摘要
目的探究lncRNA MEG3调控mTOR介导的自噬在糖尿病心肌病(diabetic cardiomyopathy,DCM)中的作用。方法构建C57BL/6 DCM小鼠模型,设置正常组和DCM组,心脏超声和Masson染色检测小鼠左心房功能结构变化。实时荧光定量聚合酶链反应(real-time quantitative polymerase chain reaction,RT-qPCR)检测lncRNA MEG3在小鼠心肌组织中的表达水平,Western blot法检测p62、LC3-Ⅱ、Beclin1、p-mTOR、mTOR表达量。分离C57BL/6小鼠乳鼠心肌细胞,高糖诱导心肌细胞损伤。转染si-NC、si-MEG3后高糖处理,设置为正常组、高糖组、高糖+si-NC组、高糖+si-MEG3组,RT-qPCR检测lncRNA MEG3在心肌细胞的表达水平,Western blot法检测p62、LC3-Ⅱ、Beclin1、p-mTOR、mTOR、p-ERK、ERK表达量。CCK-8、LDH、ROS、GSH-Px试剂盒检测心肌细胞损伤。结果DCM小鼠心功能出现异常。与正常组比较,DCM组小鼠心肌细胞p62表达显著升高,LC3-Ⅱ和Beclin1表达显著降低,p-mTOR表达升高。细胞实验中,高糖诱导的心肌细胞比正常组p62表达显著升高,LC3-Ⅱ和Beclin1表达显著降低,p-mTOR和p-ERK表达升高;细胞活力和GSH-Px活性降低,LDH和ROS活性显著升高。下调lncRNA MEG3后,高糖+si-MEG3组逆转了高糖+si-NC组自噬相关蛋白以及细胞损伤指标的变化。结论下调lncRNA MEG3抑制了ERK/mTOR信号通路的激活,高糖诱导的心肌细胞自噬得以恢复,且糖尿病心肌损伤被缓解。
Objective To explore the role of lncRNA MEG3 in regulating mTOR mediated autophagy in diabetic cardiomyopathy(DCM).Methods A C57BL/6 DCM mouse model was established.The normal group and DCM group were set up.Cardiac ultrasound and Masson staining were used to detect the changes of the functional and structure of the left atrium.The expression level of lncRNA MEG3 in mouse myocardial tissue was detected by real-time quantitative polymerase chain reaction(RT-qPCR),and the expression level of p62,LC3-Ⅱ,Beclin1,p-mTOR and mTOR were detected by Western blot.Myocardial cells of C57BL/6mouse were isolated,and high glucose induced myocardial cell damage.The cells were transfected with si-NC and si-MEG3 and treated with high glucose,the normal,high glucose,high glucose+si-NC and high glucose+si-MEG3 groups were set up.The expression level of lncRNA MEG3 was detect by RT-qPCR.The expression levels of p62,LC3-Ⅱ,Beclin1,p-mTOR,mTOR,p-ERK and ERK were detected by Western blot.The myocardial cell injury was detected by CCK-8、LDH、ROS、GSH-Px kits.Results Abnormal cardiac function was observed in DCM mouse.Compared with the normal group,the expression level of p62 in cardiomyocytes of DCM group was significantly increased,the expression levels of LC3-Ⅱand Beclin1 were significantly decreased,and the expression level of p-mTOR was increased.In the cell experiment,the expression level of p62 in cardiomyocytes induced by high glucose was significantly higher than that in the normal group.The expression levels of LC3-Ⅱand Beclin1 were significantly decreased,and the expression levels of p-mTOR and p-ERK were increased;the cell viability and GSH-Px activity were decreased.The LDH and ROS activities were significantly increased.After downregulating lncRNA MEG3,the changes of autophagy related proteins and cell damage indexes in the high glucose+si-MEG3 group were reversed.Conclusion Down-regulation of lncRNA MEG3 inhibits the activation of ERK/mTOR signaling pathway,and the autophagy induced by high glucose can be restored.The myocardial damage of diabetes mellitus can be alleviated.
作者
章睿
郑萤萤
朱悦红
ZHANG Rui;ZHENG Yingying;ZHU Yuehong(Department of Rehabilitation Medicine,Zhejiang Hospital,Zhejiang 310013,China)
出处
《医学研究杂志》
2024年第3期110-114,126,共6页
Journal of Medical Research
基金
浙江省医药卫生科技计划项目(2020KY390)
浙江省中医药科技计划项目(2021ZB007)。
