Effects of Probiotic-Fermented Deer Bone Water Extract on Immune Regulation and Gut Microbiota in Rheumatoid Arthritis via the NOTCH Signaling Pathway

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease, and current treatments are limited by significant side effects. Deer bone, which is rich in proteins and various active compounds, possesses anti-inflammatory and bone-health-promoting properties. However, its fermented product's effects on RA treatment remain unexplored. In this study, we evaluated the therapeutic effects of probiotic-fermented deer bone aqueous extract (BbF) in an adjuvant arthritis (AA) rat model, combined with LPS-stimulated RAW264.7 macrophage models. In vivo experiments showed that BbF significantly reduced paw swelling, arthritis index, and improved bone mineral density. BbF also alleviated synovial hyperplasia and inflammatory cell infiltration. It suppressed pro-inflammatory cytokines (TNF-α, IL-1β, etc.) and inhibited macrophage migration and invasion. Transcriptomic analysis revealed significant enrichment of the Notch signaling pathway, and Western blot confirmed the downregulation of Notch3, Notch4, DLL4, and Jagged1 proteins. BbF also restored gut microbiota homeostasis, increasing beneficial bacteria such as <i>Firmicutes</i> and <i>Clostridia</i>, while decreasing potential pathogens like <i>Proteobacteria</i>, <i>Gammaproteobacteria</i>, and <i>Escherichia-Shigella</i>. Furthermore, BbF enhanced short-chain fatty acids (SCFCs) production, including butyrate and caproic acid. These results indicate that BbF alleviates RA by inhibiting the Notch signaling pathway and regulating gut microbiota, providing new insights for the development of functional foods with immune-modulatory properties.