Involvement of bilitranslocase and beta-glucuronidase in the vascular protection by hydroxytyrosol and its glucuronide metabolites in oxidative stress conditions

Abstract

Olive oil vascular benefits have been attributed to hydroxytyrosol (HT). However, HT biological actions are still debated because of its high metabolisation into glucuronides (GC). The aim of this study was to test HT and GC vasculoprotective effects on aorta rings from male Wistar rats. Vasorelaxant dose-responses to acetylcholine (ACh) or sodium nitroprusside (SNP) were realized in presence/absence of tert-butylhydroperoxide (t-BHP) (30 min, 500 μM). To prove this antioxidative effect, DHE staining was realized on phenol-treated aortas in oxidative stress conditions. Implication of bilitranslocase, a phenol transporter, and β-glucuronidase, implicated in deconjugation, were assessed using phenylmethanesulfonyl fluoride (PMSF, 100 μM, 20 min) and saccharolactone (SAL, 300 μM, 60 min), respectively. In addition, β-glucuronidase protein expression was assessed by western blot after using the same experimental procedures. In presence of oxidative stress, HT and GC enhanced endothelium-dependent and -independent relaxation to ACh and SNP compared to t-BHP-treated aortas. This effect was associated with a significant reduction in DHE staining for phenol-treated aortas compared to t-BHP-treated aortas. However, this protective effect was lost for GC when a washing step was introduced between phenols incubation and t-BHP stress. Bilitranslocase inhibition with PMSF reduced significantly HT but not GC effect on the vascular function upon stress induction. Moreover, GC protective effect in oxidative stress conditions was reduced by pre-incubation of aorta rings with SAL and was not associated with a reduction in β-glucuronidase protein expression. Finally, only HT was detected by high-pressure liquid chromatography in aorta rings incubated with GC and t-BHP. These results suggest that in conditions of oxidative stress, GC has to be deconjugated into HT that is transported through the cell membrane by bilitranslocase to produce its protective effect on vascular function.