Suppressive Effects of Hydroxytyrosol on Oxidative Stress and Nuclear Factor-κB Activation in THP-1 Cells

Abstract

This study was designed to investigate whether hydroxytyrosol (HT) may ameliorate oxidative stress and nuclear factor kappaB (NF-kappaB) activation in the lipopolysaccharide (LPS)-stimulated THP-1 cell line. We measured the intracellular reactive oxygen species (ROS) formation using 2,7-dichlorofluorescein diacetate (DCFH-DA) as a fluorescent probe. Intracellular glutathione (GSH) level was estimated by fluorometric methods. Nitric oxide (NO) production was measured as nitrite (a stable metabolite of NO) concentrations using the Griess reagent system following Jiancheng Institute of Biotechnology protocols. To study the effect of HT on LPS-induced NF-kappaB activation in THP-1 cells, Western blot analysis of the nuclear fraction of cell lysates was performed. The results showed that treatment of THP-1 cells with HT significantly reduced LPS-stimulated NO production and ROS formation in a concentration-dependent manner. HT at 50 and 100 microM concentrations increased the GSH level. The specific DNA-binding activities of NF-kappaB on nuclear extracts from 50 and 100 microM HT treatments were significantly suppressed. The antioxidant N-acetylcysteine (NAC) also showed the same effects as HT on LPS-induced ROS and NO generation, change of GSH level, and NF-kappaB activation. These findings suggest that HT has antioxidant activity to suppress intracellular oxidative stress and NF-kappaB activation in THP-1 cells.