Wutou decoction ameliorates rheumatoid arthritis through inhibiting hypoxia-induced invasive phenotype of fibroblast-like synoviocytes mediated by the Notch pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Despite its long history of use in traditional medicine for joint disorders, Wutou Decoction (WTD) has not been widely adopted as a complementary therapy for rheumatoid arthritis (RA) in modern practice. This is primarily due to a lack of robust pharmacological evidence and a clear understanding of its mechanisms of action. Further investigation into the mechanistic basis of WTD is essential to validate its efficacy and safety in the treatment of RA.

AIM OF THE STUDY: Fibroblast-like synoviocytes in rheumatoid arthritis (RA-FLS) display a highly invasive phenotype, contributing to progressive bone and cartilage destruction. Evidence suggests that crosstalk between the hypoxic microenvironment and the Notch signaling pathway plays a central role in driving this pathological behavior, highlighting both as potential therapeutic targets. This study aimed to determine whether WTD exerts its therapeutic effect on RA by disrupting hypoxia-induced activation of the Notch signaling pathway.

MATERIALS AND METHODS: A collagen-induced arthritis (CIA) model in rats and hypoxia-treated RA-FLS were employed. WTD used in this study consist of Aconitum carmichaelii Debeaux. (processed root, 6 g), Astragalus mongholicus Bunge. (root, 9 g), Ephedra sinica Stapf. (herbaceous stem, 9 g), Paeonia lactiflora Pall. (root, 9 g) and Glycyrrhiza uralensis Fisch. (root, 9 g). The anti-arthritic effects of WTD were assessed through arthritis scoring, spleen index, and hematoxylin-eosin staining and Safranin O/Fast Green). The involvement of the Notch pathway and invasive RA-FLS phenotype was evaluated by immunostaining, RT-qPCR, and western blotting.

RESULTS: WTD significantly attenuated synovial hyperplasia and cartilage degradation in CIA rats. In both CIA rat synovium and hypoxia-treated RA-FLS, WTD markedly inhibited activation of the HIF and Notch signaling pathways, along with the expression of their downstream target genes. Further analyses revealed that WTD suppressed hypoxia-induced RA-FLS invasiveness, including cartilage matrix degradation and pro-angiogenic activities, by blocking Notch signaling. Molecular docking suggested that active compounds in WTD may bind to the negative regulatory region domain of Notch receptors. Notably, WTD also reversed the hypoxia response induced by forced activation of the Notch pathway.

CONCLUSIONS: These findings elucidate the mechanism by which WTD alleviates RA, firstly demonstrating that it effectively inhibits the hypoxia-induced invasive phenotype of RA-FLS by targeting the Notch signaling pathway. However, this study has several limitations: the findings are based on a single batch of WTD, the predicted binding of active compounds to Notch receptors has not been experimentally validated, and the individual contributions of the candidate quality markers to the overall therapeutic effect remain to be systematically determined.