Ferulic acid enhances nitric oxide production through up-regulation of argininosuccinate synthase in inflammatory human endothelial cells

Aim: In this study, we investigated the protective effect of ferulic acid (FA) on nitric oxide (NO) production in tumor necrosis factor (TNF)-alpha-stimulated inflammatory human umbilical vein endothelial cells (HUVECs), and elucidated the mechanism(s) involved. Main methods: The TNF-alpha-stimulated inflammatory HUVECs were treated with acetylcholine (ACh) and/or FA. NO productions were measured by monitoring nitrite and nitrate using a 2,3-diaminonaphthalene Kit. Expressions of mRNA and proteins were evaluated by RT-PCR and Western blotting, respectively. Key findings: FA treatment resulted in a dose-dependent (10-200 mu M) restoration of ACh-mediated NO production in TNF-alpha-treated HUVECs, whereas treatment with the FA analogues, coumaric acid, and apocynin resulted in no significant effect. FA treatment had no effect on O-2(center dot-) production in TNF-alpha-stimulated HUVECs. N-G-monomethyl-L-arginine acetate (a nitric oxide synthase (NOS) inhibitor) and alpha-methyl-DL-aspartic acid (an argininosuccinate synthase (ASS) inhibitor) counteracted the effects of FA on the NO production. While FA treatment did not significantly affect the protein expression of p-eNOS or eNOS, the protein expression of ASS as well as mRNA expression was restored to normal levels upon exposure to FA in TNF-alpha-stimulated HUVECs. In nucleus, FA attenuated the increase of nuclear factor-kappa B (NF-kappa B) expression by TNF-alpha. Significance: FA treatment rescues the defect in ACh-induced NO production resulting from TNF-alpha-stimulation in inflammatory HUVECs. This effect was likely due, in part, to the FA-mediated up-regulation of ASS expression via the suppression of NF-kappa B inflammatory signaling cascade. (C) 2016 Elsevier Inc. All rights reserved.