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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 study the mechanism of astragaloside IV (As-IV) on hypoxia / reoxygenation-induced cardiomyocyte injury. Methods: Primary cultured neonatal SD rat primary cardiomyocytes were cultured by trypsin digestion method. Hypoxia / reoxygenation model was established. Creatine kinase (CK-MB), a marker of myocardial injury in cultured cardiomyocytes, The activity of sarcoplasmic reticulum Ca2 + -ATPase (SERCA2a) was detected by real-time PCR. The expression of PKA C subunit α (PKA-Cα) The 16th serinephos-phorylated phospholamban (Ser16-PLN) expression level, while As-IV (30μmol / L) intervention, to observe its role. RESULTS: Hypoxia / reoxygenation (H / R) resulted in an increase in CK-MB release, a decrease of about 35% in SERCA2a activity, a 28% decrease in PKA-Cα gene expression and a decrease in Ser16-PLN The expression was down-regulated by 51% (P <0.05). As-IV intervention reversed these changes and returned to normal levels. CONCLUSIONS: Astragaloside Ⅳ can inhibit the injury of cardiomyocytes induced by hypoxia / reoxygenation by elevating the PKA-Cα gene expression and increasing the phosphorylation of 16-position serine in phosphorylated phospholamban (PLN), releasing PLN to SERCA2a Inhibition, thereby enhancing SERCA2a function.