The quantitative data are shown in f. All values are expressed as means ± SD (n = 3). Values not sharing a common superscript differ significantly. c ×100 Treating RAW 264.7 cells with kinsenoside SC75741 (10–50 μM) for 3 days did not affect cell viability, which was assessed by the MTS assay (data not shown). Figure 3b shows that kinsenoside dose-dependently inhibited RANKL-induced osteoclast

differentiation in RAW 364.7 cells. Kinsenoside inhibited osteoclast Selleckchem Emricasan formation by 20 % (p < 0.05), 60 % (p < 0.05), and 71 % (p < 0.05) at 10, 25, and 50 μM, respectively. Kinsenoside inhibited early-stage osteoclastogenesis Complete osteoclast differentiation of RAW 264.7 cells takes up to 5 days after RANKL stimulation. To identify which stage of osteoclastogenesis was affected by kinsenoside, RAW 264.7 cells were treated with 50 μM of kinsenoside on different days, from Day 0 to Day 4 after RANKL stimulation. Kinsenoside inhibited osteoclast formation by concurrent addition (Day 0) or by Day 1 after RANKL stimulation (Fig. 3c and d). When kinsenoside was added to the culture for the final 3 days (Days 2–4), it failed to suppress RANKL-induced osteoclast differentiation. Kinsenoside XAV-939 clinical trial inhibited osteoclast formation by 44 % (p < 0.05) and 32 % (p < 0.05) at Day 0 and Day 1, respectively. Kinsenoside

inhibited bone resorption RAW264.7 cells were plated on bone slices and stimulated with RANKL in the presence or absence of kinsenoside. RANKL-stimulated cells formed a number of Evodiamine pits (Fig. 3e), indicating that the bone resorption activity of RANKL-treated cells transformed them into functionally active state resembling osteoclasts. Treatment with

kinsenoside (10–50 μM) significantly reduced the number and area of resorption pits compared with RANKL treatment alone. Kinsenoside inhibited osteoclast resorption by 20 % (10 μM; p < 0.05), 34 % (25 μM; p < 0.05), and 67 % (50 μM; p < 0.05). Kinsenoside inhibited RANKL-induced NF-κB activation by electrophoretic mobility shift assay RAW 264.7 cells were pretreated with kinsenoside for 2 h and then treated with RANKL for 1 h. The prepared nuclear extracts were then assayed for NF-κB activation by the electrophoretic mobility shift assay (EMSA). Figure 4a–c show that RANKL treatment caused a significant increase in the DNA-binding activity of NF-κB (p < 0.05). Treating RAW 264.7 cells with kinsenoside (25 and 50 μM) significantly suppressed the RANKL-induced DNA-binding activity of NF-κB by 13 % (p < 0.05) and 35 % (p < 0.05), respectively. Fig. 4 Kinsenoside inhibited RANKL-induced transcriptional activity of NK-κB in RAW 264.7 cells. a EMSA results showed a supershift of complex formed in the presence of anti-p50 and anti-p65 antibodies. The p65 subunits cause a specific binding of NF-κB to consensus DNA sequence. Cold the nuclear extract was preincubated with an excess of unlabeled oligonucleotide. b RAW 264.