苦参素对缺氧/复氧损伤乳鼠心肌细胞线粒体保护作用的研究

Protective Effect of Oxymatrine on Mitochondria of Meonatal Rat Cardiomyocytes Camaged by Hypoxia/Reoxygenation

目的探索苦参素(OMT)对缺氧/复氧(H/R)所致乳鼠心肌细胞线粒体损伤的保护作用及其机制。方法分离并体外培养乳鼠心肌细胞3天后制备H/R损伤细胞模型,设正常对照组、H/R组和OMT低、中、高剂量(20、40、80μg/ml)组,各组于造模前30min给药处理。复氧2h后提取线粒体,比色法检测线粒体呼吸酶[NADH脱氢酶(ND)、琥珀酸脱氢酶(SDH)、细胞色素氧化酶(CCO)]活性、ATP含量和Na^+-K^+-ATP酶、Ca^2+-ATP酶活力,检测游离Ca2+浓度;荧光法检测膜电位(△ψm)和膜通透性转换孔(MPTP)开放度,透射电子显微镜(TEM)观察线粒体超微结构变化;生化分析法检测MDA含量和SOD、CAT活力。结果与H/R组比较,OMT中、高剂量组线粒体ND、SDH、CCO活性和ATP含量显著升高(P<0.05);Na^+-K^+-ATP酶、Ca^2+-ATP酶活力显著升高且游离Ca2+浓度显著降低(P<0.05);△ψm显著升高、MPTP开放度显著降低(P<0.01);线粒体膜破裂、脊溶解断裂等超微结构病变明显改善;线粒体MDA含量显著降低且SOD、CAT活力显著升高(P<0.05)。结论OMT对H/R所致乳鼠心肌细胞线粒体结构和功能损伤具有一定的保护作用,作用机制可能与抑制线粒体Ca2+超载、抑制氧化应激和维持膜稳定性有关。

Objective To investigate the protective effect and mechanism of Oxymatrine(OMT)on mitochondria of neonatal rat cardiomyocytes damaged by hypoxia/Reoxygenation(H/R).Methods After isolating and culturing neonatal rat cardiomyocytes in vitro for 3 days,the H/R injured cell model was prepared and set normal control group,H/R group and OMT low-,medium-and high-dose(20,40,80μg/ml)group,and the drug was given at 30 min before modeling.2 h after reoxygenation.The mitochondrial respiratory enzymes(NADH dehydrogenase(ND),succinate dehydrogenase(SDH),cytochrome oxidase(CCO))activity,ATP content and Na^+-K^+-ATP enzyme,Ca^2+-ATP enzyme activity were detected by colorimetry.The membrane potential(△ψm)and membrane permeability transition pore(MPTP)openness were detected by fluorescence detection.The ultrastructure of mitochondria was observed by transmission electron microscope(TEM).The MDA content and SOD,CAT activity were detected by biochemical analysis.Results Compared with H/Rgroup,the activity of ND,SDH,CCO and the content of ATP were significantly increased(P<0.05).The activity of Na^+-K^+-ATPase,Ca^2+-ATPase were significantly increased and the content of Ca2+was significantly decreased(P<0.05).The△ψm was increased and the MPTP opening was decreased(P<0.01).The mitochondrial membrane rupture,spinal lysis rupture and other ultrastructural lesions were significantly improved.The content of MDA was significantly decreased and the activity of SOD,CAT were significantly increased(P<0.05).Conclusion OMT has certain protective effect on mitochondrial structure and function damage of neonatal rat myocardial cells caused by H/R.The mechanism may be related to inhibition of mitochondrial Ca2+overload and oxidative stress,and maintenance of membrane stability.