Stephanine Protects Against Osteoporosis by Suppressing Osteoclastogenesis via Inhibition of the <scp>RANKL</scp>—<scp>RANK</scp> Interaction

The interaction between the receptor activator of nuclear factor-κB ligand (RANKL) and its receptor RANK is known to regulate osteoclastogenesis in bone remodelling and has become an important therapeutic target for the treatment of osteoporosis. Stephanine (SA), an isoquinoline aporphine-type alkaloid isolated from Stephania plants, possesses excellent anti-inflammatory effects and can be used for rheumatoid arthritis treatment. However, its specific role in osteoclastogenesis and osteoporosis remains unknown. In this study, we investigated the influence of SA on osteoclastogenesis in RANKL-stimulated RAW 264.7 cells and osteoporosis in an ovariectomised (OVX) mouse model and elucidated the underlying molecular mechanism. In vitro, SA can bind to RANK and RANKL with the K_D values of 3.7 and 76.47 μM, respectively, and disrupt the RANKL-RANK interaction, which inhibits RANKL-stimulated RANK-tumour necrosis factor receptor associated factor 6 (TRAF6) binding and RANK signalling pathways activation, downregulates the expression of key osteoclastogenesis-related regulatory factors in osteoclast precursors, ultimately suppresses osteoclast differentiation and activation. In vivo, SA significantly ameliorated bone loss through inhibiting osteoclastogenesis in OVX mice because of the decreased number of osteoclasts and the increased trabecular bone area. SA markedly inhibited the serum levels of tartrate-resistant acid phosphatase 5b (TRACP-5b), c-telopeptide of type I collagen (CTX-I), and RANKL, whereas it increased that of osteoprotegerin (OPG) in OVX mice. Additionally, SA strikingly downregulated the OVX-induced expression of osteoclast-specific genes and proteins. Taken together, this study elucidated that SA can effectively protect against osteoporosis by suppressing osteoclastogenesis via inhibition of the RANKL-RANK interaction, which supports the potential application of SA as a natural therapeutic agent for osteoporosis.