Therapeutic potential of xanthohumol in senile osteoporosis: mTOR-driven regulation of AKT/mTOR/p70S6K autophagy axis in D-galactose models.

Background: Senile osteoporosis (SOP) is a bone disorder characterized by bone loss, structural deterioration, and increased fracture risk. Xanthohumol (XAN), a bioactive isoflavone derived from Humulus lupulus L., shows potential bone-protective properties.

Purpose: This study aimed to evaluate the anti-SOP efficacy of XAN and clarify its mechanisms through multidisciplinary approaches.

Methods: A SOP mouse model was established using d-galactose (D-gal), followed by a 12-week XAN treatment. Animal procedures were approved by the Ethics Committee of Naval Military Medical University (No. 202130301). Pharmacological evaluations were conducted using the Morris water maze and Micro-CT. Mechanisms were predicted through integrated transcriptome analysis and metabolomics. In vitro, a d-gal-induced SOP model was established in MC3T3-E1 osteoblasts. The autophagy inhibitor 3-methyladenine (3-MA) or beclin-1 siRNA was applied to evaluate the effect of XAN on osteoblast function. Target identification was performed using drug affinity responsive target stability (DARTS), molecular docking, molecular dynamics, cellular thermal shift assay (CETSA), and microscale thermophoresis (MST) to assess the targeting action of XAN.

Results: XAN improved bone quality and cognitive function in aging mice, demonstrating its potent anti-SOP effects. Metabolomics and femur immunohistochemistry indicated that XAN mitigated bone loss primarily by AKT/mTOR/p70S6K activation. In vitro, XAN enhanced cell differentiation, promoted mineralized nodule formation, and reduced apoptosis and senescence in d-gal-injured osteoblasts. These cytoprotective effects were counteracted by autophagy inhibition with 3-MA or beclin-1 siRNA. Moreover, XAN promoted autophagosome flux toward autolysosome, up-regulated beclin-1, and down-regulated key proteins in the AKT/mTOR/p70S6K pathway. Importantly, binding assays identified mTOR as a direct target of XAN, which was further validated in vivo using the mTOR-specific agonist MHY1485 and inhibitor rapamycin.

Conclusions: Our study innovatively reveals that XAN prevents age-related bone loss by targeting mTOR and regulating the AKT/mTOR/p70S6K autophagy axis. This work provides the first mechanistic evidence supporting the potential of XAN as a natural therapeutic agent for SOP.