Genistein alleviates rheumatoid arthritis by inhibiting fibroblast-like synovial exosome secretion regulated by the Rab27/nSMase2/Mfge8 pathway

Genistein (GEN), the predominant soy isoflavone in legumes, exhibits potential anti-rheumatoid arthritis (RA) effects. This study aims to explore the role of GEN in alleviating RA by regulating exosome secretion and the inflammatory microenvironment through the Rab27/nSMase2/Mfge8 pathway in collagen-induced arthritis (CIA) mice and in vitro. In vivo studies revealed that GEN treatment significantly reduced paw swelling in CIA mice and protected the integrity of knee and ankle joints in CIA mice. GEN supplementation caused a significant decrease in the levels of MMP-9 and pro-inflammatory factors TNF-α, IL-1β and IL-6. GEN also significantly diminished the expressions of β-catenin and exosomal Dvl3 and miR-221-3p in fibroblast-like synoviocytes (FLS). Molecular docking results showed that GEN had strong binding energy with Rab27a, nSMase2 and Mfge8, the key regulators of exosome secretion, respectively, which was confirmed by CETSA and DARTS detection. In vitro mechanism analysis demonstrated that GEN treatment simultaneously downregulated the expression of Rab27a, nSMase2 and Mfge8, and phenotypic analysis verified that GEN prevented the secretion of Alix⁺Hsp70⁺CD63⁺ exosomes induced by type II collagen (CII) from FLS. Further analysis showed that GEN inhibited the expression of the Wnt signaling pathway protein β-catenin and exosomal Dvl3 in FLS. Additionally, GEN inhibited CII-induced secretion of MMP-9 and TNF-α, IL-1β and IL-6 in FLS, and GEN also significantly inhibited CII-induced FLS migration. Notably, GEN inhibited the expression of miR-221-3p in FLS exosomes and enhanced osteoblast differentiation and mineralization in vitro. Collectively, this study clarified that GEN alleviates RA by inhibiting the secretion of FLS exosomes regulated by the Rab27/nSMase2/Mfge8 pathway and by inhibiting Dvl3/β-catenin and miR-221-3p.