地佐辛对缺氧复氧诱导的大鼠心肌细胞损伤的作用及机制

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。与H/R+地佐辛+anti-miR-NC组比较,H/R+地佐辛+anti-miR-7a-5p组MDA含量、细胞凋亡率、Bax蛋白表达及TRIM10蛋白表达均升高(P＜0.05),而SOD和GSH-Px活力、Bcl-2蛋白表达均降低(P＜0.05)。结论: 地佐辛可降低H/R诱导的大鼠心肌细胞H9C2氧化应激和凋亡,其可能通过调控miR-7a-5p/TRIM10轴发挥作用。

Effects of dezocine on cardiac myocytes injury induced by hypoxia and reoxygenation in rats and its mechanism

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). Compared with the H/R group, the MDA content, apoptosis rate, the expression of Bax protein, and the expression of TRIM10 mRNA and protein in the different doses of dezocine intervention group were decreased (P＜0.05), while the activities of SOD and GSH-Px, and the expressions of Bcl-2 protein and miR-7a-5p were all increased (P＜0.05), and there were significant differences in each index between the different doses of dezocine intervention groups (P＜ 0.05). Compared with the H/R+miR-NC group, the MDA content, apoptosis rate, the protein expressions of Bax and TRIM10 in the H/R+miR-7a-5p group were decreased (P＜0.05), while the activities of SOD and GSH-Px, and the expression of Bcl-2 protein were all increased (P＜0.05). Compared with the H/R+dezocine+anti- miR-NC group, the MDA content, apoptosis rate, the protein expressions of Bax and TRIM10 in the H/R+dezocine+anti-miR-7a-5p group were all increased (P＜0.05), while the activities of SOD and GSH-Px, and the expression of Bcl-2 protein were all decreased (P＜0.05). Conclusion: Dezocine can reduce oxidative stress and apoptosis of rat cardiomyocytes H9C2 induced by H/R, which may play a role in regulating the miR-7a-5p / TRIM10 axis.