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目的探讨新型的自由清除剂依达拉奉(edaravone,EDA)能否保护H9c2心肌细胞对抗阿霉素(doxorubicin,DOX)引起的损伤。方法应用DOX(5μmol·L-1)处理H9c2心肌细胞建立DOX心肌毒性损伤模型。CCK-8比色法测定细胞存活率;Hoechst 33258核染色法观察细胞凋亡的形态学和数量改变;双氯荧光素(DCFH-DA)染色荧光显微镜照像检测细胞活性氧(ROS)水平;罗丹明123(Rh123)染色荧光显微镜照像测定线粒体膜电位(MMP);Western blot法测定caspase-3蛋白的表达水平。结果应用20、40、80μmol·L-1EDA分别预处理H9c2心肌细胞60 min,可明显地抑制5μmol·L-1DOX引起的细胞毒性,使细胞存活率升高,其中40μmol·L-1EDA的保护作用最大;应用40μmol·L-1EDA分别预处理心肌细胞30、60、90、120 min,可明显地抑制DOX引起的细胞毒性,其中预处理60 min的保护作用最大;此外,在5μmol·L-1DOX处理H9c2心肌24 h前,应用40μmol·L-1EDA预处理60 min可明显抑制DOX引起的心肌损伤作用,表现为抑制DOX引起的细胞内ROS生成增多及抑制DOX的致细胞凋亡作用(使凋亡细胞数目减少和cleaved caspase-3表达下调)和MMP的损伤作用。结论EDA能保护H9c2心肌细胞对抗DOX诱导的心肌毒性,此保护作用可能与其抑制ROS生成及减轻DOX对MMP的损伤有关。
Objective To investigate whether edaravone (EDA), a new free radical scavenger, can protect H9c2 cardiomyocytes against doxorubicin (DOX) -induced injury. Methods DOX (5μmol·L-1) H9c2 cardiomyocytes were used to establish DOX model of myocardial toxicity. Cell viability was determined by CCK-8 colorimetric assay. Morphological and quantitative changes of apoptotic cells were observed by Hoechst 33258 nuclear staining. Fluorescence microscopy was used to detect the level of reactive oxygen species (ROS) Mitochondrial membrane potential (MMP) was measured by Rh123 staining and fluorescence microscopy. The expression of caspase-3 protein was detected by Western blot. Results Pretreatment of H9c2 cardiomyocytes with 20, 40, 80μmol·L-1EDA for 60min significantly inhibited the cytotoxicity induced by 5μmol·L-1DOX and increased the cell viability. The protective effect of 40μmol·L-1EDA The pretreatment of cardiomyocytes with 30μmol·L-1EDA for 30,60,90 and 120 min, respectively, could significantly inhibit the cytotoxicity induced by DOX, of which the protective effect was the highest at 60 min. In addition, pretreatment with 5μmol·L-1DOX Pretreatment with 40 μmol·L-1 EDA for 60 min before treatment of H9c2 myocardium significantly inhibited the myocardial injury induced by DOX, which showed that the inhibition of DOX-induced increase of intracellular ROS production and the inhibition of DOX-induced apoptosis Decreased number of dead cells and decreased expression of cleaved caspase-3) and MMP. Conclusion EDA can protect H9c2 cardiomyocytes against DOX-induced myocardial toxicity. This protective effect may be related to its inhibition of ROS production and mitigation of MMP-2 damage by DOX.