苦参素对缺氧/复氧损伤乳鼠心肌细胞氧化应激和细胞凋亡的影响

摘要

目的：研究苦参素(OMT)对缺氧/复氧损伤乳鼠心肌细胞氧化应激和细胞凋亡的影响。方法无菌环境下分离 SD大鼠乳鼠心肌细胞并培养72 h后随机分为：空白对照组、缺氧/复氧(H/R)模型对照组、OMT(20μg/mL、40μg/mL和80μg/mL)干预组和舒血宁注射液(SXN,100μg/mL)干预组(阳性对照组),每组设10个复孔。药物干预6 h后,通过MTT法检测细胞存活率；检测细胞培养液中心肌酶[谷草转氨酶(AST)、磷酸肌酸激酶(CPK)、乳酸脱氢酶(LDH)]释放量；测定细胞中抗氧化酶[超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH Px)、过氧化氢酶(CAT)]活性和丙二醛(MDA)含量；通过流式细胞仪检测细胞凋亡状况并计算凋亡率,通过 RT PCR检测细胞中bcl 2mRNA、BaxmRNA表达并计算bcl 2/Bax比值,通过Western blot方法检测细胞caspase 3蛋白表达水平并进行半定量分析。结果与模型对照组比较,OMT40μg/mL、80μg/mL干预组、SXN干预组乳鼠心肌细胞存活率显著升高,培养液中 AST、CPK、LDH释放量显著降低,细胞中 SOD、CAT活性显著升高且 MDA含量显著降低,细胞凋亡状况显著改善、凋亡率显著降低,细胞中 bcl 2 mRNA 表达显著上调且 Bax mRNA 表达显著下调、bcl 2/Bax 表达比值显著升高,细胞中caspase 3蛋白表达显著升高,上述差异均有统计学意义(P<0.05或P<0.01)；其中 OMT 80μg/mL干预组细胞中 GSH Px活性显著升高(P<0.05)。结论苦参素对缺氧/复氧损伤乳鼠心肌细胞氧化应激和细胞凋亡具有抑制作用,其作用机制可能与苦参素能有效改善抗氧化酶活性、调节凋亡相关基因表达有关。%Objective To investigate the effects of oxymatrine(OMT)on oxidative stress and apoptosis in neonatal rat impaired by hy-poxia- reoxygenation(H/R).Methods Cadiocytes of neonate rats were cultivated for 72 hours and divided into six groups:normal control group,H/R model control group,OMT (20μg/mL,40μg/mL and 80μg/mL)treatment groups and Shuxuening injection(SXN, 100μg/mL)treatment group (n=10). After 6 hours of drugs intervention,the survival rate was detecd.The content of aspartate trans-aminase (AST),creatine phosphokinase (CPK),lactate dehydrogenase (LDH)in culture medium were deteced.The activity of su-peroxide dismutase (SOD),catalase (CAT),glutathione peroxidase (GSH Px)and the content of malondialdehyde(MDA)in car-diomyocytes were determined.The apoptosis was observed by flow cytometry and the apoptosis rate was calculated.The expression of bcl 2 mRNA,Bax mRNA were detected by reverse transcription polymerase chain reaction (RT PCR).The ratio of bcl 2/Bax was calculated.The expression of caspase 3 protein was detected by western blot. Results Compared with the model control group,the survival rate were significantly increased in OMT(40μg/mL,80μg/mL)treatment groups.The content of CPK,AST,LDH in culture medium were significantly decreased.The activity of SOD,CAT in cardiomyocytes were significantly increased and the content of MDA was significantly decreased.The apoptosis rate was significantly decreased.The expression of bcl 2 was significantly up regulated,the expression of Bax and caspase 3 were significantly down regulated.The ratio of bcl 2/Bax were significantly in-creased.The activity of GSH Px was significantly increased(P<0.05).Al of the difference above were significant(P<0.05 or P<0.01).Conclusion OMT had inhibitive effects on oxidative stress and apoptosis of neonatal rat impaired by H/R,which perhaps relat-ed to its effects of improving the activity of antioxidase and regulating the expression of apoptosis related genes.