脑缺血预处理通过p38 MAPK-mTOR信号通路调控神经元自噬诱导脑缺血耐受的机制研究

It has been found that cerebral ischemic preconditioning (CIP) can induce brain ischemic tolerance. It is important to study the mechanism involved in the process, because it will provide experiment fundament and new clue for the study in the prevention and treatment of brain ischemia. Autophagy is a process of bulk degradation of cytoplasmic components by lysosomes. Moderate autophagy plays protective effect, while incomplete or excessive autophagy can lead to cell death. mTOR is one of the important pathways regulating autophagy and is regulated by p38 MAPK. Our previous study has proved that p38 MAPK participates in the induction of brain ischemic tolerance induced by CIP. Our preliminary experiment results suggest that regulation of neuronal autophagy might contribute to the induction of oxygen-glucose deprivation (OGD) tolerance by OGD preconditioning. Taken together, we hypothesize that p38 MAPK-mTOR signaling pathway may participate in the regulation of neuronal autophagy for the induction of brain ischemic tolerance induced by CIP. The present project is aimed to prove the hypothesis in vivo and in vitro study by transmission electron microscope, immunohistochemistry, western blot, siRNA and other techniques. At first, we will study whether CIP induces brain ischemic tolerance by regulating autophagy. On the basis, we will further reveal the molecular mechanism of p38 MAPK-mTOR signaling pathway in regulating neuronal autophagy for the induction of brain ischemic tolerance by CIP. It will provide experiment fundament for the study in prevention and treatment of brain ischemia.

研究发现脑缺血预处理(CIP)可诱导脑缺血耐受，深入研究其机制可为脑缺血性疾病的防治研究提供新思路和线索，具有重要意义。自噬是溶酶体降解利用细胞内物质成分的过程，适度自噬发挥保护作用，但不完全或过度自噬可导致细胞死亡。mTOR是调节自噬的重要通路，且受p38 MAPK调控。我们前期研究证明，p38 MAPK在CIP诱导的脑缺血耐受中发挥作用。我们预实验结果提示氧-葡萄糖剥夺(OGD)预处理可能通过调节神经元自噬诱导OGD耐受。据此，我们提出CIP可能通过p38 MAPK-mTOR信号通路调控神经元自噬诱导脑缺血耐受。本研究将在整体水平和细胞水平，通过透射电镜、免疫组化、Western blot、siRNA等技术，首先证实CIP通过影响自噬诱导脑缺血耐受，在此基础上，进一步研究p38 MAPK-mTOR信号通路在细胞水平调控神经元自噬诱导缺血耐受的分子机制，为脑缺血疾病防治研究提供实验依据。

自噬是溶酶体降解利用细胞内物质成分的过程，适度自噬发挥保护作用，但不完全或过度自噬可导致细胞死亡。我们前期研究证明，自噬在CIP诱导的脑缺血耐受中发挥作用。但其机制尚不清楚。本课题应用免疫荧光、Western blot、电镜、siRNA等技术，进一步在整体和细胞水平深入探讨其机制。.在体和细胞水平研究均证实，自噬参与CIP或OGD预处理诱导的脑缺血耐受。Western blot结果证实在脑缺血耐受诱导过程中，Beclin-1、p62、LC3-II/LC3-I表达均上调；免疫荧光双标发现，神经元表达的LC3II明显增多；电镜结果显示神经元自噬小体数量明显增多；应用自噬的特异性抑制剂Bafilomycin A1，可抑制CIP或OGD预处理诱导脑缺血耐受。.细胞水平研究证实，p38 MAPK-mTOR信号通路通路调控神经元自噬参与OGD预处理诱导的脑缺血耐受。在该过程中p-p38 MAPK表达上调、p-mTOR表达下调；应用p38 MAPK的特异性抑制剂SB203580或其siRNA剂量依赖性抑制OGD诱导脑缺血耐受，同时抑制该过程中自噬相关蛋白Beclin-1、p62、LC3-II/LC3-I的表达上调和p-mTOR的表达下调；应用mTOR的抑制剂rapamycin或其siRNA均可引起自噬相关蛋白Beclin-1、p62、LC3-II/LC3-I的表达过度上调，同时阻断了OGD诱导的脑缺血耐受。.上述结果表明，OGD预处理通过p38 MAPK-mTOR信号通路诱导神经元自噬而发挥脑保护作用，为阐明脑缺血耐受诱导机制提供实验依据，为脑缺血性疾病的治疗提供新思路，具有重要意义。.该研究共发表全文5篇，项目负责人均为通讯作者，其中发表全文3篇(均为SCI论文)，会议论文1篇、会议报告1篇。共培养4名研究生，其中博士研究生3人，硕士研究生1人。