肌醇需求酶1信号通路在自噬改善大鼠冠心病心肌缺血损伤中的作用  

Inositol-requiring Enzyme 1 Attenuates Myocardial Ischemia Injury by Restoring the Blocked Autophagy Flux in Mice

作　　者：尹磊[1] 王剑[1] 金静[1] 章若涵 刘燕飞[1] YIN Lei;WANG Jian;JIN Jing;ZHANG Ruohan;LIU Yanfei(Department of Cardiology,The Fourth Hospital of Changsha(Changsha Hospital Affiliated to Hunan Normal University),Changsha 410006,China)

机构地区：[1]长沙市第四医院(湖南师范大学附属长沙医院)心血管内科,长沙410006

出　　处：《中国循环杂志》2024年第5期503-510,共8页Chinese Circulation Journal

基　　金：湖南省卫生健康委员会科研项目(202232151289)。

摘　　要：目的:基于探讨肌醇需求酶1(IRE1)信号通路在自噬改善大鼠冠心病心肌缺血损伤中的作用。方法:将H9c2细胞分为对照组、IRE1组、缺氧缺糖(OGD)/复氧(OGD/R)组、OGD/R+IRE1组、氯喹组、IRE1+氯喹组、OGD/R+氯喹组、OGD/R+IRE1+氯喹组、OGD组、OGD+氯喹组、OGD/R+IRE1+敲低X盒结合蛋白1(si-XBP1)组、OGD/R+IRE1+过表达X盒结合蛋白1(XBP1-OE)组。通过自噬双标腺病毒(Adv-RFP-GFP-LC3)评估各组细胞的自噬通量。通过免疫荧光和免疫印迹分析X盒结合蛋白1(XBP1)的核转位。另将32只成年雄性C57BL/6 J小鼠随机分为假手术组、缺血/再灌注(I/R)组、IRE1组和I/R+IRE1组,每组8只。通过超声心动图评估大鼠心功能。通过定量免疫印迹分析自噬相关蛋白。结果:(1)细胞试验:与OGD/R组比,OGD/R+IRE1组H9c2细胞中IRE1蛋白表达水平显著增加(P<0.001),微管相关蛋白轻链3蛋白Ⅱ(LC3Ⅱ)和泛素结合蛋白(p62)蛋白表达均显著降低(P均<0.05)。与OGD/R+氯喹组比,OGD/R+IRE1+氯喹组H9c2细胞中LC3Ⅱ和p62蛋白表达均显著增加(P均<0.05)。与对照组比,OGD/R组H9c2细胞中IRE1细胞核/细胞质荧光强度比显著增加(P<0.001);与OGD/R组比,OGD/R+IRE1组IRE1细胞核/细胞质荧光强度增加(P<0.001)。与OGD/R组比,OGD/R+IRE1组核蛋白中的XBP1水平增加(P<0.05)。与OGD/R+IRE1组比,OGD/R+IRE1+si-XBP1组黄色点状体显著减少(P<0.01),OGD/R+IRE1+XBP1-OE组黄色点状体显著增加(P<0.05)。(2)大鼠体内实验:与假手术组比,I/R组左心室射血分数和短轴缩短率均显著降低(P均<0.05)。与I/R组比,I/R+IRE1组心功能障碍改善(P均<0.05)。与假手术组比,I/R组心肌自噬空泡的数量、IRE1、LC3Ⅱ和p62表达均显著增加(P均<0.05)。与I/R组比,I/R+IRE1组心肌自噬空泡的数量、p62表达均显著降低(P均<0.05),心肌组织中IRE1、LC3Ⅱ的表达均增加(P均<0.05)。结论:IRE1通过促进XBP1的核转位恢复了OGD/R和I/R诱导的自噬通量阻断,自噬通量的恢复有�Objectives:To explore the impact of inositol-requiring enzyme 1(IRE1)signaling pathway on autophagy flux and cardiac function in mice with myocardial ischemia/reperfusion(I/R)injury.Methods:H9c2 cells were divided into control group,IRE1 group,oxygen glucose deprivation/reoxygenation(OGD/R)group,OGD/R+IRE1 group,chloroquine group,IRE1+chloroquine group,OGD/R+IRE1+chloroquine group,OGD/R+chloroquine group,OGD group,OGD+chloroquine group,OGD+IRE1+RNAi X-box binding protein 1(si-XBP1)group and OGD+IRE1+XBP1 overexpression(XBP1-OE)group.Autophagy flux of cells in each group was evaluated by autophagy double-labeled adenovirus(Adv-RFP-GFP-LC3).The nuclear translocation of X-box binding protein 1(XBP1)was analyzed by immunofluorescence and western blot.Adult male C57BL/6 J mice were randomly divided into sham operation group,I/R group,IRE1 group and I/R+IRE1 group(n=8 each).Cardiac function was evaluated by echocardiography.Quantitative western blot analysis was used to detect protein expression of autophagy-related molecules.Results:(1)Compared with OGD/R group,the expression level of IRE1 protein was significantly upregulated(P<0.001),and the expressions of microtubule-associated proteins light chain 3B(LC3Ⅱ)and sequestosome 1(p62)proteins were significantly decreased in IRE1+OGD/R group(all P<0.05).Compared with OGD/R+chloroquine group,the expression of LC3Ⅱand p62 protein was significantly increased in OGD/R+IRE1+chloroquine group(all P<0.05).Compared with control group,the fluorescence intensity ratio of IRE1 nucleus/cytoplasm was significantly increased in OGD/R group(P<0.001),and further increased in IRE1+OGD/R group(P<0.001).The level of XBP1 in the nuclear protein was significantly higher in IRE1+OGD/R group than in OGD/R group(P<0.01).Compared with OGD/R+IRE1 group,the yellow punctures was significantly decreased in OGD/R+IRE1+si-XBP1 group(P<0.01),and significantly increased in OGD/R+IRE1+XBP1-OE group(P<0.05).(2)Compared with Sham group,the left ventricular ejection fraction(LVEF)and fractional shorteni

关 键 词：肌醇需求酶1 心功能 心肌缺血/再灌注 缺氧缺糖/复氧 自噬通量 

分 类 号：R54[医药卫生—心血管疾病]