Bmal1 Attenuates Collagen-Induced Arthritis by Regulating Macrophage Polarization via Sirt1

Background: Rheumatoid arthritis (RA) is an inflammatory disease primarily affecting the joints, with macrophages playing a critical role in its pathogenesis. Bmal1 is a key regulator of macrophage polarization, but its role in RA remains unclear. This study aimed to define the role of Bmal1 in regulating macrophage polarization in RA and to elucidate the underlying molecular mechanisms. Methods: Synovial tissue and peripheral blood mononuclear cells (PBMCs) were used to assess Bmal1 expression. High-throughput transcriptome sequencing was performed to identify signaling pathways and key molecules in RAW264.7 cells following Bmal1 knockdown. The expression of Bmal1, macrophage polarization markers and pro-inflammatory cytokines was evaluated using quantitative real-time PCR, immunofluorescence, Western blotting, flow cytometry and ELISA. Results: Bmal1 expression was significantly reduced in RA patients, which was confirmed by the Gene Expression Omnibus (GEO) datasets GSE55235. Methotrexate (MTX) treatment was associated with increased Bmal1 expression. In vitro, Bmal1 overexpression inhibited M1 macrophage polarization and NF-κB signaling. Mechanistically, the JASPAR database predicted that Bmal1 binds to the Sirt1 promoter, which was confirmed by chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays. Bmal1 positively regulated Sirt1 expression in RAW264.7 cells. Sirt1 knockdown partially reversed the inhibition of M1 macrophage polarization and NF-κB signaling induced by Bmal1 overexpression. In vivo, Bmal1 expression was reduced in collagen-induced arthritis (CIA) mice, whereas its overexpression attenuated disease severity and was associated with increased Sirt1 expression and enhanced M2 macrophage polarization. Conclusion: Bmal1 attenuates inflammatory responses by regulating macrophage polarization, potentially through up-regulation of Sirt1 and inhibition of NF-κB signaling. These findings suggest that Bmal1 may represent a potential therapeutic target for RA. Keywords: Bmal1, rheumatoid arthritis, macrophage polarization, Sirt1, NF-κB signaling pathway