Wholegrain oats contain a variety of phenolic compounds thought to help maintain healthy vascular function, through the maintenance of local levels of the vasodilator nitric oxide (NO). Thus, the full molecular mechanisms involved are not yet clear. With this work we aim to understand the possible cellular mechanisms by which avenanthramides and ferulic acid derivatives, present in oats, may help maintain a healthy vascular function through the modulation of the NO pathway. Primary Human Umbilical Vein Endothelial Cells (HUVEC) were exposed to ferulic acid, isoferulic acid, hydroferulic acid, ferulic acid 4-O-glucuronide, isoferulic acid 3-O-sulfate, dihydroferulic acid 4-O-glucuronide, avenanthramide A, avenanthramide B and avenanthramide C (1 μM) or vehicle (methanol) for 24 h. Apocynin and Nω-Nitro-L-arginine (L-NNA) were additionally included as controls. NO and cyclic GMP (cGMP) levels, superoxide production and the activation of the Akt1/eNOS pathway were assessed. The statistical analysis was performed using one-way ANOVA followed by a Tukey post-hoc t-test. Apocynin and all phenolic compounds increased NO levels in HUVEC cells (increased DAF2-DA fluorescence and cGMP), and significantly reduced superoxide levels. Protein expression results highlighted an increase in the Akt1 activation state, and increased eNOS expression. Overall, our results indicated that the glucuronide metabolites do not enhance NO production through the Akt1/eNOS pathway, thus all compounds tested are able to reduce NO degradation through reduced superoxide formation.

Nitric oxide (NO) is an important vasodilator involved in the regulation of vascular homeostasis, and plays a crucial role in maintaining a normal endothelial function (1) . Dietary phenolic acids such as ferulic acid can improve vascular function (2) , through maintenance of local levels of NO. Thus the molecular events involved are not fully understood (3) and further mechanistic investigations are required. The aim of this study is to understand the precise cellular mechanisms by which ferulic acid and its metabolites maintain healthy vascular function through modulation of the NO pathway.Primary Human Umbilical Vein Endothelial Cells were exposed to ferulic acid, isoferulic acid, hydroferulic acid, ferulic acid 4-O-glucuronide, isoferulic Acid 3-O-Sulfate and Dihydroferulic acid 4-O-glucuronide (1μM) for 24 h or 2 h. Apocynin and Nω-Nitro-L-arginine (L-NNA) were used as additional controls. Superoxide production, cyclic GMP cGMP) levels, and Akt1 activation were determined. The statistical analysis of the data (at least 4 biological replicates) was conducted by one-way ANOVA followed by a Tukey post-hoc t-test using the Graphpad 7 software.Similarly to apocynin, all compounds tested showed the ability to enhance NO levels, measured as production of cGMP, and significantly decrease superoxide production, Protein expression results obtained through western blotting showed that the treatment with ferulic acid, isoferulic acid, hydroferulic acid and isoferulic Acid 3-O-Sulfate was also able to significantly increase Akt1 activation, measured as the ratio of the phosphorylated (Ser 473) / total protein. Our results indicated that all the tested ferulic acid metabolites can modulated NO balance, measured as cGMP production, by decreasing its degradation (via reduced superoxide formation), however the glucuronide-conjugated metabolites are not able to significantly enhance NO production through the Akt1/eNOS pathway.