Gut Microbiota-Derived TMAO Drives MC3T3-E1 Senescence and Osteogenic Dysfunction via cGAS-STING-NF-κB Signaling: Implications for Age-Related Bone Loss.

Age-related osteoporosis is closely associated with osteoblast dysfunction, in which cellular senescence plays a key role. Trimethylamine N-oxide (TMAO), a gut microbiota-derived metabolite, is implicated in aging and metabolic diseases and has been linked to bone metabolism. However, whether TMAO impairs bone formation by regulating osteoblast senescence remains unclear. This study investigated the effects of TMAO on osteoblast senescence and osteogenic function, focusing on the cGAS-STING-NF-κB signaling axis. MC3T3-E1 cells were treated with TMAO to evaluate proliferation, cell cycle progression, senescence, and osteogenic differentiation. Cytosolic DNA release and activation of the cGAS-STING-NF-κB axis were assessed. In vivo, a chronic TMAO exposure model was established, combined with AAV9-mediated STING knockdown, and bone microarchitecture was analyzed by micro-CT. TMAO significantly inhibited proliferation and induced G0/G1 arrest in MC3T3-E1 cells without apparent cytotoxicity. It increased SA-β-gal-positive cells and upregulated senescence-associated markers, indicating a senescent phenotype. Functionally, TMAO suppressed osteogenic differentiation and mineralization and downregulated osteogenic proteins. Mechanistically, TMAO promoted abnormal release of mitochondrial DNA into the cytosol, activated the cGAS-STING pathway, and enhanced NF-κB signaling. STING overexpression exacerbated, whereas STING knockdown alleviated, TMAO-induced senescence and osteogenic impairment. NF-κB inhibition partially reversed these effects. In vivo, TMAO exposure impaired trabecular and cortical bone microarchitecture, which was partially improved by STING knockdown. TMAO induces osteoblast senescence and impairs osteogenic function, potentially via mtDNA-mediated activation of the cGAS-STING-NF-κB axis. These findings provide insight into age-related bone loss and suggest potential therapeutic targets.