NADPH氧化酶在次氯酸修饰白蛋白诱导血管内皮细胞炎性反应中的作用

目的 阐明次氯酸修饰白蛋白（HOCl-Alb）诱导血管内皮细胞炎性反应的机制。 方法 用体外制备的HOCl-Alb与人脐静脉内皮细胞共同培养；用光泽精增强化学发光法测定NADPH氧化酶活性；用免疫沉淀和Western印迹法测定p47phox磷酸化及p47phox与p22phox结合；用免疫荧光化学染色法观察p47phox膜迁移；分别用RT-PCR和Western印迹法测定细胞间黏附分子1（ICAM-1） mRNA和蛋白表达。为了解NADPH氧化酶在HOCl-Alb上调ICAM-1表达过程中的作用，在HOCl-Alb刺激细胞前，在细胞培养液中预先加入NADPH氧化酶特异性抑制剂夹竹桃麻素（apocynin），观察ICAM-1表达的变化。 结果 HOCl-Alb激活NADPH氧化酶具有剂量和时间依赖性，200 mg/L HOCl-Alb刺激15 min使NADPH氧化酶活性增加的量是牛血清白蛋白组的6.16倍（P ＜ 0.01），并可诱导p47phox磷酸化和膜迁移，及其与p22phox结合。HOCl-Alb上调ICAM-1表达的作用可被夹竹桃麻素抑制，500 μmol/L 夹竹桃麻素对HOCl-Alb诱导的ICAM-1表达的抑制率为68.97%（P ＜ 0.01）。 结论 NADPH氧化酶是HOCl-Alb诱导ICAM-1高表达的重要途径，与血管内皮炎性反应关系密切。

Role of NADPH oxidase in the regulation of vascular endothelial cell inflammation induced by hypochlorite-modified albumin

Objective To elucidate the mechanism of inflammation in vascular endothelial cells induced by hypochlorite-modified albumin (HOCl-Alb). Methods HOCl-Alb-induced NADPH oxidase activity in human umbilical vein endothelial cells (HUVEC) was measured by lucigenin-enhanced chemiluminescence. Phosphorylation of p47phox and binding of p47phox and p22phox were measured with immunoprecipitation and Western blotting. Membrane translocation of p47phox was measured with immunofluorescence. RT-PCR and Western blotting were used to determine the intercellular adhesion molecule-1 (ICAM-1) mRNA and protein expression in the presence or absence of apocynin, respectively. Results Co-incubation of HUVEC with HOClAlb resulted in the enhancement of NAIDPH oxidase activity in time- and dose-dependent manner.Compared with bovine serum albumin group, exposure of the cells with 200 mg/L HOCl-Alb for 15min resulted in a 6.16-fold increase in NADPH oxidase activity. Phosphorylation and membrane translocation of p47phox and binding of it with p22phox were also induced by HOCl-Alb. ICAM-1expression was up-regulated after exposure to HOCl-Alb and this effect was significantly abolished by apocynin, a specific inhibiter of NADPH oxidase, in dose-dependent manner. Preincubation of the cells with 500 μmol/L apocynin inhibited the expression of ICAM-1 protein induced by HOClAlb by 68.97% (P＜0.01). Conclusion NADPH oxidase plays a central role in HOCl-Albmediated ICAM-1 expression and provides a mechanism for HOCl-Alb-related vascular endothelial inflammation.