Dehydropachymic acid decreases bafilomycin A1 induced β-Amyloid accumulation in PC12 cells 2017 Mengyao Yu.pdf

该【Dehydropachymic acid decreases bafilomycin A1 induced β-Amyloid accumulation in PC12 cells 2017 Mengyao Yu 】是由【dt83088549】上传分享，文档一共【27】页，该文档可以免费在线阅读，需要了解更多关于【Dehydropachymic acid decreases bafilomycin A1 induced β-Amyloid accumulation in PC12 cells 2017 Mengyao Yu 】的内容，可以使用淘豆网的站内搜索功能，选择自己适合的文档，以下文字是截取该文章内的部分文字，如需要获得完整电子版，请下载此文档到您的设备，方便您编辑和打印。:..Author’eptedManuscriptDehydropachymicaciddecreasesbafilomycinA1inducedβ-umulationinPC12cellsMengyaoYu,XiaoyanXu,NanJiang,WeiWei,FangLi,LimingHe,XiaLuoate/jepPII:S0378-8741(17)30079-XDOI:http://dx./.:JEP10654Toappearin:JournalofEthnopharmacologyReceiveddate:25August2016Reviseddate:29December2016Accepteddate:6January2017Citethisarticleas:MengyaoYu,XiaoyanXu,NanJiang,WeiWei,FangLi,LimingHeandXiaLuo,DehydropachymicaciddecreasesbafilomycinA1inducedβ-umulationinPC12cells,JournalofEthnopharmacology,http://dx./.,typesetting,,andalllegaldisclaimersthatapplytothejournalpertain.:..DehydropachymicaciddecreasesbafilomycinA1inducedβ-umulationinPC12cells*MengyaoYu,XiaoyanXu,NanJiang,WeiWei,FangLi,LimingHe,XiaLuoSichuanAcademyofChineseMedicalSciences,51,4thsectionofSouthRenminRoad,Chengdu,610041,PRChina*Correspondingauthor:e-mailaddress:447080215@,Tel.:862885250783,fax:862885250783AbstractEthnopharmacologicalrelevanceFuling,thesclerotiumofPoriacocos,wasfrequentlyusedintraditionalChinesemedicine(TCM)formulaeforAlzheimer'sdisease(AD),:..Toinvestigatetheeffectandcorrespondingmechanismsofdehydropachymicacid,whichisoneofthemajortriterpenesinPoriacocos,ontheclearanceofβ--APP(PC12-APP).PC12-,,25μg/mLfor4h,andthenco-treatedwith50nmol/-,Aβ1-42,LC-3,-LC3-GFP(PC12-LC3-GFP)–42umulationofAPPandAβ1–42inbafilomycinA1inducedPC12-,DPAloweredtheLC3II/LC3IratioandreducedtheGFP--:..–:..AD,Alzheimer'sdisease;ALP,autophagy-lysosomepathway;APP,amyloidprecursorprotein;ATG,autophagy-relatedgene;AVs,autophagicvacuoles;Aβ,β-amyloidpeptide;DPA,dehydropachymicacid;GAPDH,glyceraldehyde-3-phosphatedehydrogenase;GSK-3,Glycogensynthasekinase3;LC3,microtubule-associatedprotein1lightchain3;mTOR,themammaliantargetofrapamycin;PBS,phosphatebuffersolution;PS1,Presenilin-1;TCM,traditionalChinesemedicine;V-ATPase,VacuolartypeH+-ATPaseKeywordsAlzheimer'sdisease;Dehydropachymicacid;autophagy;lysosome;β-amyloid;bafilomycinA1poundsstudiedinthearticledehydropachymicacid(PubChemCID:15226717);bafilomycinA1(PubChemCID:6436223)'sdisease(AD)monneurodegenerativedisease,whichaffectsmorethan35millionpeopleworldwidewithincreasingtendency(Finder,2010).Thisdiseaseinvolvesaprogressivelossofsynapsesinthecerebralcortexandhippocampusleadingto:..impairedmemoryandthedeteriorationofcognitivefunctions(Vinters,2015).AlthoughtherearesomedebatesintheetiologyandpathogenesisofAD,amyloidβpeptide(Aβ)isconsideredasthemostimportantpathologicalfactors,whichdrivestheprogressionofneurodegenerativedisorders(Pimplikar,2009).Theautophagyisinvolvedinthedegradationoflong-livedproteins(BolandandNixon,2006).Deficitsintheautophagyresultinproteinaggregation,thegenerationoftoxicproteinspecies,anelles,whicharehallmarksofAD(Martini-Stoicaetal.,2016).AgrowingbodyofevidenceindicatesthatinductionofautophagythroughmTOR-dependentormTOR-independentpathwayscoulddecreaseAβdepositsandthenreducememorydeficitsinpreclinicalmodels(Casarejosetal.,2011;Leeetal.,2015;al.,2010;al.,2011;ZhuandWang,2015).Moreover,promotingautophagosomematuration,transport,fusionandlysosomalproteolysiswhichisfacilitativetoautophagicfluxmayalsoameliorateamyloidpathologiesandmemorydeficits(Butleretal.,2011;al.,2015;Parretal.,2012;Yangetal.,2011).Therefore,autophagy-lysosomepathway(ALP)(Schw.)