【作者】 尹晓慧；

【导师】 吴士良； 张光毅； 侯筱宇；

【作者基本信息】 苏州大学 ， 病理学与病理生理学， 2014， 博士

【摘要】 研究背景和研究目的缺血再灌注过程中，谷氨酸过度释放造成的神经兴奋毒作用是缺血性脑损伤的重要机制。谷氨酸受体GluK2亚基被过度激活后，通过脚手架蛋白PSD-95与酪氨酸蛋白激酶MLK3结合，形成GluK2-PSD-95-MLK3信号模块。在此模块中，MLK3通过交叉磷酸化而将自身激活，并作用于其下游底物，通过MAPK信号级联，最后激活转录因子c-Jun，引起FasL表达增加。这就是所说的MAPK信号通路介导凋亡的核通路。Fas是重要的死亡受体，FasL表达的增加提示FasL-Fas信号通路在GluK2-PSD-95信号模块介导的缺血性脑损伤中发挥终极作用。然而Fas信号通路是否真的在GluK2-PSD-95信号模块介导的缺血性脑损伤中发挥重要作用，以及Fas介导缺血性脑损伤的具体机制目前并不很清楚。Fas凋亡信号通路广泛存在于各种组织细胞。Fas被配体FasL激活后，其胞内区与FADD结合，并通过FADD募集凋亡蛋白caspase8，组装成DISC复合体。在此复合体中，caspase8自身裂解活化，继而启动caspase信号级联或刺激线粒体释放Cyt c而激活凋亡执行蛋白caspase3，引起DNA水解和细胞凋亡。Fas凋亡信号通路中的信号分子受到多种机制的调节。Fas的巯基亚硝基化修饰能够促进其由胞浆向胞膜转位，并在胞膜上聚集成高分子量的聚合体CD95hi，同时向胞浆内化。CD95hi能够与FADD和caspase8组装成hiDISC，hiDISC是Fas介导凋亡的更主要形式。本研究分两个部分，分别利用PSD95的PDZ1抑制性环肽和Fas shRNA，采用生物化学和组织染色的方法，证明GluK2-PSD-95信号模块通过Fas信号通路介导缺血性神经元损伤；利用生物化学的方法，研究NO供体GSNO保护缺血再灌神经元的机制，从而反映Fas信号通路介导缺血性脑损伤的机制。第一部分GluK2-PSD-95信号模块通过Fas信号通路介导缺血性神经元损伤方法1.用TUNEL法，检测PDZ1环肽阻断GluK2-PSD-95结合后，再灌注5d大鼠海马CA1区锥体神经元凋亡情况的变化。2.用免疫荧光方法，检测PDZ1环肽对缺血再灌过程中Fas和FasL表达的影响；用免疫印迹和免疫沉淀的方法，检测PDZ1对缺血再灌过程中DISC组装和下游信号蛋白活化的影响。3.用DAPI染色法检测Fas shRNA对OGD后原代大鼠海马神经元凋亡的影响。结果1.TUNEL检测显示，缺血前给予PDZ1环肽显著减少了大鼠海马CA1区神经元的凋亡。2.缺血前给予PDZ1环肽显著抑制了再灌6h FasL表达的增加、DISC的组装和下游凋亡蛋白的激活，但是对Fas表达没有明显影响。3.OGD前给予Fas shRNA显著减少了复糖复氧过程中原代海马神经元的凋亡。第二部分GSNO通过抑制Fas巯基亚硝基化和Fas信号转导在缺血性脑损伤中发挥神经元保护作用方法1.用焦油紫染色方法，检测大鼠全脑缺血前给予GSNO对海马CA1区神经元死亡的影响。2.用免疫印迹的方法，检测GSNO对缺血再灌注过程中凋亡相关蛋白活化的影响。3.用生物素转化法，检测缺血再灌注过程中Fas巯基亚硝基化的变化，及缺血前给予GSNO对Fas巯基亚硝基化的影响。4.用生物素转化法，检测缺血前给予GSNO对nNOS巯基亚硝基化的影响；NOS活性检测试剂盒和NO检测试剂盒检测GSNO对nNOS活性和NO生成的影响。5.缺血前给予nNOS抑制剂7-NI，生物素转化法检测Fas巯基亚硝基化的变化。6.用免疫印迹方法，分别检测缺血再灌过程中Fas和CD95hi在胞膜和胞浆中的表达变化，及GSNO对其影响。7.用免疫沉淀方法，检测GSNO对缺血再灌过程中DISC和hiDISC组装的影响。结果1.大鼠全脑缺血前给予GSNO显著减少了再灌注5d海马CA1区神经元的死亡。2.缺血前给予GSNO显著抑制了再灌注6h凋亡相关蛋白的活化。3.大鼠全脑缺血再灌注过程中Fas巯基亚硝基化增强，再灌注6h达高峰；缺血前给予GSNO显著抑制了再灌注6h Fas的巯基亚硝基化。GSNO对照组中Fas巯基亚硝基化也有较明显增强，但是远远低于缺血再灌注组。4.缺血再灌6h nNOS巯基亚硝基化降低，其活性及NO生成量显著增加；缺血前给予GSNO则显著增强nNOS的巯基亚硝基化，但是抑制了其活性和NO生成。5.缺血前给予7-NI，再灌6h Fas巯基亚硝基化显著降低。6.缺血再灌过程中，Fas的胞膜表达量明显增加，再灌6h达高峰；Fas的胞浆表达量相应地显著减少，以再灌6h为低谷；CD95hi的胞膜表达量也有显著增加，且再灌6h为高峰；而且CD95hi的胞浆表达量呈同样的增加趋势。缺血前给予GSNO阻止了再灌6hFas和CD95hi在胞膜和胞浆中表达的变化。7.缺血前给予GSNO显著抑制了再灌6h DISC和hiDISC组装的增强。结论鉴于以上实验结果，我们得出以下结论：1.缺血再灌过程中，GluK2-PSD-95信号模块通过Fas信号通路介导神经元损伤。2. NO供体GSNO通过使nNOS巯基亚硝基化而抑制其活性，减少NO生成，从而使Fas的巯基亚硝基化降低，抑制其膜转位、聚集和内化，以及DISC组装和下游凋亡蛋白的活化，最终实现其保护缺血再灌神经元的功能。更多还原

【Abstract】 Background and purposeOne of the crucial mechanisms of ischemia-induced brain injury is that theexcessively released glutamate during reperfusion induces excitoxicity. Whenexcessively stimulated, GluK2, one subunit of glutamate receptors, recruits MLK3through the scaffold PSD-95and assemblies GluK2-PSD-95-MLK3module. Then,MLK3auto-activates via cross phosphorylation and activates its substrates throughMAPK cascade, resulting in activation of c-Jun and