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].
Conclusions: Inhibiting mTOR pathway by rapamycin reverses the rUCCAO-induced cognitive impairment partly through the suppression of apoptosis and autophagy.
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。