自噬相关蛋白5对氧-糖剥夺/复氧损伤的心肌细胞线粒体自噬的影响  

Effects of autophagy-related protein 5 on mitophagy in oxygen-glucose deprivation/reoxygenation injured cardiomyocytes

作　　者：林品捷 林晨晗 龚瑾 欧筱雯 许昌声 方周菲 潘忞 韩英[2,3,4,5,6] LIN Pinjie;LIN Chenhan;GONG Jin;OU Xiaowen;XU Changsheng;FANG Zhoufei;PAN Min;HAN Ying(Fujian Medical University,First Clinical College,Fuzhou,Fujian 350005,China;Department of Geriatrics,The First Affiliated Hospital of Fujian Medical University;Department of Geriatrics,National Regional Medical Center,Binhai Campus of the First Affiliated Hospital,Fujian Medical University;Fujian Hypertension Research Institute,The First Affiliated Hospital of Fujian Medical University;Clinical Research Center for Geriatric Hypertension Disease of Fujian province,The First Affiliated Hospital of Fujian Medical University;Branch of National Clinical Research Center for Aging and Medicine,The First Affiliated Hospital of Fujian Medical University;Department of General Practice,the First Affiliated Hospital of Fujian Medical University)

机构地区：[1]福建医科大学第一临床学院,福建福州350005 [2]福建医科大学附属第一医院老年科 [3]福建医科大学附属第一医院滨海院区国家区域医疗中心老年科 [4]福建省高血压研究所 [5]福建省老年高血压疾病临床研究中心 [6]国家老年疾病临床医学研究中心福建分中心 [7]福建医科大学附属第一医院全科医学科

出　　处：《中华高血压杂志（中英文）》2024年第8期737-745,共9页Chinese Journal of Hypertension

基　　金：福建省卫生健康科研人才培养项目(2019-CX-29)。

摘　　要：目的 观察自噬相关蛋白5(ATG5)对氧-糖剥夺/复氧(OGD/R)的H9c2心肌细胞线粒体自噬的影响。方法 通过对H9c2心肌细胞进行OGD/R模拟心肌缺血再灌注损伤。H9c2细胞分别进行如下处理:无任何处理(正常对照组),OGD/R模型(OGD/R组),OGD/R+转染空载质粒(OGD/R-vehicle组),OGD/R+转染ATG5过表达质粒(OGD/R-ATG5组),OGD/R+无意义序列干扰RNA(OGD/R-siRNA negative组)和OGD/R+沉默ATG5基因序列处理(OGD/R-siRNA-ATG5组)。应用细胞计数试剂盒(CCK8)检测H9c2细胞增殖;荧光显微镜观察线粒体的活性;应用逆转录荧光定量聚合酶链式反应(RT-qPCR)和蛋白免疫印迹(Western blot)法检测ATG5、FUN14结构域包含蛋白1(FUNDC1)、线粒体动力相关蛋白1(DRP1)、视神经萎缩相关蛋白1(OPA1)mRNA与蛋白表达;Western blot法检测微管相关蛋白1A/1B-轻链3Ⅱ(LC3Ⅱ)、核孔糖蛋白P62(P62)蛋白表达;电镜观察H9c2细胞线粒体结构与自噬小体的形成。结果 与正常对照组比较,OGD/R组、OGD/R-vehicle组、OGD/R-ATG5组、OGD/R-siRNA negative组和OGD/R-siRNA-ATG5组的H9c2细胞增殖率降低(F=106.35,P<0.05);OGD/R组、OGD/R-vehicle组、OGD/R-siRNA negative组和OGD/R-siRNA-ATG5组线粒体活性降低,OGD/R-ATG5组线粒体活性升高(F=50.74,P<0.05);OGD/R组、OGD/R-vehicle组、OGD/R-ATG5组、OGD/R-siRNA negative组ATG5、FUNDC1、DRP1 mRNA与蛋白表达水平升高,OGD/R-siRNA-ATG5组ATG5、FUNDC1、DRP1 mRNA与蛋白表达水平降低(均P<0.05);全部组别OPA1 mRNA与蛋白表达水平与正常对照组相比降低(F=9.92、24.33,均P<0.05);OGD/R组、OGD/R-vehicle组、OGD/R-ATG5组、OGD/R-siRNA negative组的LC3Ⅱ蛋白表达水平升高,P62蛋白表达水平降低(F=18.07,17.50,均P<0.05),OGD/R-siRNA-ATG5组LC3Ⅱ蛋白表达水平降低,P62蛋白表达水平升高。与OGD/R组比较:OGD/R-ATG5组细胞增殖率、线粒体活性、ATG5、FUNDC1、DRP1、OPA1 mRNA与蛋白表达水平升高,LC3Ⅱ蛋白表达水平升高,P62与蛋白表达水平降低;OGD/R-siRNA-ATObjective To observe the effect of autophagy-related protein 5(ATG5)on mitophagy in oxygen-glucose deprivation/reoxygenation(OGD/R)-induced H9c2 cardiomyocytes.Methods OGD/R was used to simulate myocardial ischemia-reperfusion injury in H9c2 cardiomyocytes.H9c2 cells were treated as follows:no treatment(control group),OGD/R model(OGD/R group),OGD/R+transfection of empty vector(OGD/R-Vehicle group),OGD/R+transfection of ATG5 overexpression plasmid(OGD/R-ATG5 group),OGD/R+meaningless sequence interference RNA(OGD/R-siRNA-negative group)and OGD/R+silencing ATG5 gene sequence(OGD/R-siRNAATG5 group).Cell counting kit 8(CCK8)was used to detect the proliferation of H9c2 cells.Mitochondrial survival was observed under microscope.Reverse transcription-quantitative polymerase chain reaction(RT-qPCR)and western-blot were used to detect the mRNA and protein expressions of ATG5,FUN14 domain-containing protein 1(FUNDC1),dynamin-related protein 1(DRP1),and optic atrophy associated protein 1(OPA1).Western-blot was used to detect the expressions of microtubule-associated protein 1A/1B-light chain 3Ⅱ(LC3Ⅱ)and nuclear porin glycoprotein P62(P62).Results Compared with the control group,the proliferation rate of H9c2 cells in OGD/R group,OGD/R-Vehicle group,OGD/R-ATG5 group,OGD/R-siRNA negative group and OGD/R-siRNA-ATG5 group was decreased(F=106.35,P<0.05).Mitochondrial activity was decreased in OGD/R,OGD/R-vehicle,OGD/R-siRNA negative and OGD/R-siRNA-ATG5 groups,while increased in OGD/R-ATG5 group(F=50.74,P<0.05).The expression levels of ATG5,FUNDC1 and DRP1 mRNA and protein in OGD/R group,OGD/R-vehicle group,OGD/R-ATG5 group and OGD/R-siRNA negative group were increased,while the mRNA and protein expressions of ATG5,FUNDC1 and DRP1 were decreased in OGD/R-siRNA-ATG5 group(all P<0.05).The expression levels of OPA1 mRNA and protein in all groups were lower than those in the control group(F=9.924,24.334,P<0.05).The expression level of LC3Ⅱprotein in OGD/R group,OGD/R-Vehicle group,OGD/R-ATG5 group and OGD/R-siRNA negative group

关 键 词：缺血再灌注损伤 线粒体自噬 线粒体分裂 自噬相关蛋白5 心肌细胞H9c2 

分 类 号：R542.22[医药卫生—心血管疾病]