基于Wnt信号通路研究黄芩汤干预炎症微环境下结肠癌细胞上皮间质转化与细胞周期进程的作用机制  

Mechanism of Huangqin Decoction on epithelial-mesenchymal transition and cell cycle of colon cancer cells under inflammatory conditions based on Wnt signaling pathway

作　　者：马旭冉 王敦方 冯雪 刘雅清 刘滨 杨伟鹏[2] MA Xu-ran;WANG Dun-fang;FENG Xue;LIU Ya-qing;LIU Bin;YANG Wei-peng(Institute of Pharmaceutical Research,Shandong University of Traditional Chinese Medicine,Jinan 250355,China;Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China;School of Basic Medicine,Heilongjiang University of Chinese Medicine,Harbin 150006,China)

机构地区：[1]山东中医药大学药物研究院,山东济南250355 [2]中国中医科学院中药研究所,北京100700 [3]黑龙江中医药大学基础医学院,黑龙江哈尔滨150006

出　　处：《中草药》2023年第7期2155-2162,共8页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金面上项目(81473592);国家自然科学基金面上项目(82074328);中国中医科学院科技创新工程项目(CI2021B015,CI2021A04604)。

摘　　要：目的研究黄芩汤对炎症微环境下结肠癌HT-29细胞上皮间质转化与细胞周期进程的影响。方法使用脂多糖(lipopolysaccharide,LPS)诱导淋巴瘤THP-1细胞,取细胞上清液作为条件培养基,将条件培养基加入HT-29细胞培养体系中构建炎症微环境下结肠癌细胞模型,给予黄芩汤(500、250、125μg/mL)干预后,Transwell法检测细胞迁移与侵袭能力;Western blotting检测海马无翅基因3a(recombinant wingless type MMTV integration site family member 3a,Wnt3a)、β-连环蛋白(β-catenin)、E-cadherin、糖原合酶激酶-3β(glycogen synthasc kinase-3β,GSK-3β)、细胞周期蛋白依赖激酶4(cyclin dependent kinase 4,CDK4)和细胞周期蛋白D1(cyclin D1)的蛋白表达;qRT-PCR检测Wnt3a、β-catenin、E-cadherin、GSK-3β、CDK4和cyclin D1的m RNA表达。结果与对照组比较,黄芩汤组迁移与侵袭的细胞数目均显著减少(P<0.01),细胞中Ecadherin、GSK-3β蛋白和m RNA表达水平均显著升高(P<0.01),CDK4、cyclin D1、Wnt3a、β-catenin蛋白和m RNA表达水平均显著降低(P<0.01),呈剂量相关性。结论黄芩汤能够有效抑制炎症微环境下结肠癌细胞的上皮间质转化异常,其作用机制可能与降低经典Wnt通路中Wnt3a、β-catenin表达,同时促进E-cadherin、GSK-3β表达有关。黄芩汤还可通过有效抑制CDK4、cyclin D1的表达来修复周期检查点,从而调节炎症微环境下结肠癌细胞的细胞周期紊乱。Objective To study the effect of Huangqin Decoction(黄芩汤)on epithelial-mesenchymal transition and cell cycle process of colon cancer HT-29 cells in inflammatory microenvironment.Methods Lipopolysaccharide(LPS)was used to induce lymphoma THP-1 cells,and cell supernatant was used as conditioned medium.The conditioned medium was added to HT-29 cell culture system to construct colon cancer cell model in inflammatory microenvironment.After intervention with Huangqin Decoction(500,250,125μg/mL),cell migration and invasion ability were detected by Transwell method.Western blotting was used to detect recombinant wingless type MMTV integration site family member 3a(Wnt3a),β-catenin,E-cadherin,glycogen synthase kinase-3β(GSK-3β),cyclin-dependent kinase 4(CDK4)and cyclin D1 protein expressions;qRT-PCR was used to detect Wnt3a,β-catenin,E-cadherin,GSK-3β,CDK4 and cyclin D1 mRNA expressions.Results Compared with control group,number of migrating and invading cells in Huangqin Decoction group were significantly decreased(P<0.01),E-cadherin,GSK-3βprotein and mRNA expression levels were significantly increased(P<0.01),while CDK4,cyclin D1,Wnt3a,β-catenin protein and mRNA expression levels were significantly decreased in a dose-dependent manner(P<0.01).Conclusion Huangqin Decoction can effectively inhibit the abnormal epithelial-mesenchymal transition of colon cancer cells in inflammatory microenvironment,and its mechanism may be related to reducing the expressions of Wnt3a andβ-catenin in classical Wnt pathway and promoting the expressions of E-cadherin and GSK-3β.Huangqin Decoction can also repair the periodic checkpoint by effectively inhibiting the expressions of CDK4 and cyclin D1,thus regulating the cell cycle disorder of colon cancer cells in inflammatory microenvironment.

关 键 词：黄芩汤 结肠癌 炎症微环境 上皮间质转化 细胞周期 Wnt信号通路 黄芩苷 汉黄芩苷 千层纸素A苷 甘草素二糖苷 

分 类 号：R285.5[医药卫生—中药学]