雷帕霉素在大鼠局灶性脑缺血再灌注后24 h给药对脑损伤的保护作用

doi:10.3785/j.issn.1008-9292.2018.10.01

浙江大学学报（医学版）

2018, Vol. 47

Issue (5): 443-449 DOI: 10.3785/j.issn.1008-9292.2018.10.01

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雷帕霉素在大鼠局灶性脑缺血再灌注后24 h给药对脑损伤的保护作用

梁刚(

),牛育苗,李一涵,魏安怡,董静尹*(

),曾玲晖*(

)

浙江大学城市学院医学院, 浙江杭州 310015

Rapamycin treatment starting at 24 h after cerebral ischemia/reperfusion exhibits protective effect on brain injury in rats

LIANG Gang(

),NIU Yumiao,LI Yihan,Wei Anyi,DONG Jingyin*(

),ZENG Linghui*(

)

School of Medicine, Zhejiang University City College, Hangzhou 310015, China

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摘要：

目的: 探讨雷帕霉素在大鼠局灶性脑缺血再灌注后24 h给药对脑损伤的保护作用。方法: 104只Sprague Dawley大鼠随机分为手术对照组、模型对照组、建模后6 h雷帕霉素给药组（6 h给药组）和建模后24 h雷帕霉素给药组（24 h给药组）。采用Longa法建立大鼠大脑中动脉缺血再灌注模型。采用神经功能损伤程度评分（NSS）对大鼠进行神经功能评分；氯化三苯基四氮唑（TTC）染色法检测各组大鼠脑梗死体积；Fluoro-Jade B（FJB）染色法检测大鼠脑组织神经元凋亡；蛋白质印迹法检测各组大鼠mTOR信号通路磷酸化S6蛋白表达。结果: 与手术对照组比较，模型对照组大鼠NSS升高，脑梗死体积增加，FJB阳性细胞增多，半暗带磷酸化S6蛋白表达量增加（均P < 0.01）；与模型对照组比较，6 h和24 h给药组大鼠NSS均降低，脑梗死体积缩小，FJB阳性细胞减少，半暗带磷酸化S6蛋白表达量减少（均P < 0.05或P < 0.01），且24 h给药组各项指标变化与6 h给药组差异均无统计学意义（均P > 0.05）。结论: 缺血再灌注后24 h给予雷帕霉素对大鼠局灶性脑缺血再灌注所致脑损伤仍有保护作用。

关键词： 脑缺血/药物疗法; 再灌注损伤/药物疗法; 神经元; 细胞凋亡; 蛋白激酶类/生理学; 西罗莫司/药理学; 信号传导

Abstract:

Objective: To investigate whether rapamycin treatment starting at 24 h after cerebral ischemia/reperfusion(I/R) has protective effect on brain injury in rats. Methods: The rat I/R model was established by middle cerebral artery occlusion according to Longa's method. A total of 104 Sprague Dawley rats were randomly divided into sham group, model group, and rapamycin-treated groups (6 h or 24 h after modeling). Neurological function was assessed with neurological severity score (NSS). Triphenyl tetrazolium chloride (TTC) staining and Fluoro-Jade B (FJB) staining were used to examine the infarct volume and neuronal apoptosis, respectively. The expression of p-S6 protein in mTOR signaling pathway was detected by Western blot analysis. Results: Compared with sham group, NSS of the model group was significantly increased and TTC staining indicated obvious infarct area (all P < 0.01). Furthermore, significantly increased number of FJB-positive cells and p-S6 expression in the penumbra area were shown in the model group (all P < 0.01). Compared with the model group, both rapamycin-treated groups demonstrated decreased NSS, infarction volume and FJB positive cells as well as p-S6 expression in the penumbra area (P < 0.05 or P < 0.01). There was no significant difference between the groups of rapamycin administrated 6 h and 24 h after modeling (all P > 0.05). Conclusion: Rapamycin treatment starting at 24 h after I/R exhibits protective effect on brain injury in rats.

Key words: Brain ischemia/drug therapy Reperfusion injury/drug therapy Neurons Apoptosis Protein kinases/physiology Sirolimus/pharmacology Signal transduction

收稿日期: 2018-09-12 出版日期: 2019-01-23

R743

基金资助: 杭州市科技发展计划重大科技创新专项(20152013A02);浙江省公益技术应用研究（实验动物）(2017C37122)

通讯作者: 董静尹（1976—），女，硕士，副教授，主要从事神经病理学研究；E-mail:dongjy@zucc.edu.cn;https://orcid.org/0000-0002-2477-3222.曾玲晖（1972—），女，博士，教授，主要从事神经病理学研究；E-mail:zenglh@zucc.edu.cn;https://orcid.org/0000-0002-5924-4419

作者简介: 梁刚(1990-), 男, 硕士研究生, 药师, 主要从事药理学研究; E-mail:wenlinglg90@163.com; https://orcid.org/0000-0003-0679-0894

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引用本文:

梁刚, 牛育苗, 李一涵, 魏安怡, 董静尹, 曾玲晖. 雷帕霉素在大鼠局灶性脑缺血再灌注后24 h给药对脑损伤的保护作用[J]. 浙江大学学报（医学版）, 2018, 47(5): 443-449.

LIANG Gang, NIU Yumiao, LI Yihan, Wei Anyi, DONG Jingyin, ZENG Linghui. Rapamycin treatment starting at 24 h after cerebral ischemia/reperfusion exhibits protective effect on brain injury in rats. J Zhejiang Univ (Med Sci), 2018, 47(5): 443-449.

链接本文:

http://www.zjujournals.com/med/CN/10.3785/j.issn.1008-9292.2018.10.01 或 http://www.zjujournals.com/med/CN/Y2018/V47/I5/443

表 1 神经功能损伤程度评分系统[15]

图 1 雷帕霉素不同时期给药对缺血再灌注大鼠神经功能缺损评分的影响(n=10)

图 2 雷帕霉素不同时期给药对缺血再灌注大鼠脑梗死体积的影响

图 3 雷帕霉素不同时间给药对缺血再灌注大鼠神经元凋亡的影响

图 4 各组大鼠大脑正常区和缺血半暗带磷酸化S6(p-S6)和总S6(t-S6)蛋白表达及S6磷酸化水平比较(n=6)

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