电针通过AKT/mTOR/P70S6K通路促进大鼠局灶脑缺血/再灌注大脑缺血皮质区血管再生的研究

关键词：电针  局灶脑缺血/再灌注  AKT/mTOR/P70S6K  血管再生  雷帕霉素

Keywords:electroacupuncture  focal cerebral ischemia/reperfusion  angiogenesis  AKT/mTOR/P70S6K  rapamycin

摘要：

      摘要 目的：观察电针对大鼠局灶脑缺血/再灌注大脑缺血皮质区p-AKT、p-mTOR、p-P70S6K表达及CD34+微血管密度变化的影响，探讨电针促进大脑缺血皮质区血管再生的机制。 方法：140只雄性SD大鼠随机分为4组：假手术组、模型组、模型+电针组（电针组）、模型+电针+哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin，mTOR）特异性抑制剂雷帕霉素（Rapamycin，RAPA）组（抑制剂组）；采用线栓法制备SD大鼠右侧局灶脑缺血/再灌注模型，缺血2h后将假手术组、模型组、电针组和抑制剂组各分为再灌注12h、24h、48h、72h、7d五个亚组；取大鼠“百会”穴（GV 20）及左侧“四关”穴（合谷LI 4/太冲LR 3）为电针刺激穴位，刺激时间30min/次，每日1次，最长持续7d；采用Western Blot检测各组大脑缺血皮质区磷酸化的蛋白激酶B（p-protein kinase B，p-AKT）、磷酸化的哺乳动物雷帕霉素靶蛋白（p-mammalian target of rapamycin，p-mTOR）、磷酸化的核糖体蛋白S6乳激酶（p-ribosomal protein S6 kinase，p-P70S6K）蛋白的表达水平；免疫组化法检测大脑缺血皮质区VEGFR2蛋白、CD34+微血管血管的表达；Longa法检测神经功能。 结果：与假手术组相比，模型组p-AKT蛋白的表达在24h、48h和72h增加（P＜0.05），模型组p-mTOR和p-P70S6K的蛋白表达在48h、72h和7d增加（P＜0.05），与模型组相比较，电针组各个时间点p-AKT、p-mTOR和p-P70S6K蛋白的表达均显著增加（P＜0.01），在RAPA的作用下，抑制剂组中各个时间点p-AKT、p-mTOR和p-P70S6K蛋白的表达明显低于电针组（P＜0.05）；与模型组相比，电针组再灌注72h和7d大脑缺血皮质区VEGFR2蛋白、CD34+微血管的表达明显增多（P＜0.01），与电针组相比，抑制剂组再灌注72h和7d大脑缺血皮质区VEGFR2蛋白、CD34+血管减少（P＜0.05）；与模型组和抑制剂组相比，电针组再灌注72h、7d神经功能评分明显改善（P＜0.05）。 结论：电针可提高大鼠局灶脑缺血/再灌注大脑缺血皮质区p-AKT、p-mTOR和p-P70S6K的表达，可能通过AKT/mTOR/P70S6K通路促进大鼠大脑缺血皮质区血管的再生和神经功能的恢复。

Abstract:

      Abstract Objective：To explore the mechanism of electroacupuncture in promoting angiogenesis in ischemic cerebral cortex by investigating the effects of electroacupuncture on p-AKT，p-mTOR，p-P70S6K and CD34+ microvessels in ischemic cerebral cortex after focal cerebral ischemia/reperfusion． Method：A total of 140 healthy male adult Sprague-Dawley（SD）rats were randomly assigned to　shame operation group，model group，model +electroacupuncture group（electroacupuncture group）and　electroacupuncture plus RAPA（A specific antagonist of mTOR）group（antagonist group）．The model of focal cerebral ischemia/reperfusion was prepared by the thread embolism method，and all groups were divided into 12h，24h，48h，72h and 7d sub-groups after reperfusion 2 hour．Electroacupuncture was given at "Baihui"（GV 20）and left "Siguan"（Hegu LI 4／Taichong LR 3）acupoints for 30 minutes every turn，once per day．Western Blot was used to detect the protein expression of p-protein kinase B（p-AKT），p-mammalian target of rapamycin（p-mTOR），p-ribosomal protein S6 kinase（p-P70S6K）in rat cerebral ischemic cortex．The VEGFR2 protein and the CD34+ microvessels were measured by immunohistochemical method．Longa score was applied to evaluate neurobehavioral function． Result：Compared with sham operation group，the protein expression of p-AKT in model group was increased at 24h，48h and 72h，the protein expression of p-mTOR and p-P70S6K in model group were increased at 48h，72h and 7d（P＜0.05）．Compared with model group，the protein expression of p-AKT，p-mTOR and p-P70S6K were more increased significantly at each time point in electroacupuncture group（P＜0.01）．Compared with electroacupuncture group，the protein expression of p-AKT，p-mTOR and p-P70S6K at each time point were decreased in antagonist group（P＜0.05）．Compared with model group，the number of VEGFR2 protein and CD34+ microvessels were increased significantly at 72h and 7 day in electroacupuncture group（P＜0.05）．Compared with electroacupuncture group，the VEGFR2 protein and the number CD34+ microvessels were reduced at 72h and 7 day in antagonist group（P＜0.05）．Compared with model group and antagonist group，the neurobehavioral function was improved significantly at 72h and 7 day in I/RE group（P＜0.05）． Conclusion：Electroacupuncture could promote the expression of p-AKT，p-mTOR and p-P70S6K in rat cerebral ischemic cortex and may promote angiogenesis and neurobehavioral recovery via AKT/mTOR/P70S6k pathway.