白藜芦醇预处理促进miR-20b-5p抑制STIM2改善线粒体的功能减轻心肌缺血再灌注损伤  

Resveratrol pretreatment improves mitochondrial function and alleviates myocardial ischemia-reperfusion injury by up-regulating mi R-20b-5p to inhibit STIM2

作　　者：李静[1] 段群军[2] 沈健[1] LI Jing;DUAN Qun-jun;SHEN Jian(Department of Cardiology,the Second Affiliated Hospital of Medical College of Zhejiang University,Hangzhou 310003,China;Department of Cardiovascular Surgery,the Second Affiliated Hospital of Medical College of Zhejiang University,Hangzhou 310003,China)

机构地区：[1]浙江大学医学院附属第二医院心血管内科,浙江杭州310003 [2]浙江大学医学院附属第二医院心脏大血管外科,浙江杭州310003

出　　处：《中国中药杂志》2022年第18期4987-4995,共9页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(82070275)。

摘　　要：探究白藜芦醇(RES)预处理通过微小RNA-20b-5p(miR-20b-5p)抑制基质相互作用分子2(STIM2)改善线粒体的功能减轻心肌缺血再灌注(IR)损伤的作用机制。将90只大鼠随机分成假手术(sham)组、模型(IR)组、IR+RES组(50 mg·kg^(-1)RES)、IR+RES+antagomir NC组(50 mg·kg^(-1)RES+80 mg·kg^(-1)antagomir NC)、IR+RES+miR-20b-5p antagomir组(50 mg·kg^(-1)RES+80 mg·kg^(-1)miR-20b-5p antagomir),每组18只。使用冠状动脉左前降支结扎法建立IR大鼠模型,术前2周IR+RES组大鼠腹腔注射50 mg·kg^(-1)RES,sham组、IR组注射等剂量生理盐水,每日1次。超声仪检测各组大鼠左室舒张末期内径(LVIDd)、左心室收缩末期内径(LVIDs);2,3,5-三苯基氯化四氮唑(TTC)法及苏木精-伊红(HE)染色法分别检测心肌梗死面积及组织病理学情况;实时荧光定量PCR(qRT-PCR)法检测心肌组织miR-20b-5p表达。通过氧糖剥夺/复氧(OGD/R)构建H9c2心肌细胞OGD/R模型;CCK-8法检测H9c2细胞活力;H9c2细胞分为空白(control)组、OGD/R组、OGD/R+RES组(25μmol·L)、OGD/R+RES+inhibitor NC组、OGD/R+RES+miR-20b-5p inhibitor组、mimic NC组、miR-20b-5p mimic组、inhibitor NC组、miR-20b-5p inhibitor组;流式细胞仪检测细胞凋亡;Western blot法检测细胞B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、cleaved-半胱氨酸蛋白酶3(cleaved-caspase 3)、STIM2表达;根据线粒体膜电位(MMP)检测试剂盒、活性氧(ROS)检测试剂盒、三磷酸腺苷(ATP)测定试剂盒分别检测MMP、ROS、ATP水平;双荧光素酶报告基因实验验证miR-20b与STIM2靶向关系。与sham组相比,IR组心肌梗死面积、LVIDd、LVIDs、心肌组织病理程度显著增加,miR-20b-5p表达显著降低(P<0.05);与IR组相比,IR+RES组心肌梗死面积、LVIDd、LVIDs、心肌组织病理程度显著降低,miR-20b-5p表达显著增加(P<0.05);与IR+RES组相比,IR+RES+miR-20b-5p antagomir组心肌梗死面积、LVIDd、LVIDs、心肌组织病理程度显著增加,miR-20b-5p表达显著降低(P<0.05)�This study aimed to explore the mechanism of resveratrol(RES)pretreatment in improving mitochondrial function and alleviating myocardial ischemia-reperfusion(IR)injury by inhibiting stromal interaction molecule 2(STIM2)through microRNA-20 b-5 p(miR-20 b-5 p).Ninety rats were randomly assigned into sham group,IR group,IR+RES(50 mg·kg^(-1)RES)group,IR+RES+antagomir NC(50 mg·kg^(-1)RES+80 mg·kg^(-1)antagomir NC)group,and IR+RES+miR-20 b-5 p antagomir(50 mg·kg^(-1)RES+80 mg·kg^(-1)miR-20 b-5 p antagomir)group,with 18 rats/group.The IR rat model was established by ligation of the left anterior descending coronary artery.Two weeks before the operation,rats in the IR+RES group were intraperitoneally injected with 50 mg·kg^(-1)RES,and those in the sham and IR groups were injected with the same dose of normal saline,once a day.Ultrasonic instrument was used to detect the left ventricular internal diameter at end-diastole(LVIDd)and left ventricular internal diameter at end-systole(LVIDs)of rats in each group.The 2,3,5-triphenyte-trazoliumchloride(TTC)method and hematoxylin-eosin(HE)staining were employed to detect the myocardial infarction area and histopathology,respectively.Real-time quantitative PCR(qRT-PCR)was carried out to detect the expression of miR-20 b-5 p in myocardial tissue.Oxygen glucose deprivation/reoxygenation(OGD/R)was performed to establish an OGD/R model of H9 c2 cardiomyocytes.CCK-8 assay was employed to detect H9 c2 cell viability.H9 c2 cells were assigned into the control group,OGD/R group,OGD/R+RES group(25μmol·L),OGD/R+RES+inhibitor NC group,OGD/R+RES+miR-20 b-5 p inhibitor group,mimic NC group,miR-20 b-5 p mimic group,inhibitor NC group,and miR-20 b-5 p inhibitor group.Flow cytometry was employed to detect cell apoptosis.Western blot was employed to detect the expression of B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),cleaved-cysteine proteinase 3(cleaved-caspase-3),and STIM2 in cells.The mitochondrial membrane potential(MMP)assay kit,reactive oxygen species(ROS)assay kit,and a

关 键 词：白藜芦醇 微小RNA-20b-5p 基质相互作用分子2 缺血再灌注 线粒体 

分 类 号：R285.5[医药卫生—中药学]