astrocyte(星形胶质细胞)(学习资料)课件

AstrocyteReactivitytoUnconjugatedBilirubinRequiresTNF-αandIL-1βReceptorSignalingPathwaysGLIA59:14–25(2011)SCI(2010)：5.19周康康2012-7-21AstrocyteReactivitytoUnconj1Wehavereportedthattumornecrosisfactor(TNF)-αandinterleukin(IL)-1βareproducedbyculturedneuronsandmainlybyglialcellsexposedtounconjugatedbilirubin(UCB).Theeffectsofthesecytokinesaremediatedbycellsurfacereceptorsthroughanuclearfactor(NF)-κB-dependentpathwaythatwehaveshowedtobeactivatedbyUCB.Summary21Wehavereportedthattum2ExposureofastrocytestoUCBincreasedtheexpressionofbothTNF-αreceptorTNFR1andIL-1βreceptorIL-1R1,butnotTNFR2,aswellastheiractivation,observedbyaugmentedbindingofreceptors’molecularadaptors,TRAF2andTRAF6,respectively.3SilencingofTNFR1,usingsiRNAtechnology,orblockadeofIL-1βcascade,usingitsendogenousantagonist,IL-1receptorantagonist(IL-1ra),preventedUCB-inducedcytokinereleaseandNF-κBactivation.32Exposureofastrocytesto4Interestingly,lackofTNF-αsignaltransductionreducedUCB-inducedcelldeathforshortperiodsofincubation,incontrast,inhibitionofIL-1βcascadeproducedasustainedblockadeofastrocyteinjurybyUCB.5Together,ourdatashowthatinflammatorypathwaysareactivatedduringinvitroexposureofratastrocytestoUCB.ThissupportstheconceptthatinflammatorypathwaysplayaroleinbraindamagebyUCB,andthattheymayrepresentimportantpharmacologicaltargets.44Interestingly,lackofTNMaterialsandmethods1PrimaryCultureofAstrocytes：2-day-oldWistarrats2TransientTransfection：threedifferentdoublestrandedratTNFR1smallinterfering(si)RNAs(30nM),scrambledsiRNA(negativecontrol)ortheabsenceofsiRNA(mockcontrol).3CellTreatment：50μMUCBplus100μMhumanserumalbumin(HSA)(UCBtoHSAmolarratioof0.5),from15minto24h,at370C.4WesternBlot：TheproteinexpressionofTNFR1,TNFR2,andIL-1R1weredeterminedbyWesternblotanalysis.5Materialsandmethods1Prima5MeasurementofCytokineRelease：TNF-α,IL-1β,andIL-6withspecificDuoSetRELISADevelopmentkits.6DetectionofNF-κBActivation—immunofluorescencedetection：rabbitanti-p65NF-κBsubunitantibody(1:200)astheprimaryantibodies,aFITC-labeledgoatanti-rabbitantibody(1:160)asthesecondaryantibodies.7EvaluationofCellDeath—LDHAstrocyteswerethenidentifiedinfixedcellsbyanantibodydirectedagainstGFAP.（神经胶质原纤维酸性蛋白）Toidentifythetotalnumberofcells,astroglialnucleiwerestainedwithHoechstdye33258.（烟酸己可碱—DNA染料）65MeasurementofCytokineRResults1UCBIncreasestheProteinContentofTNFR1andIL-1R1,butnotofTNFR2,andInducesTheirEngagement.TNF-αcascadeismediatedthroughtheactivationoftwosurfacereceptors,TNFR1andTNFR2,whileIL-1βpathwayoccursviaIL-1R1engagement.

Engagementofcellsurfacereceptorsisfollowedbyrecruitmentofseveraladaptorproteinstothereceptorcomplex.

TRAF2andTRAF6.7Results1UCBIncreasesthe8899ThesedataindicatethatastrocytesexposedtoUCBpreferentiallyexpressTNFR1ratherthanTNFR2,atleastduringthefirst24hoftreatment,suggestingthatTNF-αactioninourstudymodeloccursessentiallythroughTNFR1.UCBincubationsignificantlyincreasedthiseffectwithmaximumlevelsat1hforTRAF2/TNFR1complexandlastingfrom1to12hforTRAF6/IL-1R1complex,indicatingthatUCBtreatmentincreasesthetransductionofTNF-αandIL-1βsignalsthroughTRAF2andTRAF6,respectively.10Thesedataindicatethatastro2TransfectionofTNFR1siRNAsSilencesTNFR1ExpressionElicitedbyUCB,WhileIL-1raPreventsIL-1R1EngagementTriggeredbyUCB.112TransfectionofTNFR1siR121213133SilencingofTNFR1andSuppressionofIL-1R1ActivityReducesUCB-InducedSecretionofTNF-α,IL-1βandIL-6.143SilencingofTNFR1andSu151516164SilencingofTNFR1andSuppressionofIL-1R1ActivityReducesUCB-InducedActivationofNF-κB.174SilencingofTNFR1andSu181819195SilencingofTNFR1andSuppressionofIL-1R1ActivityModulatesUCB-InducedCytotoxicity.205SilencingofTNFR1andSu2121222223232424Together,ourresultsdemonstratethatuponUCBexposureastrocytesmountaninflammatoryresponsethatisexacerbatedandperpetuatedintimebytheactivationofTNFR1andIL-1R1signalingpathways.——AstrocyteReactivitytoUnconjugatedBilirubinRequiresTNF-αandIL-1βReceptorSignalingPathways.25Together,ourresultsdemonstrHence,cytokinepathwa