Please wait a minute...
浙江大学学报（医学版）
浙江大学学报（医学版）  2017, Vol. 46 Issue (4): 405-412    DOI: 10.3785/j.issn.1008-9292.2017.08.10
原著     
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
张斌斌, 吴美玲, 刘露娜, 竺杨彬, 开洁静, 曾玲晖
浙江大学城市学院医学院, 浙江 杭州 310015
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
 全文: PDF(4136 KB)  
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。
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    
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 words: Sirolimus/pharmacology    Protein kinases/physiology    Signal transduction/physiology    Autophagy    Apoptosis    Brain ischemia/drug therapy    Memory/drug effects    Disease models, animal
收稿日期: 2017-04-24 出版日期: 2017-08-25
CLC:  R743  
基金资助:

杭州市科技局医疗卫生专项（20140633B37）；杭州市科技局重大专项（20152013A02）；国家自然科学基金（81371429）
通讯作者: 曾玲晖(1972-),女,博士,教授,博士生导师,主要从事神经系统疾病的发病机制及药物治疗研究;E-mail:zenglh@zucc.edu.cn;http://orcid.org/0000-0002-5924-4419     E-mail: zenglh@zucc.edu.cn
作者简介: 张斌斌(1996-),男,学士,主要从事神经系统药物研究;E-mail:zbb19960528@163.com;http://orcid.org/0000-0002-2577-8826;吴美玲(1993-),女,硕士,主要从事神经损伤性疾病的机制及药物治疗研究;E-mail:chicsoon@163.com;http://orcid.org/0000-0001-9549-9729
服务　　
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章　　

引用本文:

张斌斌 等. 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[J]. 浙江大学学报（医学版）, 2017, 46(4): 405-412.

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.

链接本文:

