摘要
目的:探究长链非编码RNA(lncRNA)母系表达基因3(MEG3)对大鼠心肌H9c2细胞缺氧/复氧(H/R)的保护作用及其与核因子E2相关因子2(Nrf2)/抗氧化反应元件(ARE)通路的关系。方法:将H9c2细胞分为Control组、H/R组、H/R+Ad-绿色荧光蛋白(GFP)组、H/R+Ad-MEG3组、H/R+Ad-MEG3+si-NC组及H/R+Ad-MEG3+si-Nrf2组。实时荧光定量逆转录酶聚合酶链式反应(RT-qPCR)检测MEG3表达;噻唑蓝(MTT)法检测细胞活力;流式细胞仪检测细胞凋亡率;2′,7′-二氯荧光黄双乙酸盐(DCFH-DA)法检测活性氧(ROS)水平;酶联免疫吸附测定法(ELISA)检测超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性;蛋白质印迹(Western Blot)检测Nrf2/ARE通路相关蛋白表达。结果:与Control组比较,H/R组细胞凋亡率、细胞中ROS水平及Nrf2、血红素氧化酶1(HO-1)和醌氧化还原酶-1(NQO-1)蛋白水平均增高,而细胞活力、细胞中MEG3表达水平、SOD和CAT活性均降低(P<0.05)。MEG3过表达可部分逆转H/R对H9c2细胞和Nrf2/ARE通路蛋白的影响(P<0.05);敲低Nrf2可削弱MEG3过表达对H/R损伤的保护作用(P<0.05)。结论:MEG3过表达可抑制氧化应激和细胞凋亡减轻H/R心肌损伤,可能与激活Nrf2/ARE抗氧化信号有关。
Objective:To explore the protective effect of maternal expression of long non-coding RNA(lncRNA)maternally expressed gene 3(MEG3)on hypoxia/reoxygenation(H/R)in rat myocardial H9c2 cells and its relationship with the nuclear factor E2 related factor 2(Nrf2)/antioxidant response element(ARE)pathway.Methods:H9c2 cells were divided into control group,H/R group,H/R+Ad-Green fluorescent protein(GFP)group,H/R+Ad-MEG3 group,H/R+Ad-MEG3+si-NC group and H/R+Ad-MEG3+si-Nrf2 group.Real-time fluorescent quantitative reverse transcriptase polymerase chain reaction(RT-qPCR)was used to detect MEG3 expression.Methyl thiazolyl tetrazolium(MTT)method was used to detect the viability of cells.Flow cytometry was used to detect the apoptosis rate.2′,7′-dichlorofluorescent yellow diacetate(DCFH-DA)method was used to detect the level of reactive oxygen species(ROS).Enzyme-linked immunosorbent assay(ELISA)was used to detect the activities of superoxide dismutase(SOD)and catalase(CAT).Western Blot was used to detect the expression levels of Nrf2/ARE pathway related proteins.Results:Compared with the control group,the H9c2 cell apoptosis rate,cell ROS level and Nrf2,heme oxidase 1(HO-1)and quinone oxidoreductase-1(NQO-1)protein levels increased in the H/R group,while the cell viability,the MEG3 expression level in the cells,SOD and CAT activities reduced(P<0.05).MEG3 overexpression partially reversed the effects of H/R on H9c2 cells and Nrf2/ARE pathway proteins(P<0.05).Knockdown of Nrf2 could weaken the protective effect of MEG3 overexpression on H/R injury(P<0.05).Conclusion:Up-regulating the expression of MEG3 could inhibit oxidative stress and apoptosis to protect H/R myocardial Injury,which may be related to the activation of Nrf2/ARE antioxidant signals.
作者
余敏
崔跃
黎鹏飞
谢丹
陈芬
YU Min;CUI Yue;LI Pengfei;XIE Dan;CHEN Fen(Wuhan Red Cross Hospital,Wuhan 430015,Hubei,China)
出处
《中西医结合心脑血管病杂志》
2024年第4期660-664,共5页
Chinese Journal of Integrative Medicine on Cardio-Cerebrovascular Disease
基金
湖北省卫健委基金项目(No.WJ2017Q039)。