18β-Glycyrrhetinic Acid Inhibits Osteoclastogenesis In Vivo and In Vitro by Blocking RANKL-Mediated RANK-TRAF6 Interactions and NF-κB and MAPK Signaling Pathways

Bone metabolism is determined by a delicate balance between bone resorption by osteoclasts and bone formation by osteoblasts. The imbalance due to over-activated osteoclasts plays an important role in various diseases. Activation of NF-kappa B and MAPK signaling pathways by receptor activator of nuclear factor-kappa B ligand (RANKL) is vital for osteoclastogenesis. Here, we for the first time explored the effects of 18 beta-glycyrrhetinic acid (18 beta-GA), a pentacyclic triterpenoid found in the Glycyrrhiza glabra L roots, on RANKL-induced osteoclastogenesis, osteoclast functions and signaling pathways in vitro and in vivo. In bone marrow monocytes (BMMs) and RAW264.7 cells, 18 beta-GA inhibited osteoclastogenesis, decreased expression of TRAP, cathepsin K, CTR and MMP-9, blocked actin ring formation and compromised osteoclasts functions in a dose-dependent manner at an early stage with minimal effects on osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). For underlying molecular mechanisms, 18 beta-GA inhibited RANKL-induced phosphorylation of p65, p50, and IkB, blocked p65 nuclear translocation and decreased the DNA-binding activity of NF-kappa B. Besides, 18 beta-GA inhibited the activation of the MAPK pathways. Co-immunoprecipitation showed that 18 beta-GA treatment blocked RANK-TRAF6 association at an upstream site. In vivo, 18 beta-GA treatment inhibited ovariectomy-induced osteoclastogenesis and reduced bone loss in mice. Overall, our results demonstrated that 18 beta-GA inhibited RANKL-induced osteoclastogenesis by inhibiting RANK expression in preosteoclasts and blocking the binding of RANK and TRAF6 which lead to the inhibition of NF-kappa B and MAPK signaling pathways. 18 beta-GA is a promising novel candidate in the treatment of osteoclast-related diseases such as postmenopausal osteoporosis.