基于Keap1/Nrf2/ARE信号通路探讨平胃胶囊对胃黏膜上皮细胞恶变的影响及其机制  

Effect of Pingwei Capsule on Malignant Transformation of Gastric Mucosal Epithelial Cells and Its Mechanism Based on Keap1/Nrf2/ARE Signaling Pathway

作　　者：王丽娟[1,2] 汪龙德[3] 汪霞 牛小英 樊泽坤[1,2] 李正菊 牛媛媛 毛兰芳 WANG Lijuan;WANG Longde;WANG Xia;NIU Xiaoying;FAN Zekun;LI Zhengju;NIU Yuanyuan;MAO Lanfang(Gansu University of Traditional Chinese Medicine,Lanzhou 730000,China;Key Laboratory of Dunhuang Medicine,Ministry of Education,Lanzhou 730000,China;Affiliated Hospital of Gansu University of Traditional Chinese Medicine,Lanzhou 730020,China)

机构地区：[1]甘肃中医药大学,兰州730000 [2]敦煌医学与转化教育部重点实验室,兰州730000 [3]甘肃中医药大学附属医院,兰州730020

出　　处：《中国现代应用药学》2025年第4期539-548,共10页Chinese Journal of Modern Applied Pharmacy

基　　金：国家自然科学基金项目(82160883);敦煌医学与转化教育部重点实验室开放课题(DHYX23-13)。

摘　　要：目的探讨平胃胶囊(Pingwei capsule,PWJN)含药血清对N-甲基-N'-硝基-N-亚硝基胍(N-methyl-N'-nitro-Nnitrosoguanidine,MNNG)诱导的人胃黏膜上皮细胞(human gastric mucosal epithelial cells,GES-1)损伤和Kelch样环氧氯丙烷相关蛋白1(Keap1)-核因子E2相关因子2(Nrf2)/抗氧化反应元件(ARE)通路的影响。方法用2×10^(−5)mol·L^(−1)的MNNG处理GES-1细胞,建立MC细胞模型,检测增殖细胞相关抗原(Ki67)及磷酸酯酶与张力蛋白同源物(PTEN)重组蛋白的表达,进行模型评价;细胞增殖与活性检测试剂盒(CCK-8)筛选PWJN含药血清及Nrf2抑制剂(ML385)最佳干预浓度及时间;将细胞分为6组,正常组、模型组、空白血清组、PWJN组、ML385组、PWJN+ML385组;采用RT-qPCR检测细胞Keap1、Nrf2、醌氧化还原酶(NQO1)、谷胱甘肽巯基转移酶(GST)的mRNA表达情况,Western blotting检测细胞Keap1、Nrf2、NQO1、GST的蛋白表达水平,ELISA检测细胞中PTEN浓度,免疫荧光染色法检测细胞中Nrf2和Ki67共表达水平,羧基荧光素二醋酸盐琥珀酰亚胺酯细胞增殖分析法检测PWJN对MC细胞增殖分裂的影响。结果PWJN含药血清最适合作用条件为5.8%干预48 h,ML385最适合作用条件为12.5μmol·L−1干预24 h。与正常组相比,模型组与空白血清组中Nrf2、NQO1、GST mRNA及蛋白表达水平、细胞增殖率显著升高(P<0.01),Keap1和PTEN表达水平显著降低(P<0.01);与空白血清组相比,模型组Keap1、Nrf2、NQO1、GST mRNA及蛋白表达水平,PTEN表达、细胞增殖率差异均无统计学意义;与空白血清组相比,PWJN组中Nrf2、NQO1、GST蛋白及mRNA表达水平、细胞增殖率显著降低(P<0.01),Keap1和PTEN表达水平显著升高(P<0.01);与ML385组相比,PWJN+ML385组中Nrf2、NQO1和GST mRNA及蛋白表达水平显著降低(P<0.01),Keap1和PTEN表达水平显著升高(P<0.01)。结论PWJN通过调节Keap1/Nrf2/ARE信号通路,降低Nrf2的转录活性及下游过表达因子,从而关闭Nrf2通路,实现正常的氧化-抗�OBJECTIVE To investigate the effects of Pingwei capsule(PWJN)medicated serum on N-methyl-N'-nitro-Nnitrosoguanidine(MNNG)-induced human gastric mucosal epithelial cells(GES-1)injury and Kelch-like epichlorohydrin-related protein 1(Keap1)-nuclear factor E2-related factor 2(Nrf2)/antioxidant response element(ARE)pathway.METHODS The GES-1 cells 2×10^(−5)mol·L^(−1)MNNG to establish a MC cell model.The expression of proliferating cell-associated antigen(Ki67)and phosphatase and tensin homolog(PTEN)recombinant protein was detected to model evaluate the MC model.The optimal intervention concentration and time of PWJN-containing serum and Nrf2 inhibitor(ML385)were screened by CCK-8 assay.The cells were divided into 6 groups:normal group,model group,blank serum group,PWJN group,ML385 group,PWJN+ML385 group;Keap1,Nrf2,quinone oxidoreductase(NQO1)and glutathione S-transferase(GST)were detected by RT-qPCR.The protein expression levels of Keap1,Nrf2,NQO1 and GST were detected by Western blotting.The concentration of PTEN in cells was detected by ELISA.The co-expression levels of Nrf2 and Ki67 in each group were detected by immunofluorescence staining.The effect of PWJN on the proliferation and division of MC cells was detected by carboxyfluorescein diacetate succinimidyl ester cell proliferation assay.RESULTS The most suitable condition for PWJN-containing serum was 5.8%intervention for 48 h,and the most suitable condition for ML385 was 12.5μmol·L−1 intervention for 24 h.Compared with the normal group,the expression levels of Nrf2,NQO1,GST mRNA and protein,and cell proliferation rate in the model group and the blank serum group were significantly increased(P<0.01),the expression levels of Keap1 and PTEN were significantly decreased(P<0.01).Compared with the blank serum group,there was no significant difference in the expression of Keap1,Nrf2,NQO1,GST mRNA and protein,PTEN expression and cell proliferation rate in the model group.Compared with the blank serum group,the expression levels of Nrf2,NQO1,GST protein and m

关 键 词：胃黏膜上皮细胞 平胃胶囊 Keap1/Nrf2/ARE 氧化应激 

分 类 号：R285.5[医药卫生—中药学]