中国中药杂志

2022, v.47(18) 4987-4995

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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

李静;段群军;沈健;
LI Jing;DUAN Qun-jun;SHEN Jian;Department of Cardiology,the Second Affiliated Hospital of Medical College of Zhejiang University;Department of Cardiovascular Surgery,the Second Affiliated Hospital of Medical College of Zhejiang University;

摘要(Abstract):

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)、IR+RES+antagomir NC组(50 mg·kg(-1) RES+80 mg·kg(-1) RES+80 mg·kg(-1) antagomir NC)、IR+RES+miR-20b-5p antagomir组(50 mg·kg(-1) antagomir NC)、IR+RES+miR-20b-5p antagomir组(50 mg·kg(-1) RES+80 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) 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(-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(-1))、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)。与control组相比,OGD/R组H9c2细胞活力显著下降(P<0.05),H9c2细胞凋亡率、Bax和cleaved-caspase 3表达、ROS水平显著增加,Bcl-2表达、MMP、ATP水平显著降低(P<0.05);与OGD/R组相比,OGD/R+RES组(25、50、100μmol·L(-1))、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)。与control组相比,OGD/R组H9c2细胞活力显著下降(P<0.05),H9c2细胞凋亡率、Bax和cleaved-caspase 3表达、ROS水平显著增加,Bcl-2表达、MMP、ATP水平显著降低(P<0.05);与OGD/R组相比,OGD/R+RES组(25、50、100μmol·L(-1))细胞活力显著增加(P<0.05),细胞凋亡率、Bax和cleaved-caspase 3表达、ROS水平显著降低,Bcl-2表达、MMP、ATP水平显著增加(P<0.05);与OGD/R+RES组相比,OGD/R+RES+miR-20b-5p inhibitor组细胞凋亡率、Bax和cleaved-caspase 3表达、ROS水平显著增加,Bcl-2表达、MMP、ATP水平显著降低(P<0.05);miR-20b-5p与STIM2存在靶向关系;与mimic NC组相比,miR-20b-5p mimic组STIM2表达显著降低(P<0.05);与inhibitor NC组相比,miR-20b-5p inhibitor组STIM2表达显著增加(P<0.05)。RES预处理可通过促进miR-20b-5p表达来抑制STIM2表达,以此改善线粒体的功能,减轻心肌IR损伤。
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))细胞活力显著增加(P<0.05),细胞凋亡率、Bax和cleaved-caspase 3表达、ROS水平显著降低,Bcl-2表达、MMP、ATP水平显著增加(P<0.05);与OGD/R+RES组相比,OGD/R+RES+miR-20b-5p inhibitor组细胞凋亡率、Bax和cleaved-caspase 3表达、ROS水平显著增加,Bcl-2表达、MMP、ATP水平显著降低(P<0.05);miR-20b-5p与STIM2存在靶向关系;与mimic NC组相比,miR-20b-5p mimic组STIM2表达显著降低(P<0.05);与inhibitor NC组相比,miR-20b-5p inhibitor组STIM2表达显著增加(P<0.05)。RES预处理可通过促进miR-20b-5p表达来抑制STIM2表达,以此改善线粒体的功能,减轻心肌IR损伤。
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) group, IR+RES+antagomir NC(50 mg·kg(-1) RES+80 mg·kg(-1) RES+80 mg·kg(-1) antagomir NC) group, and IR+RES+miR-20 b-5 p antagomir(50 mg·kg(-1) antagomir NC) group, and IR+RES+miR-20 b-5 p antagomir(50 mg·kg(-1) RES+80 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) 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(25 μmol·L(-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(25 μmol·L(-1)), 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 adenosine triphosphate(ATP) assay kit were used to detect the MMP, ROS, and ATP levels, respectively. Dual luciferase reporter gene assay was adopted to verify the targeting relationship between miR-20 b-5 p and STIM2. Compared with the sham group, the modeling of IR increased the myocardial infarction area, LVIDd, LVIDs, and myocardial pathology and down-regulated the expression of miR-20 b-5 p(P<0.05). These changes were alleviated in the IR+RES group(P<0.05). The IR+RES+miR-20 b-5 p antagomir group had higher myocardial infarction area, LVIDd, LVIDs, and myocardial pathology and lower expression of miR-20 b-5 p than the IR+RES group(P<0.05). The OGD/R group had lower viability of H9 c2 cells than the control group(P<0.05) and the OGD/R+RES groups(25, 50, and 100 μmol·L(-1)), 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 adenosine triphosphate(ATP) assay kit were used to detect the MMP, ROS, and ATP levels, respectively. Dual luciferase reporter gene assay was adopted to verify the targeting relationship between miR-20 b-5 p and STIM2. Compared with the sham group, the modeling of IR increased the myocardial infarction area, LVIDd, LVIDs, and myocardial pathology and down-regulated the expression of miR-20 b-5 p(P<0.05). These changes were alleviated in the IR+RES group(P<0.05). The IR+RES+miR-20 b-5 p antagomir group had higher myocardial infarction area, LVIDd, LVIDs, and myocardial pathology and lower expression of miR-20 b-5 p than the IR+RES group(P<0.05). The OGD/R group had lower viability of H9 c2 cells than the control group(P<0.05) and the OGD/R+RES groups(25, 50, and 100 μmol·L(-1))(P<0.05). Additionally, the OGD/R group had higher H9 c2 cell apoptosis rate, protein levels of Bax and cleaved caspase-3, and ROS level and lower Bcl-2 protein, MMP, and ATP levels than the control group(P<0.05) and the OGD/R+RES group(P<0.05). The OGD/R+RES+miR-20 b-5 p inhibitor group had higher H9 c2 cell apoptosis rate, protein levels of Bax and cleaved-caspase 3, and ROS level and lower Bcl-2 protein, MMP, and ATP levels than the OGD/R+RES group(P<0.05). miR-20 b-5 p had a targeting relationship with STIM2. The expression of STIM2 was lower in the miR-20 b-5 p mimic group than in the mimic NC group(P<0.05) and lower in the inhibitor NC group than in the miR-20 b-5 p inhibitor group(P<0.05). RES pretreatment can inhibit the expression of STIM2 by promoting the expression of miR-20 b-5 p, thereby improving the function of mitochondria and alleviating myocardial IR damage.

关键词(KeyWords): 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
resveratrol;microRNA-20b-5p;stromal interaction molecule 2;ischemia-reperfusion;mitochondria

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(82070275)

作者(Authors): 李静;段群军;沈健;
LI Jing;DUAN Qun-jun;SHEN Jian;Department of Cardiology,the Second Affiliated Hospital of Medical College of Zhejiang University;Department of Cardiovascular Surgery,the Second Affiliated Hospital of Medical College of Zhejiang University;

DOI: 10.19540/j.cnki.cjcmm.20220314.401

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