7 cells. Osteoclasts are multinucleated cells of hematopoietic origin and are the primary bone-resorbing cells [5]. TRAP is a different form of the enzyme acid phosphatase, which is found mainly in bone. Osteoclasts release TRAP during bone resorption [21]. Histological sections stained with TRAP showed that the number of osteoclasts decreased in the region of the spongiosa in kinsenoside-treated OVX mice. TRAP activity is commonly used as a histochemical

marker of identifying osteoclasts [26]. MMP-9 is required for osteoclastic migration and resorption [27]. Kinsenoside treatment inhibited the mRNA expression of femoral TRAP and MMP-9, but not ALP. These findings indicate that kinsenoside can suppress the differentiation and resorption of osteoclasts. These results agree with the findings obtained by Masuda AZD1480 price et al., who showed that the ethanolic extract of A. Omipalisib in vivo formosanus inhibited bone loss caused by OVX by suppressing osteoclast formation [18]. Osteoclasts are multinucleated cells originating from selleck screening library the fusion of mononuclear progenitors in the monocyte/macrophage family [28]. Previous research has shown that two key molecules, M-CSF and RANKL, are essential and sufficient to promote osteoclastogenesis [8]. Thus, M-CSF and RANKL were added to induce osteoclastogenesis

in the primary BM cell culture system. In the RAW 264.7 macrophage cell-cultured system, only RANKL was added to induce osteoclast differentiation. In this study, kinsenoside dose-dependently suppressed the formation of osteoclasts in BMs and a RAW 264.7 cell culture system. Results further show that RAW 264.7 cells were markedly blocked by the concurrent administration of RANKL

and kinsenoside and weakly blocked by subsequent addition of kinsenoside. This suggests that inhibition occurred during the initial stage DOK2 of osteoclastogenesis. Previous research has shown that M-CSF enhances RANKL-induced osteoclast formation [29]. To exclude the interference of M-CSF, therefore, RANKL-induced RAW 264.7 cell differentiation into osteoclastlike cells was used to assess the effects of kinsenoside on the signal transduction pathway. In addition, a BM system was used to examine the effects of kinsenoside on osteoclast precursor fusion, osteoclast formation, and resorption. Activation of the NF-κB pathway is a key factor in RANKL-induced osteoclast differentiation [10]. The results of EMSA analysis show that kinsenoside inhibits the RANKL-induced DNA binding activity of p65. Immunofluorescence staining and Western blot analysis of nuclear protein also show that kinsenoside suppressed the nuclear translocation of p65 protein. Using transient transfection with κB-luciferase as an indicator of NF-κB activity, this study shows that kinsenoside inhibits the RANKL-increased NF-κB activity.