小儿咳喘颗粒治疗儿童支气管肺炎的活性成分及作用机制预测分析

Predictive Analysis of the Active Components and Mechanism of Xiaoer Kechuan Granule in the Treatment of Children Bronchopneumonia

目的利用网络药理学、分子对接、细胞实验探索小儿咳喘颗粒治疗儿童支气管肺炎的活性成分及其作用机制。方法通过TCMSP、TCMID、STITCH数据库和文献检索,收集小儿咳喘颗粒处方中麻黄、川贝母、苦杏仁、黄芩等15味药的活性成分及其作用靶点,并在Genecards和OMIM数据库中查找儿童支气管肺炎靶点。利用STRING网络平台对共有靶点构建蛋白互作网络以及GO(Gene Ontology)功能注释和KEGG(Kyotoencyclopediaofgenesandgenomes)通路分析,并用Cytoscape3.7.2软件构建PPI(Protein-Protein interaction network,PPI network)和“疾病-药物-成分-靶点-通路”网络图,最后根据网络图的分析结果选取关键成分和靶点进行分子对接。体外细胞实验采用脂多糖(Lipopolysaccharide,LPS)诱导的RAW264.7炎症细胞模型,检测小儿咳喘颗粒对RAW264.7细胞的抗炎作用。免疫印迹(Western blot)实验检测肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)、白细胞介素-6(Interleukin 6,IL-6)、白细胞介素-1β(Interleukin-1β,IL-1β)以及血管内皮生长因子A(Vascular endothelial growth factor A,VEGFA)的表达,进一步验证其作用机制。结果收集到小儿咳喘颗粒活性成分有槲皮素、β-谷甾醇、木犀草素、山柰酚等220个,靶点有TNF、IL-6、IL-1β、VEGFA等439个,疾病靶点350个。GO功能和KEGG通路分析表明小儿咳喘颗粒治疗支气管肺炎主要包括IL-17信号通路、AGE-RAGE信号通路与TNF信号通路等,主要涉及细胞代谢、免疫反应和细胞因子转化等生物过程。分子对接结果显示小儿咳喘颗粒的关键成分槲皮素、β-谷甾醇、木犀草素、山柰酚与疾病靶点TNF、IL-6、IL-1β、VEGFA有较强的结合活性。实验结果显示,小儿咳喘颗粒给药组可显著降低炎症因子一氧化氮(NO)、TNF-α、IL-6的含量,同时抑制TNF-α、IL-6、IL-1β以及VEGFA蛋白的表达,初步验证了预测结果的可靠性。结论采用网络药理学结合分子对接及体外实验的方法,从多组分-多靶点-多通路的角度分析预测小儿咳喘颗粒治疗儿童支气管肺炎的主要活性成分及潜在作用机制,为后续实验提供参考和依据。

Objective To explore the active ingredients and their potential mechanism action of Xiaoer Kechuan Granule in the treatment of children bronchopneumonia by the method of network pharmacology,molecular docking and cell experiments.Methods Through TCMSP,TCMID,STITCH database and literature search,to collect the active ingredients and targets of 15 traditional Chinese medicines like Ephedra,Fritillaria cirrhosa D.Don,Semen Armeniacae Amarum,Scutellaria baicalensis Georgi in Xiaoer Kechuan Granule,and collect the disease targets of children bronchopneumonia in Genecards and OMIM database.Through the STRING network platform to build protein interaction network for the common targets,GO(Gene Ontology) functional enrichment and KEGG(Kyoto encyclopedia of genes and genomes) pathway enrichment analysis,and then used the Cytoscape 3.7.2 software to build Protein-Protein interaction network(PPI network) and "disease-drug-ingredients-targets-pathway" network.Finally,according the analysis results of network,make the molecular docking with the key ingredients and targets.In vitro cell test,Lipopolysaccharide(LPS) induced RAW264.7 inflammatory cell model was used to detect the anti-inflammatory effect of Xiaoer Kechuan Granule on RAW264.7 cells,and carried out western blot to detect the expression of Tumor necrosis factor-α(TNF-α),Interleukin 6(IL-6),Interleukin-1β(IL-1β) and Vascular endothelial growth factor A(VEGFA).Results There are 220 different kinds of active ingredients in Xiaoer Kechuan Granule including quercetin,β-sitosterol,luteolin,Kaempferol,etc.and 439 corresponding targets,such as TNF,IL-6,IL-1β and VEGFA.and 350disease targets.GO function and KEGG pathway analysis showed that the treatment of children bronchopneumonia with Xiaoer Kechuan Granule mainly includes IL-17 signaling pathway,AGE-RAGE signaling pathway and TNF signaling pathway,etc.which are mainly involved in biological processes such as cell metabolism,immune response and cytokine conversion.Molecular bonding results showed that quercetin,β-sitosterol,luteolin and kamanol had strong binding activities with disease targets TNF,IL-6,IL-1β and VEGFA.Experimental validation results showed that the treatment group of Xiaoer kechuan granules significantly reduced the content of inflammatory factors NO,TNF-α and IL-6.and inhibited the expression of TNF-α,IL-6,IL-1β and VEGFA proteins,which preliminatively verified the reliability of the predicted results.Conclusion Using network pharmacology combined with molecular docking and in vitro experiments to explore the mechanism of action in the Xiaoer Kechuan Granule cures children bronchopneumonia,to provide a reference and basis for subsequent experiments.