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研究

To Investigate the Effect of Pingwei Capsule on Malignant Transformation of GES-1 Cells and Its Mechanism Based on Keap1/Nrf2/ARE Signaling Pathway

关键词:  胃黏膜上皮细胞(GES-1)  平胃胶囊  Keap1/Nrf2/ARE  氧化应激

Key words:  Gastric mucosal epithelial cells (GES-1)  Pingwei capsule  Keap1/Nrf2/ARE  oxidative stress

摘要:

摘要：目的 探讨平胃胶囊（PWJN）含药血清对N-甲基-N'-硝基-N-亚硝基胍（N-methyl-n'-nitro-N-nitrosoguanidine,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2×10-5mol/L的MNNG处理细胞，建立GES-1细胞损伤模型，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A：正常组、B：模型组、C：空白血清组、D：PWJN组、E：ML385组、F：PWJN+ML385组；采用实时荧光定量聚合酶链式反应（Real-time PCR）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，蛋白质印迹法（Western blot）检测细胞Keap1、Nrf2、NQO1、GST的蛋白表达水平，酶联免疫吸附测定法（ELISA）检测细胞中PTEN浓度，免疫荧光染色法（IF）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（Carboxyfluorescein Diacetate Succinimidyl Ester，CFSE）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48h，ML385最适合作用条件为12.5μmol/L干预24h；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）；模型组与空白血清组相比，Keap1、Nrf2、NQO1、GST mRNA及蛋白表达水平，PTEN表达，细胞增殖率均无显著差异（P>0.05），不具有统计学意义；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），差异具有统计学意义。结论 平胃胶囊通过调节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-抗氧化平衡可能是治疗胃癌前病变的作用机制之一。

Abstract:

ABSTRACT: OBJECTIVE To investigate the effects of Pingwei Capsule (PWJN) medicated serum on N-methyl-N' -nitro-N-nitrosoguanidine (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 GES-1 cells were treated with 2×10-5mol/L MNNG to establish GES-1 cell injury model. The expression of proliferating cell-associated antigen (Ki67) and phosphatase and tensin homolog (PTEN) recombinant protein was detected for model evaluation.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 : A:normal group, B:model group, C:blank serum group, D:PWJN group, E:ML385 group, F:PWJN+ML385 group ;Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR) was used to detect Keap1, Nrf2, quinone oxidoreductase (NQO1) and glutathione S-transferase (GST) were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). The protein expression levels of Keap1, Nrf2, NQO1 and GST were detected by Western blot. The concentration of PTEN in cells was detected by enzyme-linked immunosorbent assay (Elisa). The co-expression levels of Nrf2 and Ki67 in each group were detected by immunofluorescence staining (IF),The effect of PWJN on the proliferation and division of MC cells was detected by Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) cell proliferation assay. RESULTS The most suitable condition for PWJN-containing serum was 5.8 % intervention for 48h, and the most suitable condition for ML385 was 12.5μmol/L intervention for 24h. 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 between the model group and the blank serum group (P>0.05), which was not statistically significant ;Compared with the blank serum group, the expression levels of Nrf2, NQO1, GST protein and mRNA and cell proliferation rate in the PWJN group were significantly decreased (P<0.01), and the expression levels of Keap1 and PTEN were significantly increased (P<0.01); Compared with ML385 group, the expression levels of Nrf2, NQO1 and GST mRNA and protein in PWJN+ML385 group were significantly decreased (P<0.01), and the expression levels of Keap1 and PTEN were significantly increased (P<0.01). CONCLUSION Pingwei Capsule can reduce the transcriptional activity of Nrf2 and downstream overexpression factors by regulating Keap1/Nrf2/ARE signaling pathway, thereby closing the Nrf2 pathway and achieving normal oxidation-antioxidation balance, which may be one of the mechanisms for the treatment of gastric precancerous lesions.