e In vitro kinase assay of IKK was done as described in “Materials and methods”. The IC50 values were approximately 57.6 μM. Values were expressed as means ± SD (n = 3) Kinsenoside inhibited HDAC inhibitor NFATc1 expression RAW 264.7 cells were incubated with RANKL in the presence

or absence of kinsenoside for 2 h. Treatment with RANKL for 24 h raised the protein expression levels of NFATc1 by Western blot analysis (Fig. 5b; p < 0.05). The expression level of NFATc1 in the RANKL group was 3.5 times greater than that in control group. Pretreatment with kinsenoside led to a 40 % (25 μM; p < 0.05) and 60 % (50 μM; p < 0.05) decrease in NFATc1 expression (Fig. 5b). Effects of kinsenoside on cytoplasmic phosphorylation levels of p-IκBα, p-p65, and p-IKKα/β in RANKL-stimulated RAW 264.7 cells RAW 264.7 cells were incubated with RANKL in the presence or absence of kinsenoside for 2 h. Treatment

with RANKL for 1 h increased the cytoplasmic phosphorylation HSP990 mouse levels of p-IκBα, p-p65, and p-IKKα/β, but not IκBα, IKKα, and IKKβ, by Western blot analysis (Fig. 5c and d). The phosphorylation levels of p-IκBα and p-p65 in the RANKL group were 182 % NU7026 in vitro (p < 0.01) and 182 % (p < 0.05), respectively, both of which were greater than those in the control group. Kinsenoside treatment did not affect the level of IκBα. Kinsenoside treatment led to 27 % (25 μM; p < 0.05) and 39 % (50 μM; p < 0.05) decreases in p-IκBα level and 16 % (10 μM; p < 0.05), 32 % (25 μM; p < 0.05), and 39 % (50 μM; p < 0.05) decrease in p-p65 level (Fig. 5c). The levels of p-IKKα/β in the RANKL group were 145 % (p < 0.05) greater than that in the control group. Kinsenoside treatment did not affect the levels of IKKα, IKKβ, and p-IKKα/β (Fig. 5d). Kinsenoside inhibited

IKK activity To determine whether kinsenoside interacts with IKK directly, this study examines the effects of kinsenoside on IKK enzymatic activity. The culture treatment of RAW 264.7 cells with 50 ng/ml RANKL for 1 h effectively increased IKK activity. Kinsenoside treatment (10–200 μM) for 2 h inhibited IKK activity in a concentration-dependent manner. The IC50 value was approximately 57.6 μM Tenoxicam (Fig. 5e). Kinsenoside did not affect the mRNA expression of RANK and TRAF6 The fragments shown in Fig. 6a reflect the pooled data for three samples. The BMs were incubated with M-CSF (20 ng/ml) for 3 days to induce the production of osteoclast precursor. Osteoclast precursors incubated with kinsenoside for 120 min were then treated with M-CSF (20 ng/ml) and RANKL (50 ng/ml) for 1 day in the presence or absence of kinsenoside. As Fig. 6a shows, RT-PCR amplified fragments of RANK and TRAF6. The RANK/GAPDH and TRAF6/GAPDH ratios in the RANKL group were 170 % (p < 0.05) and 220 % (p < 0.05), respectively, both of which are greater than those in the control group. Kinsenoside treatment (10–50 μM) did not affect the ratios of RANK/GAPDH and TRAF6/GAPDH (Fig. 6a).