晚期糖基化终产物对大鼠血管外膜成纤维细胞中烟酰胺腺嘌呤二核苷酸磷酸氧化酶p22phox亚基及活性氧表达的影响

Effect of Advanced Glycation End Products on NADPH Oxidase Subunit p22phox Expression and Reactive Oxygen Species Production in Vascular Adventitial Fibroblasts in Rats

目的:探讨晚期糖基化终产物(AGE)对大鼠血管外膜成纤维细胞(AF)中烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶p22phox亚基及活性氧表达的影响。方法:用组织贴块法培养SD大鼠的血管外膜成纤维细胞,用逆转录—聚合酶链反应、蛋白质印迹检测不同浓度的糖基化人血清白蛋白(AGE-HSA,分为对照组、100μg/ml AGE-HSA组,200μg/ml AGE-HSA组、300μg/ml AGE-HSA组)对NADPH氧化酶p22phox亚基信使核糖核酸(mRNA)及蛋白的表达的影响,并观察不同干预因素[分为对照组1、200μg/ml AGE-HSA组(200μg/ml处理组1)、200μg/ml AGE-HSA+50μg/ml抗RAGE中和抗体组(抗RAGE中和抗体组1)及200μg/ml AGE-HSA+30 nmol/L坎地沙坦组(坎地沙坦组1)对AGE-HSA上调NADPH氧化酶p22phox亚基mRNA和蛋白表达的影响。用2’,7’-二氯荧光黄双乙酸盐检测不同干预因素[空白对照组、AGE-HSA 200μg/ml组(200μg/ml处理组2)、200μg/ml AGE-HSA+50μg/ml抗RAGE中和抗体组(抗RAGE中和抗体组2)、200μg/ml AGE-HSA+30μmol/L夹竹桃素组(夹竹桃组),200μg/ml AGE-HSA+30 nmol/L坎地沙坦组(坎地沙坦组2)对血管外膜成纤维细胞内活性氧表达的影响。结果:3个浓度(100μg/ml、200μg/ml和300μg/ml)的AGE-HSA组均与对照组比较,血管外膜成纤维细胞NADPH氧化酶p22phox亚基mRNA及蛋白的表达随AGE-HSA增加呈浓度依赖性上调;抗RAGE中和抗体组1、坎地沙坦组1比200μg/ml处理组1的p22phox mRNA及蛋白表达降低;抗RAGE中和抗体组2、夹竹桃素组2及坎地沙坦组2比200μg/mlAGE-HSA处理组2血管外膜成纤维细胞内活性氧相对荧光强度降低,上述比较差异均有统计学意义(P<0.05)。结论:AGE-HSA经由RAGE影响活性氧与NADPH氧化酶p22phox亚基的表达上调,活性氧的上调与NADPH氧化酶p22phox亚基表达相关。NADPH氧化酶抑制剂及坎地沙坦可通过下调NADPH氧化酶p22phox亚基的表达减少血管外膜成纤维细胞内活性氧的产生。

Objective： To investigate the effect of advanced glycation end products （AGE） on NADPH oxidase subunit p22phox expression and reactive oxygen species （ROS） production in vascular adventitial fibroblasts （VAF） in rats. Methods ： The isolated VAF of SD rats were cultured with the adherent tissue explants method. ①The effect of AGE-HSA on p22phox mRNA and protein expression were measured by RT-PCR and Western-blot with different concentrations of AGE-HSA at 100 μg/ml, 200 μg/ml,300μg/ml and Control group respectively. ②The effect of different reagents on AGE-HSA regulating p22phox mRNA and protein expression were conducted as 200 μg/ml AGE-HSA group,200 μg/ml AGE-HSA ＋ anti-RAGE neu-tralizing antibody group, and 200 μg/ml AGE-HSA ＋ Candesartan group. ③The effect of different reagents on ROS production in AF were measured by ROS assay kit as 200 μg/ml AGE-HSA group,200 μg/ml AGE-HSA ＋ anti-RAGE neutralizing antibody group,200 μg/ml AGE-HSA ＋ Apopcin group,and 200 μg/ml AGE-HSA ＋ Candesartan group. Results： ①Compared with Control group, p22phox mRNA and protein expression were up-regulated by AGE-HSA in a dose- dependent manner. ②Compared with 200 μg/ml AGE-HSA group, p22phox mRNA and protein expression decreased in 200 μg/ ml AGE-HSA ＋ anti-RAGE neutralizing antibody group and 200 μg/ml AGE-HSA ＋ Candesartan group. ③Compared with 200 vg/ml AGE-HSA group,the ROS production in VAF decreased in 200 μg/ml AGE-HSA ＋ anti-RAGE neutralizing antibody group,200 tLg/ml AGE-HSA ＋ Apopcin group,and 200 μg/ml AGE-HSA ＋ Candesartan group. All P〈0. 05. Conclusion： The mRNA and protein expression of p22phox,the ROS production in VAF were up-regulated by AGE-HSA in rats, and those effects could be inhibited by RAGE. NADPH oxidase inhibitors and candesartan can reduce ROS by down-regula- ting p22phox expression.