黄芪甲苷Ⅳ通过影响β-actin对内皮型一氧化氮合酶的调节作用  

Astragaloside IV Regulates Endothelial Nitric Oxide Synthase Activity through Interaction with β-actin

作　　者：米琼宇[1] 邰婷[1] 潘玉琴[1] 何帮顺[1] 赵树立[1] 

机构地区：[1]南京医科大学附属南京医院中心实验室,江苏南京210000

出　　处：《现代生物医学进展》2013年第29期5644-5648,共5页Progress in Modern Biomedicine

基　　金：南京医科大学科技发展基金重点项目(2011NJMU200);南京医科大学科技发展基金面上项目(2012NJMU224);南京市医学科技发展资金资助(QRX11247)

摘　　要：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。Objoctivc： To investigate the regulation of astragaloside IV （AS-IV） on endothelial nitric oxide synthase （eNOS） and related mechanisms in human umbilical endothelial cells. Methods： Cultured human umbilical endothelial cells EA. Hy926 were admini- stered with AS-IV in the presence or absence of phalloidin, an actin filaments stabilizer. Immtmoprecipitation was used to detect the association of eNOS with globular actin, eNOS activity was assessed by measuring the conversion of L-3H-arginine to L-3H-citrullinine. Cyclic guanosine-3＇, 5＇-monophosphate （cGMP） production was measured by using ^125I-cGMP Radioimmunoassay Kits. Phosphorylation and total protein of eNOS and Protein Kinase B （Akt） were determined by westem blotting. Results： （1） 10-min of AS-IV treatment caused a significantly increasing association of globular actin with eNOS （P〈0.05 or P〈0.01）. This increase of association was abolished by pretreatment with phalloidin （P〈0.01）. （2） AS-IV remarkably increased eNOS activity （P〈0.05）, cGMP （P〈0.01）, phosphorylation of eNOS on Ser-1177 （P〈0.01） and Akt on Ser-473 （P〈0.001）. AS-IV-induced increase in eNOS activity （P〈0.05）, cGMP （P〈0.01） and phosphorylation of eNOS （P〈0.01） were all attenuated by pretreatment with phalloidin, whereas phosphorylation of Akt was not attenuated. Conclusion： Association of globular actin with eNOS plays an essential and necessary role in AS-IV-induced eNOS activation through enabling its phosphorylation by Akt at serine residue 1177.

关 键 词：黄芪甲苷Ⅳ 骨架蛋白 内皮型一氧化氮合酶 

分 类 号：R285.5[医药卫生—中药学] R54[医药卫生—中医学]