Rhusflavone Modulates Osteoclastogenesis Through RANKL-Induced AKT Signaling in Bone Marrow-Derived Macrophages

Osteoclast differentiation inhibition is a viable treatment strategy for osteoporosis because osteoclasts play a vital role in disease progression. Rhusflavone (Rhus), a biflavonoid, exhibits a sedative-hypnotic effect via the positive allosteric modulation of GABA(A) receptors. Although several biflavonoids possess activities that help prevent bone loss, the potential effects of Rhus on osteoclastogenesis have not been reported yet. In this study, we investigated the effects and underlying biological mechanisms of Rhus isolated from the dried roots of <i>Rhus succedanea</i> on osteoclastogenesis in primary cultured bone marrow-derived macrophages. No cytotoxicity was observed in bone marrow macrophages (BMMs) or during osteoclast differentiation. However, Rhus reduced the number of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear osteoclasts during receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastogenesis. The results of F-actin ring formation demonstrated that Rhus suppresses the bone resorption activity of osteoclasts. Additionally, Rhus inhibits the expression of osteoclast differentiation marker proteins, specifically c-Fos and NF-ATc1. Western blot analysis revealed that Rhus primarily attenuated RANKL-mediated key signaling pathways, particularly the AKT signaling pathway. Furthermore, we found that the AKT activator and inhibitor pharmacologically abolished and enhanced the inhibitory effects of Rhus on osteoclast differentiation, respectively. Taken together, our findings provide evidence that Rhus is a promising biologically active compound that regulates osteoclast differentiation by inhibiting the AKT signaling pathway, which may contribute to future drug development.