依达拉奉保护H9 c2心肌细胞对抗阿霉素的心肌毒性作用

Edaravone protects H9c2 cells against doxorubicin-induced cardiotoxicity

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。

Aim To explore whether edaravone （EDA）, a novel free radical scavenger, protects H9c2 cardiac cells against doxorubicin （ DOX ）-induced car-diotoxicity. Methods H9c2 cells were treated with 5μmol＆#183;L-1 DOX to establish a model of DOX cardio-toxicity. Cell viability was examined by cell counter kit （ CCK-8 ） . Changes in morphology and amount of ap-optotic cells were detected by Hoechst 33258 staining;intracellular level of reactive oxygen species （ ROS ） was measured by DCFH-DA staining and photofluorog-raphy;mitochondrial membrane potential （ MMP） was observed by rhodamine 123 （ RH123 ） staining and photoflurograph; the expression level of caspase-3 was determined by Western blot assay. Results Pretreat-ment of H9 c2 cells with 20 , 40 and 80 μmol ＆#183; L-1 EDA for 60 min markedly inhibited cytotoxicity in-duced by 5 μmol ＆#183; L-1 DOX, respectively, as evi-denced by an increase in cell viability. The protective＆amp;nbsp;effect induced by 40 μmol ＆#183; L-1 EDA was maximal. Pretreatment of H9 c2 cells with 40 μmol ＆#183; L-1 EDA for 30 , 60 , 90 and 120 min significantly attenuated DOX-induced cytotoxicity, respectively, having a max-imal protection at 60 min. Furthermore, pretreatment of H9 c2 cells with 40 μmol ＆#183; L-1 EDA for 60 min be-fore exposure to 5 μmol ＆#183; L-1 DOX for 24 h obviously reduced cardiac injuries, as evidenced by decreases in the DOX-induced intracellular ROS generation, num-ber of apoptotic cells, and expression of cleaved caspase-3, as well as loss of MMP. Conclusions EDA can protect H9 c2 cardiac cells against DOX-in-duced cardiotoxicity, this protection may be associated with inhibition of ROS production and preservation of MMP.