MADPH氧化酶抑制剂对晚期氧化蛋白产物诱导内皮细胞高通透性的影响及机制

Protective effect of diphenyleneiodonium,a NADPH oxidase inhibitor,on hyperpermeability of endothelial cells exposed to AOPP-HSA in vitro

目的:探讨NADPH氧化酶(Nox)抑制剂对晚期氧化蛋白产物(AOPP)刺激下血管内皮的保护作用。方法:体外培养人脐静脉内皮细胞进行实验,人血清白蛋白(HSA)作为阴性对照,用不同浓度(50、100和200mg/L)AOPP-HSA共同孵育8 h后,利用5-氯甲基二乙酸荧光素标记人急性单核细胞白血病细胞株THP-1的细胞渗出数量反映内皮细胞的通透性,研究不同浓度AOPP-HSA对单层细胞通透性的影响。此外,另将细胞分为HSA组、AOPP-HSA组和AOPP-HSA+二联苯碘(DPI)组,进而探讨AOPP-HSA对Nox活化水平的影响以及DPI对内皮细胞骨架重构和细胞通透性改变的作用。结果:AOPP-HSA可使血管内皮细胞通透性明显增加(P<0.05)。AOPP-HSA可导致Nox磷酸化水平上升,并呈剂量依赖性。Nox抑制剂DPI预处理组可抑制AOPP-HSA刺激下Nox磷酸化水平的上升,从而抑制血管内皮细胞通透性增加及细胞骨架重构。结论:AOPP-HSA可通过激活Nox导致血管内皮细胞通透性受损,Nox抑制剂DPI可以降低其通透性及细胞骨架重构,起到一定的保护作用。

AIM： To investigate the effect of advanced oxidation protein product-human serum albumin（ AOPP-HSA） at different concentrations on the permeability of human umbilical vein endothelial cell（ HUVEC） monolayer and the protective effect of NADPH oxidase inhibitor diphenyleneiodonium（ DPI） against AOPP-HSA exposure.METHODS： Cultured HUVECs were exposed to 200 mg / L HSA（ control） or AOPP-HSA（ 50,100 and 200 mg / L）. The permeability of the endothelial monolayer was assessed by measuring CMFDA-labeled THP-1 cells across the endothelial cells. The cultured HUVECs were treated with HSA（ 200 mg / L）,AOPP-HSA（ 200 mg / L）,or AOPP-HSA（ 200 mg / L）＋ DPI（ 100 μmol / L）,and the activation of NADPH oxidase,endothelial monolayer permeability and cytoskeleton rearrangement were evaluated. RESULTS： AOPP-HSA increased the permeability of the endothelial cell monolayer,and AOPP-HSA at 200 mg / L significantly increased the phosphorylation level of NADPH oxidase in the cells. Treatment with100 μmol / L DPI obviously attenuated AOPP-HSA-induced NADPH oxidase activation,the increase in the permeability of the cell monolayer and the cytoskeleton rearrangement. CONCLUSION： AOPP-HSA increases the hyperpermeability of HUVEC monolayer via the phosphorylation of NADPH oxidase,and the NADPH oxidase inhibitor DPI reverses such effects.