仁青芒觉对MNNG诱导的胃黏膜上皮细胞恶性转化的干预作用及机制  

Interventional Effect and Mechanisms of Renqing Mangjue on MNNG-induced Malignant Transformation of Gastric Mucosal Epithelial Cells

作　　者：陈沛萍 黄凤玉 张昕卓 孔祥英[2] 肖自青 李彦希 苏晓慧[2] 林娜[2] CHEN Peiping;HUANG Fengyu;ZHANG Xinzhuo;KONG Xiangying;XIAO Ziqing;LI Yanxi;SU Xiaohui;LIN Na(Guangzhou University of Chinese Medicine,Guangzhou 510006,China;Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China;Arura Tibetan Medicine Co.Ltd.,Xining 810003,China)

机构地区：[1]广州中医药大学,广州510006 [2]中国中医科学院中药研究所,北京100700 [3]金诃藏药股份有限公司,西宁810003

出　　处：《中国实验方剂学杂志》2025年第8期69-77,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：青海省中央引导地方科技发展资金项目(2023ZY025);中国中医科学院中药研究所技术研发项目(20230301);国家“重大新药创制”科技重大专项(2019ZX09731-002)。

摘　　要：目的:观察仁青芒觉对N-甲基-N′-硝基-N-亚硝基胍(MNNG)诱导的胃黏膜上皮细胞恶性转化的干预作用,并基于环磷酸鸟苷(cGMP)/蛋白激酶G(PKG)/丝裂原活化蛋白激酶(MEK)/细胞外信号调节激酶(ERK)信号通路探讨其预防胃癌癌前病变的分子机制。方法:首先采用MNNG诱导人胃黏膜上皮细胞GES-1恶性转化,建立胃癌前病变细胞模型(MC细胞);通过细胞增殖活性检测(CCK-8)试剂盒法筛选MNNG诱导GES-1细胞恶性转化的有效浓度,并摸索仁青芒觉抑制恶性转化的GES-1细胞增殖的有效浓度;将GES-1细胞分为空白组、模型组和仁青芒觉1、3、10、30 mg·L^(-1)组;其次通过Transwell迁移实验、细胞划痕实验、实时荧光定量聚合酶链式反应(Real-time PCR)评估仁青芒觉对GES-1细胞恶转细胞迁移能力及上皮-间充质转化(EMT)特性的影响;通过中药整合药理学计算平台(TCMIP)v2.0数据库获得仁青芒觉的化学成分候选靶点,利用美国国立生物技术信息中心基因表达(GEO)数据库胃癌癌前病变各阶段的疾病靶点,基于Metascape数据库进行通路富集分析,预测仁青芒觉的潜在作用机制;最后通过蛋白免疫印迹法(Western blot)验证仁青芒觉预防胃癌癌前病变的作用机制。结果:MNNG浓度为20μmol·L^(-1)时对GES-1细胞的抑制率约为50%(P<0.01),且在此浓度下GES-1细胞体现出恶性转化的生物学特性;仁青芒觉对正常GES-1细胞增殖影响差异无统计学意义,但能显著抑制MC细胞的增殖(P<0.01),并显著减弱MC细胞的迁移能力(P<0.01),升高GES-1细胞的EMT进程中E钙黏蛋白的mRNA表达水平(P<0.05),抑制N钙黏蛋白及转录因子Snail mRNA的表达(P<0.05,P<0.01);网络预测结果显示,仁青芒觉可能通过调节cGMP/PKG及MAPK/ERK相关信号通路,预防胃癌癌前病变;Western blot结果显示,仁青芒觉能显著上调cGMP/PKG信号通路中PKG、B型利钠肽受体(NPRB)蛋白表达(P<0.01),明显下调其下游基因MEK、ERK蛋�Objective:This study aimed to investigate the intervention effect of Renqing Mangjue on the malignant transformation of gastric mucosal epithelial cells induced by N-methyl-N′-nitro-N-nitrosoguanidine(MNNG)and to explore its molecular mechanism in preventing precancerous lesions of gastric cancer based on the cyclic guanosine monophosphate(cGMP)/protein kinase G(PKG)/mitogen-activated protein kinase(MEK)/extracellular signal-regulated kinase(ERK)signaling pathway.Methods:Human gastric mucosal epithelial cells(GES-1)were initially induced by MNNG to establish a precancerous cell model(MC cells).The effective concentration of MNNG for inducing malignant transformation in GES-1 cells was screened using the cell proliferation activity decection(CCK-8)assay,and the effective concentration of Renqing Mangjue for inhibiting the proliferation of transformed GES-1 cells was also determined.GES-1 cells were divided into a blank control group,a model group,and treatment groups with Renqing Mangjue at concentrations of 1,3,10,and 30 mg·L^(-1).Furthermore,the effects of Renqing Mangjue on the migratory ability and epithelial-mesenchymal transition(EMT)characteristics of GES-1 malignant transformed cells were evaluated using Transwell migration assays,wound healing assays,and real-time quantitative reverse transcription polymerase chain reaction(Real-time PCR).Additionally,candidate chemical components and target sites of Renqing Mangjue were obtained from the TCMIP v2.0 database,and disease targets at various stages of gastric cancer precursors were sourced from the Gene Expression Omnibus(GEO)database.Pathway enrichment analysis was performed using the Metascape database to predict the potential mechanisms of action of Renqing Mangjue.Finally,the protective mechanism of Renqing Mangjue against gastric cancer precursors was validated through Western blot analysis.Results:At a concentration of 20μmol·L^(-1),MNNG exhibited an inhibition rate of approximately 50%on GES-1 cells(P<0.01),and at this concentration,the GES-1 cel

关 键 词：仁青芒觉 胃癌癌前病变 上皮-间充质转化(EMT) 网络药理学 环磷酸鸟苷(cGMP)/蛋白激酶G(PKG)/丝裂原活化蛋白激酶(MEK)/细胞外信号调节激酶(ERK)信号通路 

分 类 号：R259[医药卫生—中西医结合] R289[医药卫生—中医内科学] R573[医药卫生—中医学]