Prim-O-glucosylcimifugin ameliorates rheumatoid arthritis by targeting GBP5 to suppress pyroptosis and inhibit synovial cell proliferation.

Rheumatoid arthritis (RA) is a chronic autoimmune disorder characterized by persistent synovial inflammation and progressive cartilage degradation, largely driven by the pathological activation of fibroblast-like synoviocytes (FLS). Prim-O-glucosylcimifugin (POG), a chromone derivative isolated from Saposhnikovia divaricata, has been shown to exert potent anti-inflammatory and anti-proliferative effects. However, its anti-RA effect and mechanism remain unclear. The present investigation aimed to evaluate the anti-RA activity of POG and delineate the underlying mechanisms. To determine the anti-proliferative effect of POG on tumor necrosis factor-alpha (TNF-α)-activated RA-FLS, cell counting kit-8 and western blot were performed. In a murine collagen-induced arthritis (CIA) model, the in vivo efficacy of POG was assessed via measurements of paw swelling, arthritis scores, and hematoxylin and eosin staining. POG attenuated RA progression by suppressing RA-FLS proliferation. Proteomics revealed that its anti-arthritic action involves modulating pyroptosis, which was examined using western blot, immunohistochemistry, enzyme-linked immunosorbent assay, and immunofluorescence. In vitro, POG reduced lactate dehydrogenase release and downregulated critical mediators of the P2X7R/NLRP3-pyroptosis axis. In the CIA model, POG decreased levels of interleukin (IL)-1β and IL-18, inhibited NLRP3 inflammasome activity, and alleviated synovial pyroptosis. Mechanistically, guanylate-binding protein 5 (GBP5) was confirmed as a POG target through molecular docking, molecular dynamics simulation, cellular thermal shift assay, cycloheximide chase assay, drug affinity responsive target stability, surface plasmon resonance, and small interfering RNA experiments. The POG-GBP5 interaction lowered GBP5 expression, diminishing its stimulatory effect on NLRP3 and ultimately blocking pyroptosis. In conclusion, POG exerts anti-arthritic effects by targeting the GBP5/P2X7R-NLRP3 axis to inhibit pyroptosis.