线粒体钾通道参与葛根素抗心肌细胞缺氧/复氧损伤的作用

The role of mitochondrial K^+ channels in the cardioprotection of puerarin against hypoxia/reoxygenation injury in rats

目的:探讨线粒体ATP敏感性钾通道和线粒体钙激活钾通道在葛根素预处理抗心肌细胞缺氧/复氧损伤中的作用。方法:采用酶解分离大鼠心肌细胞复制心肌细胞缺氧/复氧模型,台盼蓝拒染法测定心肌细胞存活率;四甲基罗丹明乙酯(TMRE)孵育测定线粒体膜电位值;分离线粒体测定线粒体渗透性转换孔开放程度。结果:与缺氧/复氧组相比,葛根素(0.24mmol/L)预处理5min可明显增加心肌细胞的存活率,线粒体ATP敏感性钾通道抑制剂5-羟基癸酸(100μmol/L,预处理20min)或线粒体钙激活钾通道阻断剂paxilline(1μmol/L,预处理5min)均可拮抗葛根素的作用。葛根素预处理可明显减弱缺氧引起的线粒体膜电位的耗损,5-羟基癸酸和paxilline都能明显拮抗其作用。在分离心肌线粒体模型上,葛根素显著减弱CaCl2诱导的线粒体在A520处吸光度降低,其作用与单独应用线粒体渗透性转换孔抑制剂环孢菌素A相似;5-羟基癸酸和paxilline可拮抗葛根素的保护作用。结论:在大鼠分离心肌细胞模型或分离线粒体模型上,葛根素预处理具有抗缺氧/复氧损伤的作用,这种保护作用可能与其促进线粒体ATP敏感性钾通道和线粒体钙激活钾通道的开放,进而稳定线粒体膜电位,抑制线粒体渗透性转换孔开放有关。

Objective：To determine whether the cardioprotection of puerarin （Pue） against hypoxia/reoxygenation injury is mediated by mitochondrial ATP-sensitive potassium channel （mitoKATP） and/or mitochondria calcium-activated potassium channel（mitoKCa）. Methods： Cardiomyocytes were isolated from male Sprague-Dawley rats and hypoxia/reoxygenation injury was induced by myocyte pelleting model. Cell viability was assessed by trypan blue exclusion and mitochondrial membrane potential was measured by loading with TMRE. The opening of mitochondrial permeability transition pore was determined spectrophotometrically. Results： Pretreatment with Pue at 0.24 mmol/L for 5 min increased the cell viability against hypoxia/reoxygenation injury,while mitochondrial ATP-sensitive potassium channel inhibitor 5-hydroxydecanoate （5-HD,100 μmol/L,20 min） or mitochondrial calcium-activated potassium channel blocker paxilline （Pax,1 μmol/L,5 min） attenuated the effect of puerarin. The pretreatment with Pue at 0.24 mmol/L for 5 min attenuated collapse of △Ψm induced by hypoxia/reoxygenation injury,5-HD and Pax abrogated the effect of Pue. In mitochondria isolated from hearts pretreated with Pue,a significant inhibition of Ca2＋-induced swelling was observed,and this inhibition was attenuated by 5-HD and Pax. Conclusion： These findings indicate that Pue protects cardiomyocytes against hypoxia/reoxygenation injury via activating mitoKATP channel and mitoKCa channel,and inhibiting mitochondrial permeability transition pore opening.