南蛇藤提取物调控Lgr5/Wnt/β-catenin信号通路逆转胃癌前病变的机制  

M echanism of Reversal of Precancerous Lesions of Gastric Cancer byCelastrus orbiculatus Extract via Regulating Lgr5/W nt/β-catenin Signaling Pathway

作　　者：张效泽 朱方圆 文俊凇 刘延庆 朱耀东 ZHANG Xiaoze;ZHU Fangyuan;W EN Junsong;LIU Yanqing;ZHU Yaodong(The First Affiliated Hospital of Anhui Medical University,Hefei 230000,China;Yangzhou University,Yangzhou 225000,China)

机构地区：[1]安徽医科大学第一附属医院,合肥230000 [2]扬州大学,江苏扬州225000

出　　处：《中国实验方剂学杂志》2024年第12期70-77,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金面上项目(82274355);安徽省自然科学基金面上项目(2208085MH278);安徽高校自然科学研究项目(KJ2020A0215)。

摘　　要：目的:基于胃类器官损伤模型探究南蛇藤提取物(COE)调控富含亮氨酸重复序列的G蛋白偶联受体5(Lgr5)/配体分泌介质(Wnt)/β-连环蛋白(β-catenin)信号通路影响胃类器官增殖分化及Lgr5表达从而逆转胃癌前病变(PLGC)的作用机制。方法:通过小鼠胃腺体干细胞建立胃类器官,使用N-甲基-N’-硝基-N-亚硝基胍(MNNG,0.02 mg·L^(-1))处理胃类器官构建胃类器官损伤模型;将胃类器官损伤模型随机分为正常组、模型组(0.02 mg·L^(-1)MNNG)、COE低、中、高质量浓度组(5、10、20 mg·L^(-1))、Wnt抑制剂Dickkopf相关蛋白1(DKK1)(0.5 mg·L^(-1))组并分别用不同药物处理24 h;镜下观察不同药物浓度胃类器官的数量与体积;采用噻唑蓝(MTT)比色法检测胃类器官损伤模型活力;采用苏木素-伊红(HE)染色法观察胃类器官形态与病理学;采用蛋白免疫印迹法(Western blot)检测不同药物浓度胃类器官中Lgr5、黏蛋白2(MUC2)、黏蛋白5AC(MUC5AC)、黏蛋白6(MUC6)、Wnt和β-catenin表达情况。结果:与正常组比较,模型组胃类器官数量显著减少(P<0.01),体积显著减小(P<0.01),活力显著降低(P<0.01),Lgr5、MUC2、Wnt与β-catenin表达显著升高(P<0.01),MUC5AC、MUC6表达显著降低(P<0.01)。与模型组比较,COE低、中、高质量浓度组胃类器官数量及体积均显著升高(P<0.01);胃类器官活力显著增强(P<0.01),且在COE质量浓度为20 mg·L^(-1)时效果最显著(P<0.01);COE中、高质量浓度组Lgr5、MUC2表达显著降低(P<0.01),COE低、中、高质量浓度组MUC5AC、MUC6的表达明显升高(P<0.05,P<0.01)。与模型组比较,Wnt抑制剂能够明显促进胃类器官MUC5AC、MUC6的表达(P<0.05,P<0.01),显著降低MUC2、Wnt与β-catenin的表达(P<0.01),COE高质量浓度与Wnt抑制剂共同使用能够促进这一趋势(P<0.01)。结论:COE通过抑制Lgr5、MUC2、Wnt、β-catenin的表达和促进MUC5AC及MUC6的表达,抑制Wnt/β-catenin通路,促进胃类器官增殖分化,逆转PLGC过Objective::This study aims to investigate the mechanism in which Celastrus orbiculatusextract(COE)affects the proliferation and differentiation of gastric organoids and the expression of Lgr5 and thus reverses the precancerous lesions of gastric cancer(PLGC)by regulating the leucine-rich repeat containing G protein-coupled receptor 5(Lgr5)/Wingless(W nt)/β-catenin signaling pathway based on a gastric organoid injury model.M ethod::Gastric organoids were established based on stem cells of the mouse gastric gland.Gastric organoid injury models were constructed by treating gastric organoids with N-methyl-N'-nitro-N-nitrosoguanidine(MNNG,0.02 mg·L^(-1)).Gastric organoid injury models were randomly divided into normal group,model group(0.02 mg·L^(-1) MNNG),low,medium,and high dose(5,10,20 mg·L^(-1))groups of COE,and W nt inhibitor Dickkopf-related protein 1(DKK1)(0.5 mg·L^(-1))group,and they were treated with respective agents for 24 h.The number and volume of gastric organoids under different drug concentrations were observed under a microscope.The viability of the gastric organoid injury models was detected by Methyl thiazolyl tetrazolium(MTT)assay.The morphology and pathology of gastric organoids were observed using Hematoxylin and Eosin(HE)staining.The expression levels of Lgr5,Mucin2(MUC2),Mucin5AC(MUC5AC),Mucin6(MUC6),W nt,andβ-catenin in gastric organoids under different drug concentrations were detected by Western blot(W B).Result::Compared with the normal group,the number,volume,and activity of gastric organoids in the model group were decreased(P<0.01),while the expressions of Lgr5,MUC2,W nt,andβ-catenin were significantly increased(P<0.01).The expressions of MUC5AC and MUC6 were significantly decreased(P<0.01).Compared with the model group,the number and volume of gastric organoids in the low,medium,and high dose groups of COE were all improved(P<0.01),and the vitality of gastric organoids was significantly enhanced(P<0.01).The effect was the most significant at a COE concentration of 20 mg·L^(-1)(P

关 键 词：南蛇藤提取物 胃癌前病变 胃类器官损伤模型 富含亮氨酸重复序列的G蛋白偶联受体5(Lgr5)/配体分泌介质(Wnt)/β-连环蛋白(β-catenin)信号通路 

分 类 号：R2-0[医药卫生—中医学] R22R242R285.5