Integrating network pharmacology and experimental verification to explore the mechanism of Tripterygium wilfordii in ankylosing spondylitis

Objective: This study aimed to validate the mechanism of triptolide in treating ankylosing spondylitis (AS) through network pharmacology, molecular docking, and in vitro experiments.Methods: We gathered AS-related genes using databases including DrugBank, OMIM, GeneCards, TTD and DisGeNET. TCMSP database was used to collect Tripterygium wilfordii (TWHF)-related data. Additionally, the potential targets of TWHF in treating AS were predicted by consulting databases such as Venny, String, Cytoscape, and Cytohubba. Subsequently, a protein-protein interaction network was created and the gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis were performed by metascape database. After selecting the most active ingredient of TWHF, molecular docking was performed to confirm the predicted results. Furthermore, we explore the potential mechanism of the most active ingredient of TWHF in the treatment of AS in vitro.Result: By integrating the results of network pharmacological analysis, 62 genes were found to be strongly associated with AS, such as STAT3, TNF, MMP9, VEGFA, CXCL8, PTGS2, etc. Triptolide (TP) is one of the most active ingredients in TWHF. The enrichment analysis indicated that 292 biological processes and 132 signaling pathways were involved, with the T helper 17 cells cell differentiation pathway as the key pathway. TP was selected for molecular docking and in vitro experiments. The molecular docking results indicated that TP had excellent affinity with 6 key targets. Further, flow cytometry, cell counting assay, and ELISA demonstrated that the serum level of IL-17 was higher in AS patients compared to XXX, and 25 mu g/mL TP was the optimal intervention concentration. RT-qPCR and Western blotting further verified that TP could inhibit the activation of ROR gamma t and the JAK2/STAT3 signaling pathway.Conclusion: In conclusion, based on network pharmacology, molecular docking, and experimental verification in vitro, we proposed that the TP can inhibit the activation of ROR gamma t and the JAK2/STAT3 signaling pathway and inhibit the differentiation of T helper 17 cells cells. The article provide a theoretical basis for further development and utilization of TWHF in AS management.