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Journal of ZheJiang University(Medical Science)  2017, Vol. 46 Issue (4): 405-412    DOI: 10.3785/j.issn.1008-9292.2017.08.10
    
Inhibiting mammalian target of rapamycin signaling pathway improves cognitive function in mice with chronic cerebral ischemia
ZHANG Binbin, WU Meiling, LIU Luna, ZHU Yangbin, KAI Jiejing, ZENG Linghui
School of Medicine, Zhejiang University City College, Hangzhou 310015, China
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Abstract  

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.



Key wordsSirolimus/pharmacology      Protein kinases/physiology      Signal transduction/physiology      Autophagy      Apoptosis      Brain ischemia/drug therapy      Memory/drug effects      Disease models, animal     
Received: 24 April 2017      Published: 25 August 2017
CLC:  R743  
Cite this article:

ZHANG Binbin, WU Meiling, LIU Luna, ZHU Yangbin, KAI Jiejing, ZENG Linghui. Inhibiting mammalian target of rapamycin signaling pathway improves cognitive function in mice with chronic cerebral ischemia. Journal of ZheJiang University(Medical Science), 2017, 46(4): 405-412.

URL:

http://www.zjujournals.com/xueshu/med/10.3785/j.issn.1008-9292.2017.08.10     OR     http://www.zjujournals.com/xueshu/med/Y2017/V46/I4/405


抑制哺乳动物雷帕霉素靶蛋白信号通路对慢性脑缺血小鼠认知功能的改善和机制

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。


关键词: 西罗莫司/药理学,  蛋白激酶类/生理学,  信号传导/生理学,  自噬,  细胞凋亡,  脑缺血/药物疗法,  记忆/药物作用,  疾病模型,  动物 

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