针刺激活Ⅲ型PI3K/Beclin-1通路调控脑缺血再灌注损伤大鼠缺血侧海马区细胞自噬  

Acupuncture regulates autophagy in hippocampal neurons of cerebral ischemia/reperfusion injured rats by activating typeⅢPI3K/Beclin-1pathway

作　　者：唐红 吕倩忆 汪红娟 江姗姗 陈楚淘[1] 田浩梅[1] 向静 李燕平[1] TANG Hong;LÜQian-yi;WANG Hong-juan;JIANG Shan-shan;CHEN Chu-tao;TIAN Hao-mei;XIANG Jing;LI Yan-ping(College of Acupuncture-Moxibustion and Tuina,Hunan University of Chinese Medicine,Changsha 410208,China;Department of Rehabilitation,The Second People’s Hospital of Chengdu,Chengdu 610021)

机构地区：[1]湖南中医药大学针灸推拿学院,长沙410208 [2]成都市第二人民医院康复科,成都610021

出　　处：《针刺研究》2023年第5期423-430,共8页Acupuncture Research

基　　金：国家自然科学基金项目(No.81874508、82274662);湖南省自然科学基金项目(No.2020JJ4065、2021JJ30490);长沙市科技局自然科学基金项目(No.kq2014094);湖南省研究生科研创新课题(No.CX20220799);湖南中医药大学研究生创新课题(No.2022CX79、2022CX103)。

摘　　要：目的:观察针刺对脑缺血再灌注损伤大鼠缺血侧海马组织Ⅲ型磷脂酰肌醇-3-羟激酶(PI3K)及Beclin-1等自噬相关指标的影响,探讨针刺调控Ⅲ型PI3K/Beclin-1通路适度激活脑缺血再灌注损伤大鼠缺血侧海马神经细胞自噬的作用机制。方法:SD大鼠按照随机数字表法分为假手术组、模型组、针刺组、模型+3-甲基腺嘌呤(3-MA)组、针刺+3-MA组,每组11只。使用改良线栓法制备大鼠大脑中动脉闭塞再灌注模型。模型+3-MA组、针刺+3-MA组于再灌注前30 min予侧脑室注射3-MA 5䥺SymbolmApL(400 nmol/5䥺SymbolmApL)。针刺组、针刺+3-MA组针刺“大椎”“水沟”“百会”,每15 min捻转1次,留针30 min,每12 h治疗1次,共7次。于再灌注后2 h、干预后进行Garcia神经功能评分,干预后通过TTC染色法观察大鼠脑组织缺血面积百分比,Western blot法检测缺血侧海马组织Ⅲ型PI3K、Beclin-1、自噬标志物微管相关蛋白1轻链3B(LC3B)、溶酶体相关膜蛋白2(Lamp2)、P62的蛋白表达水平,透射电镜法观察缺血侧海马组织神经细胞超微结构。结果:再灌注后与假手术组比较,其余各组大鼠神经功能评分降低(P<0.01)。与假手术组比较,模型组大鼠神经功能评分显著降低、脑梗死面积百分比升高(P<0.01),缺血侧海马组织Ⅲ型PI3K、Beclin-1、LC3B-Ⅱ/Ⅰ、Lamp2表达显著降低(P<0.01),P62表达显著升高(P<0.01),神经细胞水肿较重。与模型组比较,干预后针刺组神经功能评分升高、脑梗死面积百分比降低(P<0.01),缺血侧海马组织Ⅲ型PI3K、Beclin-1、LC3B-Ⅱ/Ⅰ、Lamp2表达升高(P<0.01,P<0.05),P62表达降低(P<0.01),神经元结构未见明显损伤,溶酶体增多;模型+3-MA组脑梗死面积百分比升高(P<0.05),Ⅲ型PI3K、Beclin-1表达降低(P<0.01),P62表达水平升高(P<0.05),神经细胞中重度水肿,初级溶酶体存在。与模型+3-MA组比较,针刺+3-MA组神经功能评分显著升高、脑梗死面积百分比显著降低(P<0.05Objective To observe the effects of acupuncture on the expression of typeⅢphosphatidylinositol 3-hydroxykinase(PI3K)and Beclin-1in hippocampus of rats with cerebral ischemia/reperfusion injury(CI/RI),so as to explore the mechanism of acupuncture in regulating typeⅢPI3Kpathway to activate autophagy in the hippocampal neurons of CI/RI rats.Methods SD rats were randomly divided into sham operation group(n=11)and operation group.Then after successful modeling,rats in the operation group were randomly divided into model,acupuncture,model+3-MA and acupuncture+3-MA groups,with 11rats in each group.The model of CI/RI was established by occlusion of the middle cerebral artery.Rats in the model+3-MA and acupuncture+3-MA groups were injected with 3-MA(400nmol/5μL)5μL into the lateral ventricle 30min be-fore reperfusion.Rats in the acupuncture and acupuncture+3-MA groups were punctured with filiform needles at“Dazhui”(GV14),“Shuigou”(GV26)and“Baihui”(GV20)and stimulated manually once every 15min.The acupuncture intervention was conducted for 30min each time,once every 12hfor a total of 7times.The degree of neurological impairment was evaluated 2h after reperfusion and after intervention by Garcia score.After intervention,the percentage of cerebral ischemic area was ob-served by TTC staining,the protein expression levels of typeⅢPI3K,Beclin-1,microtubule-associated protein 1light chain 3B(LC3B),lysosome associated membrane protein 2(Lamp2)and P62in ischemic hippocampal tissue were detected by Western blot,the ultrastructure of neurons in ischemic hippocampus was observed by transmission electron microscopy(TEM).Results Compared with the sham operation group,the Garcia score was decreased(P<0.01),the percentage of cerebral ischemic area was increased(P<0.01),the expression levels of typeⅢPI3K,Beclin-1,LC3B-Ⅱ/Ⅰ,Lamp2proteins were decreased(P<0.01),and the expression level of P62protein was increased(P<0.01)in ischemic hippocampal tissue in the model group.Compared with the model group,the Garcia score

关 键 词：脑缺血再灌注损伤 针刺 细胞自噬 Ⅲ型PI3K/Beclin-1通路 

分 类 号：R246.1[医药卫生—针灸推拿学]