GSK-3β/PINK1-Parkin通路调控脑缺血中线粒体自噬的作用及机制研究

Stroke is one leading cause of death worldwide. Although many clinical and basic research studies have been focused on the possible mechanisms, therapy for stroke is still underutilized with frustrating progress. Among all the factors contribute to the disease, mitochondria function has been proved to play a crucial role. As the autophagy for damaged mitochondria, mitophagy is seemingly as one promising target for central nervous system disease and ischemic injury. Previous studies proved GSK-3β involved in mitochondria functional regulation, as well as neuroprotection after ischemic injury. Moreover, in our preliminary experiment, we found GSK-3β phosphorylation upregulated Parkin expression, which is one classic mitophagy related protein. But whether GSK-3 is involved in mitophagy in cerebral ischemic injury remains unknown and the related molecular mechanisms also need further exploration. The aim of our study is to investigate the role of GSK-3β/PINK1-Parkin signaling pathway in mitophagy during cerebral ischemic injury. Using both in vivo and in vitro assays, together with morphology, molecular biology and proper intervention techniques, we assume that GSK-3β regulated PINK1-Parkin signaling pathway participants in mitophagy. Furthermore, the other possible involved molecules are also explored and the interfering strategies are constructed. Overall, by clarifying the role of GSK-3β/PINK-Parkin pathway in mitophagy during cerebral ischemic reperfusion injury, it would be helpful for searching the novel future intervention targets for this major public health burden.

探索治疗缺血性脑卒中的新靶点是重要的医学课题。线粒体参与多种生理病理进程，在脑缺血损伤中扮演重要角色。线粒体自噬能将受损的线粒体降解从而维持细胞内环境稳定，可能作为未来脑卒中干预的关键靶点。大量文献和我们以往研究工作表明GSK-3β活性对脑缺血损伤发挥关键调控作用，且GSK-3β参与线粒体能量代谢、氧化应激、凋亡等功能，我们近期也证明GSK-3β磷酸化可上调线粒体自噬相关蛋白Parkin的表达，据此提出“GSK-3β/PINK1-Parkin通路调控脑缺血损伤中线粒体自噬”的假设。本课题拟通过离体及在体模型，结合功能、形态、分子生物学等方法及多种干预手段研究GSK-3β是否参与调控脑缺血损伤中的线粒体自噬，分析PINK1-Parkin是否作为其下游信号通路发挥关键作用。通过本研究有助于深入理解脑缺血损伤中线粒体自噬分子信号调控机制，也可为脑卒中临床干预提供潜在的新靶点。

本研究拟证明“GSK-3β/PINK1-Parkin通路调控脑缺血损伤中线粒体自噬”的假设，通过离体及在体模型，结合功能、形态、分子生物学等方法及多种干预手段研究GSK-3β是否参与调控脑缺血损伤中的线粒体自噬，分析PINK1-Parkin是否作为其下游信号通路发挥关键作用。结果发现：首先，证明GSK-3β参与脑缺血损伤及线粒体自噬的调控过程，通过在体大脑缺血再灌注损伤模型（ischemic reperfusion, IR），观察到GSK-3β抑制剂TDZD-8可减轻IR脑梗死面积，运用电镜扫描观察到自噬小体在使用TDZD-8后增多，运用western blot及免疫荧光技术观察到线粒体自噬相关蛋白LC3-I、PINK1及Parkin表达增高，SQSTM1及mTOR表达降低；其次，证明GSK-3β磷酸化的保护作用部分由线粒体自噬介导，在体通过工具药抑制线粒体自噬，TDZD-8神经保护作用明显减弱，表现为脑梗死面积较TDZD-8组明显增大，线粒体自噬相关蛋白LC3-I、PINK1及Parkin表达较TDZD-8组明显减低，SQSTM1及mTOR表达升高；同时运用CCK-8检测、免疫荧光及western blot在离体原代神经元氧糖剥夺模型（Oxygen glucose deprivation, OGD）中也观察到了相似的改变；最后，证明干预PINK1/Parkin能够影响GSK-3β产生的保护作用，观察到运用PINK1/Parkin干扰RNA在离体OGD中能够逆转TDZD-8的保护作用，包括运用干扰RNA后CCK-8及凋亡增高，mPTP减低，LC3-I表达减低，SQSTM1表达增高。通过本研究有助于深入理解脑缺血损伤中线粒体自噬分子信号调控机制，也可为脑卒中临床干预提供潜在的新靶点。