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目的探讨胞外信号调节激酶1/2(ERK1/2)在依达拉奉(EDA)保护H9c2心肌细胞对抗异丙肾上腺素(ISO)损伤中的作用。方法用不同浓度的ISO处理H9c2心肌细胞,建立β1肾上腺素受体持续兴奋诱导心脏毒性的体外模型。EDA在ISO处理心肌细胞前1h加入培养基中作为预处理。PD-98059(ERK1/2抑制剂)在应用EDA前加入培养基中并作用1h,以抑制ERK1/2的磷酸化。CCK-8比色法检测细胞存活率;Western blot法检测总量及磷酸化ERK1/2蛋白的表达;罗丹明123(Rh123)染色荧光显微镜照相检测线粒体膜电位(MMP)。结果 80μmol/L ISO处理H9c2心肌细胞24h,可使磷酸化的ERK1/2及MMP的水平明显降低。EDA在10～40μmol/L浓度范围内预处理1h可以剂量依赖性地减弱80μmol/L ISO处理H9c2心肌细胞48h引起的毒性反应;40μmol/L EDA预处理1h可明显抑制ISO作用24h引起的ERK1/2磷酸化水平降低及MMP受损。ERK1/2抑制剂,PD-98059,可以取消EDA诱导的细胞保护作用,使细胞毒性及MMP受损加重。结论 EDA可保护H9c2心肌细胞对抗ISO诱导的损伤作用,其机制之一可能与拮抗ISO对ERK1/2的磷酸化抑制作用有关。
Objective To investigate the role of extracellular signal-regulated kinase 1/2 (ERK1 / 2) in protection of isoproterenol (ISO) injury induced by edaravone (EDA) in H9c2 cardiomyocytes. Methods The H9c2 cardiomyocytes were treated with ISO at different concentrations to establish an in vitro model of β1-adrenergic receptor-induced cardiotoxicity induced by sustained excitability. EDA was added to the medium as pretreatment 1 h before ISO treatment of cardiomyocytes. PD-98059 (inhibitor of ERK1 / 2) was added to the medium before EDA application for 1 h to inhibit phosphorylation of ERK1 / 2. The cell viability was detected by CCK-8 colorimetric assay. The total amount of phosphorylated ERK1 / 2 protein was detected by Western blot. Mitochondrial membrane potential (MMP) was detected by Rh123 staining. Results Hypoxic-ischemic-reperfusion (H9c2) cardiomyocytes treated with 80μmol / L of ISO for 24 hours significantly reduced the levels of phosphorylated ERK1 / 2 and MMPs. EDA pretreated with 10 ~ 40μmol / L for 1h could attenuate the toxicity induced by 80μmol / L ISO for 48h in H9c2 cardiomyocytes in a dose-dependent manner. Pretreatment with 40μmol / L EDA for 1h significantly inhibited the ERK1 / 2 phosphorylation and MMP damage. ERK1 / 2 inhibitor, PD-98059, can abolish EDA-induced cytoprotection and increase cytotoxicity and MMP damage. Conclusion EDA can protect H9c2 cardiomyocytes against ISO-induced injury. One of the mechanisms may be related to antagonizing the inhibition of phosphorylation of ERK1 / 2 by ISO.