基于网络药理学和实验验证探讨甘草附子汤治疗类风湿关节炎的作用机制

Mechanism of Gancao Fuzi Decoction(甘草附子汤)in the Treatment of Rheumatoid Ararthritis Based on Network Pharmacology and Experimental Verification

目的:本研究旨在对甘草附子汤治疗类风湿关节炎的作用机制进行网络药理学分析。方法:甘草附子汤的活性成分和靶点从中药系统药理学和药物数据库(DrugBank)中获取。网络药理学方法涉及“药物-成分-靶点”网络图的构建和靶点预测。使用京都基因和基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)数据库确定甘草附子汤和类风湿关节炎集群网络的重要信号通路。用分子对接软件(AutoDock vina)对主要活性成分与核心靶蛋白进行计算机模拟对接,预测主要成分与核心靶点的结合度。动物实验验证甘草附子汤对类风湿关节炎模型小鼠相关蛋白表达的影响。结果:筛选出山柰酚、柚皮素、甘草查耳酮A、异鼠李素、β-谷甾醇等82个有效成分,对应于甘草附子汤的151个目标。此外,1979个基因与类风湿关节炎密切相关,其中有88个与甘草附子汤的靶点重叠,被认为与治疗相关。功能富集分析表明,甘草附子汤通过调节多种途径发挥其在类风湿关节炎中的药理作用,包括细胞凋亡和自噬、软骨分化、炎症和免疫调节等。动物实验方面,与正常对照组比较,模型对照组小鼠关节肿胀明显,关节炎指数评分显著升高(P<0.01),踝关节中p-ERK1、ERK1和COX-2阳性表达均显著升高(P<0.01);与模型对照组比较,甘草附子汤4.8 g/kg组能显著抑制关节肿胀,明显降低关节炎指数评分(P<0.05或P<0.01),明显改善踝关节病理变化,踝关节中p-ERK1、ERK1和COX-2阳性表达显著降低(P<0.01)。结论:甘草附子汤是通过多组分、多靶点和多途径协同治疗类风湿关节炎。

Objective:To explore the mechanism of Gancao Fuzi Decoction(甘草附子汤)against rheumatoid arthritis based on network pharmacology.Methods:The active components and targets of Gancao Fuzi Decoction were obtained from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)and DrugBank.A drug-component-target network was constructed for target prediction.Important signaling pathways of Gancao Fuzi Decoction and rheumatoid arthritis cluster networks were identified by Kyoto Encyclopedia of Genes and Genomes(KEGG).AutoDock vina was used to perform molecular docking between main components and key targets to predict their binding.Finally,animal experiments were carried out to verify the effect of Gancao Fuzi Decoction on related protein expression of rheumatoid arthritis model mice.Results:Eighty two active components including kaempferol,naringenin,licochalcone A,isorhamnetin and beta-sitosterol were screened,corresponding to 151 targets of Gancao Fuzi Decoction.In addition,1979 genes were closely associated with rheumatoid arthritis,of which 88 overlapped with the targets of Gancao Fuzi Decoction and were considered to be therapeutically relevant.Functional enrichment analysis suggested that Gancao Fuzi Decoction exerted pharmacological effects on rheumatoid arthritis by regulating multiple pathways,including apoptosis,autophagy,cartilage differentiation,inflammation,and immune regulation.By animal experiments,the model control group had obvious joint swelling and increased arthritis index score and positive expression of p-ERK1,ERK1 and COX-2 in ankle joint as compared with the normal control group(P<0.01);Gancao Fuzi decoction 4.8 g/kg group inhibited joint swelling,reduced arthritis index score(P<0.05 or P<0.01),improved the pathological changes of ankle joint and down regulated the positive expression of p-ERK1,ERK1 and COX-2 in ankle joint as compared with the model control group(P<0.01).Conclusion:Gancao Fuzi Decoction produced therapeutic effects on rheumatoid arthritis model mice via multi-component,multi-target and multi-pathway synergy.