Rehmannioside D prevents estrogen-deficiency induced osteoporosis by interacting with c-Jun to dismantle the AP-1 complex and suppress MAPK/NF-κB signaling

Background: Postmenopausal osteoporosis (PMOP) necessitates novel therapeutics with defined targets. Rehmannioside D (RD), a bioactive compound from Rehmannia glutinosa, exhibits anti-inflammatory properties, yet its anti-osteoporotic mechanism remains unclear.

Purpose: This study integrated network pharmacology with experimental validation to elucidate the efficacy and mechanism of RD in suppressing osteoclastogenesis.

Methods: Potential targets were screened via network pharmacology. In vitro, the effects of RD on RANKL-induced osteoclast differentiation and function were assessed in bone marrow-derived macrophages (BMDMs). Target interaction was verified using Cellular Thermal Shift Assay (CETSA), molecular docking, and Co-IP. In vivo, osteoprotective efficacy was evaluated in ovariectomized (OVX) mice.

Results: Network analysis highlighted c-Jun and c-Fos as core targets. In vitro, RD significantly inhibited osteoclastogenesis and resorptive function without cytotoxicity. Mechanistically, RD exerted a dual inhibitory effect: It suppressed the activation of MAPK and NF-κB signaling pathways, thereby downregulating NFATc1 expression and inhibiting osteoclast differentiation; Distinctly, RD was identified to interact with and stabilize to c-Jun, sterically hindering its heterodimerization with c-Fos to dismantle the AP-1 complex. This disruption of AP-1 subsequently triggered intrinsic Apoptosis, evidenced by an elevated Bax/Bcl-2 ratio and Caspase-3 cleavage. In vivo, RD prevented trabecular bone loss and preserved microarchitecture in OVX mice by reducing osteoclast numbers.

Conclusion: RD attenuates bone loss via a dual mechanism: it inhibits osteoclast differentiation by suppressing MAPK/NF-κB signaling and simultaneously induces mature osteoclast Apoptosis by targeting c-Jun to disrupt the AP-1 complex.