摘要
目的 探讨环状RNA同源域相互作用蛋白激酶3(circHIPK3)在氯氮平诱导H9C2心肌细胞损伤中的作用及相关机制.方法 通过细胞转染使H9C2细胞中circHIPK3过表达或沉默circHIPK3并用氯氮平处理H9C2细胞.采用实时荧光定量PCR(qRT-PCR)检测circHIPK3的表达.采用四甲基偶氮唑盐(MTT)检测细胞存活率.采用试剂盒检测乳酸盐脱氢酶(LDH)、肌酸激酶同工酶(CK-MB)、心肌肌钙蛋白(cTnI)、丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)水平.采用流式细胞术检测细胞凋亡率和线粒体膜电位.采用蛋白质免疫印迹(Western blot)检测B细胞淋巴瘤-2相关X蛋白(Bax)、B细胞淋巴瘤-2(Bcl-2)、切割型半胱氨酸天冬氨酸蛋白水解酶-3(cleaved Caspase-3)蛋白表达.结果 氯氮平显著提高 H9C2 细胞 circHIPK3、LDH、CK-MB、cTnI、MDA、凋亡率、Bax、cleaved Caspase-3 水平(P＜0.05),显著降低H9C2细胞存活率、SOD、GSH-Px、Bcl-2、线粒体膜电位(P＜0.05)o circHIPK3过表达促进氯氮平对上述指标的影响(P＜0.05),沉默circHIPK3逆转氯氮平对上述指标的影响(P＜0.05).结论 氯氮平可通过促进circHIPK3表达诱导H9C2心肌细胞损伤,其机制可能与circHIPK3激活氧化应激与线粒体凋亡通路有关.
Abstract
Objective To investigate the effect of circular RNA(circRNA)circHIPK3 on clozapine induced injury of H9C2 my-ocardial cells and its related mechanisms.Methods CircHIPK3 was overexpressed or silenced in H9C2 cells by cell transfection and H9C2 cells were treated with clozapine.Real-time quantitative PCR(qRT-PCR)was used to detect the expression of circHIPK3.Methyl thiazolyl tetrazolium assay(MTT)was used to detect cell survival rate.Relevant kits were used to detect the levels of lactate de-hydrogenase(LDH),creatine kinase isoenzyme(CK-MB),Cardiac troponin(cTnI),Malondiadehyde(MDA),superoxide dismutase(SOD)and Glutathione peroxidase(GSH-Px).Flow cytometry was used to detect cell apoptosis rate and mitochondrial membrane poten-tial.Western blot was used to detect the protein expression of B-lymphocytoma-2-associated X protein(Bax),B-lymphocytoma-2(Bcl-2)and cleaved aspartate-specific cysteine proteinase-3(cleaved Caspase-3).Results Clozapine significantly increased circHIPK3,LDH,CK-MB,cTnI,MDA,apoptosis rate,Bax,cleaved Caspase-3 levels of H9C2 cells(P＜0.05),and significantly decreased cell survi-val rate,SOD,GSH-Px,Bcl-2 levels and mitochondrial membrane potential of H9C2 cells(P＜0.05).CircHIPK3 overexpression promot-ed the effect of clozapine on the above indexes(P＜0.05),while silencing circHIPK3 reversed the effect of clozapine on the above indexes(P＜0.05).Conclusion Clozapine can induce H9C2 myocardial cells injury by promoting circHIPK3 expression,and the mecha-nism may be related to the activation of oxidative stress and mitochondrial apoptosis pathways by circHIPK3.
维普
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