Hydroxygenkwanin exerts osteoprotective effects by regulating LRG1-PJA1-PML axis

Osteoporosis (OP), particularly postmenopausal OP (PMOP) driven by estrogen decline, is a prevalent chronic disorder causing severe bone loss and elevated fracture risk, posing a critical global public health challenge. Current therapies (hormone replacement, bisphosphonates) are limited by gastrointestinal reactions, musculoskeletal pain, ocular symptoms, renal impairment, and high costs, highlighting the need for safer, economical natural product-based treatments. Hydroxygenkwanin (HGK), a flavonoid from Daphne genkwa with antitumor, anti-inflammatory, and neuroprotective activities, is understudied in OP. RNA sequencing of femoral tissue from SHAM, ovariectomized (OVX), and OVX + HGK-treated mice identified leucine-rich alpha-2-glycoprotein 1 (LRG1) as the most significantly altered gene, prompting investigation of HGKs mechanism targeting LRG1 in OP. UbiBrowser analysis and CO-IP confirmed E3 ubiquitin ligase PJA1 mediates ubiquitin-dependent degradation of osteogenesis regulator PML. In vivo, HGK mitigates OVX-induced bone loss via the novel LRG1-PJA1-PML axis, significantly improving bone microstructural parameters and increasing the expression of the bone formation related markers. In vitro, using H2O2-treated MC3T3-E1 cells, HGK directly binds to LRG1 to upregulate its expression, enhancing LRG1-PJA1 interaction via adopting molecular docking analysis, root mean square deviation (RMSD), a cellular thermal shift assay (CETSA) and a biotin-streptavidin pull-down assay. This sequesters PJA1, inhibiting PML ubiquitination and degradation, which causes stabilized PML to promote osteogenic differentiation. These findings establish a causal link between HGK and the LRG1-PJA1-PML axis, providing preclinical evidence for HGK as a promising OP candidate with a well-defined mechanism.