基于网络药理学和实验验证探讨桔梗改善哮喘的机制研究

Mechanism of Jiegeng(桔梗)in Improving Asthma:An Exploration Based on Network Pharmacology and Experimental Verification

目的:分析探讨桔梗治疗哮喘的潜在作用机制。方法:采用TCMSP和TCMID数据库,以"OB≥30%,DL≥0.18,HL≥4 h"为限定条件,检索得到与桔梗相关的活性成分;通过Swiss TargeTCMStPrediction,SEA数据库和TTD、DisGeNET、GAD数据库相结合,获得桔梗治疗哮喘的候选靶点。使用String数据库结合Cytoscape 3.7.2软件,获得桔梗治疗哮喘的候选靶点相互作用关系及网络图。采用Cytoscape 3.7.2软件的ClueGO插件,对其进行GO分类富集分析。通过KOBAS3.0对其进行KEGG通路富集分析。使用Cytoscape 3.7.2软件构建"成分-作用靶点"及"桔梗-成分-靶点-通路"相互作用网络图。采用LPS诱导的人支气管上皮细胞(16HBE)炎症模型,RT-PCR法检测桔梗皂苷D对16HBE细胞的Tnfα、Il1β、Il1αmRNA表达的影响;采用Western Blot法检测桔梗皂苷D对16HBE细胞的MMP9蛋白表达的影响。结果:从数据库中得到桔梗活性成分40个,包括桔梗皂苷D、金合欢素、木犀草素等;桔梗治疗哮喘的候选靶点100个,包括TNF、IL-6、VEGFA、IL-1β等。GO分类富集分析,其中BP分析40个条目、CC分析37个条目、MF分析49个条目。KEGG通路富集分析,得到150条通路,主要包括神经活性配体-受体相互作用信号通路、cGMP-PKG信号通路、PI3K-AKT信号通路等。桔梗皂苷D对LPS诱导的16HBE细胞增殖有显著的抑制作用;与模型对照组比较,桔梗皂苷D可以显著下调16HBE细胞Tnfα、Il1β、Il1αmRNA表达及MMP9蛋白表达。结论:桔梗对哮喘有潜在的治疗作用,其中桔梗皂苷D是有效成分之一,其作用机制是通过降低Tnfα、Il1β、Il1αmRNA表达,抑制哮喘气道重塑的生物标志物MMP9蛋白表达来发挥治疗作用。

Objective:To analyze the potential mechanism of Jiegeng(桔梗)in the treatment of asthma.Methods:The active components of Jiegeng(桔梗)were retrieved from TCMSP and TCMID under the conditions of"OB≥30%,DL≥0.18,and HL≥4 h",and the candidate targets for Jiegeng(桔梗)against asthma from Swiss Target Prediction,TCMSP,SEA,TTD,DisGeNET,and GAD.The interactions and network of these candidate targets were constructed by String and Cytoscape 3.7.2.ClueGo plug-in of Cytoscape 3.7.2 was utilized for GO enrichment analysis,followed by KEGG pathway enrichment analysis using KOBAS3.0.Cytoscape 3.7.2 was employed for constructing the"component-target"and"Jiegeng(桔梗)-component-target-pathway"interaction networks.After the human bronchial epithelial(16 HBE)cells were treated with LPS for inducing inflammation,the effects of platycodin D on the mRNA expression levels of Tnfα,Il1β,and Il1αin 16 HBE cells were determined by RT-PCR,and its effects on MMP9 protein expression were assayed by Western Blotting.Results:A total of 40 active components of Jiegeng(桔梗)were obtained,including platycodin D,acacetin,and luteolin.There were 100 candidate targets for Jiegeng(桔梗)in the treatment of asthma,including TNF,IL6,VEGFA,and IL-1β.As revealed by GO enrichment analysis,40 entries were enriched into BP,37 into CC,and 49 into MF.KEGG pathway enrichment analysis yielded 150 pathways,mainly including neuroactive ligand-receptor interaction signaling pathway,cGMP-PKG signaling pathway,and PI3 K-AKT signaling pathway.Platycodin D significantly inhibited the proliferation of 16 HBE cells induced by LPS.Compared with the model group,platycodin D significantly down-regulated the mRNA expression levels of Tnfα,Il1β,and Il1αas well as MMP9 protein expression in 16 HBE cells.Conclusion:Platycodin D is one of the effective components responsible for the anti-asthama effects of Jiegeng(桔梗).It exerts the therapeutic effect by lowering the mRNA expression of Tnfα,Il1β,and Il1αand inhibiting the expression of MMP9 protein,a biomarker of asthma airway remodeling.