Gut microbiota-derived lithocholic acid ameliorates estrogen deficiency-induced osteoporosis via inhibiting neutrophil extracellular trap formation

Postmenopausal osteoporosis (PMOP) is an estrogen deficiency-induced metabolic bone disorder featured by progressive bone loss and elevated fracture susceptibility. Neutrophil extracellular traps (NETs) and microbially derived bile acids (BAs) have recently emerged as central modulators of bone homeostasis in diverse skeletal disorders. Nevertheless, it remains largely unclear whether and how BAs regulate NETs formation through the “gut microbiota–metabolite–osteoimmunology” axis in PMOP conditions. In this study, we aimed to determine whether lithocholic acid (LCA), a microbiota-derived secondary BA, ameliorated ovariectomy (OVX)-induced osteoporosis via suppressing NETs formation in a gut microbiota-dependent manner. Here, we observed that NETs formation was markedly increased in bone marrow neutrophils of OVX mice, and targeted clearance of NETs using DNase I notably rescued the osteoporotic phenotype. Targeted BA metabolomics identified distinct reduced fecal LCA levels in OVX mice, which was inversely correlated with NETs formation and bone loss. Further exogenous LCA supplementation, antibiotic-induced microbiota depletion, and fecal microbiota transplantation experiments collectively substantiated that LCA inhibited NETs formation and ameliorated osteoporosis in a gut microbiota-dependent manner. Subsequent 16S rRNA sequencing and untargeted metabolomics revealed that LCA treatment profoundly remodeled gut microbial composition and downregulated microbial N-formylmethionine (fMET), a core metabolite derived from methionine/cysteine metabolism. Mechanistically, fMET facilitates NETs formation by activating the FPR1/SMARCA4/HIF-1α/PADI4 signaling axis. Targeted inhibition of SMARCA4 or HIF-1α efficiently suppressed NETs formation and substantially reversed the OVX-induced osteoporotic phenotype. Collectively, our findings indicate that LCA exerts anti-osteoporotic effects by modulating the “gut microbiota–metabolite–osteoimmunology” axis, thus representing a promising therapeutic candidate for PMOP. Schematic diagram illustrating the LCA-mediated anti-osteoporotic effects. LCA treatment reshapes the gut microbiota composition and metabolic profile, downregulates fMET levels, and thereby blocks fMET's pro-NETosis effect mediated by the FPR1/SMARCA4/HIF-1α/PADI4 axis.