(TCM),Fulingisconsideredtohavetheeffectsofclearingdamp,promotingdiuresis,,binedwithothermedicinalmaterials,fortreatingvarioussymptoms,suchasimmunedysfunction,urination:..disorders,diarrhea,,aliteraturereviewbasedonthetraditionalevidencefromclinicalapplicationsdemonstratedthatPoriacocoswasthemostfrequentlyusedinthedementiainterventioninTCMformulaeoverthepast10centuries(Linetal.,2012).RecentresearchesalsofoundthatPoriacocoscouldreduceAβ1–42levelandbinationofothermedicinalmaterials(Parketal.,2009;Seoetal.,2010).Inpreviousstudies,fivetriterpenoidconstituentsincludeddehydropachymicacid,pachymicacid,poricoicacidA,3-epidehydrotumulosicacidand3-dehydrotrametenolicacid,(DPA)andpachymicacidcouldincreasethenumberofpunctainPC12cellsstabletransfectedwithpSelcect-LC3-GFP(PC12-LC3-GFP),–42contentinculturemediumofPC12cellsstabletransfectedwithpCB6-APP(PC12-APP).Nevertheless,DPAdramaticallysuppressedtheoverproductionofAβ1–42inthePC12-,weinvestigatedintracellularandextracellularAβ1–42production,amyloidprecursorprotein(APP)degradation,autophagicfluxandlysosomalacidificationinthebafilomycinA1inducedALPimpairedPC12cells,andtheresultsdemonstratedthatthesuppressionofβ-umulationby:..(-0020,,purity>90%,detectedbyHPLC)waspurchasedfromPushBiotechnology(Chengdu,China)anddissolvedinDMSO(,Sigma-Aldrich,SaintLouis,USA).Intheexperimentalprocedure,%%poloxamer188(,BASF,Ludwigshafen,Germany).AntibodiesagainstAPP(),Aβ1-42(),andGAPDH()werepurchasedfromCellSignalingTechnologies(Beverly,USA).AntibodyagainstLC3()waspurchasedfromSigma-(-6486)wasobtainedfromSantaCruz(Dallas,USA).Horseradishperoxidase-(HRP-)conjugatedsecondaryantibody(-2301andZB-2306)werepurchasedfromZSBIO(Beijing,China).BafilomycinA1()wasobtainedfromCaymanChemical(AnnArbor,USA).()wereobtainedfromthecellbankoftheChineseacademyofsciences(Shanghai,China)andmaintainedinRPMI1640(-022,Gibco,:..Carlsbad,USA)containing15%horseserum(-122,Gibco,Carlsbad,USA)%fetalbovineserum(-020,Gibco,Carlsbad,USA)at37℃with5%-LC3-GFP(-gfplc3,Invivogen,SanDiego,USA)andpCB6-APP(APPgene1-695,transcriptvariant3)(,YRGene,Chagnsha,China,)wererespectivelytransfectedintoPC12cellsbyusingLipofectamine2000(-027,Invitrogen,Carlsbad,USA)accordingtothemanufacturer’(-zn-lp,Invivogen,SanDiego,USA)or400μg/mLG418(-gn-1,Invivogen,SanDiego,USA)-LC3-GFPwasidentifiedwithfluorescencemicroscopyandwesternblotting,whilethestabletransfectantwithpCB6-APPwasidentifiedwithwesternblottingandtheassayofAβ1--APP(PC12-APP)wereseededat2×10cellsperwellina96-,,,25μg/,theplatewascentrifugatedat200gfor10min,’sbalancedsaltssolutionand10μL5mg/mLMTT(,Sigma-Aldrich,SaintLouis,USA)%SDSsolution(:..mol/LHCl)-platereader(Thermo,Boston,USA).-42inculturemedium5PC12-×10cellsperwellina12-%horseserum,1%fetalbovineserumanddifferentconcentrationsofDPA(,,25μg/mL),,bafilomycinA1wasaddedintothemediumatthefinalconcentrationof50nmol/L,-ordingtothemanufacturer’sprotocol(-62601,Wako,Osaka,Japan).ThecellswerelysedandtheproteinconcentrationwasquantitatedbyBCAassaykit(,Beyotime,Shanghai,China).-APPcellswereseededat1×10cellsperwellina6-(50mmol/LTris-,150mmol/LNaCl,1%TritonX-100,1%Sodiumdeoxycholate,%SDS,1mmol/LPMSF(,Sigma-Aldrich,SaintLouis,USA),andproteaseinhibitors(,Pierce,Carlsbad,USA))at4°,000gfor10min,,Aβ1-42,LC-3,cathepsinD:..-20%SDS-PAGE(20μgextract/lane)andthenelectro-(10mmol/LTris-HCl,150mmol/LNaCl,%Tween20)containing3%:1000overnightat4°C,–antibodycomplexwasvisualizedwithenhancedchemiluminescence(ECL)(,Beyotime,Shanghai,China),Aβ1-42,LC-3andmaturecathepsinD(mCatD)-LC3-GFP(PC12-LC3-GFP)wereseededat1×105cellsperwellina24--APPcellswereseededat1×10cellsperw