6-Hydroxyflavone Attenuates Inflammatory Osteolysis by Inhibiting Osteoclast Activation via the Nrf2 and Calcium Signaling Pathways

Inflammatory osteolysis (IO) is a pathological bone disorder characterized by excessive osteoclast activation and bone resorption driven by inflammatory mediators and oxidative stress. 6-Hydroxyflavone (6-HOF), a natural flavonoid with potent antioxidant and anti-inflammatory properties, has not been fully investigated in the context of IO. In this study, we explored the therapeutic effects and underlying mechanisms of 6-HOF in LPS-induced osteoclastogenesis and inflammatory bone loss. Network pharmacology analysis predicted that 6-HOF primarily targets oxidative stress and calcium signaling pathways. In vitro, 6-HOF inhibited multinucleated osteoclast formation in a concentration-dependent manner without affecting cell viability, downregulated osteoclast-specific genes including Nfatc1, Ctsk, Dc-stamp, and Mmp9, and significantly reduced the expression and secretion of pro-inflammatory cytokines IL-6, TNF-α, and IL-1β. Mechanistically, 6-HOF suppressed intracellular ROS accumulation, disrupted RANKL-induced Ca²⁺ oscillations, and inhibited NFATc1 signaling essential for osteoclast differentiation, while activating the Keap1/Nrf2 antioxidant pathway to restore redox homeostasis. In vivo, micro-CT analyses showed that 6-HOF treatment alleviated LPS-induced bone loss by reducing osteoclast numbers and preserving trabecular microarchitecture. Collectively, these results indicate that 6-HOF inhibits osteoclastogenesis and inflammatory responses through the dual regulation of oxidative stress, calcium signaling, and pro-inflammatory cytokine production, highlighting its potential as a promising therapeutic candidate for IO and other bone-destructive disorders.