基于网络药理学、分子对接和动物实验探讨老虎芋醇提物治疗类风湿性关节炎的作用机制

Exploring the mechanism of action of alocasia cucullata alcohol extract in treating rheumatoid arthritis based on network pharmacology, molecular docking and animal experiments

关键词:  网络药理学  动物实验  老虎芋醇提物  类风湿性关节炎  作用机制

Key words:  Network pharmacology  Animal experiments  Alocasia cucullata alcohol extract  Rheumatoid arthritis  Mechanism of action

摘要:

目的：基于网络药理学、分子对接和动物实验探讨老虎芋醇提物治疗类风湿性关节炎（RA）的作用机制。方法：利用Pubchem、Swiss Target Prediction收集老虎芋醇提物潜在活性成分及靶点；Genecards、DisGeNET数据库检索RA疾病靶点；通过韦恩图获得老虎芋醇提物与RA疾病的交集靶点；利用STRING数据库进行交集靶点的PPI蛋白互作分析；利用Cytoscape软件绘制“靶点-成分-药物-疾病”网络图；DAVID数据库进行基因本体论（GO）和京都基因与基因组百科全书（KEGG）富集分析；采用AutoDockTools软件完成关键化合物-靶点的分子模拟对接。大鼠随机分为正常组、RA组、老虎芋醇提物低剂量组、老虎芋醇提物中剂量组、老虎芋醇提物高剂量组，以完全弗氏佐剂和牛Ⅱ型胶原混合乳液诱导建立RA模型，以不同剂量老虎芋醇提物分组干预后检测各组大鼠足容积与关节炎指数（AI）；HE染色检测大鼠踝关节组织形态；ELISA检测大鼠血清炎性因子水平；免疫印迹法检测大鼠踝关节组织核心靶点蛋白表达。结果：老虎芋醇提物筛选出17个活性成分，与RA疾病共有交集靶点132个，主要调控AKT1、EGFR、SRC、BCL2、TLR4等核心靶点，涉及plasma membrane、protein binding、signal transduction等信号通路；分子对接结果显示，老虎芋醇提物主要活性成分木犀草素、山奈酚与AKT1、EGFR、SRC、BCL2、TLR4具有较好的结合活性；动物实验结果显示，老虎芋醇提物可减轻RA大鼠踝关节组织病变，降低其足容积、AI、踝关节病理评分、血清TNF-α、IL-17与IL-6水平、踝关节组织p-AKT1/AKT1、p-SRC/SRC与EGFR、BCL2、TLR4蛋白相对表达并呈剂量依赖性（P＜0.05）。结论：老虎芋醇提物治疗RA的主要活性成分为木犀草素、山奈酚，可以通过调控关键靶点蛋白AKT1、EGFR、SRC、BCL2、TLR4表达，减轻炎症反应、改善RA踝关节组织病变。

Abstract:

Objective: To explore the mechanism of action of alocasia cucullata alcohol extract in the treatment of rheumatoid arthritis (RA) based on network pharmacology, molecular docking and animal experiments. Methods: Potential active ingredients and targets of alocasia cucullata alcohol extract were collected through Pubchem and Swiss Target Prediction. Genecards and DisGeNET databases were used to search for RA disease targets. The intersection targets between the alocasia cucullata alcohol extract and RA diseases were obtained by Venn diagram. The STRING database was used for PPI protein interaction analysis of intersecting targets. Cytoscape software was used to draw a "target component drug disease" network diagram. DAVID database was used for gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. AutoDockTools software was used to perform molecular simulation docking between key compounds and targets. The rats were randomly assigned into normal group, RA group, low dose group, medium dose group and high dose group of alocasia cucullata alcohol extract, and the RA model was induced by the mixed lotion of complete Freund's adjuvant and bovine type II collagen. The foot volume and arthritis index (AI) of rats in each group were measured after the intervention of different doses of alocasia cucullata alcohol extract. HE staining was used to detect the morphology of rat ankle joint tissue. ELISA was used to detect the levels of inflammatory factors in rat serum. Western blot was used to detect the protein expression of core target in rat ankle joint tissue. Results: A total of 17 active ingredients were screened out from the alocasia cucullata alcohol extract, which shared 132 intersection targets with AR disease, mainly regulating core targets such as AKT1, EGFR, SRC, BCL2 TLR4 and involves plasma membrane, protein binding, signal transduction and other signaling pathways. Molecular docking results showed that luteolin and kakaferol, the main active components of alocasia cucullata alcohol extract, had good binding activity with AKT1, EGFR, SRC, BCL2 and TLR4. Animal experiment results showed that the alocasia cucullata alcohol extract could alleviate ankle joint tissue lesions in RA rats, reduce foot volume, AI, ankle joint pathological score, serum TNF-α, IL-17, and IL-6 levels, and the relative expression of p-AKT1/AKT1, p-SRC/SRC and EGFR, BCL2, TLR4 proteins in ankle joint tissue, in a dose-dependent manner (P<0.05). Conclusion: The main active ingredients alocasia cucullata alcohol extract in the treatment of RA are luteolin and kaferol, which can reduce inflammatory response and improve RA ankle tissue lesions by regulating the expression of key target proteins AKT1, EGFR, SRC, BCL2 and TLR4.