Monotropein synergizes with methotrexate to attenuate synovial inflammation in adjuvant-induced arthritis mice and fibroblast-like synoviocyte via GSK-3β

Background: Methotrexate (MTX) is the preferred drug for the treatment of rheumatoid arthritis (RA), but the toxic effects of long-term use limited its widespread clinical practice. The combination therapy of MTX with natural products is an effective strategy to reduce the toxic effects of MTX and enhance the therapeutic efficacy. Monotropein (MON), an iridoid glycosides, has potential therapeutic effects in inflammatory bone loss and osteoarthritis, and liver injury caused by MTX. However, whether MON can increase the therapeutic effects of MTX on RA and the related mechanism is not clear.

Purpose: This study aimed to clarify the MON enhancement on MTX in the treatment of RA, and also explore the target and mechanism of MON in anti-RA.

Methods: The adjuvant-induced arthritis (AA) mice and RA-fibroblast-like synoviocytes (RA-FLSs) were used to evaluate the alleviating effects of MON, MTX and their combination on RA. The network pharmacology analysis, molecular docking and surface plasmon resonance (SPR) were utilized to predict the potential therapeutic targets and mechanism of MON on RA. The interference RNA was used to knockdown the target gene in TNF-α-stimulated FLSs and AA mice to validate the mechanism of MON on RA.

Results: MON synergizes with MTX to suppress synovial inflammation in AA mice and inhibit TNF-α-induced FLSs proliferation, migration, inflammatory factors production, as well as the activation of NF-κB and JAK2/STAT3 signaling pathways. Network pharmacology predicted GSK-3β as a potential target of MON, and the molecular docking, CETSA, and SPR assays exhibited that MON had a good affinity with GSK-3β. Furthermore, GSK-3β knockdown suppressed TNF-α-induced FLSs proliferation, migration, matrix metalloproteinase 2 (MMP2) and MMP3 expression, and the activation of NF-κB and STAT3 signaling, while MON lost its inhibitory effects in GSK-3β knockdown FLSs. In addition, joint tissue-specific GSK-3β knockdown in AA mice alleviated synovial inflammation, decreased paw swelling and arthritis score, suppressed the phosphorylation of p65 and STAT3 of joint tissue in AA mice, while MON lost efficacy in GSK-3β-knockdown mice.

Conclusion: MON synergizes with MTX to inhibit synovial inflammation in AA mice and RA-FLSs inflammation by targeting GSK-3β to suppress NF-κB and STAT3 pathways.

Fibroblast-like synoviocytes; GSK-3β; Methotrexate; Monotropein; Rheumatoid arthritis.