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

Zhao, Jian, Suyama, Aki, Chung, Hsuan, Fukuda, Toshihiko, Tanaka, Mitsuru, Matsui, Toshiro
Life sciences 2016 v.145 pp. 224-232
Western blotting, acetates, acetylcholine, argininosuccinate synthase, dose response, endothelial nitric oxide synthase, ferulic acid, gene expression, human umbilical vein endothelial cells, humans, messenger RNA, monitoring, nitrates, nitric oxide, nitrites, protective effect, protein synthesis, reverse transcriptase polymerase chain reaction, superoxide anion, transcription factor NF-kappa B, tumor necrosis factors
In this study, we investigated the protective effect of ferulic acid (FA) on nitric oxide (NO) production in tumor necrosis factor (TNF)-α-stimulated inflammatory human umbilical vein endothelial cells (HUVECs), and elucidated the mechanism(s) involved.The TNF-α-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.FA treatment resulted in a dose-dependent (10–200μM) restoration of ACh-mediated NO production in TNF-α-treated HUVECs, whereas treatment with the FA analogues, coumaric acid, and apocynin resulted in no significant effect. FA treatment had no effect on O2⁻ production in TNF-α-stimulated HUVECs. Nᴳ-monomethyl-l-arginine acetate (a nitric oxide synthase (NOS) inhibitor) and α-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-α-stimulated HUVECs. In nucleus, FA attenuated the increase of nuclear factor-kappa B (NF-κB) expression by TNF-α.FA treatment rescues the defect in ACh-induced NO production resulting from TNF-α-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-κB inflammatory signaling cascade.