“通督调神”电针预处理介导miR-124-3p/GSK-3β/Cyp-D信号通路对脑缺血再灌注损伤大鼠脑皮质线粒体通透性转换孔的影响及机制探讨  

Effect and Mechanism of Tongdu Tiaoshen Electroacupuncture Pretreatment-mediated MiR-124-3p/GSK-3β/Cyp-D Signaling Pathway on Mitochondrial Permeability Transition Pore in Cerebral Cortex of Rats with Cerebral Ischemiareperfusion Injury

作　　者：张国庆[1] 童婷婷 王颖[1] 李奎武 张利达 吴晓晴 李成龙 汪俊丽 张君宇[4] 韩为[1] ZHANG Guoqing;TONG Tingting;WANG Ying;LI Kuiwu;ZHANG Lida;WU Xiaoqing;LI Chenglong;WANG Junli;ZHANG Junyu;HAN Wei(Department of Encephalopathy,the Second Affiliated Hospital of Anhui University of Chinese Medicine,Hefei 230061,China;The Second Clinical Medical College,Anhui University of Chinese Medicine,Hefei 230061,China;Department of TCM,Brain Hospital Affiliated to Guangzhou Medical University,Guangzhou 510370,China;Department of Geriatrics,the Second Affiliated Hospital of Anhui University of Chinese Medicine,Hefei 230061,China)

机构地区：[1]安徽中医药大学第二附属医院脑病科,安徽省合肥市230061 [2]安徽中医药大学第二临床医学院,安徽省合肥市230061 [3]广州医科大学附属脑科医院中医科,广东省广州市510370 [4]安徽中医药大学第二附属医院老年病科,安徽省合肥市230061

出　　处：《中国全科医学》2023年第24期3050-3060,共11页Chinese General Practice

基　　金：国家自然科学基金面上项目(81973933);安徽省第十三批115产业创新团队“针药结合防治脑病创新团队”项目(皖人才办[2020]4号);安徽省名中医王颖工作室建设项目(中发展[2022]5号)。

摘　　要：背景缺血性脑卒中发病率、死亡率、致残率均较高,溶栓后的再灌注损伤对患者影响较大,针刺是治疗本病的特色疗法,但作用机制尚不明确。目的探讨“通督调神”电针预处理对脑缺血再灌注损伤(CIRI)大鼠miR-124-3p/糖原合成酶激酶β(GSK-3β)/亲环素D(Cyp-D)信号通路及线粒体膜通透性转换孔(MPTP)的影响,探讨其防治CIRI的可能机制。方法2022年6—8月,将100只清洁级SD大鼠随机分为假手术组、模型组、电针组、抑制剂组和电针+激动剂组,每组20只。造模前干预7 d,电针组、电针+激动剂组选取通督调神穴组:百会、风府、大椎穴进行电针干预,1次/d,连续7 d;造模前24 h电针+激动剂组和抑制剂组分别侧脑室注射miR-124激动剂和抑制剂(5 nmol)。除假手术组,余组采用改良线栓法制备大鼠右侧脑缺血再灌注模型;造模成功后,取大鼠右侧脑皮质,采用改良神经功能损伤评分量表(mNSS)、TTC染色观察各组大鼠神经功能损伤程度,TUNEL染色以及透射电镜观察神经细胞损伤情况;免疫荧光染色、Western blotting、实时荧光定量PCR检测各组大鼠脑皮质GSK-3β、p-GSK-3β、Cyp-D、细胞色素C(Cyt-C)、半胱氨酸天冬氨酸蛋白酶3(Caspase-3)表达;流式细胞检测MPTP开放程度,MPTP阳性细胞率越高,代表MPTP开放程度越大。结果除假手术组,各组大鼠均造模成功。与假手术组比较,模型组大鼠mNSS评分及相对脑梗死体积均增加,线粒体结构破坏严重,细胞凋亡指数升高,p-GSK-3β、Cyp-D阳性表达分别减弱和增强,miR-124-3p及Cyp-D、Cyt-C、Caspase-3 mRNA表达增高,p-GSK-3β/GSK-3β值降低,Cyp-D、Cyt-C、Caspase-3蛋白相对表达量增高,MPTP开放程度增加(P<0.05)。与模型组比较,电针组、抑制剂组mNSS评分及相对脑梗死体积均降低,线粒体结构破坏减轻,p-GSK-3β/GSK-3β增加,Caspase-3蛋白相对表达量降低;电针+激动剂组GSK-3βmRNA表达增高(P<0.05);电针组、抑制剂Background The incidence,mortality and disability rates of ischemic stroke are high,and the reperfusion injury after thrombolytic therapy has a great impact on patients.Acupuncture is a characteristic therapy for the treatment of the disease,but the action mechanism remains unclear.Objective To observe the effect of Tongdu Tiaoshen electroacupuncture(EA)pretreatment on miR-124-3p/glycogen synthase kinaseβ(GSK-3β)/cyclin D(Cyp-D)signaling pathway and mitochondrial permeability transition pore(MPTP)of cerebral ischemia-reperfusion injury(CIRI)rats,and explore its possible mechanism of prevention and control of CIRI.Methods From June to August 2022,a total of 100 clean SD rats were randomly divided into the sham operation group,model group,EA group,agonist group and EA+inhibitor group,with 20 rats in each group.For 7 d of intervention before modeling,in the EA group and EA+inhibitor group,"Baihui"(GV 20),"Fengfu"(GV 16)and"Dazhui"(GV 14)were selected to perform electroacupuncture 1 time a day for 7 days.For 24 h before modeling,miR-124 agonist and inhibitor(5 nmol)were injected into the lateral ventricles in the EA+agonist group and inhibitor group.Except for the sham operation group,the right cerebral ischemia-reperfusion model of rats was prepared by the modified suture method in the rest groups.The right cerebral cortex of rats was taken,the degree of neurological impairment in each group was observed using mNSS scale and TTC staining,the nerve cell injury was observed by TUNEL staining and transmission electron microscopy.The expressions of GSK-3β,p-GSK-3β,Cyp-D,Cyt-C and Caspase-3 in cerebral cortex of each group were detected by immunofluorescence staining,Western blot and real-time quantitative PCR.The degree of MPTP openness was detected by flow cytometry,the higher rate of MPTP-positive cells indicated greater degress of MPTP openness.Results Except for the sham operation group,all rest groups of rats were successfully modeled.Compared with the sham operation group,mNSS score and infarct volume of brain

关 键 词：再灌注损伤 脑缺血 卒中 线粒体通透性转换孔 通督调神 电针疗法 miR-124-3p 穴 百会 穴 风府 穴 大椎 

分 类 号：R619.9[医药卫生—外科学]