3-Methoxychrysazin suppresses RANKL-induced osteoclastogenesis by modulating Notch2 signaling

Bone homeostasis is maintained by a delicate balance between osteoblast and osteoclast activity, and disruption of this equilibrium leads to bone loss and associated disorders. Oxidative stress and dysregulation of key signaling pathways, such as Notch2 and NF-κB, play pivotal roles in promoting osteoclastogenesis and pathological bone resorption. As part of our ongoing search for bioactive natural products, this study investigated the EtOAc extract of the culture broth of the bioluminescent bacterium Photorhabdus luminescens, leading to the HPLC-purified isolation of 3-methoxychrysazin (3-MC), which was structurally characterized by NMR spectroscopy and HR-ESI-MS. This study subsequently evaluated the effects of 3-MC on RANKL-induced osteoclast differentiation and oxidative stress in RAW264.7 cells. Treatment with 3-MC significantly and dose-dependently inhibited osteoclast formation, function, and migration, as evidenced by reduced numbers of TRAP-positive multinucleated cells, impaired F-actin ring formation, and suppressed expression of osteoclast-related genes, including dcstamp, ctsk, acp5, and atp6v0d2. In addition, 3-MC attenuated oxidative stress by lowering intracellular ROS levels and restoring the expression of antioxidant enzymes such as SOD and CAT. Mechanistically, 3-MC inhibited the Notch2 signaling pathway, demonstrated by the downregulation of Notch2 and Hes1, and suppressed the NF-κB pathway by reducing phosphorylation of p65 and IKKα. Taken together, these findings indicate that 3-MC mitigates osteoclast differentiation and oxidative stress, accompanied by downregulation of the Notch2 signaling axis, highlighting its potential as a promising therapeutic candidate for bone-related disorders.