Urolithin A supplementation alleviates osteogenic disfunction and promotes bone fracture healing in inflammatory environments

Excessive and chronic inflammation can cause osteogenic dysfunction and disrupt the balance of the osteoimmune microenvironment, thereby increasing the risk of fracture non-union. Urolithin A (UA), a gut microbiota-derived metabolite produced from dietary sources, has been reported to inhibit RANKL-induced osteoclastogenesis and alleviate postmenopausal osteoporosis. However, the effect of UA on osteogenesis, particularly under pathogenic inflammatory conditions, remains unclear. In this study, mouse bone marrow-derived mesenchymal stromal cells (mBMSCs) were used to evaluate osteogenesis in vitro, and RAW 264.7 cells were used as macrophages in vitro. Tumour necrosis factor (TNF)-α was used to establish an inflammatory environment. In vivo, a mouse femur fracture model with local TNF-α injection was established, and UA or vehicle was administered by intragastric gavage. The UA showed no obvious effect on cell viability at concentrations ranging from 0 to 10 μM and had no direct effect on the osteogenic differentiation of mBMSCs. TNF-α treatment significantly decreased the expression of osteogenesis-related genes and proteins and inhibited calcium deposition, whereas UA reversed this inhibitory effect in a dose-dependent manner. Mechanistically, UA inhibited activation of the TNF-α-induced nuclear factor-κB signalling pathway. Furthermore, UA reduced pro-inflammatory cytokine levels and inhibited type-1 macrophage polarisation under TNF-α-induced inflammatory conditions. Conditioned medium derived from RAW 264.7 cells stimulated with TNF-α after UA treatment promoted the osteogenic differentiation of mBMSCs. In vivo, local administration of TNF-α significantly impaired bone fracture healing in the mouse femur fracture model, whereas intragastric supplementation with UA improved fracture healing and reduced pro-inflammatory responses. Collectively, these findings demonstrated that UA alleviates osteogenic dysfunction through inhibition of the nuclear factor-κB signalling pathway and regulation of macrophage-mediated inflammation under TNF-α–induced inflammatory conditions, thereby promoting osteogenesis and fracture healing.