基于网络药理学、分子对接和实验验证探讨蛇床子素治疗结直肠癌的作用机制  

Mechanism of osthole against colorectal cancer based on network pharmacology,molecular docking,and experimental validation

作　　者：蒋子寅 韩昌鹏[1] JIANG Zi-yin;HAN Chang-peng(Yueyang Hospital of Integrated Traditional Chinese and Western Medicine,Shanghai University of Traditional Chinese Medicine,Shanghai 200437,China)

机构地区：[1]上海中医药大学附属岳阳中西医结合医院,上海200437

出　　处：《中国中药杂志》2024年第21期5752-5761,共10页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(81874468,82174393)。

摘　　要：通过体内外实验,结合网络药理学及分子对接技术,探究蛇床子素治疗结直肠癌(CRC)的作用机制。检索Swiss-TargetPrediction、SuperPred药物数据库及GeneCards、OMIM疾病数据库,分别获取蛇床子素(osthole)和CRC的相关靶点;利用STRING数据库及Cytoscape 3.8.0软件构建蛋白-蛋白相互作用(PPI)网络并筛选出核心靶点。对共同靶点进行基因本体(GO)和京都基因与基因组百科全书(KEGG)富集分析。利用AutoDock Vina软件,选取核心靶点与蛇床子素进行分子对接验证。分别设置不同浓度蛇床子素组处理HCT116细胞,采用cell counting kit-8(CCK-8)法及细胞克隆形成法检测细胞增殖能力,Transwell实验检测细胞迁移能力,Western blot、RT-qPCR检测半胱天冬蛋白酶3(CASP3)、缺氧诱导因子1A(HIF1A)、核因子κB亚基1(NFKB1)、糖原合成酶激酶-3β(GSK3B)、磷酸化GSK3B(p-GSK3B)、蛋白激酶B(Akt)、磷酸化Akt(p-Akt)、哺乳动物雷帕霉素靶蛋白(mTOR)、磷酸化mTOR(p-mTOR)的表达情况。构建HCT116细胞裸鼠皮下瘤模型,将裸鼠随机分为模型组,蛇床子素低(20 mg·kg^(-1))、中(40 mg·kg^(-1))、高(60 mg·kg^(-1))剂量组,给药18 d后观察裸鼠瘤体的生长情况,取材后测量瘤体大小和质量,并用苏木素-伊红(HE)染色观察各组肿瘤的组织形态学改变。网络药理学分析表明,蛇床子素治疗CRC主要涉及106个治疗靶点和113条治疗通路,其中关键通路为PI3K/Akt信号通路、MAPK信号通路等。分子对接结果显示,蛇床子素与核心靶点具有较强的关联性。体外研究表明,蛇床子素能显著抑制HCT116细胞的增殖和迁移能力;Western blot和RT-qPCR实验表明,与模型组相比,蛇床子素给药组的NFKB1、HIF1A、p-Akt、p-mTOR、GSK3B表达均显著降低,CASP3、p-GSK3B(Ser9)表达均显著上升。体内研究表明,与模型组相比,蛇床子素能够明显减少肿瘤的质量与体积,抑制肿瘤的生长,促进肿瘤的凋亡,且结果呈剂量依赖性趋Through in vitro and in vivo experiments,combined with network pharmacology and molecular docking techniques,this study investigated the mechanism of action of osthole in the treatment of colorectal cancer(CRC).The relevant targets of osthole and CRC were retrieved from the SwissTargetPrediction and SuperPred in drug databases,as well as GeneCards and OMIM in disease databases.Protein-protein interaction(PPI)networks were constructed using the STRING database and Cytoscape 3.8.0 software,and core targets were screened.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses were performed on common targets.Molecular docking validation of core targets with osthole was conducted using AutoDock Vina software.HCT116 cells were treated with different concentrations of osthole,and cell proliferation was detected using the CCK-8 assay and the clonogenic assay.Cell migration ability was assessed using Transwell assay.Western blot and RT-qPCR were performed to detect the expression of caspase-3(CASP3),hypoxia-inducible factor 1 alpha(HIF1A),nuclear factor kappa B subunit 1(NFKB1),glycogen synthase kinase-3 beta(GSK3B),phosphorylated-GSK3B(p-GSK3B),protein kinase B(Akt),phosphorylated-Akt(p-Akt),mammalian target of rapamycin(mTOR),and phosphorylated-mTOR(p-mTOR).A subcutaneous tumor model of HCT116 cells in nude mice was established,and the mice were randomly divided into the model group,low-dose osthole group(20 mg·kg^(-1)),medium-dose osthole group(40 mg·kg^(-1)),and high-dose osthole group(60 mg·kg^(-1)).After 18 days of administration,the growth of tumor xenografts was observed,and the size and weight of tumors were measured after excision.Hematoxylin-eosin(HE)staining was performed to observe the histological changes in tumors in each group.Network pharmacology analysis revealed that osthole treatment of CRC mainly involved 106 treatment targets and 113 treatment pathways,with key pathways including the PI3K/Akt signaling pathway and MAPK signaling pathway.Molecular docking results show

关 键 词：蛇床子素 结直肠癌 网络药理学 细胞凋亡 PI3K/AKT信号通路 裸鼠成瘤 

分 类 号：R285[医药卫生—中药学]