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、抑制剂组;共聚焦显微镜观察线粒体膜电位(ΔΨm ),100 μmolL-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 50 μmolL-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L-NAME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。
Abstract
OBJECTIVE To investigate the cardioprotective effect of astragaloside IV on the mitochondrial pathway, and to explore its possible mechanism. METHODS Rat cardiomyocytes were isolated enzymatically, and then randomly divided into control group, astragaloside IV group, astragaloside IV plus inhibitor group and inhibitor group. The mitochondrial permeability transition pore (mPTP) opening was evaluated by measuring mitochondrial membrane potential (ΔΨm). Nitric oxide (NO) generation was measured by 4-amino-5-methylamino-2′, 7′-difluorofluorescein (DAF-FM) diacetate. Fluorescence images were obtained with confocal microscopy.GSK-3β and VASP phosphorylation were determined with Western blot. RESUTLS 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 fluorescence intensity, which were reversed by ODQ, KT5823 and L-NAME. CONCLUSION Astragaloside IV protects H2O2-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.
关键词
黄芪甲苷 /
心肌细胞 /
线粒体 /
一氧化氮 /
线粒体通透性转移孔
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Key words
astragaloside IV /
myocardium /
mitochondria /
NO /
mitochondrial permeability transition pore
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中图分类号：
R965
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参考文献
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脚注
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基金
河北省自然科学基金资助项目(H2012401019);河北省中医药管理局科研计划项目 (2012123);河北联合大学杰出青年基金项目(JP201302)
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