壮药双路通脑方对缺血-再灌注脑损伤大鼠神经元自噬和凋亡的影响  

Effects of Zhuang Medicine Shuanglu Tongnao Decoction on Autophagy and Apoptosis of Neuronal Cells in Rats with Cerebral Ischemia/Reperfusion Injury

作　　者：梅小平 翟阳 王凯华[2] 滕红丽 郑光珊 杨鹏 邹敏 MEI Xiaoping;ZHAI Yang;WANG Kaihua;TENG Hongli;ZHENG Guangshan;YANG Peng;ZOU Min(Department of Medical Affairs,Guangxi International Zhuang Medical Hospital,Nanning 530200,China;Department of Encephalopathy,Guangxi International Zhuang Medical Hospital,Nanning 530200,China;Guangxi University of Traditional Chinese Medicine,Nanning 530200,China;Department of Science and Technology,Guangxi International Zhuang Medical Hospital,Nanning 530200,China;Department of Pediatrics,Guangxi International Zhuang Medical Hospital,Nanning 530200,China)

机构地区：[1]广西国际壮医医院医务部,南宁530200 [2]广西国际壮医医院脑病科,南宁530200 [3]广西中医药大学,南宁530200 [4]广西国际壮医医院科技部,南宁530200 [5]广西国际壮医医院儿科,南宁530200

出　　处：《医药导报》2023年第6期781-789,共9页Herald of Medicine

基　　金：国家自然科学基金资助项目(81874453);广西壮族自治区中医药管理局自筹经费科研课题(20210360,20210506);广西科技厅自然科学基金资助项目(2021JJB140540);广西中医药重点学科中医脑病学(GZXK-Z-20-14);广西国际壮医医院“青苗工程”人才培育项目资助;广西中医药大学第二批“岐黄工程”高层次人才团队培育项目(2021008);广西国际壮医医院院级课题(GZ2021007,GZ2021052);广西中医药大学校级青年基金资助项目(2018QN035)。

摘　　要：目的探讨壮药双路通脑方对缺血-再灌注(I/R)脑损伤大鼠神经元自噬和凋亡的影响及可能机制。方法SD大鼠随机分为假手术组,模型对照组,小、中、大剂量双路通脑方组,大剂量双路通脑方+AMPK激活剂AICAR组,每组18只。按照分组给予不同的药物干预7 d后,采用线栓法制备大脑中动脉栓塞模型。再灌注24 h后,评估大鼠神经功能缺损的程度;2,3,5-三苯基氯化四氮唑(TTC)染色观察脑梗死体积;苏木精-伊红(HE)染色观察缺血半影区海马CA1区神经元形态学变化;TUNEL染色检测神经元凋亡;透射电子显微镜观察自噬小体的形成;免疫荧光(IF)染色检测微管相关蛋白1轻链3(LC-3)-Ⅱ的表达;Western blotting检测自噬标志基因Beclin-1、LC3A/B、p62及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)及其磷酸化蛋白的表达。结果与假手术组比较,模型对照组神经功能缺损评分、脑梗死体积、神经元凋亡率、自噬小体数量、LC3-Ⅱ阳性表达、海马组织Beclin-1蛋白水平和LC3-II/LC3-I、p-AMPK/AMPK、p-ULK1(S317)/ULK1比值显著升高,p62蛋白水平和p-mTOR/mTOR、p-ULK1(S757)/ULK1比值显著降低(P<0.05);与模型对照组比较,小、中、大剂量双路通脑方组大鼠神经功能缺损评分、脑梗死体积、神经元凋亡率、自噬小体数量、LC3-Ⅱ阳性表达、海马组织Beclin-1蛋白水平和LC3-II/LC3-I、p-AMPK/AMPK、p-ULK1(S317)/ULK1比值显著降低,p62蛋白水平和p-mTOR/mTOR、p-ULK1(S757)/ULK1比值显著升高(P<0.05),且呈剂量依赖性;AICAR可明显减弱大剂量双路通脑方对脑I/R大鼠神经元自噬和凋亡的抑制作用。结论双路通脑方可能通过调控AMPK/mTOR信号通路,抑制神经元的过度自噬,减少神经元凋亡,进而发挥对脑I/R大鼠的神经保护作用。Objective To investigate the impact and potential mechanism of Shuanglu Tongnao decoction on the autophagy and apoptosis of neuronal cells in rats with ischemia/reperfusion(I/R)brain injury.Methods SD rats were randomly divided into sham operation group,model control group,low-dose,medium-dose and high-dose Shuanglu Tongnao decoction groups,high-dose Shuanglu Tongnao decoction+AMPK activator AICAR group,with 18 rats in each group.According to the groups,the middle cerebral artery occlusion(MCAO)model was established by thread embolism method after 7 d of different drug interventions to groups.After 24 hours of reperfusion,the degree of neurological deficit in rats was evaluated;2,3,5-triphenyltetrazolium chloride(TTC)staining was used to observe the volume of cerebral infarction.Hematoxylin-eosin(HE)staining was used to observe the morphological changes of neurons in the hippocampal CA1 area of the ischemic penumbra.Neuronal apoptosis was detected by TUNEL staining and transmission electron microscopy was used to observe the formation of autophagosomes.Immunofluorescence(IF)staining was used to detect the expression of microtubule-associated protein 1 light chain 3(LC-3)-Ⅱ;Western blotting was used to detect the expression of autophagy marker genes Beclin-1,LC3A/B,p62,adenylate-activated protein kinase(AMPK),mammalian target of rapamycin(mTOR),Unc-51-like kinase 1(ULK1)and their phosphorylated proteins.Results Compared with the sham operation group,the neurological deficit score,cerebral infarction volume,neuron apoptosis rate,number of autophagosomes,LC3-Ⅱpositive expression,hippocampal Beclin-1 protein level and LC3-Ⅱ/LC3-Ⅰ,p-AMPK/AMPK,p-ULK1(S317)/ULK1 ratios were obviously increased in the model control group,and the p62 protein level and p-mTOR/mTOR and p-ULK1(S757)/ULK1 ratios were obviously decreased(P<0.05).Compared with the model control group,the neurological deficit score,cerebral infarction volume,neuron apoptosis rate,number of autophagosomes,LC3-Ⅱpositive expression,hippocampal Beclin-1 pr

关 键 词：壮药 双路通脑方 缺血性脑卒中 自噬 凋亡 腺苷酸活化蛋白激酶/哺乳动物雷帕霉素靶蛋白信号通路 

分 类 号：R965[医药卫生—药理学]