糖基化终产物对人脐静脉内皮细胞eNOS活性和表达的影响

Effects of advanced glycation end products on activity and expression of eNOS in human umbilical vein endothelial cells

目的 研究糖基化终产物 (AGEs)对内皮型一氧化氮合酶 (eNOS)的活性及蛋白表达的影响。方法 用糖孵育法制备糖基化修饰的牛血清白蛋白 (AGE BSA) ,用胶原酶法分离培养人脐静脉内皮细胞 (HUVECs)。将内皮细胞与不同浓度的AGE BSA在体外分别培养 3 ,6,12 ,2 4,48h ,用同位素二步色谱法检测基础状态下与组胺刺激后的eNOS活性 ,用蛋白免疫印迹法 (Western blotting)检测eNOS蛋白表达水平。结果 基础状态下 ,HUVECs的eNOS有部分激活 ,组胺刺激后eNOS活性明显增加 (P <0 .0 0 1)。AGE BSA明显抑制基础状态下的eNOS活性和组胺刺激后的eNOS活性增高(P <0 .0 0 1) ,这种抑制作用呈时间、浓度依赖性。AGE BSA与HUVECs共同孵育 2 4h后的eNOS表达水平明显降低 (P <0 .0 0 1) ,且随着孵育时间的延长 ,eNOS表达水平进一步降低 (P <0 .0 5 ,48hvs 2 4h) ,这种抑制作用与AGE BSA的浓度有关。结论 AGE BSA明显抑制eNOS基础状态和组胺刺激后的活性增高 ,AGE BSA明显抑制HUVECs的eNOS表达 ,AGE BSA对eNOS活性的抑制作用可能与降低eNOS表达有关。

Objective To investigate the effects of advanced glycation end products (AGEs) on activity and expression of endothelial nitric oxide synthase in cultured human umbilical vein endothelial cells (HUVECs). Methods AGE-modified bovein serum albumin (AGE-BSA) was prepared by incubated BSA with glucose at 37?℃ in vitro. HUVECs were co-incubated with different concentrations of AGE-BSA for 3, 6, 12, 24 and 48 hours respectively. The activity of eNOS is determined by the convertion of radiolabelled 3H-L-arginine to 3H-L-citrulline and eNOS expression levels were measured by Western blotting method. Results The activity of eNOS in HUVECs was partially activated under basic condition, and it was markedly increased by histamine (P<0.001). AGE-BSA significantly inhibited the activity of eNOS in a concentration and time-dependent manner. The expression of eNOS had a significant reduction when HUVECs were incubated with AGE-BSA (200?mg·L -1) for 24 hours (P<0.001)and a further decrease (P<0.05, 48?h vs 24?h) for 48 hours. Conclusions AGE-BSA significntly inhibit the activity of eNOS,and this inhibited effect may be partially due to reduced eNOS expression.