基于AMPK/mTOR/ULK1信号通路探讨电针对脑缺血再灌注损伤模型大鼠神经功能的影响  

Effect of electroacupuncture on neurological function in rats with cerebral ischemia-reperfusion injury based on AMPK/mTOR/ULK1 signaling pathway

作　　者：徐小雯 徐钒锋 朱炎贞 詹杰 詹乐昌[1,2,3,4] 陈红霞 XU Xiaowen;XU Fanfeng;ZHU Yanzhen;ZHAN Jie;ZHAN Lechang;CHEN Hongxia(Clinical Medical College of Acupuncture-Moxibustion and Rehabilitation,Guangzhou University of CM,Guangzhou 510006,Guangdong Province,China;Second Clinical College of Guangzhou University of CM,Guangzhou 510120,Guangdong Province;Department of Rehabilitation,Guangdong Provincial Hospital of Chinese Medicine,Guangzhou 510120,Guangdong Province;Guangdong Provincial Key Laboratory of TCM Prevention and Treatment of Intractable Chronic Diseases,Guangzhou 510120,Guangdong Province)

机构地区：[1]广州中医药大学针灸康复临床医学院,广东广州510006 [2]广州中医药大学第二临床医学院,广东广州510120 [3]广东省中医院康复科,广东广州510120 [4]广东省中医药防治难治性慢病重点实验室,广东广州510120

出　　处：《中国针灸》2024年第12期1418-1425,共8页Chinese Acupuncture & Moxibustion

基　　金：广东省基础与应用基础研究基金项目:2022A1515220025;广东省中医药防治难治性慢病重点实验室项目:2019KT1145;广东省重点领域研发计划项目:2020B1111100008。

摘　　要：目的:探讨电针调控腺苷酸活化蛋白激酶(AMPK)/哺乳动物雷帕霉素靶蛋白(mTOR)/Unc-51样激酶1(ULK1)信号通路对脑缺血再灌注损伤(CIRI)模型大鼠神经功能的影响。方法:33只雄性SD大鼠,随机分为假手术组、模型组、电针组,每组11只。模型组、电针组大鼠采用线栓法制备右侧大脑中动脉闭塞/再灌注(MCAO/R)模型,假手术组血管分离后不插入线栓。造模成功后,电针组行电针干预,穴取“百会”及患侧“足三里”,疏密波,频率2 Hz/15 Hz,每次20 min,每日1次,连续干预3 d。分别于术后1、3 d评定大鼠改良神经功能缺损评分(mNSS)。干预结束后,采用氯化三苯基四氮唑(TTC)法测定大鼠脑梗死面积,尼氏染色法观察大鼠缺血侧皮层神经元形态,透射电镜观察大鼠缺血侧皮层神经元超微结构,免疫荧光法检测大鼠缺血侧皮层微管相关蛋白1轻链3(LC3)、苄氯素-1(Beclin-1)阳性表达,Western blot法检测大鼠缺血侧皮层p-AMPK、AMPK、p-mTOR、mTOR、p S757-ULK1、ULK1、螯合体1(p62)蛋白表达。结果:与假手术组比较,模型组大鼠m NSS评分升高(P<0.01);脑梗死面积百分比升高(P<0.01);缺血侧皮层神经元分布松散,核固缩,呈空泡化现象,神经元数量减少(P<0.01),细胞肿胀破裂,线粒体收缩,电子密度增加,有大量自噬碎片;缺血侧皮层LC3、Beclin-1阳性表达均升高(P<0.01),p-mTOR/mTOR、pS757-ULK1/ULK1及p62蛋白表达降低(P<0.01)。与模型组比较,电针组大鼠m NSS评分降低(P<0.05);脑梗死面积百分比降低(P<0.01);缺血侧皮层神经元分布较为规律,数量增多(P<0.01),线粒体结构较清晰,嵴断裂减轻,神经元损伤情况改善;缺血侧皮层LC3、Beclin-1阳性表达均降低(P<0.01),p-AMPK/AMPK降低(P<0.05),p-mTOR/mTOR、p S757-ULK1/ULK1及p62蛋白表达升高(P<0.01)。结论:电针“百会”“足三里”可能通过调控AMPK/mTOR/ULK1信号通路,抑制自噬,改善CIRI大鼠神经功能缺损和脑组织病理损伤,发挥Objective To investigate the effect of electroacupuncture(EA)on neurological function in rats with cerebral ischemia-reperfusion injury(CIRI)by regulating adenosine monophosphate-activated protein kinase(AMPK)/mammalian target of rapamycin(m TOR)/Unc-51-like kinase 1(ULK1)signaling pathway.Methods Thirty-three male SD rats were randomly divided into a sham-operation group,a model group and an EA group,with 11 rats in each group.The right middle cerebral artery occlusion/reperfusion(MCAO/R)model was prepared by thread occlusion method in the model group and the EA group.In the sham-operation group,no thread was inserted after vascular separation.After the success of modeling,in the EA group,EA was applied to"Baihui"(GV 20)and"Zusanli"(ST 36)on the affected side,with disperse-dense wave,the frequency of 2 Hz/15 Hz,for 20 min,once a day.EA was delivered continuously for 3 days.On day 1 and day 3 of operation,the score of the modified neurological deficit scale(m NSS)was evaluated.After intervention completion,the cerebral infarction area was measured by the thiazolyl blue tetrazolium chloride(TTC)method.Nissl staining was used to observe the damage of cortical neurons on the ischemic side in each group.Using transmission electron microscopy,the ultrastructure of cortical neurons on the ischemic side was observed.With the immunofluorescence method adopted,the positive expression of the related protein 1 light chain 3(LC3)and benzyl chloroform(Beclin-1)on the ischemic side was detected.The protein expression of p-AMPK,AMPK,p-m TOR,mTOR,pS757-ULK1,ULK1 and chelating ligand 1(p62)in the ischemic cortex was detected using Western blot method.Results(1)Compared with the sham-operation group,in the model group,the mNSS score increased(P<0.01),the percentage of infarction area was increased(P<0.01);the cortical neurons on the ischemic side were loosely distributed,with karyopyknosis and vacuolization,and the number of neurons was reduced(P<0.01);the cells were swollen and ruptured,mitochondrial shrunk,electron density highe

关 键 词：脑缺血再灌注损伤 电针 AMPK/mTOR/ULK1信号通路 自噬 

分 类 号：R245.97[医药卫生—针灸推拿学]