自噬在凝血酶预处理诱导脑缺血耐受过程中的作用及机制研究

Due to secondary brain injury, ischemic stroke could cause severe functional defects like hemiparesis. Pretreatment with a low dose of thrombin attenuates the brain edema and significantly reduces the infarct size in the rat middle cerebral artery occlusion model. Interestingly, autophagy occurs after thrombin treatment in the brain. .Our preliminary data showed that cortical co-injection of thrombin and 3-methyladenine (3MA, autophagy inhibitor) aggravated the cerebral infarction. In contrast, co-injection of thrombin and rapamycin (autophagy agonist) reduced the infarct size after cerebral ischemia. These results indicated that the autophagy activities were negatively correlated with the extent of ischemic brain damages. We thereby hypothesized that the protective effect of thrombin preconditioning after ischemic stroke was achieved through the autophagy..In this study, we will investigate the relations between autophagy activities and the extent of brain damage after thrombin preconditioning. The autophagy activities, regulated by 3MA and rapamycin, respectively, will be assessed by monitoring LC3 I/II conversion rate, beclin1 activities, and cellular ultrastructure. Infarct size, brain edema and neurological deficits will be applied to evaluate brain damage. Meanwhile, we will measure the changes of the heat shock protein (HSP)27 and HSP32, activated P44/42 mitogen-activated protein kinase (MAPK) and c-Jun amino-terminal kinase (JNK), which were reported to be up-regulated by thrombin preconditioning. Furthermore, to clarify the roles of MAPK and JNK cell signaling pathways in thrombin preconditioned ischemia, we will evaluate the brain damages after the level of P44/42 MAPK and/or JNK are inhibited..Our studies will provide novel insights into the role and mechanisms of the autophagy in thrombin preconditioning induced cerebral ischemic tolerance and thereby shed lights on the studies of clinical treatment and therapeutic targets for ischemic stroke.

脑缺血后继发性脑损伤是病情加重以及影响预后的重要原因。在中枢神经系统，缺血、缺氧等预处理都可以诱导脑缺血的耐受。缺血预处理的脑保护作用可以通过诱导自噬实现。凝血酶预处理也有脑保护作用，也可以诱导自噬，我们设想，凝血酶预处理诱导的脑保护作用也是通过自噬实现的。.本研究采用动物实验随机对照的方法，应用自噬活性药物，调节自噬的活性，评估自噬活性变化对凝血酶预处理脑保护效应的影响。在此基础上，深一步研究其中的病理机制。凝血酶预处理减轻脑缺血后脑损伤过程中上调了热休克蛋白27和32，P44/42 MAPK和JNK细胞信号传导通路也在此过程中被激活。那么，自噬调节凝血酶预处理脑保护作用的过程是否和上述蛋白有关？是否通过上述细胞信号传导通路起作用？需要我们进一步的验证。通过本研究，我们预期：自噬是凝血酶预处理诱导脑缺血耐受的关键因素；自噬调节凝血酶预处理脑保护作用的过程是通过相关细胞信号传导通路实现的。

研究背景： 缺血性卒中是严重威胁人类健康的疾病，目前仍然缺乏有效治疗手段，脑缺血后继发性脑损伤是病情加重以及影响预后的重要原因，其相关病理机制的研究成为迫切需要。研究提示，缺血预处理诱导的自噬可以减轻脑缺血后脑损伤，而凝血酶预处理也可以减轻脑缺血后脑损伤，凝血酶又能在脑内诱导细胞自噬，本课题应用大鼠脑缺血模型，研究凝血酶预处理减轻的脑缺血后脑损伤和自噬的关系以及更深层次的机制。.主要研究内容：成年SD大鼠随机分为对照的生理盐水组、凝血酶预处理组、凝血酶+雷帕霉素组和凝血酶+3MA组。（1）大鼠在注射7天建脑缺血模型，行永久性大脑中动脉阻塞手术。造模后24h测量脑梗塞面积体积,脑组织水含量以及行为学测试评估脑损伤；（2）电镜下直接观察大鼠自噬细胞，Western blot实验检测自噬指标LC3和beclin1，通道蛋白HSP27、HSP32、P44/42 MAPK和JNK；（3）在前面的基础上，预处理的同时可加入通道蛋白阻滞剂，再按前文所述的方法评估大鼠脑缺血后的脑损伤以及检测自噬的活性。.结果： 我们成功建立了大鼠脑缺血模型。凝血酶预处理后，脑梗塞的面积体积比较对照组减小，水含量降低，行为学好转。加入自噬激动剂雷帕霉素后，该作用得到进一步的增强，自噬抑制剂3MA可以降低凝血酶的这些作用。电镜下可见，凝血酶组的大鼠脑内可见典型的自噬细胞，Western blot结果表明，LC3的转化和beclin1的表达上升明显，HSP27和P44/42 MAPK的水平也有升高。雷帕霉素可以进一步加强LC3的转化，升高beclin1、HSP27以及P44/42 MAPK的水平；而3MA则降低了LC3的转化，beclin1、HSP27以及P44/42 MAPK的水平。预处理的同时加入P44/42 MAPK 抑制剂PD98058后，明显削弱了凝血酶的脑保护和激活自噬的作用。.结论： 凝血酶预处理减轻脑缺血后脑损伤的作用和自噬相关，增强自噬活性可以加强凝血酶脑保护的作用，该作用通过了P44/42 MAPK细胞通路，这些结论都加深了我们对脑缺血后脑损伤病理机制的理解。