Modulation of nitric oxide production by olive oil phenols and their metabolites

Nitric oxide (NO) is an important signaling molecule involved in many physiological and pathological processes. NO contributes to vessels vasodilatation and blood flow increasing, but also to the vessel homeostasis by inhibiting vascular smooth muscle contraction and growth, platelet aggregation, and leukocyte adhesion to the endothelium. In the intestinal epithelium, NO may play, at low levels, a protective role against the inflammatory process, while after acute or chronic exposure a toxic mechanism of action leads to injurious effects. Dietary polyphenols and their metabolites have been found to be directly involved in the modulation of the intracellular signals that lead to the production of NO. The purpose of this study was to investigate the mechanism of the modulatory action of extra-virgin olive oil (EVOO) phenylethanoids, tyrosol (Tyr) and hydroxytyrosol (HT), and hydroxycinnamates, ferulic acid (FA), hydroferulic acid (DHFA) and isoferulic acid (IFA), in comparison to their glucuronide and sulfate metabolites, on the production of NO both at vascular and intestinal level. The first part of this investigation was assessed in endothelial cells (HUVEC and HAEC) to evaluate the superoxide production, the release of NO and cGMP and the activation of eNOS via Akt phosphorylation in endothelial cells. It was observed that the tested phenolic compounds enhanced NO concentration inhibiting its depletion caused by superoxide overproduction. Moreover, some of them enhanced its production through the modulation of Akt activation and of eNOS phosphorylation. DHFA and IFA showed better effectiveness than FA, as well as HT, which worked better than Tyr either as superoxide inhibitor or as eNOS enhancer. Interestingly, some of the tested metabolites worked in the same way of their parent compounds: it is the case of HT, whose metabolites were significantly effective in Akt and eNOS activation, as well as in superoxide counteraction. Overall, obtained data showed that these compounds promote NO production and availability, so their intake by EVOO dietary consumption can help in the prevention of cardiovascular diseases such as hypertension, thrombosis, hypercholesterolaemia and atherosclerosis. The second part of this study was focused on the modulation of NO and cGMP production in intestinal cells (Caco-2), with the aim of investigating the ability of EVOO phenolics to inhibit iNOS activation and the underlying mechanism of action. Treatments with pathological concentrations of lypopolysaccharide (LPS) were carried out to stimulate iNOS activation pathway, which involves NF-ĸB activation through IĸBα and Akt phosphorylation, and evaluate the transepithelial resistance changes on Caco-2 monolayers. It was overally observed that all the tested compounds inhibited NO release induced by LPS, acting as inhibitors of iNOS expression. Considering hydroxycinnamates, they were able to down-regulate Akt phoshorylation. IFA sulf was surprisingly the most effective in the Akt inhibition, while DHFA and its glucuronide better inhibited IĸBα degradation and iNOS. No one of the phenylethanoids instead were able to inhibit Akt activation. However, they were effective in the inhibition of IĸBα degradation, suggesting a modulatory action on a parallel mechanism upstream NF-ĸB translocation to the nucleus. All the tested phenolic compounds counteracted the deleterious effect of LPS on Caco-2 cell monolayer permeability, suggesting the ability of preserving intestinal barrier integrity. Thus, it can be assumed that reaching relevant concentrations of EVOO phenols in the gut lumen may exert beneficial effects against intestinal inflammation, which is one of the major features of many intestinal diseases.