环孢素A对柔红霉素损伤H9c2心肌细胞的保护作用

The protective effect of cyclosporine A on daunorubicin injured H9c2 cardiomyoblasts

目的研究环孢素A(cyclosporin A,CsA)对柔红霉素(daunorubicin,DNR)损伤大鼠H9c2心肌细胞的保护作用。方法CsA预处理2 h,1μmol·L^(-1) DNR共处理24 h。MTS法检测细胞生存率;苏木精-伊红染色观察细胞形态,生化试剂盒分析乳酸脱氢酶、肌酸激酶活性,细胞免疫化学检测心房利尿钠肽;DCF-DA测定细胞内活性氧;Hoechst 33342分析细胞凋亡,GreenNuc测定caspase-3活性,蛋白免疫印迹结果CsA提高细胞生存率;减少乳酸脱氢酶、肌酸激酶和心房利尿钠肽含量,改善细胞形态;降低活性氧含量,增加Bcl-2表达,抑制caspase-3和细胞凋亡率。抑制NFAT,CsA不能进一步增加细胞生存率。结论CsA抑制DNR处理H9c2细胞氧化应激,减少凋亡,减轻损伤,提高生存率,可能与抑制CaN及NFAT有关。

Aim To study the protective effect of cyclosporine A(CsA)on daunorubicin(DNR)injured H9c2 cardiomyoblasts.Methods H9c2 cells were pre-treated with CsA for 2 hours,then co-cultured with 1μmol·L^(-1) DNR for 24 h.The cell viability was determined by MTS method,morphological changes were detected by HE staining,LDH and CK activities were detected by biochemical kit,atrial natriuretic peptide(ANP)was measured by immunochemical staining,the intracellular reactive oxygen species were detected by of DCF-DA,the protein contents of Bax and Bcl-2 were determined by Western blot,caspase-3 activity was determined by GreenNuc caspase-3 activity kit,and the apoptotic rate was determined by Hoechst33342 staining.The relationship between CsA and NFAT was analyzed by VIVIT peptide co-treatment.Results CsA increased the viability of DNR injured H9c2 cardiomyoblasts,depressed LDH leakage,reduced CK and ANP content in cardiomyocytes,and mitigated DNR-induced morphology changes.CsA also induced decrement in the production of ROS and caspase-3 activity,while up-regulated Bcl-2 content,and thus inhibited DNR-induced apoptosis in H9c2 cardiomyoblasts.In the context of NFAT inhibition by VIVIT peptide,CsA could not further improve the viability of DNR injured H9c2 cells.Conclusions CsA depresses DNR induced oxidative stress and cell injury,and alleviates apoptosis and cell death in H9c2 cells.The inhibition of CaN and downstream NFAT may be involved in the protective effect of CsA.