Liquiritigenin suppresses osteoclastogenesis via multi-target mechanisms involving NF-κB/PI3K-AKT signaling pathways and metabolic reprogramming.

Osteoporosis (OP) is a prevalent systemic metabolic disease characterized by reduced bone density and compromised skeletal integrity, leading to increased fragility and fractures. This study investigated the therapeutic potential of Liquiritin (LIQ) in inhibiting osteoclastogenesis through integrated computational and experimental approaches. Molecular docking and dynamics simulations were employed to explore the interactions between LIQ and key osteoclastogenic proteins RANK and RANKL. In vitro experiments assessed LIQ's inhibitory effects on mature osteoclast (OC) formation using RNA sequencing, cellular immunofluorescence, surface plasmon resonance (SPR), cellular thermal shift assays (CETSA), and Western blot analysis. In vivo studies validated the effects of LIQ on OC formation and bone loss. The results demonstrated that LIQ (≤20 μM) exhibited no cytotoxicity to bone marrow-derived macrophages (BMMs) while potently suppressing mature OC formation. LIQ downregulated OC-specific proteins and inhibited phosphorylation in the MAPK, NF-κB, and PI3K pathways. RNA sequencing revealed that LIQ modulates mitochondrial oxidative phosphorylation and induces OC precursor apoptosis, which was confirmed by immunofluorescence and OCR/ECAR assays indicating metabolic reprogramming. Molecular docking and dynamics simulations demonstrated stable LIQ binding to RANK/RANKL, disrupting their interaction and downstream TRAF2/RIPK signaling, as verified by SPR, CETSA, and Western blot analyses. In vivo experiments confirmed that LIQ able significantly attenuated bone loss in an OVX mouse model. These findings indicate that LIQ inhibits OC formation by binding to RANK/RANKL, suppressing MAPK/NF-κB/PI3K pathway activation, and inducing metabolic reprogramming and apoptosis in OC precursors, supporting its potential as a therapeutic candidate for OP.