利拉鲁肽对缺氧和高糖诱导的心肌细胞氧化应激损伤的保护作用

摘要

Objective:To explore the effects and possible mechanism of liraglutide on hypoxia and high glucose-induced oxidative stress injury in cardiomyocytes. Methods:The neonatal rat cardiomyocytes were separated and cultured in vitro. The hypoxia and high glucose-induced injury model was established in neonatal rat cardiomyocytes. The cells were divided into six groups:the normal control group, liraglutide control group, hypoxia and high glucose model group, liraglutide treatment group, GLP-1R antagonist group and hyperosmotic control group. The metabolic ability of the cells was detected by MTT assay, the activities of LDH and CK-MB were detected by colorimetric method,SOD activity and MDA content were determined by xanthine oxidase method and thiobarbituric acid method,ROS level was measured by chemiluminescence method. The mRNA and protein expression of adaptor protein p66Shc was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Results:Compared with those in the normal control group, the cells in hypoxia and high glucose model group had poorly metabolic ability,the content of LDH, CK-MB, MDA and ROS increased (P < 0.01), the activity of SOD decreased (P <0.01), and the expression of adaptor protein p66Shc greatly increased(P <0.01). After the treatment with liraglutide,the above mentioned parameters were all improved(P < 0.01). Exendin(9-39),an antagonist of GLP-1R,attenuated the protective effect of liraglutide. Conclusion:Liraglutide has a protective effect on cardiomyocytes by down-regulating adaptor protein p66Shc expression and reducing ROS formation.%目的:探讨利拉鲁肽对缺氧和高糖诱导的心肌细胞氧化应激损伤的保护作用及其可能机制.方法:体外分离和培养原代乳鼠心肌细胞,建立缺氧和高糖氧化应激损伤模型.实验分为正常对照组、利拉鲁肽对照组、缺氧高糖模型组、利拉鲁肽处理组、胰高血糖素样肽-1受体(GLP-1R) 抑制药组、高渗对照组.采用噻唑蓝(MTT) 法检测细胞增殖活力,比色法测定乳酸脱氢酶(LDH) 和肌酸激酶同工酶(CK-MB) 的活性,黄嘌呤氧化酶法检测细胞内超氧化物歧化酶(SOD) 活性,硫代巴比妥酸法测定丙二醛(MDA) 含量,化学荧光法检测细胞内活性氧(ROS) 水平,逆转录-聚合酶链反应(RT-PCR) 和蛋白印迹法(Western blot)分析衔接蛋白(adaptin p66Shc)的表达.结果:与正常对照组比较,缺氧高糖模型组细胞增殖活力、SOD活性明显降低(P < 0.01),LDH、CK-MB活性和MDA、ROS水平显著升高(P < 0.01),p66Shc mRNA和蛋白表达量显著升高(P <0.01);而利拉鲁肽处理组细胞上述指标较缺氧高糖模型组均明显改善(P < 0.01);GLP-1受体抑制药exendin(9-39) 可拮抗利拉鲁肽的上述保护作用(P < 0.05).结论:利拉鲁肽对缺氧和高糖诱导的心肌细胞氧化应激损伤具有明显的保护作用,其机制可能与提高细胞抗氧化能力,抑制p66Shc表达,减少ROS 的生成有关.