基于网络药理学探究滋补肝肾与益气养血中药配伍干预脊髓损伤的分子作用机制

Exploring Molecular Mechanism of Action of Traditional Chinese Medicine Combination of Invigorating Liver and Kidney and Supplementing Qi and Nourishing Blood for Intervention of Spinal Cord Injury Based on Network Pharmacology

目的基于网络药理学探究滋补肝肾与益气养血中药配伍干预脊髓损伤的分子作用机制。方法基于中药系统药理学数据库及分析平台获取当归、黄芪、枸杞、熟地的有效活性成分,采用TCMID及Uniprot数据库进行靶点预测;检索多个常用疾病数据库及文献补充获得脊髓损伤相关靶基因并根据相关度进行筛选;利用jvenn平台将两组靶基因进行映射并绘制韦恩图;利用String数据库构建蛋白质-蛋白质相互作用网络并将分析文件导入Cytoscape 3.7.2软件,根据自由度进行筛选,得到中药治疗脊髓损伤的核心作用靶基因。将核心靶点输入David 6.8数据库中进行基因本体(GO)与京都基因和基因组百科全书(KEGG)信号通路富集分析。结果筛选出当归、黄芪、枸杞、熟地的有效活性成分65个(包括槲皮素、豆甾醇、β-谷甾醇、山柰酚等);对应靶基因148个,与脊髓损伤相关靶基因420个,映射出关键靶点32个,平均节点度15.8,自由度≥19的核心靶点14个,包括白细胞介素(IL)-6、肿瘤坏死因子(TNF)-α、血管内皮生长因子A(VEGFA)、IL-1β、CC趋化因子配体2、前列腺素过氧化物合酶2、表皮细胞生长因子、促分裂原活化的蛋白激酶1、髓过氧化物酶、肿瘤蛋白53、一氧化氮合酶3、表皮细胞生长因子受体、基质金属蛋白酶2、过氧化物酶体增殖物激活受体γ;分析得到GO生物过程98个,KEGG信号通路68条,按照P值升序排列后,分别筛选出符合条件的20项GO生物学过程(包括细胞迁移的正调控、蛋白激酶B信号转导的正调控、细胞因子活性等)和KEGG信号通路[包括缺氧诱导因子-1信号通路、恰加斯病(美洲锥虫病)、膀胱癌、癌症途径等]。结论滋补肝肾与益气养血中药配伍通过IL-6、TNF、VEGFA、IL-1β、CC趋化因子配体2等核心靶点协同作用于多条信号通路发挥改善炎症反应、降低氧化应激水平、调控血管生成、抑制细胞凋亡等作用,从而促进脊髓损伤修复。

Objective To explore the molecular action of mechanism of the traditional Chinese medicine(TCM)combination of invigorating liver and kidney and supplementing qi and nourishing blood in the intervention of spinal cord injury based on network pharmacology.Methods The effective active ingredients of Angelica sinensis,Astragalus membranaceus,Lycium barbarum and Radix Rehmanniae were obtained based on traditional Chinese medicine systems pharmacology database and analysis platform,the TCMID and UniProt databases were used for target prediction;target genes related to spinal cord injury were obtained by using several common disease databases and literature supplement and screened according to the correlation degree;and two groups of target genes were mapped using jvenn platform and Venn map was made;protein-protein interaction network diagram was constructed by using String database,and the analysis file was imported into the software of Cytoscape 3.7.2,and according to the degree of freedom,the core target genes of TCM in treating spinal cord injury were obtained.The core targets were input into the database David 6.8 for gene ontology(GO)analysis and Kyoto encyclopedia of genes and genomes(KEGG)signal pathway enrichment analysis.Results 65 active ingredients(including quercetin,stigmasterol,β-sitosterol,kaempferol,etc.)from the four herbs were screened out;148 corresponding target genes,420 target genes related to spinal cord injury,32 key targets mapped,with an average node degree of 15.8,and 14 core targets with a free degree≥19,including interleukin(IL)-6,tumor necrosis factor(TNF)-α,vascular endothelial growth factor A(VEGFA),IL-1β,chemokine(C-C motif)ligand 2,prostaglandin peroxidase synthase 2,epidermal growth factor,mitogen-activated protein kinase 1,myeloperoxidase,tumor protein p53,nitric oxide synthase 3,epidermal growth factor receptor,matrix metalloproteinase-2,peroxisome proliferative-activated receptorγ;through analysis,98 GO biological processes and 68 KEGG signal pathways were obtained,and 20 GO biological processes(including positive regulation of cell migration,positive regulation of protein kinase B signal transduction,and cytokine activityetc.)and KEGG signal pathways[including hypoxia inducible factor-1 signal pathway,Chagas disease(trypanosomiasis americana),bladder cancer,cancer pathway,etc.]were screened out according to the ascending order of P value.Conclusion The combination of TCM for invigorating liver and kidney and supplementing qi and nourishing blood plays a synergistic role in multiple signal pathways through IL-6,TNF,VEGFA,IL-1β,chemokine(C-C motif ligand 2)and other core targets,to improve inflammatory response,reduce oxidative stress,regulate angiogenesis,and inhibit apoptosis,thus promote the repair of spinal cord injury.