Trifolirhizin alleviates bone destruction in rheumatoid arthritis by inhibiting osteoclast differentiation through the activation of FPR2

Background: Progressive bone erosion in rheumatoid arthritis (RA) is a major driver of irreversible disability, yet current disease-modifying antirheumatic drugs (DMARDs) are insufficient to prevent this destructive process. Trifolirhizin (Trif), a pterocarpan-type flavonoid glycoside isolated from Sophora tonkinensis Gagnep., inhibits osteoclast differentiation and prevents inflammatory bone loss in mice. However, the effect of Trif on RA-associated bone destruction and its underlying mechanism remain unclear.

Purpose: We aimed to evaluate the antiarthritic bone destruction activity of Trif and explore its mechanism in collagen-induced arthritis (CIA) mice and in RANKL-induced osteoclasts.

Methods: To evaluate the antiarthritic effect of Trif, we analysed the incidence and score of arthritis. The inhibitory effect of Trif on arthritis-related bone destruction in vivo was systematically evaluated using micro-CT, histological analysis, and Western blot. The effects of Trif on RANKL-induced osteoclast differentiation and function were validated in vitro through tartrate-resistant Acid Phosphatase (TRAP) staining, F-actin ring staining, and bone resorption assays. Transcriptomic analysis was performed to investigate the mechanism by which Trif ameliorates bone destruction in RA. Furthermore, the potential molecular targets through which Trif regulates osteoclast differentiation were further elucidated using quantitative Real-Time PCR (qPCR), Western blot, immunofluorescence, pharmacological inhibition, and siRNA knockdown.

Results: In CIA mice, Trif markedly attenuated synovial inflammation, reduced osteoclast accumulation within joint tissues, and preserved trabecular bone architecture, thereby limiting structural destruction. In vitro, Trif suppressed osteoclast bone resorption by decreasing TRAP-positive cell formation, disrupting F-actin ring assembly, inhibiting osteoclast differentiation, and downregulating osteoclast-specific transcriptional programs. Integrated RNA Sequencing, qPCR validation, molecular dynamics simulation, and cellular thermal shift assay (CETSA) data consistently identified formyl peptide receptor 2 (FPR2) as a key mediator of the protective effects of Trif. Mechanistically, Trif enhanced FPR2 expression and blocked the activation of the TRAF6/MAPK/NFATc1 signalling axis, thereby restraining osteoclastogenesis. Importantly, pharmacological inhibition of FPR2 with WRW4 in CIA mice or siRNA-mediated FPR2 silencing in osteoclast precursors abolished the therapeutic benefits of Trif, indicating that FPR2 is an indispensable target.

Conclusion: These findings reveal that Trif, through activating FPR2, plays a previously unrecognized role in suppressing osteoclast-driven bone destruction, underscoring its therapeutic potential in RA.

Bone destruction; Formyl peptide receptor 2; Rheumatoid arthritis; Trifolirhizin.