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 200μg/mL组、AGEs+siRNA-RAGE组、AGEs+apocynin组。采用Annexin V-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p22^(phox)、p47^(phox)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p22^(phox)、p47^(phox)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单独处理组相比,细胞凋亡率显著下降,NADPH氧化酶亚单位p22^(phox)、p47^(phox)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 possible 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 oxy- gen 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 the expression of RAGE in a dose dependent manner. Treatment with AGEs induced a significant increase in the expression of p22phox, p47phox and the activity of ROS , caused up-regulation of Bax and down-regulation of Bcl-2, which enhanced the apoptosis of GLU- Tag 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 expression of NADPH oxidase-derived ROS and its down-steam signaling pathway p53/Bax by targeting RAGE, leading to the apoptosis of GLUTag cells.
出处
《实用医学杂志》
CAS
北大核心
2017年第3期358-362,共5页
The Journal of Practical Medicine
基金
国家自然科学基金青年基金项目(编号:81500623)
广东省科技计划项目(编号:2014A020212489)
关键词
糖基化终产物
肠道L细胞
凋亡
NADPH氧化酶
活性氧
Advanced glycation end-products
GLUTagcells
Apoptosis
NADPH oxidase
Reactiveoxidative species
