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1、中国应用生理学杂志,2011,27(2)175*神经活性甾体别孕烯醇酮对脑皮质神经元损伤的保护作用李贤慧,张新昌,王刚,刘海玲,夏时海(武警医学院分子生物学教研室,天津300162)摘要目的:利用N甲基D天门冬氨酸(NMDA)诱发新生小鼠脑皮质神经元损伤模型,探讨神经活性甾体别孕烯醇酮对脑皮质神经元的保护作用及其机制。方法:应用RTPCR和Westernblot法检测别孕烯醇酮对2氨基丁酸受体(2GABAR)表达和对蛋白激酶B(PKB,又称为Akt)磷酸化的影响。应用Western
2、blot和DNALadder方法检测NMDA诱发的神经元凋亡及别孕烯醇酮对NMDA诱发凋亡的影响。结果:Westernblot和RTPCR分析表明0.666510mol/L510mol/L别孕烯醇酮使Akt磷酸化增加并促进2GABARmRNA的表达。110mol/L别孕烯醇6酮预处理小鼠脑皮质神经元有抗凋亡作用,但510mol/L别孕烯醇酮预处理小鼠脑皮质神经元使NMDA诱发的DNALadder减弱明显,并能有效抵抗NMDA诱发的活化型PRAP、Caspase3、Caspase9
3、的增加。结论:别孕烯醇酮可通过促进2GABAR表达和增加Akt磷酸化抵抗NMDA诱发的脑皮质神经元凋亡。关键词神经活性甾体;别孕烯醇酮;细胞凋亡;脑皮质神经元中图分类号R285.5文献标识码A文章编号10006834(2011)0217504EffectsofneuroactivesteroidallopregnanoloneonthedamageofcorticalneuronsLIXianhui,ZHANGXinchang,WANGGang,LIUHaili
4、ng,XIAShihai(DepartmentofMolecularBiology,MedicalCollegeofChinesePeopleSArmedPoliceForce,Tianjing300162,China)ABSTRACTObjective:ToinvestigatetheprotectivemechanismofneuroactivesteroidallopregnanoloneonNmethylDaspartate(NMDA)inducedtoxicityinprimarymous
5、ecorticalneurons.Methotls:Primaryculturedmousecorticalneuronsweresubjectedtoallopregnanolone,theexpressionofaminobutyricacidreceptor2subunit(2GABAR)mRNAswasdetectedbyRTPCRandAktphosphorylationwasassayedbyWesternblotusingAktphosphoserine473specificant
6、ibody.AftertheculturedmousecorticalneuronswerepretreatedwithorwithoutallopregnanolonepriortotreatmentwithNMDA,DNAisolatedwasanalyzedbyagarosegelelectrophoresisandproteinscollectedwereanalyzedbyWesternblotwithanticleavedPARP,anticleavedcaspase3,andanticl
7、eavedcaspase9antibodies.Results:Whenculturedmousecorticalneuronswereexposedtoallopregnanoloneboththeexpressionof2GABARmRNAsandAktphosphorylationincreased.AllopregnanoloneinhibitedtheNMDAinducedapoptosisanddecreasedthelevelofactivePARP,activecaspase3a
8、ndactivecaspase9notablyatafinalconcen6trationof510mol/L.Conclusion:PretreatmentwithallopregnanolonemaybeneuroprotectiveonNMDAinducedneuronalcellsapoptosisbyincreasing2GABARexpres
