糖基化终末产物对大鼠肾脏微血管内皮细胞的损伤及普罗布考的保护作用

Effect of Advanced Glycation End Products on Injuring of Rat Renal Microvascular Endothelial Cells and Protective Effect of Probucol

目的探讨糖基化终末产物对大鼠肾脏微血管内皮细胞功能的损伤以及抗氧化剂普罗布考的保护作用及分子机制。方法体外分离培养大鼠肾脏微血管内皮细胞,将其分为正常对照组、糖基化终末产物损伤组和普罗布考干预组;应用TBA法、硝酸还原酶法检测各组细胞丙二醛和一氧化氮浓度;采用逆转录聚合酶链反应法及Western-blot检测细胞内皮型一氧化氮合酶mRNA水平和NADPH氧化酶蛋白表达;应用荧光显微镜观察核因子κB活化。结果与正常对照组相比,损伤组内皮细胞丙二醛浓度上升、内皮型一氧化氮合酶mRNA水平降低、细胞质NADPH氧化酶蛋白表达减少(P<0.05)。糖基化终末产物作用时间在30min和6h时培养基中一氧化氮浓度升高(P<0.05或P<0.01),而12h后一氧化氮浓度随着糖基化终末产物作用时间延长而降低(P<0.05或P<0.01)。普罗布考在10、20、50、100μmol/L范围内降低细胞丙二醛水平,抑制NADPH氧化酶蛋白活化,上调一氧化氮和内皮型一氧化氮合酶mRNA水平(P<0.05或P<0.01)。糖基化终末产物刺激微血管内皮细胞后核因子κB表达部位由细胞质移到核内,作用30min时核内表达达高峰,而后持续高表达。结论糖基化终末产物导致的大鼠肾脏微血管内皮细胞损伤和功能下降的机制可能包括:①引发脂质过氧化;②活化NADPH氧化酶,增加活性氧的生成;③内皮型一氧化氮合酶mRNA水平异常,影响一氧化氮生成;④激活核因子κB。普罗布考的作用机制可能是通过拮抗糖基化终末产物导致的脂质过氧化,核因子κB、NADPH氧化酶活化和内皮型一氧化氮合酶mRNA表达异常来保护肾脏微血管内皮细胞功能。

Aim To explore the effect of advanced glycation end products （ AGE ） on injuring of rat renal microvascular endothelial cells （RMEC）and protective effect of probncol. Methods Mierovascular endothelial cells isolated and cultured from rat renal were divided into 3 groups： normal control group, AGE group and probueol group. The levels of malondialdehyde （MDA） and niuie oxide （NO） were determined by the assay TBA and nitrate reduetase method respectively. The expression of endothelial nitric oxide synthase （eNOS） mRNA and nieotinamide-adenine dinueleotide phosphate （NADPH） oxidase protein was detected by reverse transcription polymerase chain reaction （RT-PCR） and Westem-blot respectively. The intracellular disposition of nuclear factor kappa B （NF-κB） was observed by immunostaining microscopy. Results In AGE group, the level of MDA increased but the expression of NADPH oxidase protein and eNOS mRNA decreased compared with control. The level of NO in culture medium was increased after exposure to AGE-BSA for 30 min and 6 h,but after 12 h, NO level was decreased. MDA level and the expression of NADPH oxidase, protein were decreased but the expression of eNOS mRNA and NO level were upregulated by probucol with a dose-dependent effect in the concentrations of 10, 20, 50, 100 μmol/L. The localization of NF-κB shifted from cytoplasm to nucleus after mierovascular endothelial cells were exposed to AGE-BSA. After exposed for 30 min, the expression in nucleus reached the peak and then maintained high level. Conclusion AGE can cause the injury and dysfunction of RMEC via the following possible mechanisms： ①inducing lipid preoxidation; ②activating NADPH oxidase and increasing the production of reactive oxygen species （ROS） ; ③abnormal expression of eNOS mRNA and further affecting NO production; ④ activatin of NF-κB. Probueol can protect mierovascular entothehal cells probably via antagonisting the lipid preoxidation, activating of NF-κB and NADPH oxidase, and increasing the AGE-induced abnormal expression of eNOS caused by AGE.