基于PI3K-AKT-mTOR信号通路探讨益阴通络方对缺血性脑卒中大鼠的影响  

Effect of Yiyin Tongluo Formula on Ischemic Stroke Rats Based on the PI3K-AKT-mTOR Signaling Pathway

作　　者：巩子汉 游顺华 杨婧雯 陈懿娜 杨苗苗 易顺 邹大道 马社会 王立平 GONG Zi-han;YOU Shun-hua;YANG Jing-wen;CHEN Yi-na;YANG Miao-miao;YI Shun;ZOU Da-dao;MA She-hui;WANG Li-ping(Ningxia Chinese Medicine Research Center,Yinchuan Ningxia 750021;Institute of Basic Theory for Chinese Medicine,China Academy of Chinese Medical Sciences,Beijing 100700)

机构地区：[1]宁夏回族自治区中医医院暨中医研究院,宁夏银川750021 [2]中国中医科学院中医基础理论研究所,北京100700

出　　处：《世界中西医结合杂志》2024年第11期2121-2127,2154,共8页World Journal of Integrated Traditional and Western Medicine

基　　金：宁夏自然科学基金项目(2022AAC03400)。

摘　　要：目的观察益阴通络方对缺血性脑卒中(Cerebral ischemic stroke,CIS)大鼠脑组织的保护作用及其机制。方法将72只2月龄SPF级Wistar大鼠随机分为假手术组10只及造模组62只,采用Longa线栓法复制大鼠CIS动物模型,按随机数字表法分为模型组,益阴通络方低、中、高剂量组及尼莫地平组,每组各9只,给药干预7 d。采用2,3,5-氯化三苯基四氮唑(2,3,5-Triphenyltetrazolium chloride,TTC)染色法检测各组大鼠脑组织梗死体积,酶联免疫吸附法(Enzyme linked immunosorbent assay,ELISA)检测各组大鼠脑组织超氧化物歧化酶(Superoxide dismutase,SOD)、丙二醛(Malondialdehyde,MDA)及谷胱甘肽过氧化物酶(Glutathione peroxidase,GSH-Px)含量,透射电镜观察脑组织自噬微结构,实时荧光定量PCR法(Real-time polymerase chain reaction,RT-PCR)检测脑组织苄氯素1(Beclin-1)、微管相关蛋白1-轻链3(Microtubule associated protein 1 light chain 3,LC-3)、磷脂酰肌醇-3-激酶(Phosphatidylin-ositol-3-kinase,PI3K),蛋白激酶(Protein kinase B,AKT)及哺乳动物雷帕霉素靶蛋白(mTOR)基因表达水平,蛋白免疫印迹法(Western blot)检测脑组织Beclin-1、LC-3、PI3K,AKT及mTOR蛋白表达水平。结果与假手术组比较,模型组大鼠脑组织梗死面积比明显升高(P<0.01),超微结构改变明显,病灶组织SOD、GSH-Px含量明显降低(P<0.01),MDA含量明显升高(P<0.05),Beclin-1、LC-3基因及蛋白表达水平明显升高(P<0.01),PI3K、AKT、mTOR表达水平明显降低(P<0.01);与模型组比较,尼莫地平及益阴通络方高剂量组大鼠脑组织梗死面积比明显降低(P<0.01),病理形态较规则及超微结构较完整,SOD、GSH-Px含量明显升高(P<0.05),MDA含量明显降低(P<0.05),Beclin-1、LC-3基因及蛋白表达水平明显降低(P<0.05,P<0.01),PI3K、AKT及mTOR表达水平明显升高(P<0.05,P<0.01)。结论益阴通络方对缺血性脑卒中大鼠脑损伤具有保护作用,其机制可能与调节PI3K/AKT/mTOR信号通路关键蛋白表达,�Objective To observe the protective effect and mechanisms of Yiyin Tongluo Formula on cerebral tissue in a rat model of cerebral ischemic stroke(CIS).Methods Seventy-two 2-month-old SPF-grade Wistar rats were randomly divided into a sham group(n=10)and an experimental group(n=62).The CIS rat model was established using the Longa suture method.The experimental rats were further divided into a model group,low-,medium-,and high-dose Yiyin Tongluo Formula groups,and a nimodipine group,with 9 rats in each group.The intervention lasted for 7 days.The cerebral infarct volume was assessed using the 2,3,5-triphenyltetrazolium chloride(TTC)staining method.Levels of superoxide dismutase(SOD),malondialdehyde(MDA),and glutathione peroxidase(GSH-Px)in brain tissue were measured using enzyme-linked immunosorbent assay(ELISA).Autophagic ultrastructure in brain tissue was observed using transmission electron microscopy.The mRNA expression levels of Beclin-1,microtubule-associated protein 1 light chain 3(LC-3),phosphatidylinositol-3-kinase(PI3K),protein kinase B(AKT),and mammalian target of rapamycin(mTOR)in brain tissue were detected using real-time polymerase chain reaction(RT-PCR).Protein expression levels of Beclin-1,LC-3,PI3K,AKT,and mTOR were analyzed using Western blotting.Results Compared with the sham group,the model group showed a significantly increased cerebral infarct area(P<0.01),marked ultrastructural changes,significantly decreased SOD and GSH-Px levels(P<0.01),significantly increased MDA levels(P<0.05),significantly elevated expression levels of Beclin-1 and LC-3 genes and proteins(P<0.01),and significantly reduced expression levels of PI3K,AKT,and mTOR(P<0.01).Compared with the model group,the nimodipine group and the high-dose Yiyin Tongluo Formula group showed significantly reduced cerebral infarct areas(P<0.01),more regular pathological morphology,and better-preserved ultrastructure.These groups also demonstrated significantly increased SOD and GSH-Px levels(P<0.05),significantly decreased MDA levels(P<0.05),r

关 键 词：缺血性脑卒中 益阴通络方 PI3K/AKT/mTOR信号通路 自噬活性 

分 类 号：R285.5[医药卫生—中药学]