基于网络药理学及实验验证探究黄连-厚朴药对抗幽门螺杆菌感染的作用机制

Study on the Mechanism of Rhizoma Coptidis - Magnolia Officinalis on Inhibition of Helicobacter pylori Infection based on Network Pharmacology and Experimental Verification

关键词:  关键词：黄连  厚朴  幽门螺杆菌感染  网络药理学  体外抑菌  动物实验

Key words:  KEY WORDS：rhizoma coptidis  magnolia officinalis  helicobacter pylori infection  network pharmacology  in vitro bacteriostasis  experiments on animals

摘要:

摘要：目的 基于网络药理学及实验验证的方法探究黄连-厚朴药对抗幽门螺杆菌（Helicobacter pylori，Hp）感染的作用机制。方法 利用TCMSP数据库筛选出黄连、厚朴的主要活性成分以及作用靶点，通过Drugbank、Genecards、DisGeNET数据库筛选出Hp感染的相关靶点采用Cytoscape 3.7.2软件构建药物-成分-潜在靶点网络图。利用String数据库构建蛋白互作网络，筛选出核心靶点。借助Metascape数据库对交集靶点进行GO和KEGG的富集分析。通过Autodock vina 1.1.2软件对黄连-厚朴药对的主要活性成分和对应的关键靶点进行分子对接。最后通过体外抑菌实验验证黄连-厚朴的抑菌效力，动物实验采用酶联免疫吸附法（ELISA）法及蛋白质印迹法（Western blot）法对大鼠胃黏膜相关炎症因子及蛋白进行验证。结果 黄连和厚朴主要活性成分23种，作用靶点185个，与疾病交集靶点73个，核心成分为槲皮素、小檗碱、厚朴酚、和厚朴酚等，核心作用靶点为TP53、IL6、VEGFA、TNF、AKT1。富集分析结果显示黄连-厚朴药对抗Hp感染主要涉及TNF信号通路、IL-17信号通路、Toll 样受体信号通路、C型凝集素受体信号通路。分子对接显示，槲皮素、小檗碱、厚朴酚、和厚朴酚与核心靶点TP53、IL6、VEGFA、TNF、AKT1均具有较好的结合活性。体外抑菌实验发现黄连-厚朴药对的抑菌圈与克拉霉素相近，最小抑菌浓度为31.3mg/ml，其具有较强的体外抑菌效果；动物实验结果显示，与模型组相比，黄连、厚朴药对组血清中IL-1β、IL-6、TNF-α的含量显著下降（P＜0.01），同时药对组中P53、AKT1、TNF-α蛋白表达均呈下降趋势（P＜0.05）。结论 本研究证实了黄连-厚朴药对抗Hp感染多成分、多靶点、多途径的作用机制，主要是通过抑制细菌生长、参与炎症反应、细胞迁移和增殖等途径来发挥其效用，为黄连-厚朴药对抗Hp感染的机制研究提供依据。

Abstract:

ABSTRACT：OBJECTIVE Study on the mechanism of Rhizoma coptidis - Magnolia officinalis on Inhibition of Helicobacter pylori infection based on network pharmacology and experimental verification.METHODS The main active components and target of Magnolia officinalis and Coptis chinensis were screened out by TCMSP database, and the related targets of Hp infection were screened out by Drugbank, Genecards and DisGeNET database and Cytoscape 3.7.2 software was used to construct the drug-components-potential target network. The protein interaction network was constructed using String database to screen out the core targets. Metascape database was used to enrich GO and KEGG of intersection targets. Autodock Vina 1.1.2 was used todock the main active ingredients and corresponding key targets of coptis chinensis and Magnolia officinalis drug pairs. Finally, the bacteriostatic effect of Rhizoma coptidis - Magnolia officinalis was verified by in vitro bacteriostatic experiment, in animal experiments,enzyme-linked immunosorbent assay (ELISA) and Western blotting were used to verify the related inflammatory factors and proteins in gastric mucosa of rats.RESULTS There were 23 main active components of Coptis chine and Magnolia officinae, 185 targets, and 73 intersection targets with diseases. The core components were quercetin, berberine, magnolol and magnolol, and the core targets were TP53, IL6, VEGFA, TNF and AKT1. The results of enrichment analysis showed that coptise-Magnolia officinae drugs against Hp infection mainly involved TNF signaling pathway, IL-17 signaling pathway, Toll-like receptor signaling pathway and C-type lectin receptor signaling pathway. Molecular docking showed that quercetin, berberine, magnolol and magnolol had good binding activities with core targets TP53, IL6, VEGFA, TNF and AKT1. The bacteriostatic zone of coptise-Magnolia officinalis pair was similar to that of clarithromycin, and the minimum bacteriostatic concentration was 31.3mg/ mL, which showed strong bacteriostatic effect in vitro; the results of animal experiments showed that compared with the model group, the contents of IL-1β, IL-6 and TNF-α in the serum of the Coptidis rhizoma and Magnolia officinalis drug treatment groups were significantly decreased (P<0.01), and the protein expressions of P53, AKT1 and TNF-α in the medicine pair group were decreased (P<0.05). CONCLUSION This study confirmed the multi-component, multi-target and multi-pathway mechanism of Rhizoma coptidis and Magnolia officinalis drug pair against Hp infection, mainly by inhibiting bacterial growth, participating in inflammatory response, cell migration and proliferation, providing a basis for the study of the mechanism of Coptidis and Magnolia officinalis against Hp infection.