芍药甘草汤治疗葡萄膜炎的网络药理学研究与实验验证

Mechanism of Shaoyao Gancao Decoction against Uveitis:Based on Network Pharmacology and Experimental Verification

目的:通过网络药理学预测芍药甘草汤主要活性成分治疗葡萄膜炎的作用机制,并选择T细胞受体信号通路所介导T细胞转化后的效应分子γ干扰素(IFN-γ)、白细胞介素(IL)-10进行验证。方法:检索中药系统药理学数据库与分析平台(TCMSP)、中药分子机制生物信息学分析工具库(BATMAN-TCM),筛选芍药甘草汤成分、靶点和葡萄膜炎靶点。使用Cytoscape 3.7.2软件构建成分靶点网络、成分疾病靶点网络、靶点相互作用网络。拓扑分析筛选关键靶点后使用软件插件ClueGO进行基因本体(GO)、京都基因和基因组百科全书(KEGG)分析。采用光感受器间维生素A类结合蛋白混合完全弗氏佐剂及结核杆菌皮下注射复制实验性自身免疫性葡萄膜炎大鼠模型,HE染色检测视网膜炎症变化,酶联免疫吸附试验(ELISA)检测血清IFN-γ、IL-10含量。结果:芍药甘草汤中47种候选成分(白芍35种、炙甘草12种),涉及治疗葡萄膜炎作用靶点164个,拓扑分析关键靶点33个。富集分析结果表明,芍药甘草汤治疗葡萄膜炎可能与细胞对活性氧的反应、多种细胞增殖与分化、一氧化氮与神经递质等分子的代谢调节、IL-6的细胞反应等生物过程有关,涉及IL-17信号通路、TNF信号途径、Th17细胞分化、T细胞受体信号通路、Toll样受体信号通路、PI3K-AKT信号通路等。芍药甘草汤可缓解大鼠视网膜炎症,降低炎症评分(P<0.01),减少IFN-γ和增加IL-10的含量(均P<0.01)。结论:芍药甘草汤治疗葡萄膜炎过程涉及多个重要炎症与免疫通路,这将成为研究其作用机制的重要参考。

Objective:This study predicted the targets of the active compounds of Shaoyao Gancao Decoction(SGD)through network pharmacology,to explore the mechanism of SGD against uveitis.Moreover,the effectors interferon(IFN)-γand interleukin(IL)-10 after T cell receptor signaling pathway-mediated T cell transformation were selected for the verification.Methods:The components and targets of SGD and the targets of uveitis were searched from databases.The component-target network,component-disease target network,and protein-protein interaction network were constructed with Cytoscape 3.7.2.The key targets were screened by topological analysis,followed by Gene Ontology(GO)term and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment of the targets with Cytoscape 3.7.2 plug-in ClueGO.Autoimmune uveitis was induced in rats through subcutaneous injection of the mixture of inter-photoreceptor retinoid-binding protein and complete Freund′s adjuvant,and tubercle bacillus.The changes of retinal inflammation were detected based on hematoxylin and eosin(HE)staining and the content of IFN-γand IL-10 in serum was measured by enzyme-linked immunosorbent assay(ELISA).Results:A total of 47 candidate components(35 from Radix Paeoniae Alba and 12 from Radix Glycyrrhizae Preparata)of SGD,164 targets against uveitis,and 33 key targets were screened out.The key targets were mainly involved in the biological processes such as cell response to reactive oxygen species,proliferation and differentiation of various cells,metabolic regulation of nitric oxide and neurotransmitters,and cellular response of IL-6,and the pathways of IL-17 signaling pathway,tumor necrosis factor(TNF)signaling pathway,Th17 cell differentiation,T cell receptor signaling pathway,Toll-like receptor signaling pathway,phosphatidylinositol-3-kinase-protein kinase B(PI3K-AKT)signaling pathway,etc.SGD can relieve retina inflammation,decrease inflammation score(P<0.01),lower IFN-γlevel,and raise IL-10 content(Ps<0.01),which validated part of the prediction results of network pharmacology.Conclusion:SGD exerts therapeutic effect on uveitis through multiple important immune and inflammatory pathways,which can serve as a reference for exploring the mechanism.