甘草泻心汤调控Keap1-Nrf2通路改善HT29细胞氧化应激损伤的机制  

Mechanisms of Gancao Xiexin Decoction on improving oxidative stress injury of HT29 cells by regulating Keap1-Nrf2 signaling pathway

作　　者：沈雁 郑春娅 倪思忆 刘勇攀 钟继红 刘英超 凌平 SHEN Yan;ZHENG Chun-ya;NI Si-yi;LIU Yong-pan;ZHONG Ji-hong;LIU Ying-chao;LING Ping(The Second Affiliated Hospital of Zhejiang Chinese Medical University,Hangzhou 310005,China;Zhejiang Chinese Medical University,Hangzhou 310053,China)

机构地区：[1]浙江中医药大学附属第二医院,浙江杭州310005 [2]浙江中医药大学,浙江杭州310053

出　　处：《时珍国医国药》2024年第14期3179-3188,共10页Lishizhen Medicine and Materia Medica Research

基　　金：浙江省自然科学基金探索项目(LY21H270007);浙江省中医药科技计划项目(2023ZL060)。

摘　　要：目的探讨甘草泻心汤(GXD)调控Keap1-Nrf2通路改善HT29细胞氧化应激(OS)损伤的机制。方法高分辨液质联用技术检测GXD中化合物。H2O2诱导HT29细胞建立OS态模型。细胞分为正常对照组、应激模型组、低和高剂量GXD组,观察存活率、凋亡率、周期分布以及凋亡相关蛋白表达,以验证GXD的保护作用。细胞分为si-NC组、si-NC+应激组、si-NC+应激+GXD组和si-Nrf2+应激+GXD组,检测ROS、MDA、SOD和T-AOC水平以及Keap1-Nrf2通路蛋白的表达和分布情况,以明确该通路在GXD减轻OS损伤中的机制。结果GXD中检测出20523个化合物,10个主要化合物。与应激模型组比较,GXD组存活率、G2期细胞比例升高(P<0.05),凋亡率降低(P<0.05),Bax和cleaved-caspase-3表达减少(P<0.05),Bcl-2表达增加(P<0.01)。与si-NC+应激组比较,si-NC+应激+GXD组的SOD、T-AOC、总Nrf2、核内Nrf2、HO-1和NQO1水平升高(P<0.01),ROS、MDA和Keap1水平降低(P<0.01);与si-NC+应激+GXD组比较,si-Nrf2+应激+GXD组上述指标均呈相反趋势变化。结论GXD通过激活Keap1-Nrf2通路信号转导,恢复氧化-抗氧化体系平衡,减轻细胞损伤凋亡,这可能是其改善OS损伤的作用机制。Objective To investigate the mechanism of Gancao Xiexin Decoction(GXD)in improving oxidative stress(OS)damage of HT29 cells by regulating Keap1-Nrf2 signaling pathway.Methods UHPLC-Q-TOF-MS high-resolution LC/MS approach was applied to identify compounds in GXD.HT29 cells were induced by H2 O2 to establish OS cell model.We divided cells into normal control group,stress model group,GXD low-and high-dose group;we assessed their survival rate,apoptosis rate,cycle distribution,and the expressions of apoptosis-related proteins,to verify the protective effect of GXD.We divided cells into si-NC group,si-NC+stress group,si-NC+stress+GXD group and si-Nrf2+stress+GXD group;we detected the levels of ROS,MDA,SOD and T-AOC,the expression and distribution of proteins in Keap1-Nrf2 signaling pathway,to clarify the mechanism of Nrf2-ARE signaling pathway in reducing OS damage of cells by GXD.Results A total of 20523 compounds were identified in GXD,including 10 main compounds.Compared with stress model group,GXD treatment increased the survival rates and the proportions of cells in G2 phase(P<0.05),as well as the expressions of Bcl-2(P<0.01),while decreased the apoptosis rates(P<0.05)and the expressions of Bax and cleaved-caspase-3(P<0.05).Compared with si-NC+stress group,the levels of SOD,T-AOC,total Nrf2,Nrf2,HO-1 and NQO1 in si-NC+stress+GXD group were increased(P<0.01),the levels of ROS,MDA and Keap1 were decreased(P<0.01).Compared with si-NC+stress+GXD group,the above indexes in the si-Nrf2+stress+GXD group showed an opposite trend.Conclusion By activating Keap1-Nrf2 pathway signal transduction,GXD restores the oxidation-antioxidant system balance and alleviates cell injury and apoptosis,which may be the mechanism of its ameliorating OS injury.

关 键 词：甘草泻心汤 溃疡性结肠炎 氧化应激 Keap1-Nrf2信号通路 HT29细胞 

分 类 号：R285.5[医药卫生—中药学]