Expression of TRAF6 and TAK1 in cell lysates was determined by immunoblotting. B, The level of co-immunoprecipitated TAK1 or TRAF6 was quantified and normalized to total TRAF6 or TAK1, respectively. Data are expressed as the fold change vs. the untreated control. Values are the means SD of three independent experiments. Rhinacanthin C inhibits LPS-induced osteoclastogenesis and bone resorption Bacterial infection is associated with bone destruction in periodontitis. LPS, a cell 
wall component of Gram-negative bacteria, also induces bone resorption. We also demonstrated the effects of rhinacanthin C on LPS-induced osteoclastogenesis from BMMs and bone resorption of mouse calvaria. RANKL priming is needed for LPS-induced osteoclastogenesis. We treated BMMs with RANKL for 24 h followed by LPS treatment with or without rhinacanthin C for 3 days. LPS significantly PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19703425 increased TRAP activity and the numbers of MNCs in RANKL-primed BMMs but not in non-primed BMMs. Rhinacanthin C provided dose-dependent inhibition of LPS-stimulated osteoclastogenesis from BMMs. Finally, we tested the inhibitory effects of rhinacanthin C on LPS-stimulated calvarial bone erosion in vivo using normal and OPG-/- mice. Osteoprotegerin-deficient mice exhibit osteoporosis due to enhanced osteoclastogenesis caused by a lack of soluble decoy receptor for RANKL. As shown in Fig 8, LPS significantly increased TRAP PR 619 web staining of whole calvariae in normal and OPG-/- mice. Simultaneous administration of rhinacanthin C reduced LPS-induced osteoclast formation. CT analysis showed that LPS induced bone destruction in OPG-/- mice. Rhinacanthin C ameliorated LPS-induced calvarial bone resorption of normal and OPG knockout mice. Thus, rhinacanthin C inhibits LPS-induced and RANKL-induced osteoclastogenesis. 10 / 17 Rhinacanthin C Suppresses Osteoclastogenesis Fig 6. Protective effects of rhinacanthin C on RANKL-induced mouse calvarial osteolysis. Vehicle or rhinacanthin C with or without RANKL was daily injected into the subcutaneous tissue overlying the calvaria of 8-week-old ddy mice. The mice were sacrificed on day 5. A, TRAP staining of whole calvaria and high magnification PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19704080 of TRAP stain on calvaria. Bar, 400 m. B, TRAP+ area was measured by densitometry using Image J.C, Three-dimensional micro-CT image of calvaria. D, Bone volume/total tissue volume ratio. E, Trabecular separation. F, Trabecular bone pattern factor.Discussion Identification and characterization of natural anti-osteoclastogenic compounds from herbal sources support the development of novel therapeutics for patients suffering from abnormal bone lysis and exploration of the regulatory mechanisms underlying osteoclast formation and activation. Previously, we found that rhinacanthin C is a potent inhibitor of RANKL-induced osteoclast formation from BMMs. In this study, we demonstrated the inhibitory mechanisms of rhinacanthin C and its effects in vivo. Rhinacanthin C suppressed the osteoclast-specific master transcription factor, NFATc1, which regulates osteoclast specific/related gene expression. Indeed, c-Src and integrin, which are late osteoclastogenesis genes involved in bone resorption, were down-regulated by 11 / 17 Rhinacanthin C Suppresses Osteoclastogenesis Fig 7. Rhinacanthin C inhibits LPS-induced osteoclastogenesis in RANKL-primed BMMs. A, BMMs were primed with RANKL for 24 h, then the culture medium were washed away and the cells were cultured with or without LPS or LPS plus rhinacanthin C. After 3 days,
