摘要
目的:探讨地佐辛通过调控微小RNA-7a-5p(miR-7a-5p)/泛素E3连接酶10(TRIM10)表达影响缺氧复氧(H/R)诱导的大鼠心肌细胞H9C2氧化应激和凋亡的作用机制。方法:将H9C2细胞分为对照组(细胞正常培养)、H/R组(缺氧处理3 h,复氧培养4 h)、不同剂量地佐辛干预组(分别采用10^(-7)、10^(-6)、10^(-5) mmol/L的地佐辛预处理H9C2细胞24 h,再进行H/R处理)、H/R+miR-7a-5p组(转染miR-7a-5p mimics至H9C2细胞,然后进行H/R处理)、H/R+miR-NC组(转染miR-NC至H9C2细胞,然后进行H/R处理)、H/R+地佐辛+anti-miR-7a-5p组(10^(-5) mmol/L的地佐辛预处理转染anti-miR-7a-5p的H9C2细胞24 h,再进行H/R处理)、H/R+地佐辛+anti-miR-NC组(10^(-5) mmol/L的地佐辛预处理转染anti-miR-NC的H9C2细胞24 h,再进行H/R处理),每组细胞设置3个复孔,实验重复3次。酶联免疫吸附法检测细胞中氧化应激指标丙二醛(MDA)含量、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)活力,流式细胞术检测细胞凋亡,蛋白印迹(Western blot)法检测B淋巴细胞瘤-2(Bcl-2)、B淋巴细胞瘤-2相关蛋白(Bax)和泛素E3连接酶10(TRIM10)蛋白表达,实时荧光定量PCR(RT-qPCR)检测miR-7a-5p和TRIM10 mRNA表达。双荧光素酶报告基因实验验证miR-7a-5p与TRIM10调控关系。结果:与对照组比较,H/R组MDA含量、细胞凋亡率、Bax蛋白表达及TRIM10的mRNA和蛋白表达均升高(P<0.05),而SOD和GSH-Px活力、Bcl-2蛋白表达和miR-7a-5p表达均降低(P<0.05)。与H/R组比较,不同剂量地佐辛干预组MDA含量、细胞凋亡率、Bax蛋白表达及TRIM10的mRNA和蛋白表达均降低(P<0.05),而SOD和GSH-Px活力、Bcl-2蛋白表达和miR-7a-5p表达均升高(P<0.05),且不同剂量地佐辛干预组间各指标两两比较差异均显著(P<0.05)。与H/R+miR-NC组比较,H/R+miR-7a-5p组MDA含量、细胞凋亡率、Bax蛋白表达及TRIM10蛋白表达均降低(P<0.05),而SOD和GSH-Px活力、Bcl-2蛋白表达均升高(P<0.05)。miR-7a-5p靶向负调控TRIM10表达�
Objective:To investigate the mechanisms of dezocine on regulating H9C2 oxidative stress and apoptosis of rat cardiac myocytes induced by hypoxia-reoxygenation(H/R)by regulating the expressions of microRNA-7a-5p(miR-7a-5p)/ubiquitin E3 ligase tripartite motif 10(TRIM10).Methods:H9C2 cells were divided into control group(cultured normally),H/R group(treated with hypoxia for 3 h and then reoxygenation for 4 h),different doses of dezocine intervention group(H9c2 cells were pretreated with dezocine at the concentrations of 10^(-7),10^(-6) and 10^(-5) mmol/L for 24 h,and then treated with H/R),H/R+miR-7a-5p group(H9C2 cells were transfected with miR-7a-5p mimics and then treated with H/R),H/R+miR-NC group(H9C2 cells were transfected with miR-NC and then treated with H/R),H/R+Dezocine+anti-miR-7a-5p group(H9c2 cells transfected with anti-miR-7a-5p were pretreated with 10^(-5) mmol/L dezocine for 24 h,and then treated with H/R),H/R+dezocine+anti-miR-NC Group(H9c2 cells transfected with anti-miR-NC were pretreated with 10^(-5) mmol/L dezocine for 24 h,and then treated with H/R).Each group of cells was set with 3 replicate wells,and the experiment was repeated 3 times.The content of malondialdehyde(MDA)and activity of superoxide dismutase(SOD)and glutathione peroxidas(GSH-Px)were detected by the enzyme-linked immunosorbent assay.The cells apoptosis was detected by flow cytometry.The protein expressions of B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax)and TRIM10 were detected by Western blot,and the expressions of miR-7a-5p and TRIM10 mRNA were detected by real-time quantitative PCR(RT-qPCR).The double luciferase reporter gene experiment was used to verify the regulatory relationship between miR-7a-5p and TRIM10.Results:Compared with the control group,the MDA content,apoptosis rate,the expression of Bax protein,and the expression of TRIM10 mRNA and protein in the H/R group were all increased(P<0.05),while the activities of SOD and GSH-Px,and the expressions of Bcl-2 protein and miR-7a-5p were all decreased(P<0.05)
作者
王春奎
刘希明
陶宏
段冶
刘伟
WANG Chun-kui;LIU Xi-ming;TAO Hong;DUAN Ye;LIU Wei(Department of Anesthesiology,Central People's Hospital of Tengzhou,Tengzhou 277500,China)
出处
《中国应用生理学杂志》
CAS
CSCD
北大核心
2021年第5期548-554,共7页
Chinese Journal of Applied Physiology
