丙烯醛对缺氧复氧H9c2心肌细胞损伤的影响及机制初探

目的研究丙烯醛对缺氧复氧H9c2心肌细胞损伤的影响,并探讨其促凋亡作用机理。方法建立H9c2心肌细胞缺氧复氧(H/R)损伤模型,分为正常对照组、丙烯醛组(ACR)、缺氧复氧组(H/R)、丙烯醛+缺氧复氧组(ACR+H/R)。用10μmol/L丙烯醛预处理H9c2心肌细胞30min后2h缺氧16h复氧,采用MTT法检测细胞存活率,4′,6-二脒基-2-苯基吲哚(DAPI)荧光染色检测细胞核凋亡的形态学改变,流式细胞术检测细胞凋亡,Western blot检测凋亡相关蛋白cytochrome c(cyt c)、caspase 9和caspase 3的表达水平。结果与H/R组相比,ACR+H/R组H9c2细胞存活率下降、凋亡增加(P<0.05),cyt c的线粒体蛋白表达水平下调、细胞浆蛋白表达水平上调,caspase 9和caspase 3蛋白表达水平下调并出现明显裂解蛋白。结论作为一种来源于人类生活环境的心血管毒物,丙烯醛能加剧缺氧复氧H9c2心肌细胞的损伤,可能与cyt c由线粒体释放到细胞浆、激活caspase级联反应导致线粒体功能损伤有关。

Effects of Acrolein on Apoptosis of H9c2 Cardiacmyocytes with Hypoxia/Reoxygenation Injury

Objective To determine the cytotoxic effects of acrolein on hypoxia/ reoxygenation(H/R) injury in H9c2 cardiacmyocytes and investigate the intracellular signaling pathways.Methods Hypoxia/reoxygenation(H/R) injury model was established with H9c2 cells.The H9c2 cells were divided into four groups,the control group,acrolein group(ACR),H/R group,acrolein+H/R group(ACR+H/R).H9c2 cells pretreated with or without acrolein(10 μmol/L) for 30 min were exposed to 2 h hypoxia and 16 h reoxygenation.The effect of acrolein on the cellular viability and apoptosis of H9c2 cells was measured by MTT assay,DAPI stainning and flow cytometry(FCM) respectively.The expression of apotosis-related proteins(cytochrome c,caspase 9 and caspase 3) in the H9c2 cells was detected by Western blot.Results Compared with mere H/R treatment,the decrease in cell viability and increase in the number of apoptotic cells in H9c2 cells subjected to H/R were significantly exacerbated in the presence of acrolein(P<0.05).The liberation of cytochrome c from mitochondria to cytosol,the cleavages of the initiator caspase 9 and the effector caspase 3 have been observed after pretreatment with acrolein followed by H/R in H9c2 cells.Conclusion Acrolein could aggravate H/R injury and that this effect may be related,in part,to the modification of proteins involved the release of cytochrome c from mitochondria to cytosol and activation of caspases cascade reaction.