Kaempferol alleviates inflammatory osteolysis by directly activating NRF2 in osteoclasts and modulating the immune microenvironment.

The mechanisms underlying inflammatory osteolysis are complex, with osteoclasts playing a pivotal role. The specific mechanism by which kaempferol (KE) acts on osteoclasts remains unclear. This study systematically investigated the mechanism of KE's action on osteoclasts. The results demonstrated that KE effectively inhibited osteoclast differentiation, function, and the expression of related genes. To elucidate the underlying mechanism, RNA sequencing and GSEA confirmed significant activation of the NRF2/HO-1 antioxidant pathway, while ROS and oxidative phosphorylation pathways were suppressed. Using NRF2 siRNA and the inhibitor ML385 to knock down or inhibit NRF2 function, we observed a marked attenuation of KE's inhibitory effect on osteoclasts, further confirming that its action primarily depends on this pathway. We confirmed the binding of KE to the NRF2 protein through SPR(KD = 7.03 μM), molecular docking (binding free energy of -28.2292 kcal/mol), and CETSA. Mechanistically, KE disrupted the KEAP1-NRF2 interaction, reduced NRF2 ubiquitination, and promoted its nuclear translocation. To validate the in vivo efficacy, in an LPS-induced mouse calvarial osteolysis model, KE treatment alleviated bone loss, inhibited osteoclast activity, and reduced local oxidative stress by activating the NRF2/HO-1 signaling pathway-all of which were reversed by ML385. Furthermore, considering the critical role of the immune microenvironment in inflammatory osteolysis, we confirmed through in vitro and in vivo experiments that KE also modulates macrophage polarization, thereby inhibiting inflammatory cytokine levels. In summary, this study identifies NRF2 as a direct molecular target of KE. The protective effects are achieved primarily through NRF2/HO-1 axis activation to inhibit osteoclastogenesis. This provides a solid experimental and theoretical foundation for KE as a potential therapeutic candidate for inflammatory osteolytic diseases.