基于miR-126-5p/Bcl-2/mPTP通路探究稳心汤对缺氧/复氧诱导H9c2心肌细胞损伤修复的作用机制  

The Effect of Wenxin Formula on the Damage and Repair of H9c2 Myocardial Cells Induced by Hypoxia/Reoxygenation Based on miR-126-5p/Bcl-2/mPTP Regulatory Pathway

作　　者：解紫从 刘咏梅[1] 陈恒文[1] 谭雨晴 李军[1] XIE Zicong;LIU Yongmei;CHEN Hengwen;TAN Yuqing;LI Jun(Guang′anmen Hospital,China Academy of Chinese Medicine Science,Beijing 100053,China)

机构地区：[1]中国中医科学院广安门医院,北京100053 [2]北京中医药大学,北京100029

出　　处：《中西医结合心脑血管病杂志》2023年第15期2754-2765,共12页Chinese Journal of Integrative Medicine on Cardio-Cerebrovascular Disease

基　　金：国家自然科学基金资助项目(No.81973836);中国中医科学院科技创新工程重大攻关项目(No.CI2021A00902);首都卫生发展科研专项(No.2020-1-4151)。

摘　　要：目的:观察稳心汤对缺氧/复氧诱导的H9c2心肌细胞损伤的影响,并基于miR-126-5p/Bcl-2/心肌细胞线粒体通透性转换孔(mPTP)调控通路明确其作用机制。方法:使用5%CO_(2)、1%O_(2)、94%N 2培养箱构建H9c2心肌细胞缺氧/复氧模型,并通过细胞转染技术达到miR-126-5p的过表达与抑制。待干预结束后观察心肌细胞上清液乳酸脱氢酶(LDH)释放水平、心肌细胞活性氧(ROS)含量;原位末端转移酶标记技术(TUNEL)检测心肌细胞凋亡;流式细胞仪检测心肌细胞线粒体mPTP开放水平;蛋白免疫印迹法(Western Blot)检测细胞色素C(Cyt-C)释放水平与Bcl-2、Caspase-3、Caspase-9蛋白表达水平;实时荧光定量逆转录聚合酶链式反应(qRT-PCR)检测miR-126-5p及Bcl-2、Caspase-3、Caspase-9的RNA表达水平。结果:与miR-126-5p低表达组比较,miR-126-5p过表达可显著降低LDH释放水平;减少心肌细胞ROS含量;改善心肌细胞凋亡率;提高心肌细胞CalceinAM水平,减少mPTP开放。miR-126-5p过表达可以减少H9c2心肌细胞Cyt-C释放水平,miR-126-5p低表达可部分减少H9c2心肌细胞Cyt-C释放水平。miR-126-5p过表达可上调Bcl-2蛋白与基因表达,下调Caspase-3与Caspase-9蛋白与基因表达。miR-126-5p低表达可部分上调miR-126-5p表达水平与Bcl-2蛋白与基因表达;部分下调Caspase-3与Caspase-9蛋白与基因表达。结论:稳心汤改善缺氧/复氧诱导的H9c2心肌细胞损伤可能与调控miR-126-5p/Bcl-2/mPTP信号通路有关,从而减轻氧化应激反应,降低线粒体膜通透性,抑制心肌细胞凋亡,延缓心肌损伤。Objective:To observe the effect of Wenxin Formula on H9c2 myocardial cell injury induced by hypoxia/reoxygenation,and to clarify its mechanism based on the regulatory pathway of miR-126-5p/Bcl-2/mitochondrial permeability conversion pore(mPTP).Methods:The hypoxia and reoxygenation model of H9c2 myocardial cells were constructed in a three-gas incubator,and the overexpression and inhibition of miR-126-5p were achieved by cell transfection.After the intervention,the release level of lactate dehydrogenase(LDH)and the content of reactive oxygen species(ROS)in cardiomyocytes were observed.Myocardial cell apoptosis was detected by TUNEL.The open level of mPTP was detected by flow cytometry.The release level of cytochrome-C(Cyt-C)and the expression levels of Bcl-2,Caspase-3,and Caspase-9 were detected by Western Blot.Real-time quantitative fluorescence polymerase reaction(qRT-PCR)was used to detect the expression levels of miR-126-5p,Bcl-2,Caspase-3 and,Caspase-9 RNA.Results:Compared with miR-126-5p low expression group,miR-126-5p overexpression significantly reduced the LDH release level;reduced ROS content in cardiomyocytes;improved myocardial cell apoptosis rate;calceinam level increased and mPTP opening decreased.Overexpression of miR-126-5p could reduce the Cyt-C release level of H9c2 cardiomyocytes,and low expression of miR-126-5p could partially reduce the Cyt-C release level of H9c2 cardiomyocytes.Overexpression of miR-126-5p could up-regulate the expression of Bcl-2 protein and gene,and down-regulate the expression of Caspase-3 and Caspase-9 protein and gene.When miR-126-5p expression was low,the expression level of miR-126-5p and Bcl-2 could be partially up-regulated.Partially down-regulated the expression levels of Caspase-3 and Caspase-9.Conclusion:The Wenxin Formula could improve H9c2 myocardial cells injury induced by hypoxia/reoxygenation which may be related to the regulation of miR-126-5p/Bcl-2/mPTP signaling pathway,so as to reduce oxidative stress response,reduce mitochondrial membrane permeability,in

关 键 词：稳心汤 H9C2心肌细胞 miR-126-5p/Bcl-2/心肌细胞线粒体通透性转换孔(mPTP) 缺氧/复氧 心肌损伤 

分 类 号：R285[医药卫生—中药学]