基于网络药理学探讨姜知母干预肺寒湿咳的作用机制

Prediction of mechanism of ginger-processed Anemarrhenae Rhizoma in intervening in cough due to cold and dampness in lung based on network pharmacology

基于网络药理学、分子对接技术和实验验证探讨姜知母干预“肺寒湿咳”的作用机制,为姜知母治疗肺寒湿咳这一传统用法提供现代药理学依据。使用中药系统药理学数据库分析平台(TCMSP),根据类药性原则选择口服生物利用度(OB≥30%)、药物相似性(DL≥0.18)作为筛选条件并结合相关文献来获得姜知母活性成分,建立姜知母活性成分库;采用SwissTargetPrediction数据库预测活性成分的潜在靶点;通过SymMap数据库筛选到中医证候“肺寒湿咳”对应西医症状为急性支气管炎;从人类基因数据库(GeneCards)和在线人类孟德尔遗传数据库(OMIM)获得急性支气管炎疾病的相关靶点;利用Venny数据库获取成分与疾病的交集靶点;利用STRING平台和Cytoscape软件构建交集靶点的蛋白-蛋白相互作用(PPI)网络;借助DAVID数据库对潜在治疗靶点进行GO功能富集和KEGG通路分析;采用AutoDock等软件对部分活性成分与潜在靶点进行分子对接验证;最后采用烟熏加寒冷刺激法制备小鼠急性支气管炎疾病模型进行验证。该研究共筛选出姜知母活性成分潜在作用靶点557个,药物-疾病共同靶点157个,富集分析得到1072个GO功能富集条目和169个KEGG通路富集条目;分子对接结果显示活性成分和MAPK14、MMP3蛋白有较好的结合能力,活性成分中结合能力最强的是timosaponin AⅢ;体内动物实验表明,除生知母低剂量组、生知母高剂量组、姜汤组外,其余各给药组均可减轻小鼠肺组织病变,减轻炎症细胞浸润,降低小鼠支气管肺泡灌洗液(BALF)中IL-1β、IL-18、TNF-α炎症因子水平,下调小鼠肺组织MAPK14、MMP3 mRNA及MAPK14、MMP3蛋白的表达,且姜知母的改善效果优于生知母。该研究利用网络药理学和实验验证初步揭示了姜知母可通过调控IL-17、TNF等信号通路,抑制炎症反应,达到治疗肺寒湿咳的作用,验证了姜知母治疗肺寒湿咳这一传统用法的科学性,为姜汤浸知母传统特色炮制工艺的传承和临床应用提供科学依据。

This study employed network pharmacology,molecular docking,and animal experiments to investigate the mechanism of ginger-processed Anemarrhenae Rhizoma in treating cough due to cold and dampness in lung,aiming to provide a modern pharmacological basis for this therapy.The active ingredients of ginger-processed Anemarrhenae Rhizoma were retrieved from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)with the oral bioavailability(OB≥30%)and drug likeness(DL≥0.18)as the screening conditions and from the relevant literature.A library of the active ingredients of ginger-processed Anemarrhenae Rhizoma was established,and SwissTargetPrediction was used to predict the potential targets of the active ingredients.The western medical symptom corresponding to cough due to cold and dampness in lung was identified as acute bronchitis based on SymMap,and the targets related to acute bronchitis were obtained from GeneCards,and the Online Mendelian Inheritance in Man(OMIM).Venny was utilized to obtain the common targets between the active ingredients and the disease,and STRING and Cytoscape were used to establish the protein-protein interaction(PPI)network for the common targets.The Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses of potential therapeutic targets were performed with DAVID.AutoDock was used to simulate the molecular docking between the active ingredients with potential targets.Finally,a mouse model of acute bronchitis was prepared with the smoke plus cold stimulation method for validation.In this study,a total of 557 potential targets of the active ingredients of ginger-processed Anemarrhenae Rhizoma were retrieved,including 157 common targets shared with acute bronchitis.The enrichment analysis yielded 1072 GO terms and 169 KEGG pathways.The results of molecular docking showed that the active ingredients had good binding affinity with MAPK14 and MMP3,and timosaponin AⅢshowcased the strongest binding affinity among the active ingredients.In the animal experiments,other treatment groups except the low-and high-dose raw Anemarrhenae Rhizoma and the ginger decoction groups showed reduced pathological changes in the lung tissue,attenuated inflammatory cell infiltration,lowered levels of interleukin(IL)-1β,IL-18,and tumor necrosis factor(TNF)-a in the bronchoalveolar lavage fluid(BALF),and down-regulated mRNA and protein levels of MAPK14 and MMP3 in the lung tissue.Moreover,ginger-processed Anemarrhenae Rhizoma outperformed raw Anemarrhenae Rhizoma.This study preliminarily revealed that ginger-processed Anemarrhenae Rhizoma can inhibit inflammation by regulating the IL-17,TNF,and other signaling pathways to treat cough due to cold and dampness in lung,verifying the rationality of this therapy.The findings provide a basis for the inheritance and application of the characteristic ginger processing of Anemarrhenae Rhizoma.