JNK介导的Bcl-2磷酸化调控神经细胞缺氧缺糖再复氧复糖所致的自噬性细胞死亡

Oxygen-glucose-deprivation/Reoxygenation(O/R)-induced autophagic cell death depended on JNK-Mediated phosphorylation of Bcl-2

目的:探讨自噬在神经细胞缺氧缺糖再复氧复糖损伤过程中的机制。方法:通过改良Takei和Endo的方法分离培养Sprague-Dawley大鼠皮层神经细胞并鉴定。培养至7 d后,将细胞随机分为4组,均培养细胞至0.5、2.0、6.0及12.0 h:(1)空白对照组;(2)实验组:即缺氧缺糖再复氧复糖组;(3)JNK抑制剂处理对照组:JNK特异性抑制剂SP600125处理神经细胞0.5 h;(4)JNK抑制剂预处理预处理+实验组:应用JNK特异性抑制剂SP600125预处理0.5 h后进行缺氧缺糖再复氧复糖。结果:噻唑蓝(MTT)结果示神经细胞的活性在实验组中随复氧复糖时间的延长而逐渐下降;JNK抑制剂预处理+实验组在12 h才出现明显降低(P<0.05)。电镜结果表明:实验组在0.5 h及2.0 h时,神经细胞内可见大量自噬泡及自噬溶酶体形成,6 h时自噬泡数量短暂降低后,在12 h又可见新"自噬潮"和大量已排空的自噬泡;JNK抑制剂预处理+实验组中,自噬泡在0.5 h和2.0 h时大量形成,随后随着复氧复糖时间的延长而逐渐减少。实验组和JNK抑制剂预处理+实验组,LC3Ⅱ蛋白表达在6 h前无差异,均表现为先增高再降低;而实验组LC3Ⅱ蛋白表达量在12 h却再次增加,JNK抑制剂预处理+实验组则持续降低。实验组JNK、Bcl-2的磷酸化水平及Beclin-1的蛋白表达量均随着复氧复糖时间的延长而逐渐增加;而在JNK抑制剂预处理+实验组,仅在12 h时才增加(P<0.05)。同时,Bcl-2/Beclin-1复合物在实验组呈逐渐分离的趋势,JNK抑制剂则明显抑制了这种分离。结论:JNK/Bcl-2/Beclin-1信号的调节可能是缺氧缺糖后复氧复糖诱导神经细胞自噬性细胞死亡的机制之一。

Objective：The purpose of this study was to investigate the role of autophagy in oxygen-glucose-deprivation/reoxygenation（OGD/R）injury in rat neurons. Methods：Cortical neurons were isolated from Sprague-Dawley rats and identified by immunofluorescence. The cortical neurons were randomly assigned to four groups：control group（Ⅰ）,experimental group（OGD/R group,Ⅱ）,JNK inhibitor pretreatment group（Ⅲ）and JNK inhibitor pretreatment＋OGD/R group（Ⅳ）. Results：Neuronal cell viability significantly decreased after 6 h and 12 h of reoxygenation in Group Ⅳ（P〈0.05）. Electron microscopy results showed the presence of many autophagic vacuoles and the formation of autolysosomes in the neurons;the number of autophagic vacuoles decreased transiently at 6 h,while a new autophagic flux and a large number of empty autophagic vacuoles were observed at 12 h. In Group Ⅳ,a large number of autophagic vacuoles were present at 0.5 h and 2 h of reoxygenation,which gradually decreased with increasing reoxygenation time. No significant differences in the expression of the LC3Ⅱprotein were detected between the Group Ⅱ and Ⅳ prior to 6 h of reoxygenation,and LC3Ⅱ expression showed an overall rise-decline pattern. However,LC3Ⅱ protein expression increased in GroupⅡ at 12 h of reoxygenation,whereas a continuous decline was observed in Group Ⅳ. The levels of phosphorylated JNK and Bcl-2 and the expression of Beclin-1 increased gradually as the reoxygenation time going in Group Ⅱ,whereas they increased at 12 h of reoxygenation in Group Ⅳ（P〈0.05）. In addition,progressive dissociation of the Bcl-2/Beclin-1 complex was observed in the GroupⅡ,while JNK inhibitor suppressed this dissociation. Conclusion：The regulation of the JNK/Bcl-2/Beclin-1 signaling pathway may be one of the mechanisms underlying the OGD/R-induced autophagic cell death of neurons.