Icariin abrogates osteoclast formation through the regulation of the RANKL-mediated TRAF6/NF-kappa B/ERK signaling pathway in Raw264.7 cells

Abstract

Background: Icariin is pharmacologically active prenylated flavonoid glycoside that has various biologic effects such as antioxidant, anticancer, and anti-inflammatory activities. In addition, icariin has been used in Chinese medicine for thousands of years to treat osteoporosis and it is still being used today. However, direct mechanism of icariin in the treatment of bone disease is not understood. Purpose: The purpose of this study is to investigate whether icariin influences RANKL-induced osteoclast formation in murine macrophages. Methods: Osteoclastogenesis was determined by TRAP staining and activity assay. Inhibition of signaling pathways and marker protein expression were evaluated by western blot analysis. The NF-kappa B (p65) nuclear localization was detected by immunofluorescence assay, and NF-kappa B/DNA-binding activity was detected by electrophoretic mobility shift assay (EMSA). Results: In our study, icariin inhibited the differentiation of pre-osteoclast cells into osteoclasts and suppressed expression of various genes involved in osteoclast formation and bone resorption. Also, icariin blocked the osteoclastogenesis induced by MCF7 and MDA-MB-231 breast cancer cells through inhibition of NF-kappa B activation. We found that icariin inhibited RANKL-stimulated TRAF-6 expression, and subsequently suppressed the phosphorylation of ERK, but icariin did not show an effect on p38, JNK, and Akt activation. Conclusion: These results indicate that icariin is likely to be a candidate for bone-related disease treatment and that icariin provides insights into the molecular mechanisms that influence RANKL-induced osteoclast differentiation.