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1、AstrocyteReactivitytoUnconjugatedBilirubinRequiresTNF-αandIL-1βReceptorSignalingPathwaysGLIA59:14–25(2011)SCI(2010)：5.19周康康2012-7-211Wehavereportedthattumornecrosisfactor(TNF)-αandinterleukin(IL)-1βareproducedbyculturedneuronsandmainlybyglialcellsexposed
2、tounconjugatedbilirubin(UCB).Theeffectsofthesecytokinesaremediatedbycellsurfacereceptorsthroughanuclearfactor(NF)-κB-dependentpathwaythatwehaveshowedtobeactivatedbyUCB.Summary22ExposureofastrocytestoUCBincreasedtheexpressionofbothTNF-αreceptorTNFR1andIL-
3、1βreceptorIL-1R1,butnotTNFR2,aswellastheiractivation,observedbyaugmentedbindingofreceptors’molecularadaptors,TRAF2andTRAF6,respectively.3SilencingofTNFR1,usingsiRNAtechnology,orblockadeofIL-1βcascade,usingitsendogenousantagonist,IL-1receptorantagonist(IL
4、-1ra),preventedUCB-inducedcytokinereleaseandNF-κBactivation.34Interestingly,lackofTNF-αsignaltransductionreducedUCB-inducedcelldeathforshortperiodsofincubation,incontrast,inhibitionofIL-1βcascadeproducedasustainedblockadeofastrocyteinjurybyUCB.5Together,
5、ourdatashowthatinflammatorypathwaysareactivatedduringinvitroexposureofratastrocytestoUCB.ThissupportstheconceptthatinflammatorypathwaysplayaroleinbraindamagebyUCB,andthattheymayrepresentimportantpharmacologicaltargets.4Materialsandmethods1PrimaryCultureo
6、fAstrocytes：2-day-oldWistarrats2TransientTransfection：threedifferentdoublestrandedratTNFR1smallinterfering(si)RNAs(30nM),scrambledsiRNA(negativecontrol)ortheabsenceofsiRNA(mockcontrol).3CellTreatment：50μMUCBplus100μMhumanserumalbumin(HSA)(UCBtoHSAmolarra
7、tioof0.5),from15minto24h,at370C.4WesternBlot：TheproteinexpressionofTNFR1,TNFR2,andIL-1R1weredeterminedbyWesternblotanalysis.55MeasurementofCytokineRelease：TNF-α,IL-1β,andIL-6withspecificDuoSetRELISADevelopmentkits.6DetectionofNF-κBActivation—immunofluore
8、scencedetection：rabbitanti-p65NF-κBsubunitantibody(1:200)astheprimaryantibodies,aFITC-labeledgoatanti-rabbitantibody(1:160)asthesecondaryantibodies.7EvaluationofCellDeath—LDHAstrocyteswerethenidentifiedinfixedcellsbyananti
