2'-O-Galloylhyperin attenuates osteoclastogenesis and estrogen-deficiency osteoporosis by targeting MLK3-mediated NF-κB signaling.

Estrogen deficiency-induced osteoporosis is largely driven by excessive osteoclast formation and bone resorption, but the direct pharmacological targets of flavonoid derivatives in osteoclast differentiation remain unclear. Here, we investigated the anti-osteoclastogenic activity and molecular mechanism of 2'-O-Galloylhyperin (2'-O-GH), a structurally defined flavonoid derivative. The effects of 2'-O-GH on RANKL-induced osteoclastogenesis were evaluated in bone marrow-derived macrophages and RAW264.7 cells using TRAP staining, qPCR, western blotting, F-actin staining, immunofluorescence, and bone resorption assays. Target identification was performed using a biotinylated 2'-O-GH probe coupled with pull-down and LC-MS/MS analysis, followed by molecular docking and microscale thermophoresis. In vivo efficacy was assessed in an ovariectomy-induced osteoporosis mouse model. 2'-O-GH inhibited RANKL-induced formation of TRAP-positive multinucleated osteoclasts without evident cytotoxicity at effective concentrations. It downregulated osteoclast-related markers, including NFATc1, c-FOS, SRC, ACP5, ATP6V0D2, and CTSK, and impaired F-actin ring formation and bone resorption. Mechanistically, 2'-O-GH restrained RANKL-induced NF-κB p65 nuclear translocation. Chemical proteomics identified MAP3K11/MLK3 as a candidate target, and molecular docking and microscale thermophoresis supported its direct interaction with 2'-O-GH. 2'-O-GH further suppressed RANKL-induced phosphorylation of MLK3 and IKKα/β, whereas MLK3 overexpression partially rescued NF-κB activation and osteoclast differentiation. In ovariectomized mice, 2'-O-GH attenuated bone loss without obvious effects on body or uterine weight. These findings identify MLK3 as a pharmacological target of 2'-O-GH and suggest that 2'-O-GH suppresses osteoclastogenesis through the MLK3/IKK/NF-κB axis.