Geniposide alleviates rheumatoid arthritis by inhibiting pyroptosis by regulating the miR-223-3p/NLRP3 Axis

Abstract

Geniposide (GE) has anti-inflammatory effects; however, its mechanism of action in RA is unclear. This study examined the protective properties of GE against RA and explored its possible molecular mechanisms. The effectiveness of treatment of GE was measured using a using a type II collagen-induced arthritis (CIA) model. Paw swelling, arthritis score, and histopathological alterations in the ankle joint, serum levels of autoantibodies inflammatory cytokines, and Western blotting (WB), were used to determine GE's efficacy in CIA rats. In vitro experiments were performed using flow cytometry, TUNEL assays, and WB to detect expression levels of NLRP3, GSDMD-N, Cleaved-Caspase-1. Direct binding of miR-2233p to NLRP3 was experimentally validated using dual-luciferase reporter systems. Subsequently, RT-qPCR and WB were used to measure protein levels. Our results demonstrated that GE significantly alleviated the symptoms of arthritis in CIA rats, with reduced paw swelling, lower arthritis scores, and attenuated histopathological joint damage. GE treatment suppressed serum levels of anti-CII autoantibodies, TNF-α, IL-1β, and IL-18, while downregulating NLRP3, Caspase-1, and GSDMD expression in the ankle joint. These findings were corroborated in LPS/ATP induction of RAW264.7 macrophages, where GE similarly attenuated pyroptosis markers. Dual-luciferase reporter assays confirmed that NLRP3 was a key target point of miR-223-3p. Furthermore, Western blot and RT-qPCR test results confirmed miR-223-3p as the key target of GE suppression of NLRP3 inflammasome activation and pyroptosis. Collectively, our findings indicate that GE Alleviates RA by inhibiting pyroptosis through the regulation of the miR-2233p/NLRP3 axis, highlighting its potential as a valid therapeutic agent for RA.