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

[1] 肖俊杰,詹青.慢性脑缺血免疫炎症损伤机制与相关治疗研究进展[J].脑与神经疾病杂志,2009,17(2):154-155. XIAO Junjie, ZHAN Qing. Research progress on mechanism and treatment of chronic cerebral ischemia[J]. Journal of Brain and Nervous Diseases,2009,17(2):154-155. (in Chinese)
[2] 刘汉兴,章军建.慢性脑缺血与认知功能障碍[J].国际脑血管病杂志,2004,12(4):278-281. LIU Hanxing, ZHANG Junjian. Chronic cerebral ischemia and cognitive dysfunction[J]. International Journal of Cerebrovascular Diseases,2004,12(4):278-281. (in Chinese)
[3] KOH P O, CHO J H, WON C K, et al. Estradiol attenuates the focal cerebral ischemic injury through mTOR/p70S6 kinase signaling pathway[J]. Neurosci Lett,2008,436(1):62-66.
[4] ZARE M F, ABOUTALEB N, HABIBEY R, et al. Increased phosphorylation of mTOR is involved in remote ischemic preconditioning of hippocampus in mice[J]. Brain Res,2013,1526:94-101.
[5] CHAUHAN A, SHARMA U, JAGANNATHAN N R, et al. Rapamycin protects against middle cerebral artery occlusion induced focal cerebral ischemia in rats[J]. Behav Brain Res,2011,225(2):603-609.
[6] YIN L, YE S, CHEN Z, et al. Rapamycin preconditioning attenuates transient focal cerebral ischemia/reperfusion injury in mice[J]. Int J Neurosci,2012,122(12):748-756.
[7] CHI O Z, BARSOUM S, VEGA-COTTO N M, et al. Effects of rapamycin on cerebral oxygen supply and consumption during reperfusion after cerebral ischemia[J]. Neuroscience,2016,316:321-327.
[8] MAO L, JIA J, ZHOU X, et al. Delayed administration of a PTEN inhibitor BPV improves functional recovery after experimental stroke[J]. Neuroscience,2013,231:272-281.
[9] SANGHERA K P, MATHALONE N, BAIGI R, et al. The PI3K/Akt/mTOR pathway mediates retinal progenitor cell survival under hypoxic and superoxide stress[J]. Mol Cell Neurosci,2011,47(2):145-153.
[10] YIN L, YE S, CHEN Z, et al. Rapamycin preconditioning attenuates transient focal cerebral ischemia/reperfusion injury in mice[J]. Int J Neurosci,2012,122(12):748-756.
[11] URBANEK T, KUCZMIK W, BASTA-KAIM A, et al. Rapamycin induces of protective autophagy in vascular endothelial cells exposed to oxygen-glucose deprivation[J]. Brain Res,2014,1553:1-11.
[12] KIM Y C, GUAN K L. mTOR:a pharmacologic target for autophagy regulation[J]. J Clin Invest,2015,125(1):25-32.
[13] WEI K, WANG P, MIAO C Y. A double-edged sword with therapeutic potential:an updated role of autophagy in ischemic cerebral injury[J]. CNS Neurosci Ther,2012,18(11):879-886.
[14] XU Y, TIAN Y, TIAN Y, et al. Autophagy activation involved in hypoxic-ischemic brain injury induces cognitive and memory impairment in neonatal rats[J]. J Neurochem,2016,139(5):795-805.
[15] KOIKE M, SHIBATA M, TADAKOSHI M, et al. Inhibition of autophagy prevents hippocampal pyramidal neuron death after hypoxic-ischemic injury[J]. Am J Pathol,2008,172(2):454-469.
[16] 刘辉玉,陈锐,李亚男,等.慢性间歇低氧对小鼠海马CA1区神经元自噬的影响[J].中华结核和呼吸杂志,2011,34(6):467-469. LIU Huiyu, CHEN Rui, LI Yanan, et al. Effects of chronic intermittent hypoxia on neuron autophagy in hippocampal CA1 region of mice[J]. Chinese Journal of Tuberculosis and Respiratory Diseases,2011,34(6):467-469. (in Chinese)
[17] SHI R, WENG J, ZHAO L, et al. Excessive autophagy contributes to neuron death in cerebral ischemia[J]. CNS Neurosci Ther,2012,18(3):250-260.
[18] CARLONI S, GIRELLI S, SCOPA C, et al. Activation of autophagy and Akt/CREB signaling play an equivalent role in the neuroprotective effect of rapamycin in neonatal hypoxia-ischemia[J]. Autophagy,2010,6(3):366-377.
[19] SEKIGUCHI A, KANNO H, OZAWA H, et al. Rapamycin promotes autophagy and reduces neural tissue damage and locomotor impairment after spinal cord injury in mice[J]. J Neurotrauma,2012,29(5):946-956.
[20] YANG X, HEI C, LIU P, et al. Inhibition of mTOR pathway by rapamycin reduces brain damage in rats subjected to transient forebrain ischemia[J]. Int J Biol Sci,2015,11(12):1424-1435.
[21] CHONG Z Z, YAO Q, LIN H H. The rationale of targeting mammalian target of rapamycin for ischemic stroke[J]. Cell Signal,2013,25(7):1598-1607.
[22] CHEN H, XIONG T, QU Y, et al. mTOR activates hypoxia-inducible factor-1α and inhibits neuronal apoptosis in the developing rat brain during the early phase after hypoxia-ischemia[J]. Neurosci Lett,2012,507(2):118-123.
[1] 田华 等. CD97免疫表位对乳腺癌细胞株MDA-MB231生物学行为的影响[J]. 浙江大学学报（医学版）, 2017, 46(4): 341-348.
[2] 邹继霞,章程燕,王苹莉. 肿瘤过继T细胞免疫治疗应用进展[J]. 浙江大学学报（医学版）, 2017, 46(2): 211-217.
[3] 王莉,王瑜,武海英. 尼克酰胺降低妊娠期糖尿病大鼠的血糖水平以及调控线粒体超氧水平研究[J]. 浙江大学学报（医学版）, 2017, 46(2): 179-185.
[4] 王莉 等. 尼克酰胺降低妊娠期糖尿病大鼠的血糖水平以及调控线粒体超氧水平研究[J]. 浙江大学学报（医学版）, 2017, 46(2): 179-185.
[5] 王晏鹏,黄琼晓,徐盛,舒静. 采用吸宫和电凝方法建立小鼠子宫内膜损伤模型的比较[J]. 浙江大学学报（医学版）, 2017, 46(2): 186-191.
[6] 王晏鹏 等. 采用吸宫和电凝方法建立小鼠子宫内膜损伤模型的比较[J]. 浙江大学学报（医学版）, 2017, 46(2): 186-191.
[7] 邹继霞 等. 肿瘤过继T细胞免疫治疗应用进展[J]. 浙江大学学报（医学版）, 2017, 46(2): 211-217.
[8] 张力三,陈冠峰,陈洁芳,何旭东,胡兴越. 组胺改善癫痫所致记忆形成障碍的实验研究[J]. 浙江大学学报（医学版）, 2017, 46(1): 1-6.
[9] 高思倩,沈咏梅,耿福能,李艳华,高建青. 糖尿病溃疡动物模型的建立及相关治疗研究进展[J]. 浙江大学学报（医学版）, 2017, 46(1): 97-105.
[10] 郑艳榕,张翔南,陈忠. Nix介导的线粒体自噬机制的研究进展[J]. 浙江大学学报（医学版）, 2017, 46(1): 92-96.
[11] 侯甲福,武蕾蕾,才玉婷,高长久,安锦丹,于忠诚. 锦花清热胶囊抗内毒素作用实验研究[J]. 浙江大学学报（医学版）, 2017, 46(1): 74-79.
[12] 马婷婷,王毅,陈晓倩,赵筱萍. 液相色谱-质谱联用导向的黄葵胶囊肾保护活性物质研究[J]. 浙江大学学报（医学版）, 2017, 46(1): 66-73.
[13] 陈立颖,汪仪,陈忠. 颞叶癫痫与海马成体神经再生[J]. 浙江大学学报（医学版）, 2017, 46(1): 22-29.
[14] 汪锡婷,唐藕凤,叶夷露,郑鸣之,胡珏,陈忠,钟恺. 藏红花素对小鼠电点燃癫痫的影响[J]. 浙江大学学报（医学版）, 2017, 46(1): 7-14.
[15] 屈涛 等. 丹参素对去势大鼠骨质量的影响[J]. 浙江大学学报（医学版）, 2016, 45(6): 587-591.