Ursolic Acid Ameliorates Osteoporosis via Gut Microbiota Remodeling and Enrichment of Probiotic Species

Background: The association between osteoporosis (OP) and gut microbiota (GM) has been extensively investigated to identify novel probiotics for disease management for effective OP treatment remains imperative. Ursolic acid (UA), a natural triterpenoid compound, has been shown to reduce bone loss caused by ovariectomy (OVX); however, the contribution of GM in this reduction remains unclear. Therefore, this study investigates the impact of UA on OP and demonstrates that it ameliorates disease by remodeling the GM, with metagenomic sequencing identifying potential probiotics that likely mediate these effects. Methods: OVX mice received daily UA (200 mg/kg) or vehicle for 7 weeks. Bone mass and microarchitecture were assessed via microcomputed tomography (micro-CT) and Alisin Blue Haematoxylin/Orange G (ABH) staining. Bone formation (Runx2, alkaline phosphatase (ALP), osteocalcin (OCN) immunohistochemistry) and resorption (tartrate-resistant acid phosphatase (TRAP) staining) were evaluated in these treated mice. Fecal microbiota transplantation (FMT) from UA-treated donors to antibiotic-pre-treated OVX recipients was performed to test GM causality. The gut microbial composition was analyzed using metagenomic sequencing, with key species identified via Linear Discriminant Analysis Effect Size (LEfSe) and correlated with bone parameters. To further investigate the impact of <i>Parabacteroides goldsteinii</i> (<i>P. goldsteinii</i>) on bone mass, OVX mice were administered either live <i>P. goldsteinii</i>, pasteurized <i>P. goldsteinii</i> or PBS via oral gavage daily for 6 weeks. Micro-CT analysis was used to evaluate the effect of <i>P. goldsteinii</i> on bone mass. Results: UA treatment significantly increased bone mineral density (BMD, <i>p</i> < 0.01), bone volume fraction (BV/TV, <i>p</i> < 0.05), and trabecular thickness (Tb.Th, <i>p</i> < 0.001), while decreasing trabecular separation (Tb.Sp, <i>p</i> < 0.01) in OVX mice. Critically, FMT from UA-treated donors recapitulated these osteoprotective effects in recipient OVX mice, confirming the mediating role of GM. Metagenomic analysis revealed that UA significantly altered GM structure, enriching <i>Akkermansia muciniphila</i> (<i>A. muciniphila</i>, <i>p</i> < 0.01) and <i>P. goldsteinii</i> (<i>p</i> < 0.01). Abundance of <i>A. muciniphila</i> strongly correlated with improved BV/TV (<i>p</i> < 0.01), and Tb.Th (<i>p</i> < 0.05), and reduced Tb.Sp (<i>p</i> < 0.01). <i>P. goldsteinii</i> abundance also showed a significant positive correlation with BMD (<i>p</i> < 0.05) and a negative correlation with Tb.Sp (<i>p</i> < 0.01). Furthermore, <i>P. goldsteinii</i> significantly increased the BMD of the distal femur (<i>p</i> < 0.05), BV/TV (<i>p</i> < 0.05), and Tb.Th (<i>p</i> < 0.01), while decreasing Tb.Sp (<i>p</i> < 0.001) in OVX mice. Conclusion: GM contributes to the protective effect of UA against osteoporosis, mediated by the enrichment of specific probiotics such as <i>A. muciniphila</i> and <i>P. goldsteinii</i>. This study provides the first direct evidence that <i>P. goldsteinii</i> supplementation protects against bone loss, making it a promising probiotic candidate for managing OP.