Wuetal.,1996).Theovariectomynotonlyactivatesosteoclasts,butalsoinhibitsthefunctionofosteoblasts(Hodsmanetal.,2005;Russelletal.,2008).TVAPLmayinhibittheactivityofosteoclaststhroughtheregulationofosteoblasts.Inthisstudy,weobservedtheeffectsofTVAPL,nVAP,andsTVAPontheproliferationofrabbitcostalcartilagecells,humanfetalarticularcartilagecells,andchickenfetalosteoblast-likecellsinvitro.OurresultsshowedthattheVAswerestronglyeffectiveinimprovingthemitosisofcartilageandosteoblast-likecells,withapparentdose-dependenteffects.ItwassuggestedthatTVAPLinhibitedbonelossinOVXratsthroughitsdualeffectsofinhibitingosteoclastactivityandactivatingosteoblasts.BonelossintheOVXmodeliscloselyrelatedtocytokinesIL-1,TNFandIL-6,whichplayimportantrolesinbonemetabolismandbonereconstruction(Wuetal.,1996;Kobayashietal.,2000).Theyareimportantcytokinesinbonelossduringestrogendeficiency.IL-1andTNFcandirectlyinducethemitosisofosteoclastpre-cursors,increasetheamountofosteoclasts,andinhibitthefunctionofosteoblasts.IL-1increasesboneresorption,andtrans-genicmicedeficientfortheIL-1receptorareresistanttoovariectomy-inducedboneloss(Lorenzoetal.,1998;Xingetal.,2003).IL-6isanecessary“downstreamcytokine”involvedinthedifferentiationandrecruitmentofosteoclasts.ItisinducedbyIL-1andTNF,andplaysanimportantroleintherapiddecompositionofbone.ThemainroleofIL-6isitseffectonosteoclastogenesisandboneresorption(Kotakeetal.,1996).ThemechanismofIL-6inpromotingboneresorptionisduetoitsstimulatoryeffectsontheproductionanddifferentiationofosteoclastprecursors,andimprovementoftheactivityandnumberofosteoclasts(Jilkaetal.,1992).OurstudyindicatesthattheactivitiesofIL-1andIL-6notablyincreasedintheOVXmodel,andTVAPLeffectivelyinhibitedtheiractivation,therebyinhibitingthedifferentiationofosteoclastprecursorsandformationofosteoclasts.ThisisanotherimportantmechanismunderlyingtheabilityofTVAPLtoinhibitbonelossinOVXrats.nVAPandsVAPimprovedtheproliferationofepidermiccells,mechanocytes,cartilageandosteoblast-likecells(Wengetal.,2001a;Wangetal.,2003),andinhibitedIL-1αsecretionfromTHP-1cells.ThissuggeststhatnVAPandsVAPhaveanti-osteoporosiseffects.WecomparedthestructureandthepropertiesofnVAPandsVAP(Xinetal.,2013),andfoundthattheyhaveanidenticalsequence,N-terminus:VLSAADKSNVKAAWGKVGGNAPAFGAEALLRM(Wengetal.,2001a).ThemolecularweightofsVAP(3200Da)isonly16DalessthantheactualmolecularweightofnVAP(3216Da,mainlybecausenaturalmethionineexistsinanoxidationstate)(GÖranssonetal.,1999;Wengetal.,2002).ThisstudyprovedthatnVAPistheactivecomponentinTVAPL,andsVAPhasthesamebioactivityasnVAP.TheseresultsindicatethatthesmalldifferenceinstructurehadnoeffectonthebioactivityofsVAP.ThereareverylowlevelsofnVAPintheCELantler,anditisverydifficulttoobtainenoughCELforsufficientnVAPforproduction.However,sVAPwaschemicallysynthesized,hasthesameprimarystructureasnVAP,andcanbemasslyproduced.Therefore,sVAPhasthepotentialtobedevelopedasanalternativeanti-osteoporosisagent.5.ConclusionTheresultsofourstudyindicatethatTVAPLrestorestrabecularbonearchitecture,inhibitsdecreasesofBWC,BMD,andBMCinducedbyOVX.Theanti-osteoporoticeffectofTVAPLisattributedtothepromotionofmitosis,andinhibitionofIL-1andIL-6.nVAPistheactivecomponentofTVAPL,andsVAPhasthesameactivityasnVAP;thus,sVAPmaybeanalternativeremedyfortreatingpostmenopausalosteoporosis.186L.-Z.Zhangetal./JournalofEthnopharmacology150(2013)181–186AcknowledgmentsThisworkwassupportedbyaGrantno.39870929fromtheNationalNaturalScienceFoundationofChina.ReferencesAuwerx,J.,1991.Thehumanleukemiacellline,THP-1:amultifacettedmodelforthestudyofmonocyte–macrophagedifferentiation.Experientia1,22–31.Duan,L.X.,Ma,J.S.,Weng,L.,Wang,L.J.,Chen,S.W.,Liu,Y.Q.,Wang,B.X.,Zhou,Q.L.,2007.Preventiveandtherapeuticeffectoftotalantlerpolypeptidesonosteoporosisinducedbyretinoicacidinrats.ChinesePharmaceuticalJournal4,2–267.GÖransson,U.,Luijendijk,T.,Johansson,S.,Bohlin,L.,Claeson,P.,1999.SevennovelmacrocyclicpolypeptidesfromViolaaruensis.JournalofNaturalProducts2,283–286.Guo,Y.J.,Zhou,Q.L.,Liu,P.,Wang,Y.,Fang,J.R.,Wang,B.X.,1998.Theresearchofpiloseantlerpolypeptidespromotingosteoblastprecursorcellsandchondro-cytesproliferation.ChineseJournalofBiochemicalPharmaceutics2,74–76.Guo,S.H.,Li,H.C.,Zou,C.H.,Xu,Z.Q.,Yang,D.Z.,Lin,R.P.,Lei,Y.X.,Gao,J.,Wu,K.,Sun,X.H.,Xu,Y.C.,Chi,H.H.,1995.Correlationbetweenkidneydeficiencysyndromeandbonedensity.ChineseJournalofIntegratedTraditionalandWesternMedicine11,655–657.Hodsman,A.B.,Bauer,D.C.,Dempster,D.W.,Dian,L.,Hanley,D.A.,Harris,S.T.,Kendler,D.L.,McClung,M.R.,Miller,P.D.,Olszynski,W.P.,Orwoll,E.,Yuen,C.K.,2005.Parathyroidhormoneandteriparatideforthetreatmentofosteoporosis:areviewoftheevidenceandsuggestedguidelinesforitsuse.EndocrineReviews5,688–703.Jilka,R.L.,Hangoc,G.,Girasole,G.,Passeri,G.,Williams,D.C.,Abrams,J.S.,Boyce,B.,Broxmeyer,H.,Manolagas,S.C.,1992.Increasedosteoclastdevelopmentafterestrogenloss:mediationbyinterleukin-6.Science257,88–91.Ju,D.H.,Zhang,C.Y.,Wang,A.M.,Lv,A.P.,Xu,S.J.,Cui,W.,Teng,J.R.,Wang,S.J.,Li,Y.,2000.Effectofprescriptionforwarmingandrecuperatingthekidney-yangontheactivityofIL-1andIL-6intheovariectom-inducedosteoporosisrats.ChineseJournalofBasicMedicineinTraditionalChineseMedicine6,5–9.Kato,Y.,Hiraki,Y.,Inoue,H.,Kinoshita,M.,Yutani,Y.,Suzuki,F.,1983.Differentialandsynergisticactionsofsomatomedin-likegrowthfactors,fibroblastgrowthfactorandepidermalgrowthfactorinrabbitcostalchondrocytes.EuropeanJournalofBiochemistry/FEBS3,685–690.Kimble,R.B.,Bain,S.,Pacifici,R.,1997.ThefunctionalblockofTNFbutnotofIL-6preventsbonelossinovariectomizedmice.JournalofBoneandMineralResearch:theOfficialJournaloftheAmericanSocietyforBoneandMineralResearch6,935–941.Kimble,R.B.,Vannice,J.L.,Bloedow,D.C.,Thompson,R.C.,Hopfer,W.,Kung,V.T.,Brownfield,C.,Pacifici,R.,1994.Interleukin-1receptorantagonistdecreasesbonelossandboneresorptioninovariectomizedrats.TheJournalofClinicalInvestigation5,1959–1967.Kobayashi,K.,Takahashi,N.,Jimi,E.,Udagawa,N.,Takami,M.,Kotake,S.,Nakagawa,N.,Kinosaki,M.,Yamaguchi,K.,Shima,N.,Yasuda,H.,Morinaga,T.,Higashio,K.,Martin,T.J.,Suda,T.,2000.TumornecrosisfactoralphastimulatesosteoclastdifferentiationbyamechanismindependentoftheODF/RANKLRANKinterac-tion.JournalofExperimentalMedicine2,275–286.Kotake,S,Sato,K.,Kim,K.J.,Takahashi,N.,Udagawa,N.,Nakamura,I.,Yamaguchi,A.,Kishimoto,T.,Suda,T.,Kashiwazaki,S.,1996.Interleukin-6andsolubleinterleukin-6receptorsinthesynovialfluidsrheumatoidarthritispatientsareresponsibleforosteoclast-likecellformation.JournalofBoneandMineralResearch1,88–95.Lee,H.S.,Kim,M.K.,Kim,Y.K.,Jung,E.Y.,Park,C.S.,Woo,M.J.,Lee,S.H.,Kim,J.S.,Suh,H.J.,2011.StimulationofosteoblasticdifferentiationandmineralizationinMC3T3-E1cellsbyantlerandfermentedantlerusingCordycepsmilitaris.JournalofEthnopharmacology2,710–717.Lei,T.,Zhang,X.Z.,He,M.,Xie,N.Z.,Zhao,J.S.,Chen,Y.Q.,2001.Establishmentofexperimentalanimolmodelofosteoporosis.JournalofTongJiUniversity(MedicalScience)22,12–13.Lelovas,P.P.,Xanthos,T.T.,Thoma,S.E.,Lyritis,G.P.,Dontas,I.A.,2008.Thelaboratoryratasananimalmodelforosteoporosisresearch.ComparativeMedicine5,424–430.Li,Y.J.,Kim,T.H.,Kwak,H.B.,Lee,Z.H.,Lee,S.Y.,Jhon,G.J.,2007.Chloroformextractofdeerantlerinhibitsosteoclastdifferentiationandboneresorption.JournalofEthnopharmacology2,191–198.Lorenzo,J.A.,Naprta,A.,Rao,Y.,Alander,C.,Glaccum,M.,Widmer,M.,Gronowicz,G.,Kalinowski,J.,Pilbeam,C.C.,1998.MicelackingthetypeIinterleukin-1receptordonotlosebonemassafterovariectomy.Endocrinology6,3022–3025.Mohan,S.,Jennings,J.C.,Linkhart,T.A.,Baylink,D.J.,1986.Isolationandpurificationofalow-molecular-weightskeletalgrowthfactorfromhumanbones.Biochi-micaetBiophysicaActa2,234–242.Pfeilschifter,J.,Chenu,C.,Bird,A.,Mundy,G.R.,Roodman,G.D.,19.Interleukin-1andtumornecrosisfactorstimulatetheformationofhumanosteoclastlikecellsinvitro.JournalofBoneandMineralResearch:theOfficialJournaloftheAmericanSocietyforBoneandMine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