OBJECTIVETo investigate the effects and related mechanisms of bezafibrate, a ligand of peroxisome proliferator-activated receptors alpha (PPARalpha), on endothelial nitric oxide synthase (eNOS).

METHODSFirstly, in cultured bovine aorta endothelial cells (BAEC), the effects of bezafibrate on eNOS mRNA and protein levels were investigated by Northern blot and Western blot. The half-life of eNOS mRNA and NO production determined from BAECs treated with bezafibrate were performed by real-time quantitative PCR and NO assay. Next, the effects of bezafibrate on signal pathways in BAEC, through inhibitors of PPARalpha, PI3 kinase and MAPK, were investigated by Western blot. Then luciferase reporter gene driven by human eNOS promoter were cloned and transfected endothelial cells to see the effects of bezafibrate on eNOS promoter-driven luciferase activity.

RESULTSIn cultured BAEC, bezafibrate significantly upregulated eNOS expressions at protein and mRNA levels in a concentration-dependent fashion (50 - 200 micromol/L) (P < 0.05), and increased nitric oxide (NO) production, respectively[control (14.97 +/- 1.29) micromol/L, different concentration groups (25.12 +/- 1.25) micromol/L, (30.12 +/- 1.85) micromol/L, (33.47 +/- 1.22) micromol/L], and enhanced phosphorylation of eNOS-ser-1179 site (P < 0.05), but not eNOS-thr-497 site. Through real-time quantitative PCR, bezafibrate prolonged eNOS mRNA half-life from 3.1 hour to 6.1 hour were demonstrated. Further studies showed that bezafibrate treatments significantly enhanced dose-dependently luciferase activity (relative luciferase activity in 100 micromol/L and 200 micromol/L groups 4429.43 +/- 391.41 and 6187.5 +/- 307.53), compared with untreated luciferase reporter gene group (3361.81 +/- 316.85) (P < 0.05 and P < 0.01, respectively), and induced MAPK phosphorylation expression (P < 0.05 and P < 0.01, respectively). Then these effects of bezafibrate upregulated eNOS expressions could be blocked by PPARalpha antagonist, MAPK and PI3K inhibitor while not affected by PKC and MEK inhibitor (P < 0.01).

CONCLUSIONSBezafibrate can upregulate eNOS expression, enhance eNOS-ser-1179 site phosphorylation, increase NO production and stabilize eNOS mRNA through PPARalpha dependent pathway and PPARalpha independent MAPK and PI3K pathways. This mechanism provides additional anti-atherosclerotic and anti-hypertension benefits of bezafibrate in addition of lipid-lowering effects.