基于网络药理学及实验验证探讨喉咽清治疗咽炎的作用机制

Mechanism of Houyanqing in Treating Pharyngitis Based on Network Pharmacology and Experimental Verification

目的运用网络药理学并结合动物实验探究喉咽清治疗咽炎的物质基础及作用机制。方法通过中药系统药理学分析平台(TCMSP)数据库检索喉咽清的化学成分,并通过文献检索补充潜在活性成分,再经TCMSP及Swiss Target Prediction数据库预测成分作用靶点,建立靶点数据;通过Gene Cards数据库筛选出抗炎相关靶点;用Cytoscape 3.9.0软件构建“化合物-靶点(基因)”网络可视化关系图;采用STRING数据库对靶点进行蛋白质-蛋白质相互作用(PPI)网络分析,使用Cytoscape 3.9.0软件中的Cyto NCA插件对STRING数据库导出的PPI网络进行degree值算法分析,将degree值排名前30的网络靶点作为喉咽清的抗炎核心靶点,并构建“核心靶点-活性成分”网络图;通过DAVID分析平台进行基因本体(GO)分析和基于京都基因与基因组百科全书(KEGG)通路富集分析,探讨喉咽清抗炎的潜在生物过程及其通路;利用Autodock软件进行半柔性分子对接反向验证。在动物实验中,以15%的浓氨水诱导咽炎大鼠模型,经喉咽清灌胃处理后,ELISA法检测大鼠血清一氧化氮(NO)、肿瘤坏死因子(TNF-α)、白细胞介素-6(IL-6)、白细胞介素1-beta(IL-1β)的表达,HE染色观察大鼠咽部组织病理学变化。结果从喉咽清中共筛选得到65个活性成分,关键靶点涉及PTGS2、AKT1、MAPK3、STAT3、IL-6、TNF、NFKBIA等。KEGG通路富集结果显示喉咽清主要作用于Pathways in cancer、Hepatitis B、TNF signaling pathway等信号通路。分子对接结果显示β-蜕皮甾酮、齐墩果酸、山奈酚、竹节参皂苷IVa等核心化合物与关键靶点同时满足空间匹配与能量匹配。动物实验表明,与正常对照组相比,模型对照组大鼠咽部炎性改变明显,血清IL-6、IL-1β水平升高;与模型组相比,地塞米松5 mg·kg^(-1)组、喉咽清1.34 g·kg^(-1)、2.67 g·kg^(-1)组大鼠咽部炎症减轻,地塞米松5 mg·kg^(-1)组、喉咽清1.34 g·kg^(-1)、2.67 g·kg^(-1)组血清IL-6含有量降低(P<0.05)。结论喉咽清可能是通过调控肿瘤合成途径、乙型肝炎、肿瘤坏死因子等信号通路,对细胞代谢的多个过程产生影响,抑制炎症因子的表达,发挥抗炎作用。初步推测并验证了喉咽清治疗咽炎的主要靶蛋白与通路,为后续进一步验证喉咽清靶向抗炎提供新思路。

Objective To explore the material basis and mechanism of Houyanqing in the treatment of pharyngitis by network pharmacology combined with animal experiments study.Methods The chemical constituents of Houyanqing were searched by Traditional Chinese Medicine System Pharmacology Analysis Platform(TCMSP)database,and the potential active components were supplemented by literature retrieval.Then the targets of components were predicted by TCMSP and SwissTargetPrediction database,and the target data were established.GeneCards database screened anti-inflammatory targets;the compound-target(gene)network visualization diagram was constructed by Cytoscape 3.9.0 software;the protein-protein interaction(PPI)network of the target was analyzed by STRING database.The CytoNCA plug-in in Cytoscape 3.9.0 software was used to analyze the degree algorithm of the PPI network exported by STRING database.The top 30 network targets of the degree value were used as the anti-inflammatory core targets of the throat clear,and the core target-active ingredient network diagram was constructed.Gene Ontology(GO)analysis and pathway enrichment analysis based on Kyoto Encyclopedia of Genes and Genomes(KEGG)were carried out by DAVID analysis platform to understand the potential biological processes and pathways of anti-inflammation in Houyanqing.Autodock software was used for reverse verification of semi-flexible molecular docking.In the animal experiment study,the rat model of pharyngitis was induced by 15%concentrated ammonia water.After intragastric administration of Houyanqing,the expressions of serum nitric oxide(NO),tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and interleukin-1β(IL-1β)were detected by ELISA.HE staining was used to observe the pathological changes of rat pharynx.Results A total of 65 active components were screened from Houyanqing,and the key targets were PTGS2,AKT1,MAPK3,STAT3,IL-6,TNF and NFKBIA.KEGG pathway enrichment results showed that Houyanqing mainly acted on pathways in cancer,hepatitis B,TNF signaling pathway.Molecular docking results showed that core compounds such asβ-ecdysterone,oleanolic acid,kaempferol,chikusetsusaponin Iva and key targets met both spatial and energy matching.Animal experiments showed that compared with the normal control group,the pharyngeal inflammation in the model control group was significantly changed,and the levels of serum IL-6 and IL-1βwere increased.Compared with the model group,the pharyngeal inflammation of rats in the dexamethasone 5 mg·kg^(-1)group,Houyanqing 1.34 g·kg^(-1)and 2.67 g·kg^(-1)groups was alleviated,and the serum IL-6 contents in the dexamethasone 5 mg·kg^(-1)group,Houyanqing 1.34 g·kg^(-1)and 2.67 g·kg^(-1)groups were decreased(P<0.05).Conclusion Houyanqing may affect multiple processes of cell metabolism,inhibit the expression of inflammatory factors and play an anti-inflammatory role by regulating tumor synthesis pathway,hepatitis B and TNF signaling pathways.The main target proteins and pathways of Houyanqing in the treatment of pharyngitis were preliminarily speculated and verified,which provided new ideas for further verification of targeted anti-inflammation of Houyanqing.