Fig. 6

Effects of AGEs on activation of MAPKs in HCAECs. a MAPK p38 and ERK1/2 phosphorylation. Cells were treated with AGEs (100 μg/ml) for 24 h, then phosphorylation of MAPKs [p38, c-Jun NH2-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) 2] by using Bio-Rad Bioplex luminex immunoassay. HCAECs were treated with AGEs (100 ug/ml) for different time points. b Effects of MAPK inhibitors on eNOS mRNA levels. Cells were treated with AGEs (100 μg/ml) in the presence or absence of p38 inhibitor (SB239036, 1 μM) or ERK1/2 inhibitor (PD98059, 40 μM) for 24 h, and eNOS mRNA levels were determined by real-time PCR analysis. c Effect of p38 inhibitor (SB239036) on eNOS protein levels. Cells were treated with AGEs (100 μg/ml) in the presence or absence of p38 inhibitor (SB239036, 1 μM) for 24 h, and eNOS protein levels were determined by Western blot. Full-length blots are presented. d Effect of ERK1/2 inhibitor (PD98059) on eNOS protein levels. Cells were treated with AGEs (100 μg/ml) in the presence or absence of ERK1/2 inhibitor (PD98059, 40 μM) for 24 h, and eNOS protein levels were determined by Western blot. Full-length blots are presented. e Effects of AGEs (100 μg/ml), MAPK inhibitors, and TTFA on O₂ ⁻ production. Cells were treated with AGEs (100 μg/ml) in the presence or absence of MAPK inhibitors (p38 and ERK2) or TTFA for 24 h. O₂ ⁻ production was determined by DHE staining and flow cytometric analysis. *P < 0.05 compared with the control. ^#P < 0.05 compared with AGEs treatment. n = 3. Data are means and SE of multiple experiments (n)