Epimedin A inhibits the PI3K/AKT/NF-κB signalling axis and osteoclast differentiation by negatively regulating TRAF6 expression

Objective : Epimedin A (EA) has been indicated to suppress the extensive osteoclastogenesis and bone resorption, of which the effects remain incompletely understood. The aim of our study was to investigate the effects of EA on osteoclastogenesis and bone resorption, so as to explore its corresponding signal pathway. Methods : Rats were randomly received sham-operation and ovariectomy, with alendronate as the positive group. The therapeutic effect of EA on osteoporosis was systematically analyzed by measuring bone mineral density and bone biomechanical properties. In vitro , RAW264.7 cells were treated with receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) to induce osteoclast differentiation. Cell viability assay, tartrate-resistent acid phosphatase (TRAP) staining, and immunofluorescence were used to elucidate the influences of EA on osteoclastogenesis. In addition, the expression of bone differentiation related proteins or genes were evaluated using western blot analysis or quantitative polymerase chain reaction (PCR), respectively. Results : After 3 months of oral EA intervention, OVX rats showed increased bone density, relative bone volume, trabecular thickness, and trabecular number, along with reduced trabecular separation. EA dose-dependently improved bone density and trabecular microarchitecture in de-ovulated rats. Additionally, EA inhibited expressions of TRAP and NFATc1 in OVX rats. In addition, the in vitro results indicated that EA may inhibit osteoclast differentiation by suppressing the TRAF6/PI3K/AKT/NF-κB pathway. Further studies revealed that osteoclast differentiation, which was originally inhibited by EA, was reversed when the TRAF6 gene was overexpressed. Conclusion : The findings indicated that EA may hold promise as a potential therapeutic agent for the treatment of osteoporosis.