Abstract

Purpose: Estrogen deficiency drives bone loss through a complex interplay of endocrine, oxidative, inflammatory, and bone-remodeling disturbances. Ergothioneine (EGT) is a diet-derived thiol/thione antioxidant, yet its effects on the estrogen-deficient skeleton remain unknown. This study evaluated whether EGT-alone or combined with vitamin K2, vitamin D3, and magnesium L-threonate-attenuates the skeletal and systemic consequences of ovariectomy (OVX) in mice. Methods: Forty-eight female C57BL/6J mice underwent either sham surgery or OVX. For 12 weeks, the mice received a daily oral gavage of one of the following: vehicle, alendronate (1.53 mg/kg), EGT (30 mg/kg/day), EGT with vitamin K2 (40 g/kg/day) and vitamin D3 (500 IU/kg/day), or EGT with vitamin K2, magnesium L-threonate (350 mg/kg/day), and vitamin D3 (n = 5-6 per group). Outcomes evaluated included the uterine index, tibial micro-computed tomography (micro-CT), distal-femoral histology, serum bone turnover markers (CTX-I, PINP, osteocalcin), sex hormones, TNF-;, IL-6, SOD, and MDA. Results: OVX significantly lowered the uterine index and induced tibial trabecular deterioration. This was accompanied by increased CTX-I, TNF-;, IL-6, and MDA, alongside decreased PINP, osteocalcin, and SOD (all P < 0.01 vs. sham). Alendronate successfully restored tibial micro-CT bone-volume fraction (BV/TV) and trabecular number (P < 0.01 vs. OVX). While the EGT-based regimens did not significantly restore tibial micro-CT BV/TV, trabecular thickness, or trabecular number (all P > 0.05 vs. OVX), they significantly increased the trabecular area in distal-femoral histology (14.2-15.0% across regimens vs. 7.6% in OVX; P < 0.05). Furthermore, EGT treatments lowered CTX-I, TNF-;, IL-6, and MDA, increased SOD, and partially restored PINP and osteocalcin (P < 0.05 -0.01 vs. OVX). Apparent increases in serum estradiol were assay-dependent and are considered exploratory. Conclusion: EGT-based nutritional regimens improved the systemic oxidative, inflammatory, and bone -turnover environments associated with estrogen-deficient bone loss and successfully preserved the distal-femoral trabecular area. However, tibial three -dimensional microarchitecture was not restored on micro -CT. Given that histological and micro -CT endpoints were evaluated at different skeletal sites, structural interpretations should be made with caution. These findings support the further evaluation of EGT as a dietary adjunct, warranting subsequent mechanistic and dose-optimization studies.