Effect of HNE on PGE2 release and COX-2 expression.(a) Osteoblasts were treated with HNE (0 to 20 μM) for 48 hours, and PGE2 release was evaluated in culture medium by PGE2 enzyme immunoassay kit. (b, c) Osteoblasts were treated with HNE (0 to 20 μM) for 48 or 4 hours for protein and mRNA determination, respectively. COX-2 protein expression (b) and mRNA expression (c) were evaluated by Western blot and real-time reverse transcriptase-polymerase chain reaction, respectively. Quantifications of COX-2 protein and mRNA levels were normalised, respectively, to those of β-actin protein and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNA. (d) Cells were preincubated in the absence or presence of p38 MAPK inhibitor SB202190 (10 μM) for 30 minutes, followed by incubation by HNE (20 μM) for 48 hours. PGE2 secretion was evaluated as described above. Data are means ± standard error of the mean of n = 3 and expressed as a percentage of untreated cells. Statistics: Student unpaired t test; *p < 0.05, ***p < 0.001. COX-2, cyclooxygenase-2; HNE, 4-hydroxynonenal; MAPK, mitogen-activated protein kinase; PGE2, prostaglandin E2.