Figure 4

RAGE is the corresponding receptor for HMGB1 in hDPCs. Blocking RAGE (RAGE-FC) inhibited the effects of HMGB1 on prostaglandin metabolism. Cultured hDPCs were pre-treated with or without 10 μg/ml RAGE-FC for 30 min and then incubated with 200 ng/ml HMGB1 for 30 min. (a) The protein levels of COX-1, COX-2, mPGES-1, and mPGES-2 were measured using western blot analysis. (b) Protein bands were analysed using densitometry, and β-actin served as a loading control. The results are expressed as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with control group using one-way ANOVA followed by Bonferroni’s test. (c) PGE₂ production from cultured hDPCs was measured using ELISA. Cultured hDPCs were pre-treated with 10 μg/ml RAGE-FC for 30 min and then incubated with 200 ng/ml HMGB1 for 4 h. Data are representative of three independent experiments. The results are expressed as mean ± SD of three independent experiments. **p < 0.01 and ***p < 0.001 compared with control group using one-way ANOVA. Representative images of immunofluorescence staining for (d) COX-1 (red), (e) COX-2 (red), and mPGES-1 (green) in hDPCs pre-treated with 10 μg/ml RAGE-FC for 30 min and 200 ng/ml HMGB1 treatment for another 30 min. 4′-6-diamidino-2-phenylindole (DAPI; blue) was used to counterstain the nuclei. White arrowheads mark the expression of each enzyme in perinuclear region of hDPCs. Data are representative of three independent experiments. Scale bar = 20 μm.