ESRa-Mediated Redox Reprogramming by Daidzein Limits Osteoclast-Driven Bone Loss

Excessive osteoclast activation driven by estrogen deficiency is a central pathological feature of postmenopausal osteoporosis. Although daidzein (DZ), a soy-derived phytoestrogen, has been suggested to exert bone-protective effects, its precise molecular mechanism in osteoclast regulation remains incompletely defined. Here, we demonstrate that DZ markedly suppresses RANKL-induced osteoclast differentiation and resorptive activity without cytotoxicity. DZ downregulated osteoclast-specific genes and proteins, including NFATc1, c-Fos, MMP9, and CTSK, and inhibited NFATc1 nuclear translocation. Transcriptomic profiling revealed significant enrichment of redox-related pathways, suggesting modulation of oxidative stress. Mechanistically, DZ reduced intracellular and mitochondrial reactive oxygen species (ROS) accumulation by promoting Nrf2 nuclear translocation and enhancing antioxidant defense. Attenuation of ROS was accompanied by selective inhibition of NF-kB activation, as evidenced by reduced p65 phosphorylation and nuclear translocation, whereas MAPK signaling remained largely unaffected. Molecular docking, protein stability assays, and cellular thermal shift analysis identified estrogen receptor alpha (ESRa) as a direct functional target of DZ. Pharmacological inhibition of ESRa reversed the suppressive effects of DZ on ROS production, NF-kB activation, and osteoclastogenesis. In ovariectomized mice, oral DZ administration preserved trabecular bone mass, reduced osteoclast activity, restored redox balance, and exhibited no detectable systemic toxicity. Collectively, these findings establish an ESRa-centered redox-regulatory mechanism underlying the anti-osteoporotic action of DZ.