连接蛋白43介导的ATP释放调控神经元线粒体自噬在缺血性脑损伤中的作用和机制研究

Mitochondrial abnormalities are the core of neuronal degeneration. It is associated with neuronal death. Mitophagy increased in penumbra neurons after cerebral ischemia / reperfusion. It has neuroprotective effects in early stage, but it may be harmful in late stage after cerebral ischemia / reperfusion. The mechanism of the regulation of neuronal mitophagy is not clear. Previous studies demonstrated that autophagy is energy dependence and connexin 43 (Cx43) half channel is an important way for astrocytes to release ATP into neurons. The expression of Cx43 decreased firstly and then increased after cerebral ischemia / reperfusion. Our previous studies showed that astrocyte proliferation and neuronal autophagy occurred in the same place after cerebral ischemia. So we put forward the hypothesis that the changes of ATP release, which was mediated by the changes of Cx43 expression, regulated neuronal mitophagy dynamically through AMPK-mTOR pathway after cerebral ischemia reperfusion and resulted in neuroprotection. In this study, the relationship among ATP release mediated by Cx43, neuronal mitophagy and neuronal survival was examined by Western-blot, immunofluorescence, transmission electron microscopy and other pathological method, so as to clarify the molecular mechanisms of ATP release mediated Cx43 regulating neuronal mitophagy and promoting neuronal survival after cerebral ischemia. It will provide a new idea for the research on the protection for ischemic brain injury.

线粒体异常是神经元变性的核心，与神经元死亡相关。有研究证实脑缺血/再灌注后早期缺血半暗带神经元线粒体自噬增加，与神经元存活相关。再灌注后期自噬可能有害，但其调节机制不清。既往研究证实，自噬具有能量依赖性，连接蛋白（Cx43）半通道是星形胶质细胞向神经元释放ATP的重要途径，脑缺血后Cx43表达先降低后升高，本课题组前期研究发现脑缺血后星形胶质细胞增生部位和神经元自噬部位相同，因此我们提出假说脑缺血再灌注后Cx43表达变化引起的ATP释放改变，通过AMPK-mTOR途径对神经元线粒体自噬产生动态调节，从而产生神经保护作用。本研究拟应用Western-blot、免疫荧光、流式分析等技术检测CX43介导的ATP释放、神经元线粒体自噬和神经元存活之间的关系，初步揭示脑缺血后Cx43介导的ATP释放影响神经元线粒体自噬促进神经元存活的分子机制，为缺血性脑损伤保护研究提供一个新的思路。

线粒体异常是神经元变性的核心，与神经元死亡相关。有研究证实脑缺血/再灌注后早期缺血半暗带神经元线粒体自噬增加，与神经元存活相关。再灌注后期自噬可能有害，但其调节机制不清。既往研究证实，自噬具有能量依赖性，连接蛋白（Cx43）半通道是星形胶质细胞向神经元释放ATP的重要途径。本研究通过Western-blot、免疫荧光、ATP检测等技术检测CX43介导的ATP释放、AMPK-mTOR通路改变、神经元线粒体自噬和神经元存活之间的关系，发现在脑缺血/再灌注后p-CX43表达变化增加，ATP释放减少，AMPK表达增加，mTOR表达减少，细胞线粒体自噬增加。干预CX43-AMPK-mTOR通路的任何环节都可能对下游分子表达、神经元线粒体自噬和神经元存活产生影响，且这种影响因干预时间不同、药物剂量不同，产生的效果也不同，研究结果表明CX43通过介导ATP释放的改变，影响AMPK-mTOR信号通路活性，调节神经元线粒体自噬，影响神经元存活。线粒体自噬在脑缺血/再灌注后不同时间的作用不同，要选择合适的时机进行干预才能增强神经保护作用。