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目的探讨依达拉奉(EDA)能否保护H9c2心肌细胞对抗异丙肾上腺素(ISO)诱导的氧化应激和内质网应激(ERS)。方法用ISO处理H9c2心肌细胞,建立β1肾上腺素受体持续兴奋诱导心肌细胞毒性的体外模型。EDA在ISO处理心肌细胞前1 h加入培养基中作为预处理。CCK-8比色法检测细胞存活率;双氯荧光素(DCFH-DA)染色/荧光显微镜照相检测细胞内活性氧(ROS)的含量;罗丹明123(Rh123)染色/荧光显微镜照相检测线粒体膜电位(MMP);Western blot法检测葡萄糖调节蛋白78(GRP78)的表达。结果 ISO在20～100μmol.L-1浓度范围内处理H9c2心肌细胞48 h,呈剂量依赖性地降低细胞存活率;80μmol.L-1ISO处理H9c2心肌细胞可使细胞内ROS含量明显增多及MMP明显降低;80μmol.L-1 ISO处理H9c2心肌细胞0～24 h,可时间依赖性地上调内质网应激蛋白GRP78的表达,其中12 h达到高峰。分别用10、20和40μmol.L-1 EDA预处理1 h可以减弱80μmol.L-1 ISO处理H9c2心肌细胞48h引起的细胞存活率降低,500、1 000和2 000μmol.L-1氧自由基清除剂NAC分别预处理1 h也可减轻ISO诱导的心肌细胞毒性反应;40μmol.L-1的EDA预处理1 h可明显减轻ISO诱导的胞内ROS堆积及MMP降低,并明显抑制ISO引起的GRP78的表达上调。结论 EDA可保护H9c2心肌细胞对抗ISO诱导的损伤作用,其机制可能与抗氧化及抑制内质网应激反应有关。
Objective To investigate whether edaravone (EDA) protects H9c2 cardiomyocytes against isoproterenol (ISO) -induced oxidative stress and endoplasmic reticulum stress (ERS). Methods The H9c2 cardiomyocytes were treated with ISO to establish an in vitro model of myocardial cytotoxicity induced by sustained excitability of β1 adrenergic receptor. EDA was added to the medium as a pretreatment 1 h before ISO treatment of cardiomyocytes. Cell viability was detected by CCK-8 colorimetric assay. ROS content was detected by DCFH-DA staining and fluorescence microscopy. Mitochondrial membrane was detected by rhodamine123 (Rh123) staining / fluorescence microscopy Potential (MMP); Western blot was used to detect the expression of glucose regulated protein 78 (GRP78). Results ISO treatment of H9c2 cardiomyocytes in the concentration range of 20 ~ 100μmol.L-1 for 48 h reduced the cell viability in a dose-dependent manner. Treatment of H9c2 cardiomyocytes with 80μmol·L-1 ISO significantly increased intracellular ROS and MMP Hypoxic-ischemic-reperfusion (H9c2) cardiomyocytes were treated with 80μmol·L-1 of ISO for 0-24 h, and the expression of endoplasmic reticulum stress protein GRP78 was up-regulated in a time-dependent manner. Pretreatment with 10, 20 and 40 μmol·L-1 EDA for 1 h decreased the cell viability induced by 80 μmol·L-1 ISO treatment for 48 h, and 500, 1000 and 2 000 μmol.L-1 oxygen free radicals 1 h pretreatment with scavenger NAC also reduced ISO-induced cardiomyocyte cytotoxicity. Pretreatment with 40 μmol·L-1 EDA for 1 h significantly reduced ISO-induced intracellular ROS accumulation and MMP, and markedly inhibited ISO-induced GRP78 expression is up-regulated. Conclusion EDA can protect H9c2 cardiomyocytes against ISO-induced injury. The mechanism may be related to anti-oxidation and inhibition of endoplasmic reticulum stress.