Isonardosinone suppresses osteoclastogenesis through TRAF2/TRAF6-mediated disruption of calcium signaling and coordinate inhibition of MAPK/NF-κB pathways.

Background: Osteoporosis (OP), a prevalent age-related skeletal disorder, is characterized by increased osteoclast (OC) activity, leading to bone loss, reduced bone mineral density, and elevated fracture risk. Through combined in vivo and in vitro experiments, this study aimed to elucidate the mechanisms by which isonardosinone (INS) inhibits OC differentiation, thereby providing new insights for potential therapeutic applications. Methods: Initially, the optimal non-cytotoxic concentration of INS for bone marrow-derived macrophages (BMMs) was determined. The dose-dependent inhibitory effects of INS on OC differentiation were quantitatively assessed using tartrate-resistant acid phosphatase (TRAP) staining. Osteoclast-specific gene and protein expression profiles under INS treatment were analyzed via reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. Furthermore, fluorescence assays demonstrated that INS substantially inhibited the formation of F-actin rings and the nuclear translocation of phosphorylated p65 (p-p65), while also profoundly modulating the mitochondrial membrane potential in OC precursors and promoting the induction of apoptosis. Notably, these effects were accentuated with increasing concentrations of INS. To investigate the underlying mechanisms of INS-mediated suppression of mature OC formation, Western blot analysis was employed to evaluate the modulation of relevant signaling pathways. For translational validation, an ovariectomy (OVX)-induced osteoporosis murine model was established, with bone microarchitecture and molecular markers subsequently characterized using micro-computed tomography (Micro-CT) and immunohistochemistry. Results: INS at concentrations ≤20 μM demonstrated no cytotoxic effects on BMMs. TRAP staining revealed dose-dependent inhibition of mature OC formation, with maximal suppression observed at the highest tested concentration. RT-PCR and Western blot analyses confirmed significant reductions in OC differentiation markers, including CTSK, MMP9, NFATc1, and c-Fos.Concurrently, INS upregulates antioxidant defense enzymes (Nrf2, HO-1, SOD-1, catalase), contributing to its anti-osteoclastogenic effects. Mechanistically, INS exerted bidirectional regulation of the TRAF2-RIPK1-Ca²⁺ signaling axis, concurrently modulating intracellular calcium flux and extracellular calcium-sensing pathways to disrupt OC maturation. Micro-CT quantification and immunohistochemical profiling demonstrated that INS administration significantly preserved trabecular bone mass and reduced osteoclast activity in OVX mice, thereby corroborating the in vitro findings. Conclusion: INS aptly mitigates the process of osteoclastogenesis, leading to a notable enhancement in bone density, effectively exemplifying its pivotal role in the management of OP.