Oral delivery of NOD2-activating Lactobacillus peptidoglycans prevents postmenopausal osteoporosis via gut microbiota modulation

Excessive osteoclast activity contributes to osteoporosis, a condition marked by reduced bone density. While certain probiotics have shown potential in enhancing bone mass, the specific bioactive components and their therapeutic efficacy remain unclear. In this study, we isolated peptidoglycans (PGNs) from eleven Lactobacillus species and evaluated their effects on ovariectomized (OVX) mouse models of osteoporosis. Oral administration of PGNs, particularly from Lactiplantibacillus plantarum, Lacticaseibacillus casei, and Lactobacillus ruminis, significantly restored trabecular bone volume in OVX mice. Among them, L. plantarum PGN (Lp.PGN) demonstrated the most potent effect, enhancing bone volume when administered intraperitoneally or intravenously. Histological analysis revealed increased Runx2-positive osteoblasts and reduced TRAP-positive osteoclasts in Lp.PGN-treated mice. Calcein double labeling confirmed enhanced new bone formation. In vitro, Lp.PGN promoted osteoblast mineralization and suppressed osteoclast differentiation in co-culture systems. Notably, Lp.PGN failed to improve bone mass in NOD2-deficient OVX mice, indicating that NOD2 signaling is essential for its osteoprotective effects. Additionally, Lp.PGN reduced pro-inflammatory cytokines and the RANKL/OPG ratio, and modulated gut microbiota composition by decreasing Proteobacteria and increasing Firmicutes, resembling the profile of sham-operated controls. In conclusion, NOD2-activating PGN attenuates bone loss under osteoporotic conditions by increasing osteoblast differentiation and suppressing osteoclast differentiation. Lp.PGN could play an important role in enhancing bone mass through NOD2 signaling, and NOD2 ligands could be considered as postbiotic-based therapeutic agents for the treatment of bone diseases.