黄芪皂苷Ⅳ通过PKA途径减轻缺氧/复氧心肌损伤的机制研究

The mechanism of astragaloside IV alleviation of hypoxia/reoxygenation induced cultured cardiomyocyte injury via PKA pathway

目的:研究黄芪皂苷IV(astragaloside IV,As-IV)减轻缺氧/复氧所致心肌细胞损伤的机制。方法:胰酶多次消化法培养新生SD大鼠原代心肌细胞,建立缺氧/复氧模型,测定培养心肌细胞上清心肌损伤标志物肌酸激酶同工酶(creatine kinase,CK-MB)含量,检测心肌细胞肌浆网钙ATP酶(sarcoplasmic reticulum Ca2+-ATPase,SERCA2a)活性、Real-Time PCR法测定PKA催化亚单位α(PKA C subunitα,PKA-Cα)基因的表达水平以及Western blot检测第16位丝氨酸磷酸化受磷蛋白(Ser16 phos-phorylated phospholamban,Ser16-PLN)的表达水平,同时以As-IV(30μmol/L)干预,观察其作用。结果:与正常组相比,缺氧/复氧(hypoxia/reoxygenation,H/R)引起心肌细胞CK-MB释放增加,SERCA2a活性降低约35%、PKA-Cα基因表达下调28%以及Ser16-PLN表达下调51%,P值均﹤0.05,As-IV干预则可逆转上述变化,基本恢复至正常水平。结论:黄芪皂苷Ⅳ抑制缺氧/复氧所致心肌细胞损伤的机制可能是通过上调PKA-Cα基因表达,提高受磷蛋白(phosphorylated phospholamban,PLN)16位丝氨酸的磷酸化水平,解除PLN对SERCA2a的抑制,从而增强SERCA2a的功能。

Objective： To investigate the mechanism of astragaloside IV（As-IV） on alleviation of hypoxia/reoxygenation induced myocardial injury.Methods： Cultured cardiomyocytes from neonatal SD rats were exposed to 6 h of hypoxia followed by 3 h of reoxygenation（H/R）.Meanwhile,As-IV（30 ？滋mol/L） was treated in H/R cardiomyocytes.Myocytes injury was determined by the release of creatine kinase（CK-MB） in supernatant.Myocardial SERCA2a activity was measured,PKA C subunit α（PKA-Cα） gene expression and Ser16 phosphorylated phospholamban（Ser16-PLN） protein expression level were detected by real-time PCR and Western blot respectively.Results： Cultured cardiomyocytes exposed to hypoxia/reoxygenation presented statistically higher CK-MB in supernatant,decreased myocardial SERCA2a activity,reduced PKA-Cα gene and Ser16-PLN protein expressions（P 0.05）.But As-IV treatment significantly prevented the alterations in H/R cardiomyocytes mentioned above.Conclusion： These results suggest that the cardioprotective effects of As-IV may be associated with upregulation of PKA-Cα gene and Ser16-PLN protein expressions,thus restoring the SERCA2a function in hypoxia/reoxygenation injury.