糖基化终产物通过其受体激活NADPH氧化酶及其下游通路诱导L细胞的凋亡

摘要

目的:探讨糖基化终产物(AGEs)与其受体(RAGE)及其下游信号通路在肠道L细胞(GLUTag)凋亡中的作用.方法:首先将不同浓度的AGEs作用于GLUTag细胞24 h后,RT-PCR及Western blot检测各组细胞RAGE mRNA及蛋白表达水平.然后将细胞分为BSA对照组、AGEs 200 μg/mL组、AGEs+ siRNA-RAGE组、AGEs+apocynin组.采用AnnexinV-FITC/PI检测细胞凋亡率,荧光探针检测细胞内活性氧水平,Western blot检测NADPH氧化酶亚单位p22phox、p47pbox磷酸化水平和Bcl-2、Bax蛋白表达水平.结果:AGEs干预后GLUTag细胞RAGE mRNA及蛋白表达水平呈剂量依赖性增加.AGEs单独处理组与BSA对照组相比,细胞凋亡率显著增加,NADPH氧化酶亚单位p22phox、p47phox的表达增多,细胞内ROS水平升高,促凋亡蛋白Bax表达上调,抗凋亡蛋白Bcl-2表达下调.AGEs+apocynin组、AGEs+siRNA-RAGE组与AGEs单独处理纽相比,细胞凋亡率显著下降,NADPH氧化酶亚单位p22phox、p47phox的表达减少,细胞内ROS水平下降,促凋亡蛋白Bax表达下调,抗凋亡蛋白Bcl-2表达上调.结论:AGEs作用于GLUTag,可上调细胞RAGE受体表达,激活NADPH氧化酶,使细胞内ROS生成增多,进而通过p53/Bax途径诱导GLUTag凋亡.%Objective To investigate the effects of AGEs-RAGE on the apoptosis of GLUTag cells and explore the possiblc mechanism.Methods GLUTag cells treated with 0、100、200、300μg/ml of AGEs for 24h were examined for gene and protein expression of RAGE using RT-PCR and western blotting,respectively.GLUTag cells were randomly divided into four groups:control,200μg/ml AGEs,AGEs+siRNA-RAGE and AGEs+apocynin.The protein expression of p22phox、p47phox 、Bcl-2、Bax in the cells were detected with western blotting.The reactive oxygen species (ROS) levels were examined using 2'7'-dichlorodihydroflur-rescein diacetate (DCFH-DA) and the apoptosis of L cells were tested by AnnexinV-FITC/PI.Results AGEs increased thc cxpression of RAGE in a dose dependent manner.Treatment with AGEs induced a significant increase in the expression of p22phox,p47phoxand the activity of ROS,caused up-regulation of Bax and down-regulation of Bcl-2,which enhanced the apoptosis of GLUTag cells.Apocynin,the inhibitor of NADPH oxidase,prevented those responses and the effects caused by AGEs were abolished by inhibition of RAGE activity with siRNA.Conclusion AGEs positively regulate the exprcssion of NADPH oxidase-derived ROS and its down-steam signaling pathway p53/Bax by targeting RAGE,leading to the apoptosis of GLUTag cells.