基于网络药理学和分子对接探讨桃红四物汤治疗慢性胆囊炎的机制

Mechanism of Tao-Hong-Si-Wu Decoction in Treating Chronic Cholecystitis Based on Network Pharmacology and Molecular Docking

目的:基于网络药理学和分子对接探讨桃红四物汤治疗慢性胆囊炎的机制,为新药研发节约成本,使实验更具有针对性。方法:通过TCMSP数据库寻找药物有效成分,在wiss Target Prediction中预测其对应的靶点,再通过Gene Cards数据库预测慢性胆囊炎的靶点,建立药物和疾病的靶点数据库后绘制PPI网络图、药物成分-靶点图以及GO、KEGG富集分析。根据度值,筛选出核心成分的靶点,再做分子对接验证两者结合情况,最后通过脂多糖刺激人胆囊上皮细胞诱导炎症,使用ELISA试剂盒检测上清液炎症介质的浓度,在使用不同浓度THSWD处理后,采用Western blot法检测AKT和p-AKT的表达。结果:筛选了桃红四物汤49个活性成分共计3,262个潜在靶点,去重后获得了665个潜在靶点;疾病靶点共681个。GO富集分析结果显示靶蛋白主要参与平滑肌细胞增殖、MAPK级联反应、炎症反应、蛋白激酶B信号的调控等。KEGG富集分析发现靶点参与了140条通路,主要涉及AGERAGE信号通路、HIF-1信号通路、PI3K-Akt信号通路等。根据度值筛选的核心靶点为AKT1、TNF、TP53、VEGFA、IL-1B,核心成分为山柰酚、木犀草素、槲皮素。选择了度值最高的AKT1和山柰酚进行了分子对接,并将结果可视化。CCK-8实验结果表明12.5μg/m L和25μg/m L的THSWD对胆囊上皮细胞无明显毒性作用,Elisa法检测结果发现使用脂多糖刺激后白介素-6水平显著升高,而使用THSWD处理后其水平明显下降。Western blot结果显示LPS诱导组p-AKT表达升高,THSWD处理组p-AKT表达下降。结论:本研究基于网络药理学、分子对接和细胞实验首次发现了桃红四物汤对慢性胆囊炎的治疗有效,且AKT1和山柰酚可能分别是其靶点和有效成分之一。

Objective To explore the mechanism of Tao-Hong-Si-Wu Decoction(THSWD)in treating chronic cholecystitis based on network pharmacology and molecular docking,so as to save the cost for the research and development of new drugs and make the experiment more targeted.Methods The effective components of drugs were searched by TCMSP database,and their corresponding targets were predicted in wiss Target Prediction.Then the target of chronic cholecystitis is predicted by GeneCards database.After establishing the target database of drugs and diseases,PPI network diagram,drug composition-target diagram and G0 and KEGG enrichment analysis were drawn.According to the degree value,the target of the core component was screened out,and then the combination of the two was verified by molecular docking.Finally,LPS stimulated human gallbladder epithelial cells to induce inflammation,and Elisa kit was used to detect the concentration of inflammatory mediators in supernatant.Western blot was used to detect the expression of AKT and p-AKT after treatment with different concentrations of THSWD.Results 3,262 potential targets of 49 active components of THSWD were screened,and 665 potential targets were obtained after weight removal.There are 681 disease targets,and Go enrichment analysis shows that the target proteins are mainly involved in smooth muscle cell proliferation,MAPK cascade reaction,inflammatory reaction,protein kinase B signal regulation and so on.KEGG enrichment analysis found that the target was involved in 140 pathways,mainly involving AGE-RAGE signaling pathway,HIF-1 signaling pathway,PI3K-Akt signaling pathway and so on.The core targets screened according to the degree value are AKT1,TNF,TP53,VEGFA and IL-1B,and the core components are kaempferol,luteolin and quercetin.AKT1 with the highest degree was selected for molecular docking with kaempferol,and the results were visualized.CCK-8 experimental results showed that THSWD of 12.5ug/ml and 25ug/ml had no obvious toxic effect on gallbladder epithelial cells.The results of Elisa showed that the level of IL-6 increased significantly after lipopolysaccharide stimulation,but decreased significantly after THSWD treatment.Western blot showed that the expression of p-AKT increased in LPS-induced group and decreased in THSWD-treated group.Conclusion Based on the network pharmacology,molecular docking and cell experiments,it is found for the first time that THSWD is effective in the treatment of chronic cholecystitis,and AKT1 and Kaempferol may be its target and one of its effective components respectively.