RARRES2 deficiency inhibits postmenopausal osteoporosis by promoting ferroptosis-mediated osteoclast differentiation via Nrf2/GPX4 axis inhibition

Background This study aimed to investigate the effects of retinoic acid receptor responder 2 (RARRES2) on osteoclast differentiation in postmenopausal osteoporosis (PMOP) and to elucidate the underlying mechanisms.Methods A rat model of PMOP was induced by bilateral ovariectomy (OVX) method, and an in vitro osteoclast differentiation model was induced by treating RAW264.7 cells with nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Femoral bone structure, biomechanical properties, histology, osteoclast activity, co‑localization of RARRES2/CMKLR1 with TRAP+ osteoclasts and Perilipin‑1+ bone marrow adipocytes, gene expression, Fe2+ levels, reactive oxygen species (ROS) level, and lipid peroxidation level were detected.Results OVX model rats exhibited significant trabecular bone loss, compromised bone biomechanical properties, increased osteoclast numbers, elevated osteoclast differentiation markers, and upregulated RARRES2 expression compared with Sham controls. Co‑localization immunofluorescence revealed that RARRES2 was predominantly co‑localized with TRAP+ osteoclasts and Perilipin‑1+ bone marrow adipocytes. In vitro, RARRES2 knockdown markedly suppressed osteoclast differentiation, as evidenced by decreased osteoclast-related markers (NFATc1, CTSK, c-Fos, and F-actin) and reduced TRAP activity. Mechanistically, RARRES2 knockdown increased ferroptosis in osteoclasts, manifesting by decreased Nrf2, GPX4, HO-1, NQO1, and FTH1, along with increased ACSL4, Fe2+ accumulation , ROS production, and lipid peroxidation. These findings were also confirmed in vivo. Activation of Nrf2 with DMF partially reversed the ferroptotic alterations and osteoclast‑inhibiting effects induced by RARRES2 knockdown both in vitro and in vivo.Conclusion RARRES2 knockdown inhibits osteoclast differentiation and alleviates PMOP progression by suppressing the activation of Nrf2/GPX4 and promoting osteoclast ferroptosis.