Carpaine ameliorates synovial inflammation by promoting p65 degradation and inhibiting the NF-κB signalling pathway.

Aims: Osteoarthritis (OA) is a chronic and debilitating joint disease. Despite its prevalence, especially in ageing and obese populations, effective treatments targeting the molecular mechanisms of OA are limited. This study aimed to investigate the role of carpaine (CP), a major alkaloid from the Carica papaya leaf, in inhibiting articular cartilage destruction and synovitis during OA progression, and to elucidate the underlying molecular mechanisms.

Methods: CP (purity > 98%) was dissolved in dimethyl sulfoxide (DMSO). Various antibodies and reagents were sourced from Sigma-Aldrich, Abcam, and other suppliers. Peritoneal macrophages (pMACs) were cultured in Dulbecco's Modified Eagle Medium (DMEM) and treated with CP to assess its effects on inflammatory cytokine production and nuclear factor-kappa B (NF-κB) signalling. A total of 40 ten-week-old male C57/BL6 mice underwent destabilization of the medial meniscus (DMM) surgery to induce OA. Post-surgery, mice were treated with CP (0.5 or 3 mg/kg) or vehicle via intra-articular injections for up to ten weeks. Cartilage degradation and synovitis were evaluated using Safranin O, Fast Green staining, haematoxylin and eosin (H&E) staining, immunohistochemistry, and quantitative polymerase chain reaction (PCR).

Results: CP treatment significantly reduced cartilage degeneration and maintained hyaline cartilage thickness compared to the vehicle group. Indicators of cartilage degeneration, such as collagen X (Col X) and matrix metallopeptidase 13 (MMP13), were markedly decreased in the CP-treated group. CP-treated mice exhibited significantly lower synovitis scores at both five and ten weeks post-DMM surgery. CP prominently decreased the production of proinflammatory cytokines (interleukin (IL)-1β, IL-6) in M1 polarized macrophages both in vitro and in vivo. CP impeded NF-κB signalling by promoting p65 degradation through the E3 ubiquitin ligase LRSAM1. The defensive effect of CP was reversed by Lrsam1 small interfering RNA (siRNA), confirming the role of LRSAM1 in CP-mediated NF-κB inhibition.

Conclusion: CP acts as a 'physiological brake' on NF-κB activation, thereby mitigating synovial inflammation and cartilage destruction in OA. These findings suggest that targeting synovitis via CP could be a promising therapeutic strategy for OA.