摘要：
目的:探索哺乳动物雷帕霉素靶蛋白(mTOR)抑制剂雷帕霉素在慢性脑缺血中的作用及机制.方法:6 周龄的ICR 小鼠通过右颈总动脉结扎诱导慢性脑缺血模型,采用蛋白质印迹法检测造模后不同时期(1、3、6、24 h,3、7 d,2、4、6 周)小鼠大脑皮层和海马组织中mTOR 信号通路mTOR、S6K 和S6 蛋白表达及其磷酸化水平.造模24 h 后腹腔注射雷帕霉素(3.0 mg/kg),经Fluoro-Jade B 染色观察细胞凋亡情况,蛋白质印迹法检测mTOR 信号通路的变化和对细胞自噬的影响,并采用Morris 水迷宫和Y 迷宫试验测定小鼠学习记忆功能.结果:模型小鼠大脑皮层和海马组织中mTOR 信号通路被异常激活,从6 h 开始一直持续到6 周,表现为mTOR、S6K 和S6 蛋白磷酸化增加.雷帕霉素可逆转由慢性脑缺血所致的mTOR 信号通路激活,并显著减少细胞凋亡(146.1 ±16.3与84.5 ±9.6,P <0.05).雷帕霉素还能显著逆转慢性脑缺血所导致的Beclin1和LC3-Ⅱ蛋白的表达.Morris 水迷宫和Y 迷宫试验结果显示,雷帕霉素组较模型组平台潜伏期缩短[(11.1 ±2.3)s 与(8.1 ±1.8)s,P <0.05]、游泳距离延长[(672.8 ±128.5) cm 与(558.2 ±124.9) cm,P <0.05]、穿越平台次数减少(2.8 ±0.9 与5.2 ±0.8,P <0.05)、正确反应率提高[(38.5 ±9.2)%与(64.9 ±7.9)%,P <0.05].结论:抑制mTOR 信号通路能改善由慢性脑缺血所致的学习记忆功能下降,其机制可能与抑制细胞凋亡和自噬相关.%Objective: To investigate the effect of mammalian target of rapamycin (mTOR)inhibitor—rapamycin on cognitive function after chronic cerebral ischemia in mice and its molecular mechanism.Methods: The chronic cerebral ischemia model was induced by ligation of right common carotid artery(rUCCAO)in 6-week-old ICR mice.The expressions of mTOR,S6K,S6 and corresponding phosphorylated proteins were detected by Western blotting at different time interval(1 h, 3 h, 6 h, 24 h, 3 d,7 d,2 w,4 w,6 w)after rUCCAO to determine the changes of mTOR signaling pathway.Rapamycin was administrated i.p.at the dose of 3.0 mg/kg 24 h after rUCCAO.Fluoro Jade B staining was used to detect the apoptotic cells.The expressions of Beclin and LC3-Ⅱ were detected by Western blotting to determine the status of autophagy.Morris water maze test and Y maze test were performed to evaluate cognitive functions.Results: The mTOR signaling pathway was abnormally activated from 6 h to 6 w after rUCCAO in mouse cortex.The activation of mTOR signaling pathway induced by rUCCAO was reversed by administration of rapamycin, and the apoptotic cell number was significantly decreased(146.1 ±16.3 vs 84.5 ± 9.6, P<0.05).Meanwhile,the elevation of Beclin and LC3-Ⅱ protein induced by rUCCAO was reversed by rapamycin administration.Furthermore, compared with vehicle-treated mice, the latent period[(11.1 ±2.3)s vs(8.1 ±1.8)s, P <0.05]and swimming distance[(672.8 ±128.5)cm vs(558.2 ±124.9)cm,P<0.05]were significantly decreased and the number of crossing the platform quadrant in Morris water maze increased(2.8 ±0.9 vs 5.2 ±0.8, P<0.05)in rapamycin-treated mice.Correct response rate in the Y maze was also increased significantly in rapamycin-treated mice[(38.5 ±9.2)% vs(64.9 ±7.9)%, P<0.05]. Conclusion: Inhibiting mTOR pathway by rapamycin reverses the rUCCAO-induced cognitive impairment partly through the suppression of apoptosis and autophagy.
