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目的:研究黄芪甲苷IV(AS-IV)对体外培养脐静脉内皮细胞中内皮型一氧化氮合酶(eNOS)的调节作用及可能的机制。方法:培养人脐静脉内皮细胞系EA.Hy926,用AS-IV进行干预,同时给予或不给予骨架蛋白β-actin聚合稳定剂phalloidin,用免疫共沉淀方法检测eNOS与单体β-actin结合状态的变化,用L-3H-精氨酸转化为L-3H-瓜氨酸的同位素法测定eNOS活性,I125环一磷酸鸟苷(cGMP)放射免疫法检测细胞内cGMP水平,Western blotting方法检测细胞中eNOS和蛋白激酶B(Akt)磷酸化水平,总蛋白水平。结果:①AS-IV作用10 min后,细胞内单体β-actin与eNOS的结合明显增加(P<0.05或P<0.01),预先给予phalloidin显著抑制了AS-IV引起的两者结合的增加(P<0.01)。②AS-IV明显增加了eNOS活性(P<0.05)、cGMP含量(P<0.01)、eNOS Ser-1177磷酸化水平(P<0.01)、Akt Ser-473磷酸化水平(P<0.001),预先给予phalloidin明显降低了AS-IV引起的eNOS活性(P<0.05)、cGMP含量(P<0.01)和磷酸化水平的增加(P<0.01),但对Akt的磷酸化没有影响。结论:单体β-actin与eNOS的结合在AS-IV激活eNOS的过程中起着不可或缺作用,其主要是通过促进Akt对eNOS Ser-1177的磷酸化来实现的。
AIM: To investigate the regulatory effect of astragaloside Ⅳ (AS-IV) on endothelial nitric oxide synthase (eNOS) in cultured human umbilical vein endothelial cells and its possible mechanism. Methods: The human umbilical vein endothelial cell line EA.Hy926 was cultured and treated with AS-IV with or without the phalloidin, a β-actin polymerization stabilizer. The binding status of eNOS and β-actin was detected by co-immunoprecipitation The eNOS activity was measured by the isotope method of converting L-3H-arginine into L-3H-citrulline, cGMP level was detected by radioimmunoassay with I125 cyclic guanosine monophosphate (cGMP), and the level of cGMP was detected by Western blotting Phosphorylation levels of eNOS and protein kinase B (Akt), total protein levels. Results: ① After 10 min of AS-IV treatment, the binding of β-actin to eNOS increased significantly (P <0.05 or P <0.01). Pre-administration of phalloidin significantly inhibited the increase of AS-IV P <0.01). ② AS-IV significantly increased eNOS activity (p <0.05), cGMP content (p <0.01), eNOS Ser-1177 phosphorylation (p <0.01) and Akt Ser-473 phosphorylation (p <0.001) Phalloidin significantly reduced the eNOS activity (P <0.05), cGMP content (P <0.01) and phosphorylation (P <0.01) induced by AS-IV, but had no effect on Akt phosphorylation. Conclusion: The binding of monomeric β-actin to eNOS plays an indispensable role in the activation of eNOS by AS-IV, which is mainly achieved by promoting the phosphorylation of eNOS Ser-1177 by Akt.