晚期糖基化终产物通过p38信号通路抑制内皮细胞合成一氧化氮的研究

Advanced glycation end products inhibit production of nitric oxide by human endothelial cells through activation of the p38 signal pathway

目的 研究晚期糖基化终产物 (AGE)修饰蛋白对内皮细胞生成一氧化氮 (NO)的作用及p38丝裂素活化蛋白激酶 (MAPK)信号传导通路在此病理过程中的作用。方法 用来自培养的人脐静脉内皮细胞 (HUVEC)和人内皮细胞株ECV30 4。将内皮细胞与不同浓度的AGE修饰人血清白蛋白(AGE HSA)、AGE修饰牛血清白蛋白 (AGE BSA)在体外共同培养。用Griess法检测培养上清的NO水平 ;用免疫沉淀 激酶活性测定法测定细胞p38 MAPK活性。结果 AGE HSA和AGE BSA以时间和剂量依赖的方式抑制内皮细胞生成NO ,同时导致p38信号通路激活 ;未经修饰的HSA和BSA无此作用 ,AGE HSA和AGE BSA的抑制作用亦无明显差异 (P >0 .0 5 ) ;p38通路特异阻断剂SB 2 0 35 80完全阻断AGE修饰蛋白对内皮细胞生成NO的抑制效应 ,提示AGE修饰蛋白对内皮细胞的这一生物学作用是由p38通路介导的。AGE修饰蛋白激活HUVECp38通路。在AGE修饰蛋白作用 10min时 ,p38激酶磷酸化活性明显升高 ,在 30min时到达高峰 ,在 12 0min时回到基础水平。p38磷酸化激酶的磷酸化作用随着AGE修饰蛋白浓度的增高而加强。以ECV30 4细胞株为靶细胞时 ,AGE修饰蛋白对p38通路的活化作用与对HUVEC相同。AGE BSA对内皮细胞p38通路的活化作用与AGE HSA相同。结论本研究证实 ,AGE修饰蛋白能?

Objective To study the effect of advanced glycation end products (AGE) modified protein on nitric oxide (NO) production by human endothelial cells and the possible role of signal transduction pathway in this pathological procedure. Methods Human umbilical vein endothelial cells (HUVECs) and HUVEC-derived cell line (ECV304) were cultured in vitro with AGE modified human serum albumin (AGE-HSA) or AGE modified bovine serum albumin (AGE-BSA) of the concentrations of 12.5, 25, and 50 μg/ml for 2,4,8,12, and 24 hours. NO levels in the supernatant at each time point were determined by using Griess reagents. HUVECs and ECV304 cells incubated with HAS or BSA of the same concentrations were used as controls. The phosphorylation activity of cellular p38 mitogen-activated protein kinase (p38-MAPK) was analyzed by Western blot using a phospho-specific antibody after the cells were incubated with AGE-HAS or AGE-BSA of the concentration of 12.5, 25, 50, and 100 μg/ml for 5,15, 30, 60, and 120 minutes. HUVECs were incubated with SB 203580, a specific inhibitor of p38, of the concentrations of 0.075, 0.15, and 0.3 μmol/L for 4 hours, then the culture with SB 203580 was extracted. The endothelial cells were cleansed with RPMI 1640 culture and then were incubated with AGE-HAS or AGE-BSA for 8 hours. The No level in this supernatant was examined after the above-mentioned method. Results The NO level in the supernatant of culture of HUVECs incubated with AGE-HAS or AGE-HAS for 2 hours were 92.2% ±10.1% and 94.1%±12.5% of the controls respectively. The NO levels reached the lowest values, 72.4%±7.14% and 68.3%±10.9% of the values of the controls respectively after 8-hour incubation with AGE-HAS or AGE-HAS. AGE-HSA and AGE-BSA decreased the NO levels in the supernatant in a dose and time-dependant manner. The unmodified HSA and BSA did not influence the NO production by HUVECs or ECV304 cells. There was no difference in inhibition effect on NO production between AGE-HSA and AGE-BSA (P>0.05). There was no difference in response to AGE-HSA and AGE-BSA between HUVECs and ECV304 cells (P>0.05). In the culture of HUVECs or of ECV304 cells incubated with AGE-HSA or AGE-BSA, the p38 phosphorylation activity was obviously enhanced 10 minutes after dose-dependently, reached its peak 30 minutes after, and returned to its basic level in 120 minutes. SB203580 of the concentration of 0.3 μmol/L completely abolished the inhibitive effect of AGE-albumin on NO production by endothelial cells. Conclusion AGE modified protein inhibits the production of NO by human endothelial cells through activation of the p38 signal pathway. This AGE modification-induced pathobiological cascade may be involved in the pathogenesis of atherosclerosis seen in AGE-associated diseases.