葛根素抗心肌细胞过氧化氢损伤的线粒体相关机制

MITOCHONDRIAL MECHANISM OF CARDIOPROTECTIVE EFFECT OF PUERARIN AGAINST H_2O_2-STRESS IN RATS

目的:探讨葛根素(puerarin,Pue)预处理抗过氧化氢(H2O2)应激损伤的作用是否与线粒体渗透性转换孔和/或线粒体钙激活钾通道有关。方法:采用酶解分离大鼠心肌细胞模型,台盼蓝拒染法测定心肌细胞存活率;Rhodamine123孵育测定线粒体膜电位值,分离线粒体测定mPTP孔开放程度。结果:与H2O2应激组相比,Pue(0.24mmol/L)预处理5min可明显对抗H2O2应激引起的心肌细胞存活率的降低,线粒体钙激活钾通道阻断剂paxilline(Pax,1μmol/L,预处理30min)、线粒体渗透性转换孔开放剂atractyloside(20μmol/L,预处理20min)或PKC抑制剂chelerythrine(5μmol/L,预处理30min)可拮抗Pue的作用。Pue预处理或钙激活钾通道开放剂NS1619(10μmol/L,10min)都明显减弱H2O2应激引起的线粒体膜电位的去极化,线粒体渗透性转换孔开放剂atractyloside能明显减弱Pue的作用。在分离心肌线粒体模型上,Pue(0.24mmol/L,5min)显著减弱CaCl2诱导的线粒体在A520处吸光度降低,Pax(1μmol/L,5min)可拮抗Pue的作用。结论:在大鼠分离心肌细胞模型或分离线粒体模型上,Pue预处理具有抗过氧化氢应激损伤的作用,这种保护作用可能与其抑制线粒体渗透性转换孔的开放和促进线粒体钙激活钾通道的开放有关。

Aim： To determine whether the cardioprotection of puerarin （Pue） against H2O2-stress is mediated by mitochondrial transmembrane pores and/or channels. Methods. Cardiomyocytes were isolated from male Sprague-Dawley rats. Cell viability was assessed by trypan blue exclusion and mitochondrial membrane potential was measured by loading with Rhodamine 123. 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 H2O2-stress, while mitochondrial calcium-activated potassium channel blocker paxilline （Pax, 1μMol/L, 5 min） or PKC inhibitor（chelerythrine, 5 μmol/L, 30 min） attenuated the effect of puerarin. The pretreatment with Pue at 0.24 mmol/L for 5 min or mitochondrial calcium-activated potassium channel opener NS 1619（ 10μmol/L, 10 min）attenuated mitochondrial depolarization induced by H2O2-stress, while mitochondrial permeability transition pore opener atractyloside （20 μMol/L, 20 min） abrogated the effect of Pue. In mitochondrial isolated from hearts pretreated with 0.24 rnmol/L Pue for 5 min, a significant inhibition of Ca2＋ -induced swelling was observed, which inhibition was attenuated by Pax（ 1 μmol/L, 5 min）. Conclusion： These findings indicate that Pue protects cardiomyocytes against H2O2-stress via inhibiting mitochondrial permeability transition pore opening and activating mitochondrial calcium-activated potassium channel.