miR-200a通过上调Nrf2对老年糖尿病心肌病大鼠心肌的保护作用  

Protective Effect of miR-200a on Myocardium of Elderly Diabetic Cardiomyopathy Rats by Up-regulating Nrf2

作　　者：杨晓净 肖婷[2] 曹晨 邓向群 YANG Xiaojing;XIAO Ting;CAO Chen;DENG Xiangqun(Wuhan Third Hospital,Wuhan 430000,Hubei,China)

机构地区：[1]武汉市第三医院,武汉430000 [2]华中科技大学医院

出　　处：《中西医结合心脑血管病杂志》2024年第10期1777-1782,共6页Chinese Journal of Integrative Medicine on Cardio-Cerebrovascular Disease

基　　金：湖北省自然科学基金面上项目(No.ZRMS2017001349)。

摘　　要：目的:探讨微小RNA-200a(miR-200a)对老年糖尿病大鼠心肌的保护作用及机制。方法:采用高脂饮食+链脲菌素(STZ)诱导大鼠糖尿病心肌病模型,分为对照组和STZ组。通过注射携带miR-200a腺相关病毒9(AAV9)在心肌内过表达miR-200a,分为对照+AAV9-control组、对照+AAV9-miR-200a组、STZ+AAV9-control组、STZ+AAV9-miR-200a组,每组8只。体外培养大鼠心肌细胞H9c2,分为对照组(5.5 mmol/L葡萄糖培养24 h)、高糖+AAV9-control+si RNA组(转染AAV9-control和si RNA 48 h后用33 mmol/L葡萄糖培养24 h)、高糖+AAV9-control+si核因子E2相关因子2(Nrf2)组(转染AAV9-control和si Nrf248 h后使用33 mmol/L葡萄糖培养24 h)、高糖+AAV9-miR-200a+siRNA组(转染AAV9-miR-200a和si RNA 48 h后用33 mmol/L葡萄糖培养24 h)、高糖+AAV9-miR-200a+si Nrf 2组(转染AAV9-miR-200a和si Nrf248 h后用33 mmol/L葡萄糖培养24 h)。使用酶联免疫吸附法(ELISA)试剂盒检测超氧化物歧化酶(SOD)、肌酸激酶同工酶(CK-MB)、乳酸脱氢酶(LDH)、丙二醛(MDA)含量;实时荧光定量聚合酶链式反应(PCR)检测miR-200a表达水平,蛋白免疫印迹法检测相关蛋白表达水平;2′,7′-二氯二氢荧光素二乙酸酯(DCFH-DA)染色检测细胞活性氧(ROS)水平;免疫荧光检测Nrf2表达。结果:与对照组比较,STZ组心肌组织miR-200a表达下调(P<0.05)。与对照+AAV9-control组比较,STZ+AAV9-control组心肌组织LDH、CK-MB、MDA水平升高,SOD水平、血红素氧化酶1(HO-1)、细胞核Nrf2蛋白、细胞质Nrf2蛋白表达及Nrf2荧光密度降低(P<0.05)。与STZ+AAV9-control组比较,STZ+AAV9-miR-200a组心肌组织LDH、CK-MB、MDA水平及TUNEL阳性细胞率降低,SOD水平、HO-1、细胞核Nrf2蛋白、细胞质Nrf2蛋白表达及Nrf2荧光密度升高(P<0.05)。高糖处理可降低H9c2细胞存活率,提高ROS、MDA、LDH水平(P<0.05);在此基础上转染miR-200a后上述指标被抑制;同时转染miR-200a和si Nrf2后,ROS、MDA、LDH升高,细胞存活率降低(P<0.05)。结论:miR-200Objective:To investigate the protective effect of microRNA-200a(miR-200a)on myocardium of elderly diabetic rats and its mechanism.Methods:The rats diabetic cardiomyopathy model was induced by high-fat diet+streptococcin(STZ)and divided into control group and STZ group.miR-200a was overexpressed in myocardium by injecting miR-200a-carrying adeno-associated virus 9(AAV9),which was divided into control+AAV9-control group,control+AAV9-miR-200a group,STZ+AAV9-control group,and STZ+AAV9-miR-200a group,with 8 rats in each group.The cardiomyocytes H9c2 of rats were cultured in vitro and divided into control group(5.5 mmol/L glucose for 24 h),high glucose+AAV9-control+si RNA group(transfected with AAV9-control and si RNA for 48 h and then cultured with 33 mmol/L glucose for 24 h),high glucose+AAV9-control+si nuclear factor E2-related factor 2(Nrf2)group(transfected with AAV9-control and si Nrf2 for 48 h and incubated with 33 mmol/L glucose for 24 h),high glucose+AAV9-miR-200a+si RNA group(transfected with AAV9-miR-200a and si RNA for 48 h and incubated with 33 mmol/L glucose for 24 h),high glucose+AAV9-miR-200a+si Nrf2 group(transfected with AAV9-miR-200a and si Nrf2 for 48 h and then incubated with 33 mmol/L glucose for 24 h).The levels of superoxide dismutase(SOD),creatine kinase isoenzyme(CK-MB),lactate dehydrogenase(LDH),and malondialdehyde(MDA)were detected using enzyme-linked immunosorbent assay(ELISA).miR-200a expression was detected by real-time fluorescence quantitative polymerase chain reaction(PCR).The protein expression was detected by immunoblotting.The level of reactive oxygen species(ROS)was detected by staining with 2′,7′-dichlorodihydrofluorescein diacetate(DCFH-DA),and Nrf2 expression was detected by immunofluorescence.Results:Compared with the control group,miR-200a expression of myocardial tissue downregulated in the STZ group(P<0.05).Compared with the control+AAV9-control group,levels of LDH,CK-MB,and MDA in the STZ+AAV9-control group were significantly higher,and SOD,heme oxidase-1(HO-1),cytosoli

关 键 词：糖尿病心肌病 微小RNA-200a 核因子E2相关因子2 氧化应激 大鼠 实验研究 

分 类 号：R587.2[医药卫生—内分泌] R542.2[医药卫生—内科学]