Lacticaseibacillus rhamnosus LGG Suppresses Osteoclastogenesis via TLR6/NF-κB Modulation and Attenuates Ovariectomy-Induced Bone Loss in Mice

Osteoporosis is characterized by decreased bone mass and disrupted microarchitecture. Gut microbiota-derived factors may regulate bone homeostasis. This study investigated the effects and mechanisms of Lacticaseibacillus rhamnosus LGG, conditioned medium (LCM) on osteoclastogenesis and bone loss. RANKL-induced osteoclast differentiation in bone marrow-derived macrophages (BMMs) was assessed by TRAP staining, F-actin ring imaging, resorption pit assay, qRT-PCR, and Western blotting. Ovariectomized (OVX) mice received oral LCM for 8 weeks. Bone architecture was analyzed by micro-CT and histology (H&E, TRAP, immunohistochemistry). Serum bone turnover markers and toxicity indicators were measured by ELISA. Transcriptome sequencing was performed on LCM-treated BMMs, followed by differential expression and KEGG enrichment analyses. Pathway involvement was validated via pharmacological inhibition. LCM demonstrated favorable biocompatibility while significantly reducing TRAP-positive cell number, F-actin ring formation, and bone resorption area in RANKL-treated BMMs. The expression of osteoclastogenic markers was markedly downregulated. In OVX mice, LCM treatment preserved the trabecular microarchitecture of lumbar vertebrae and femur, increased BV/TV, Tb.Th, and Tb.N, and reduced osteoclast number. Serum bone resorption marker (β-CTx) decreased, while bone formation markers (BALP and P1NP) showed no significant change. No adverse effects were observed in body weight or liver and kidney function indices. Transcriptome analysis revealed NF-κB pathway suppression. Western blotting confirmed that LCM reduced phosphorylation of IKKα, IκBα, and p65. Regulation of TLR6 can restore NF-κB activation and osteoclast function. LCM alleviates bone loss by inhibiting osteoclastogenesis mediated via the TLR6/NF-κB signaling pathway. LGG shows promise as a potential therapeutic agent, warranting further clinical investigation.