Figure 6.

Effects of KAG-308 on TNF secretion from FLS. (a) mRNA levels of Tnf and Mmp13 in mouse and human FLS. After 8 hr-starvation, cells were simultaneously treated with 10 ng/ml LPS and KAG-308 for 6 hrs. Symbols represent individual points; long and short bars show the mean and SD of three wells per group, respectively. *P < 0.05, **P < 0.005, ***P < 0.0005 vs LPS + /KAG-308− by ANOVA followed by Dunnett’s post hoc test. (b) TNF levels in culture media of mouse FLS at 0, 1, 3, 6, 12, and 24 hrs after LPS treatment with or without 10 nM KAG-308 determined by ELISA. Data are expressed as the mean of three wells per group. Error bars indicate SD. *P < 0.05 at each time point by Welch’s t test. TNF levels in the culture media of synovial tissue obtained from sham or OA mice treated with vehicle or 3 mg/kg KAG-308 for 2 wks (c), and mRNA levels of Mmp13 and Adamts5 in mouse articular chondrocytes after 24-hr-culture in the conditioned medium from synovium (d). Symbols represent individual specimens; long and short bars show the mean and SD, respectively. *P < 0.05 by ANOVA followed by Tukey’s post hoc test. (e) Immunoblotting of c-Fos and Actin at 0, 1, 3, and 6 hrs after treatment with LPS and/or 10 nM KAG-308. Lower graph indicates quantitative densitometry analysis of immunoblots (n = 5). c-Fos values were normalized to Actin. Long and short bars show the mean and SD, respectively. **P < 0.005 vs 0 hour by ANOVA followed by Dunnett’s post hoc test. (f) mRNA levels of Tnf in mouse FLS treated with LPS, KAG-308 (10 nM), and either EP₄ antagonist L-161982 (10 µM) or PKA inhibitor H-89 (10 µM) for 6 hrs. KAG-308 and LPS were simultaneously added one hr after L-161982 or H-89 treatment. Symbols represent individual points; long and short bars show the mean and SD of three wells per group, respectively. ***P < 0.0005 vs LPS + /KAG-308−/L-161982−/H-89− by ANOVA followed by Dunnett’s post hoc test.