基于网络药理学、分子对接和实验验证探讨加味活络效灵丹干预结直肠癌的作用机制  

Mechanism of Modified Huoluo Xiaoling Pills against colorectal cancer based on network pharmacology,molecular docking,and experimental validation

作　　者：江威 赵秋萍 黄琳 姜家旺 李明莉 陈小春 徐燕[3] 王国娟[2] 邓兰 张磊昌[2] 李志明 JIANG Wei;ZHAO Qiu-ping;HUANG Lin;JIANG Jia-wang;LI Ming-li;CHEN Xiao-chun;XU Yan;WANG Guo-juan;DENG Lan;ZHANG Lei-chang;LI Zhi-ming(School of Clinical Medicine,Jiangxi University of Chinese Medicine,Nanchang 330004,China;the Affiliated Hospital of Jiangxi University of Chinese Medicine,Nanchang 330019,China;Longhua Hospital,Shanghai University of Traditional Chinese Medicine,Shanghai 200032,China)

机构地区：[1]江西中医药大学临床医学院,江西南昌330004 [2]江西中医药大学附属医院,江西南昌330019 [3]上海中医药大学附属龙华医院,上海200032

出　　处：《中国中药杂志》2024年第13期3657-3667,共11页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(82160925);江西省教育厅科学技术研究项目(GJJ211215);第五批全国中医临床优秀人才研修项目(国中医药人教函[2022]1号);2023年度江西中医药大学校级研究生创新专项(JZYC23S46)。

摘　　要：借助网络药理学和分子对接预测加味活络效灵丹抗结直肠癌(colorectal cancer,CRC)可能的作用靶点及相关信号通路,并通过实验验证。采用中药系统药理学数据库与分析平台(TCMSP)获取加味活络效灵丹的活性成分及靶点,利用GeneCards、DrugBank、OMIM、TTD数据库获取CRC的相关靶点,Cytoscape软件构建药物-活性成分-靶点网络,STRING数据库构建蛋白-蛋白互作(PPI)网络,DAVID平台对靶点进行基因本体论(Gene Ontology,GO)功能和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路富集分析,AutoDock Vina进行分子对接。设置不同浓度加味活络效灵丹含药血清组分别处理HCT 116细胞,利用cell counting kit-8(CCK-8)检测各组HCT 116细胞增殖抑制情况,Transwell检测HCT 116细胞侵袭能力,蛋白免疫印迹法(Western blot)检测β-连环蛋白(β-catenin)、细胞周期蛋白D1(cyclinD1)、V-Myc骨髓细胞瘤病毒癌基因同源物(c-Myc),以及上皮间质转化(EMT)标志蛋白E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)、波形蛋白(vimentin)、基质金属蛋白酶2(MMP2)、基质金属蛋白酶7(MMP7)、基质金属蛋白酶9(MMP9)、转录因子TWIST的表达水平。网络药理学分析,得到加味活络效灵丹活性成分242个,CRC靶点1844个,CRC与加味活络效灵丹交集靶点127个,与CRC相关的信号通路涉及PI3K-Akt、TNF、HIF-1、IL-17、Wnt等。分子对接显示,主要活性成分与核心蛋白有较稳定的结合构象。CCK-8检测显示,加味活络效灵丹能显著抑制HCT 116细胞的增殖;Transwell检测显示,随着加味活络效灵丹含药血清浓度的增加,抑制HCT 116细胞的侵袭能力越明显;经含药血清处理后,β-catenin、cyclinD1、c-Myc、N-cadherin、vimentin、MMP2、MMP7、MMP9和TWIST蛋白表达受抑制,E-cadherin表达上调。结果表明,加味活络效灵丹通过多成分、多靶点、多途径抑制结直肠癌细胞的增殖、侵袭和转移,其机制可能与This study aims to predict the possible targets and related signaling pathways of Modified Huoluo Xiaoling Pills against colorectal cancer(CRC)by both network pharmacology and molecular docking and verify the mechanism of action by experiments.TCMSP was used to obtain the active ingredients and targets of Modified Huoluo Xiaoling Pills,and GeneCards,DrugBank,OMIM,and TTD were employed to acquire CRC-related targets.Cytoscape software was utilized to construct the drug-active ingredient-target network,and the STRING database was applied to establish the protein-protein interaction(PPI)network.DAVID platform was adopted to investigate the targets in terms of GO function and KEGG pathway enrichment analysis.Molecular docking was performed in AutoDock Vina.HCT 116 cells were intervened by different concentrations of Modified Huoluo Xiaoling Pills-containing serum,and CCK-8 was used to detect the proliferation inhibition of HCT 116 cells in each group.Transwell was employed to show the invasive abi-lity of HCT 116 cells,and Western blot was taken to reveal the expression levels ofβ-catenin,cyclinD1,c-Myc,as well as epithelial-mesenchymal transition(EMT)marker proteins E-cadherin,N-cadherin,vimentin,MMP2,MMP7,MMP9,and TWIST in HCT 116 cells.The network pharmacological analysis yielded 242 active ingredients of Modified Huoluo Xiaoling Pills,1844 CRC targets,and 127 overlapping targets of CRC and Modified Huoluo Xiaoling Pills,and the signaling pathways related to CRC involved PI3K-Akt,TNF,HIF-1,IL-17,Wnt,etc.Molecular docking showed that the key active ingredients had a stable binding conformation with the core proteins.CCK-8 indicated that Modified Huoluo Xiaoling Pills significantly inhibited the proliferation of HCT 116 cells.Transwell assay showed that with increasing concentration of Modified Huoluo Xiaoling Pills containing serum,the invasive ability of HCT 116 cells was more obviously inhibited.The expression ofβ-catenin,cyclinD1,c-Myc,N-cadherin,vimentin,MMP2,MMP7,MMP9,and TWIST proteins were suppressed,and t

关 键 词：加味活络效灵丹 结直肠癌 网络药理学 分子对接 细胞实验 上皮间质转化 

分 类 号：R285[医药卫生—中药学]