菊花-槐花抗乙型肝炎炎症机制的网络药理学分析及实验验证

Network phamacology analysis and experimental verification of anti-hepatitis B inflammatory mechanisms of chrysanthemum-sophora japonica

目的基于网络药理学、分子对接和细胞实验探讨菊花-槐花的抗炎效果和作用机制。方法采用中药系统药理学数据库与分析平台(TCMSP)获取菊花-槐花的活性成分以及相关靶点,利用GeneCards、Drugbank、OMIM、TTD数据库获取乙型肝炎(HBV)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-蛋白相互作用(PPI)网络,筛选核心靶点,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,Auto Dock Vina进行分子对接,预测核心成分与关键靶点的结合能力。利用脂多糖(LPS)刺激RAW264.7细胞产生急性炎症反应构建体外炎症研究模型,设置对照组(CK)、模型组(LPS)、菊花水提物组(J)、槐花水提物组(H)、菊花-槐花水提物组(J+H),采用CCK-8细胞活力检测以及Griess法、酶联免疫吸附法(ELISA)对NO、TNF-α、ILs等进行测定,以此验证菊花-槐花的抗炎效果和作用机制。结果PPI拓扑参数分析得到AKT1、TP53、IL-6、TNF-α、CASP3前5个关键靶点,“药物成分-靶点-通路-疾病”网络拓扑参数分析筛选出前5个核心成分是槲皮素、山萘酚、β-甾醇、异鼠李素与木犀草素,分子对接结果显示5个核心成分与IL-6、TNF-α关键靶点具有较高的亲和力。CCK-8检测显示菊花-槐花水提物可明显促进RAW264.7细胞的增殖,炎症因子检测显示菊花-槐花水提物能显著降低NO、TNF-α、IL-6、IL-1β的水平。结论菊花-槐花共用具有明显的抗炎效果,其机制具有多成分-多靶点-多途径的特点,可能与作用于TNF-α、IL-6靶点从而抑制细胞炎症因子的发生有关。

Objective To investigate the anti-inflammatory effects and mechanisms of chrysanthemum-sophora japonica based on network pharmacology,molecular docking and cellular experiments.Methods The active components and related targets of chrysanthemum-sophora japonica were obtained by the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP).The related targets of hepatitis B virus(HBV)were retrieved from GeneCards,Drugbank,OMIM and TTD databases.Cytoscape software was used to construct a drug-active component-target network,and the STRING database was employed to build a protein-protein interaction(PPI)network to screen for core targets.The DAVID platform was utilized for gene ontology(GO)function and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis of the targets.AutoDock Vina was applied for molecular docking to predict the binding capabilities of the core components with key targets.An extracorporal inflammatory model was established by stimulating RAW264.7 cells with lipopolysaccharides(LPS)to induce acute inflammatory responses.Five groups were set up:the control group(CK),the model group(LPS),chrysanthemum water extract group(J),sophora japonica water extract group(H)and chrysanthemum flos sophora water extract group(J+H).CCK-8 cell viability assay,Griess method and enzyme-linked immunosorbent assay(ELISA)were used to measure NO,TNF-αand ILs to verify the anti-hepatitis B inflammatory effects and mechanisms.Results PPI topological parameter analysis identified AKT1,TP53,IL-6,TNF-α,and CASP3 were the top five key targets.Network topology parameter analysis of the"drug component-target-pathway-disease"showed revealed quercetin,kaempferol,β-sterol,isorhamnetin and luteolin were the top five core components.Molecular docking results showed that these five core components had high affinity with the IL-6 and TNF-αkey targets.CCK-8 assays demonstrated that chrysanthemum flos-sophora japonica water extracts significantly promoted the proliferation of RAW264.7 cells,and inflammatory factor measurements indicated that these extracts significantly reduced the levels of NO,TNF-α,IL-6 and IL-1β.Conclusion The combination of chrysanthemum-sophora japonica exhibits significant anti-inflammatory effects.Its mechanism exhibits the characteristics of multiple components,multiple targets and multiple pathways.It may be related to acting on the targets of TNF-αand IL-6 to inhibit the occurrence of cellular inflammatory factors.