subsequent increased FasLexpression. This procedure has been named the nuclear pathway mediated by MAPKsignaling pathway. Since Fas is one of the important death receptors, the increase ofFasL expression after ischemia suggests that FasL-Fas pathway might act at theexecution phase. However, this postulation requires further investigations. Themechanisms of Fas-mediated ischemic brain injury also remain to be clarified.Fas-induced apoptosis pathway ubiquitously functions in various tissues and cells.When stimulated by its ligand FasL, the cytosolic DD of Fas interacts with FADD,which subsequently recruits caspase8and assemblies DISC. DISC formation facilitatesthe cluster and auto-activation of caspase8. Once activated, caspase8initiates caspasecascade or induces Cyt c release from mitochondria, which activate the excecutorcaspase, caspase3, and resulting in DNA cleavage and apoptosis. Multiple mechanismsregulate Fas apoptosis-inducing pathway. S-nitrosylation of Fas facilitates itstrans-localization to cell membrane, aggregation into CD95hiand internalization intoplasma. CD95hican form hiDISC with FADD and caspase8, which is the preferredmode that Fas mediate apoptosis.In this study, we investigated ischemic brain injury in two sections. We adoptedPDZ1domain inhibitor peptide and Fas shRNA to prove that GluK2-PSD-95modulemediates ischemic brain injury via Fas pathway. Then, we elucidated the mechanisms that GSNO protects neurons from ischemia injury to reflect the mechanisms that Fasmediate ischemic brain injury.Part Ⅰ GluK2-PSD-95mediates brain injury after ischemiavia Fas pathwayMethods1. Use TUNEL method to test the effect of PDZ1peptide on cell apoptosis in rathippocampal CA1region at5d of reperfusion.2. Use immumofluence method to test the effect of PDZ1peptide on Fas andFasL expression during reperfusion; use immunoblot and immunopricipitation to testthe effect of PDZ1peptide on DISC assembly and the activation of downstreammolecules during reperfusion.3. Use DAPI staining to examine the effect of Fas shRNA on apoptosis ofprimary hippocampal neurons after OGD.Results1. TUNEL staining showed that pretreatment of PDZ1peptide significantlyattenuated neuronal apoptosis in rat hippocampal CA1region.2. Pretreatment of PDZ1peptide attenuated the increase of FasL expression,DISC assembly and the activation of downstream molecules at6h of reperfusion,without effect on Fas expression.3. Pretreatment of Fas shRNA significantly reduced primary hippocampal neuronapoptosis after OGD.Part Ⅱ Neuroprotection of S-nitrosoglutathione againstischemia injury by down-regulating Fas S-nitrosylationand downstream signalingMethods1. Use cresyl viollet staining to examine the effect of GSNO pretreatment onneurons death in rat hippocampal