Tatarinan O, A Lignin-like Compound From Acorus Tatarinowii Schott Effect On Treatment Of Osteoporosis Mice Model

III Excessed number of osteoclasts leads to bone loss in the body. Thus, Inhibiting osteoclastogenesis is the major therapeutic approach to the treatment of osteoporosis and other bone loss diseases.Osteoclasts are bone-resorbing multinucleated cells formed by the fusion of mononuclear progenitors of the monocyte/macrophage hematopoietic lineage cells. RANKL-RANK signaling pathway,is the principal pathway to modulate osteoclast differentiation, RANKL binding to its receptor RANK on the surface of osteoclast precursor cells leads to the recruitment of TNF receptor-associated factor 6(TRAF6) and the subsequent activation of mitogen-activated protein kinases(MAPKs), including ERK, p38, and JNK, as well as transcription factors, such as nuclear factor-κB(NF-κB), nuclear factor of activated T-cells, cytoplasmic 1(NFATc1), and c-Fos. The activation of above signaling pathways directly regulates the expression of osteoclastic genes, such as Cts K, MMP-9, and TRAP, which regulate the formation of bone resorption pits during osteoclast differentiation.Traditional Chinese medicine,rhizoma acori tatarinowii, is often used in the clinical treatment of rheumatoid arthritis and other bone diseases. It is a commonly used anti-inflammatory traditional Chinese medicine, and pharmacological research focus on its chemical composition in recent years. Recently we found a new ligninlike compounds from rhizoma acori tatarinowii, named Tatarinan O(TO). We have confirmed that the compounds have an anti-inflammatory effect on LPS induced inflammatory macrophage models. Because inflammatory cytokines such as TNF-α, IL-6 and IL-1 positively influence osteoclastogenesis, we hypothesized that TO may suppress RANKL-mediated osteoclastogenesis.In this study, we first set-up RANKL-induced osteoclast model and explored the inhibitory effect of TO on osteoclast formation. We evaluated whether TO influence osteoclast differentiation using TRAP staing assay. Furthermore, we examined the effect of TO on bone resorption activity of osteobalst by observing pit formation in Corning Osteo Assay Surface 24-well plates. The m RNA expression of osteoclastogenesis-related marker genes including Cts K, MMP-9, TRAP, NFATc1, c-Fos was detected by real-time PCR. The effects of TO on activation of NF-κB and MAPK, the protein levels of c-Fos and NFATc1, were determined by western blot. To further evaluate the therapeutic effects of TO on osteoporosis,,we examined whether TO can improve bone loss in bilateral ovarectomy osteoporosis mice. We examined the effects of TO on bone tissue structure and osteoclast activity in the model mice by HE staining and TRAP staing, reoectively. Finally, we assessed the effect of TO on bone microstructure in mice by means of Micro-CT.In vitro study indicated that TO suppress osteoclast differentiation from RANKL-stimulated mouse bone marrow macrophages(BMMs) without significant cytotoxicity. TO also dose-dependently suppressed bone resorption activity of mature osteoclasts. Additionally, TO apparently inhibited the expression of osteoclastic marker genesexpression, such as MMP-9, Cts K and TRAP. Furthermore, our results showed that TO decreased RANKL-induced expression increases of c-Fos and NFATc1 without influencing NF-κB activation and MAPK phosphorylation. In vivo study demonstrated that TO improved bone loss in bilateral ovarectomy osteoporosis mice. Through these experiments, we found that the lignin-like component isolated from Acorus gramineus,TO has a therapeutic effect on osteoporosis, provides a theoretical basis for bone loss disease treatment in the future...