GEO基因芯片分析结合网络药理学及分子对接技术探析“黄连-木香-肉豆蔻”组方治疗溃疡性结肠炎的分子机制

Exploration of molecular mechanism of Huanglian-Muxiang-Roudoukou formula in treating ulcerative colitis based on gene expression omnibus chip data mining combined with network pharmacology and molecular docking

背景“黄连-木香-肉豆蔻”(Huanglian-Muxiang-Roudoukou,HMR)组方出自《小儿药证直诀》,能改善溃疡性结肠炎(ulcerative colitis,UC)临床症状,采用网络药理学等方法研究其具体的生物学作用机制有助于更好地挖掘中医药资源,促进中药防治UC的现代化开发与应用.目的通过基因表达综合数据库(gene expression omnibus,GEO)中的基因芯片分析结合网络药理学及分子对接方法探究HMR治疗UC的潜在分子机制.方法采用GEO数据库获取UC芯片数据,运用R语言综合分析筛选出疾病差异表达基因,获得UC的疾病靶标数据库;利用中药系统药理学数据库与分析平台(traditional Chinese medicine systems pharmacology database and analysis platform,TCMSP)分别检索HMR的活性成分及其作用靶点;取疾病差异表达基因与药物作用靶点间的交集基因,通过Cytoscape 3.9.1软件构建“中药-活性成分-疾病-靶点”网络和蛋白互作(protein-protein interaction,PPI)网络并进行拓扑分析,以此筛选出主要活性成分及核心靶点;同时利用Metascapes数据库进行基因本体(gene ontology,GO)富集分析和京都基因与基因组百科全书通路(kyoto encyclopedia of genes and genomes,KEGG)富集分析;最后使用AutoDock vina软件对主要活性成分和核心靶点进行分子对接.结果共筛选得到UC差异表达基因967个,HMR活性成分29种,对应的靶点163个.“中药-活性成分-疾病-靶点”网络中有活性成分24个,主要活性成分涉及槲皮素、豆甾醇、小檗碱、β-谷甾醇、巴马汀等;PPI网络中有蛋白26个,核心靶点涉及白介素1β(interleukin-1β,IL-1β)、白介素6(interleukin-6,IL-6)、CC趋化因子配体2[chemokine(C-C motif)ligand 2,CCL2]、肿瘤坏死因子(tumor necrosis factor,TNF)、基质金属蛋白酶9(matrix metallopeptidase 9,MMP9)等;GO富集分析主要涉及脂多糖刺激变化等生物过程,质膜外侧面等细胞成分,细胞因子活性等分子功能;KEGG通路分析主要涉及TNF信号通路、核转录因子κB(nuclear factor kappa-B,NF-κB)通路、Toll样受体(toll-like receptor,TLR)信号通路等;运用分子对接模拟证实排名前5的主要活性成分与核心靶点之间均具有较强的结合活性.结论HMR可能是通过槲皮素、豆甾醇及小檗碱等多成分,作用于IL-1β、IL-6、CCL2等多靶点,调节TNF信号通路、NF-κB信号通路、TLR信号通路等多条信号通路,从抗炎和调节肠道免疫等方面发挥对UC的治疗作用.

BACKGROUND Huanglian-Muxiang-Roudoukou(HMR)formula was recorded in the book of“Pediatric Medicine Prescription”.It can improve the clinical symptoms of ulcerative colitis(UC).Using network pharmacology and other methods to study its biological mechanism is helpful to better explore the resources of traditional Chinese medicine and promote the modern development and application of traditional Chinese medicine in the prevention and treatment of UC.AIM To explore the potential molecular mechanism of HMR in the treatment of UC based on gene expression omnibus(GEO)chip data mining,network pharmacology,and molecular docking technology.METHODS The GEO database was used to obtain UC gene expression data.Then,differentially expressed genes were identified using R software.The active components of Myristicae Semen(Roudoukou),Aucklandiae Radix(Muxiang),and Coptidis Rhizoma(Huanglian)were searched by using the traditional Chinese medicine systems pharmacology database and analysis platform(TCMSP)database,and the corresponding targets were identified.The intersections of HMR and ulcerative colitis disease targets were obtained.The“drug-active ingredients-disease-target”network and protein-protein interaction(PPI)network were constructed to screen out the core components and targets using Cytoscape 3.9.1 software.Gene ontology(GO)and Kyoto encyclopedia of genes and genomes(KEGG)enrichment analyses were performed for the intersection targets by Metascapes database.Molecular docking between main active components and core targets was performed using AutoDock vina software.RESULTS A total of 967 differential genes were identified,and there were 29 active components and 163 active targets in UC.There were 24 active components in the“drug-active ingredients-disease-target”network,including quercetin,stigmasterol,berberine,beta-sitosterol,palmatine,and so on.There were 26 proteins in the PPI network,including interleukin-1β(IL-1β),interleukin-6(IL-6),C-C motif chemokine 2(CCL2),tumor necrosis factor(TNF),matrix metallopeptidase 9(MMP9),and so on.GO enrichment analysis demonstrated that the intersection targets were mainly concentrated in biological processes such as lipopolysaccharide response,in cellular components such as external side of plasma membrane,and in molecular functions such as cytokine activity.KEGG analysis demonstrated that the intersection targets mainly involved the TNF signal pathway,nuclear factor kappa-B(NF-κB)pathway,Toll-like receptor(TLR)signal pathway,and so on.The results of molecular docking showed that the top five main active components had strong binding ability with the core targets IL-1β,IL-6,CCL2,TNF,and MMP9.CONCLUSION HMR may act on the expression of IL-1β,IL-6,CCL2,and other proteins through quercetin,stigmasterol,berberine,and other active components,regulate the TNF signaling pathway,and play a role in the treatment of UC via many mechanisms such as anti-inflammatory and intestinal immunity regulation.