CA1region after ischemia.2. Use immunoblot to examine the effect of GSNO pretreatment on activation of 3. Use Biotin-switch method to examine the S-nitrosylation of Fas duringreperfusion and the effect of GSNO pretreatment on Fas S-nitrosylation.4. Use Biotin-switch method to examine the effect of GSNO pretreatment on theS-nitrosylation of nNOS. Use NOS activity testing kit and NO testing kit to examine theeffect of GSNO pretreatment on NOS activity and NO creation.5. Use Biotin-switch method to examine the effect of7-NI pretreatment on theS-nitrosylation of Fas.6. Use immunoblot to test the expression of Fas and CD95hiat cell membrane andin plasma at vary reperfusion times and the effect of GSNO pretreatment on them.7. Use immunoprecipitation to examine the effect of GSNO pretreatment onDISC and hiDISC assembly.Results1. Pretreament of GSNO before ischemia significantly attenuated neurons deathin hippocampal CA1region.2. Pretreament of GSNO before ischemia significantly inhibited the activation ofapoptosis-associating proteins at6h of reperfusion.3. Fas S-nitrosylation was significantly enhanced during reperfusion and peakedat6h. Pretreament of GSNO before ischemia significantly inhibited Fas S-nitrosylationat6h of reperfusion. Although Fas S-nitrosylation was also increased in GSNO controlgroup, it was extremely lower than ischemia/reperfusion group.4. nNOS S-nitrosylation was significantly decreased at6h of reperfusion,however, its activity and NO creation was enhanced. Pretreament of GSNO beforeischemia significantly enhanced nNOS S-nitrosylation, but inhibited its activity and NOcreation.5. Pretreament of7-NI before ischemia significantly decreased FasS-nitrosylation at6h of reperfusion.6. Fas expression at cell membrane was significantly increased during reperfusion,and peaked at6h of reperfusion. Consistently, Fas expression in plasma decreased andreached the lowest level at6h. CD95hiexpression at cell membrane was alsosignificantly increased during reperfusion, and peaked at6h. However, CD95hiexpression in plasma showed the same increase. Pretreament of GSNO beforeischemia significantly blocked the variation of Fas and CD95hi. 7. Pretreament of GSNO before ischemia significantly blocked the assembly ofDISC and hiDISC.ConclusionsBased on the above results, we conclude as below:1. GluK2-PSD-95mediates ischemic brain injury via Fas pathway.2. GSNO inhibits nNOS activation and NO creation after brain ischemia throughS-nitrosylation on nNOS. GSNO-induced NO reduction during reperfusion causesdecrease of Fas S-nitrosylation, subsequent membrane translocation, aggregation andinternalization, which attenuates DISC assembly and activation of apoptosis proteins,thus protecting neurons from ischemic injury.更多还原