Bifidobacterium animalis RH exopolysaccharide bidirectionally modulates inflammatory bone metabolism disorders

Inflammatory dysregulation of bone metabolism underlies a range of skeletal pathologies, including osteoporosis, osteoarthritis, and periprosthetic loosening. Although probiotic metabolites demonstrate multifaceted nutritional benefits, the development of Bifidobacterium-derived extracellular polysaccharides remains limited. Consequently, we extracted extracellular polysaccharides from Bifidobacterium animalis RH (designated EPS, 21.3 kDa) and explored their therapeutic potential and mechanisms in inflammatory bone disorders. For in vitro models, we employed lipopolysaccharide (LPS)-stimulated MC3T3-E1 osteoblasts and RANKL-induced RAW 264.7 osteoclast precursors treated with increasing EPS concentrations. EPS restored LPS-impaired osteoblast function, enhancing proliferation, differentiation, mineralization, and collagen synthesis. This restoration was evidenced by increased ALP activity, calcium nodule formation, collagen expression, and suppressed pro-inflammatory cytokine secretion (IL-6, IL-1β, IL-17a/f). EPS mediated this osteogenic recovery by inhibiting the TLR4/NF-κB pathway and activating RUNX2 transcription. Concurrently, EPS attenuated osteoclastogenesis by reducing inflammatory cytokines (IL-6, IL-1β, TNF-α), inhibiting ROS generation, downregulating RANK expression, and disrupting the MITF/PU.1/NFATc1 transcriptional network governing key osteoclast-specific genes (e.g., TRAP, CTSK). This study systematically elucidated the dual regulatory mechanisms of EPS in inflammatory bone disorders, highlighting their promising therapeutic potential for skeletal pathologies and providing a foundation for developing in vivo models and dietary biotherapeutics.