中文摘要：

目的 研究黄芪甲苷（astragaloside IV）对大鼠心肌细胞线粒体保护作用及其信号转导机制。方法 酶消化法分离大鼠心肌细胞;实验随机分为对照组、黄芪甲苷组、黄芪甲苷＋抑制剂组[cGMP抑制剂ODQ、PKG抑制剂KT5823、一氧化氮合酶（NOS）抑制剂L-NAME、线粒体ATP敏感性钾离子通道（mKATP）抑制剂5-HD]、抑制剂组;共聚焦显微镜观察线粒体膜电位（ΔΨm）,100μmol·L-1H2O2造成心肌细胞氧化损伤,进而确定线粒体通透性转移孔（mPTP）的开放程度;DAF-FM荧光探针观察一氧化氮（NO）产生情况;Western blot检测磷酸化糖原合成酶激酶-3β（GSK-3β）和血管扩张剂刺激磷蛋白（VASP）蛋白表达。结果 50μmol·L-1黄芪甲苷能够模拟环孢素抑制线粒体通透性转移孔开放的作用,此作用被ODQ及KT5823所逆转;黄芪甲苷使磷酸化GSK-3β蛋白表达明显增多,此作用被ODQ、KT5823及L-NAME所阻断,5-HD未改变此作用;黄芪甲苷明显增加磷酸化血管扩张剂刺激磷蛋白表达,此作用被ODQ、KT5823所抑制;黄芪甲苷增加一氧化氮的释放,此作用被LNAME所逆转。结论 黄芪甲苷通过一氧化氮合酶产生一氧化氮,激活cGMP/PKG,使GSK-3β失活进而抑制mPTP的开放,发挥H2O2所致大鼠心肌细胞线粒体损伤的保护作用,线粒体ATP敏感性钾离子通道可能未参与此过程。

英文摘要：

OBJECTIVE To investigate the cardioprotective effect of astragaloside IV on the mitochondrial pathway, and to ex- plore its possible mechanism. METHODS Rat cardiomyocytes were isolated enzymatically, and then randomly divided into control group, astragaloside IV group, astragaloside IV plus inhibitor group [ cGMP inhibitor ODQ, PKG inhibitor KT5823, NOS inhibitor L- NAME and mitochondrial ATP sensitive potassium channel （mKATP） inhibitor 5-HD] and inhibitor group. The mitochondrial permea- bility transition pore （mPTP） opening was evaluated by measuring mitochondrial membrane potential （△ψm）. Nitric oxide （NO） gen- eration was measured by 4-amino-5-methylamino-2＇, 7＇-difluorofluorescein （DAF-FM） diacetate. Fluorescence images were obtained with confocal microscopy. GSK-3 β and VASP phosphorylation were determined with Western blot. RESULTS Astragaloside IV could mimic mPTP inhibitor cyclosporin A and showed a significant increase in TMRE fluorescence intensity, which were reversed by ODQ and KT5823. Western blot showed that astragaloside IV significantly enhanced phosphorylation of GSK-3β, which was blocked by ODQ, KT5823 and L-NAME, but not 5-HD. Astragaloside IV significantly increased VASP phosphorylation and DAF-FM green fluo- rescence intensity, which were reversed by ODQ, KT5823 and L-NAME. CONCLUSION Astragaloside IV protects H2 O2-induced mitochondrial damage in rat myocardium by modulating the mPTP opening through the inactivation of GSK-3β. NOS is responsible for NO generation and activate the cGMP/PKG pathway, mKATP may not be involved in the action of astragaloside IV.