基于网络药理学和细胞实验探讨陈皮-紫苏子药对治疗慢性阻塞性肺疾病的作用机制

Mechanism of Paired Drugs of Dried Tangerine Peel and Purple Perilla Seed in Treatment of COPD Based on Network Pharmacology and Cell Experiment

目的:基于网络药理学和细胞实验探讨陈皮-紫苏子药对治疗慢性阻塞性肺疾病(chronic obstructive pulmonary disease,COPD)的作用机制。方法:通过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)检索陈皮、紫苏子的化学成分与作用靶点。利用Cytoscape 3.7.2软件构建“药物-活性成分-作用靶点”网络,将度值排名前5位的化合物作为陈皮-紫苏子药对的核心成分。通过GeneCards数据库检索COPD相关靶点,借助微生信平台获取药物与COPD的交集靶点。将交集靶点上传至STRING数据库,构建蛋白质-蛋白质相互作用(protein-protein interaction,PPI)网络,进而筛选核心靶点。通过Metascape数据库进行基因本体(Gene Ontology,GO)和京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)分析(P<0.05)。最后,利用Autodock软件进行分子对接验证。THP-1细胞分为空白组、模型组、木犀草素组、β-谷甾醇组、柚皮素组和川陈皮素组。除空白组外,其余各组加入脂多糖(2 mg·L^(-1))建立体外细胞炎症模型;在此基础上,各药物组加入相应剂量的药物进行培养。培养24 h后,ELISA检测各组细胞白细胞介素-6(interleukin-6,IL-6)、肿瘤坏死因子-α(tumor necrosis factorα,TNF-α)含量。结果:陈皮活性成分5个(谷甾醇、柚皮素、川陈皮素等),作用靶点91个;紫苏子活性成分14个(豆甾醇、花生四烯酸、木犀草素等),作用靶点223个。拓扑分析显示,陈皮-紫苏子药对的核心成分可能是木犀草素、β-谷甾醇、花生四烯酸、柚皮素、川陈皮素。COPD相关靶点288个,药物与COPD的交集靶点34个。根据PPI网络筛选得到16个核心靶点,包括IL-6、TNF、血管内皮生长因子A(vascular endothelial growth factor A,VEGFA)等。KEGG分析得到13条通路,主要包括白细胞介素-17(interleukin-17,IL-17)信号通路、细胞因子受体互作通路、内吞与铁死亡等。分子对接结果显示,核心成分与核心靶点(IL-6、TNF)的结合能均小于-4.25 kcal·mol^(-1),说明两者具有较好的结合活性。细胞实验结果显示,与正常组比较,模型组IL-6、TNF-α含量升高(P<0.01);与模型组比较,各浓度柚皮素组、川陈皮素组、β-谷甾醇组、木犀草素组、花生四烯酸组的IL-6、TNF-α含量降低(P<0.01,P<0.05)。结论:陈皮-紫苏子药对可通过多成分、多靶点、多途径发挥治疗COPD的作用,其机制可能与抑制炎症反应有关。

Objective:Exploring the mechanism of paired drugs of Chenpi(dried tangerine peel)and Zisuzi(purple perilla seed)in the treatment of chronic obstructive pulmonary disease(COPD)based on network pharmacology and cell experiment.Method:The chemical composition and target of action of the above-mentioned drug pair were retrieved through the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP).A network of"active ingredients and target points of action"for drugs was constructed by using Cytoscape 3.7.2 software,in which the top 5 compounds with degree values was considered as the core components of the drug pair.Retrieve COPD related targets through the GeneCards database and obtain intersection targets between drugs and COPD through the microbiome platform.Upload the intersection targets to the STRING database and construct a protein-protein interaction(PPI)network to screen for core targets.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis were conducted using the Metascape database(P<0.05).Finally,molecular docking validation was performed using Autodock software.THP-1 cells were divided into blank group,model group,luteolin group,β-sitosterol group,naringenin group and nobiletin group.Except for the blank group,all other groups were added with lipopolysaccharide(2μg·mL^(-1))to establish an in vitro cellular inflammation model;On that basis,each drug group was added with corresponding doses of drugs for cultivation.After 24 hours of cultivation,ELISA was used to detect content of interleukin-6(IL-6)and tumor necrosis factorα(TNF-α)in each group of cells.Results:There are 5 active ingredients in Chenpi(such as sitosterol,naringenin,and nobiletin,etc.)with 91 targets of action;There are 14 active ingredients in Zisuzi(such as stigmasterol,arachidonic acid,luteolin,etc.),with 223 targets of action.Topological analysis shows that the core component of the drug pair may be luteolin,β-sitosterol,arachidonic acid,naringenin and nobiletin.There are 288 COPD related targets and 34 intersection targets between drugs and COPD.According to the PPI network screening,16 core targets were identified,including IL-6,TNF,vascular endothelial growth factor A(VEGFA),etc.KEGG analysis identified 13 pathways,mainly including the IL-17 signaling pathway,cytokine receptor interaction pathway,endocytosis and ferroptosis.The molecular docking results showed that the binding energies between the core components and the core target were both less than-4.25 kcal·mol^(-1),indicating good binding activity between the two.The cell experiment results showed that compared with the normal group,IL-6 and TNF-αlevels went up in model group(P<0.01);Compared with the model group,IL-6 and TNF content was decreased in different concentrations of naringenin group,nobiletin group,β-sitosterol group,luteolin group,arachidonic acid group(P<0.01,P<0.05).Conclusion:the paired drugs of Chenpi(dried tangerine peel)and Zisuzi(purple perilla seed)have a therapeutic effect in the treatment of COPD through multiple components,targets and pathways,which mechanism may be related to the inhibition of inflammatory response.