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李琼莉,蒋 锦,马宏梅,詹 剑,张 莹,罗 勇.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[J].中国康复医学杂志,2018,(4):379~404
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    点此下载全文
李琼莉  蒋 锦  马宏梅  詹 剑  张 莹  罗 勇
重庆医科大学附属第一医院神经内科,重庆,400016,重庆医科大学附属第一医院神经内科,重庆,400016
基金项目:教育部“高等学校博士学科点专项科研”联合资助课题(20095503110001);重庆市卫生局中医药科研重点项目(ZY20131027);重庆市卫生局中医药科研项目(渝中医2005-B-24)
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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。
关键词:电针  局灶脑缺血/再灌注  AKT/mTOR/P70S6K  血管再生  雷帕霉素
Effects of electroacupuncture on angiogenesis via AKT/mTOR/P70S6K pathway in rats ischemic cerebral cortex after focal cerebral ischemia/reperfusion    Download Fulltext
The First Affiliated Hospital of Chongqing Medical University,400016 and The First Affiliated Hospital of Chongqing Medical University,400016
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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.
Keywords:electroacupuncture  focal cerebral ischemia/reperfusion  angiogenesis  AKT/mTOR/P70S6K  rapamycin
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