基于UPLC-LTQ-Orbitrap-MS和网络药理学探索黄芪桂枝五物汤治疗类风湿关节炎的作用机制及实验验证

Mechanism of Huangqi Guizhi Wuwu Decoction in treatment of rheumatoid arthritis based on UPLC-LTQ-Orbitrap-MS, network pharmacology, and cell experiment

该研究采用超高效液相色谱-离子阱-静电场轨道阱质谱(UPLC-LTQ-Orbitrap-MS)对黄芪桂枝五物汤(HQGZ)的主要活性成分进行分析,从黄芪桂枝五物汤中共鉴定出活性成分50种,基于鉴定的化学成分通过网络药理学分析共得到潜在作用靶点108个,其中关键靶点87个;在此基础上,对活性成分进行靶标预测、功能研究及疾病富集。关键靶点的功能富集分析结果表明,黄芪桂枝五物汤可能主要通过鞘脂信号传导途径、肿瘤坏死因子(TNF)信号通路等途径,以及核糖核酸(RNA)聚合酶Ⅱ启动子转录的正调控、炎症反应等生物过程发挥治疗作用。在HQGZ的活性成分进行网络药理学分析及预测的基础上,结合类风湿关节炎的疾病特点,该研究对TNF信号通路中的关键蛋白进行细胞学实验进行验证,实验结果表明,HQGZ可以显著降低TNF-α诱导的人类风湿关节炎成纤维样滑膜细胞中的CASP3、TNF、RELA和IKBKB蛋白表达。UPLC-LTQ-Orbitrap-MS、网络药理学和实验验证结果表明,HQGZ中活性成分可能通过调控TNF信号通路中的关键蛋白,从而发挥抑制炎症反应、调节免疫功能及调控细胞凋亡的作用来治疗类风湿关节炎。

In this study, ultra-high performance liquid chromatography-linear ion trap/electrostatic field orbit trap combined-type mass spectrometry(UPLC-LTQ-Orbitrap-MS) was used to analyze the main active components of Huangqi Guizhi Wuwu Decoction(HQGZ). A total of 50 active components were identified from HQGZ and 108 potential targets of the components related to the treatment of rheumatoid arthritis were retrieved based on network pharmacology, including 87 key targets, followed by Gene Ontology(GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment of the targets. The result indicated that HQGZ may exert therapeutic effects mainly through the sphingolipid signaling pathway, tumor necrosis factor(TNF) signaling pathway, as well as the positive regulation of ribonucleic acid(RNA) polymerase Ⅱ promoter transcription, inflammatory response and other biological processes. At the same time, cell experiment was performed to verify the key proteins in the TNF signaling pathway. The results demonstrated that HQGZ significantly reduced the expression of caspase-3(CASP3), TNF, relaxed(RELA) protein, and IkappaB kinase beta(IKBKB) in fibroblast-like synoviocytes induced by TNF-α. The results of UPLC-LTQ-Orbitrap-MS, network pharmacology and cell experiment showed that the active components in HQGZ may inhibit inflammatory response and regulate immune function and cell apoptosis by modulating key proteins in TNF signaling pathway to treat rheumatoid arthritis.