黄蜀葵花中5种黄酮类化合物对肠道L细胞AGEs/RAGE/p38MAPK/NF-κB信号通路的调节作用

摘要

目的 探讨黄蜀葵花中5种黄酮类化合物对肠道L细胞AGEs/RAGE/p38MAPK/NF-κB信号通路的调节作用.方法 200 mg·L-1 AGEs作用肠道于L细胞株GLUTag细胞,Western blot检测细胞中RAGE、p22Phox、p47Phox、p53、Bax蛋白的相对表达量,ELISA法检测TNF-α、IL-1、IL-6、caspase-3、caspase-9的含量.实验分为7组:空白对照组(NG)、模型组(AGEs)、槲皮素组(QT)、异槲皮苷组(IQT)、金丝桃苷组(HY)、槲皮素-3′-O-葡萄糖苷组(QG)、棉皮素-8-O-葡萄糖醛酸苷组(GG).NG细胞培养于DMEM低糖培养基中;AGEs细胞培养于含200 mg·L-1 AGEs的DMEM低糖培养基中;给药组细胞分别培养于含50 μmol·L-1各黄酮单体及含200 mg·L-1 AGEs的DMEM低糖培养基中.各组细胞经药物干预24 h后,Western blot检测细胞中RAGE、p22Phox、p47Phox、p53、Bax、p-p38MAPK、NF-κB蛋白的相对表达量,ELISA法检测TNF-α、IL-1、IL-6的含量及caspase-3、caspase-9的活性.结果 模型组细胞中RAGE、p22Phox、p47Phox、Bax、caspase-3、caspase-9、Phospho-p38MAPK、NF-κB相对表达量及细胞上清液中TNF-α、IL-1、IL-6的分泌量明显上调(P<0.01),p53相对表达量明显下调(P<0.01).50 μmol·L-1槲皮素、异槲皮苷、金丝桃苷、槲皮素-3′-O-葡萄糖苷、棉皮素-8-O-葡萄糖醛酸苷分别给药后,各蛋白的相对表达量及炎症因子的分泌量接近正常组.结论 黄属葵花中5种黄酮类化合物能明显抑制肠道L细胞AGEs/RAGE/p38MAPK/NF-κB信号通路.%Aim To investigate the regulation of five flavonoids from Abelmoschl manihot on the AGEs/RAGE/p38MAPK/NF-κB signaling pathway in GLUTag cells.Methods With GLUTag cells treated with 200 mg·L-1 AGEs,Western blot analysis was applied to determine the expression of RAGE, p22Phox, p47Phox, p53 and Bax proteins and ELISA analysis was used to detect the levels of TNF-α, IL-1, IL-6, caspase-3, caspase-9.The cells were divided into seven groups: the normal group(NG), model group(AGEs), quercetin-treated group(QT), isoquercitrin-treated group(IQT), hyperoside-treated group(HY), quercetin-3′-O-glucoside-treated group(QG), gossypetin-8-O-glucuronide-treated group(GG).NG cells were cultured in DMEM low-glucose culture medium without adding any other intervention factors;model group cells were cultured in DMEM low-glucose medium containing 200 mg·L-1 AGEs;cells in five treatment groups were cultured in DMEM low-glucose culture medium containing 200 mg·L-1 AGEs, which were respectively added 50 μmol·L-1 QT, IQT, HY, QG, GG.Western blot analysis was applied to determine the expression of RAGE, p22Phox, p47Phox, p53 and bax proteins and ELISA analysis was used to detect the levels of TNF-α, IL-1, IL-6, caspase-3, caspase-9.Results The contents of RAGE, p22Phox, p47Phox, bax, caspase-3, caspase-9, phospho-p38MAPK, NF-κB and TNF-α, IL-1 and IL-6 significantly increased in model group cells(P<0.01), and the relative expression of p53 was significantly down-regulated(P<0.01) in model group cells.After treated with 50 μmol·L-1 quercetin, isoquercitrin, hyperoside, quercetin-3′-O-glucoside, gossypetin-8-O-glucuronide, the content of proteins and inflammatory factors were close to normal group.Conclusion The five flavonoids in Abelmoschus manihot(L.) Medicus can inhibit AGEs/RAGE/p38MAPK/NF-κB signaling pathway in GLUTag cells.