Effect of alpha-zearalanol on the generation of reactive oxygen species (ROS) and ROS-activated signal transduction in tumor necrosis factor alpha-stimulated human endothelial cells

Abstract

To investigate the effects of alpha-zearalanol (alpha-ZAL) on the generation of reactive oxygen species (ROS) and ROS-activated signal transduction in the tumor necrosis factor (TNF-alpha)-stimulated human umbilical vein endothelial cells (HUVECs). HUVECs were cultured and divided into 4 groups: (1) normal control group, (2) TNF-alpha stimulated group, undergoing TNF-alpha stimulation for 24 h, (3) alpha-ZAL retreatment group, undergoing re-treatment with alpha-ZAL of the concentrations of 1 x 10(-8), 1 x 10(-7), or 1 x 10(-6) mol/L for 1 h, then stimulation of TNF-alpha for 24 h, and (4) plasmid transfection group, transfected with p47(phox) siRNA for 24 h to block the NADPH oxidase protein subunit p47(phox) in the HUVECs, or transfected with blank plasmid as control. The intracellular ROS production was detected by using 2, 7-dichlorofluorescin diacetate as probe. Semi-quantitative RT-PCR and immunocytochemistry were used to detect the mRNA and protein expression of p47(phox). The activation of extracellular signal-regulated kinase (ERK) and nuclear translocation of nuclear factor-kappaB (NF-kappaB), stimulatory protein (SP)-1, and activator protein (AP)-1 were assessed with Western blotting. The ROS level in the HUVECs of the TNF-alpha group was higher than that of the control group by 155.4%, and alpha-ZAL reduced the ROS level dose-dependently. TNF-alpha treatment up-regulated the p47(phox) mRNA expression by 212.8%, and obviously increased the p47(phox) protein expression; and alpha-ZAL pretreatment attenuated the TNF-alpha-induced p47(phox) mRNA expression by 63.0%, and also markedly inhibited the p47(phox) protein expression. No obvious ROS was found in the HUVECs stimulated by TNF-alpha after the transfection of p47(phox) siRNA. The ERK activation and nuclear translocation of transcription factors SP-1 and NF-kappaB induced by TNF-alpha were abolished or markedly inhibited by alpha-ZAL pretreatment. alpha-ZAL has a potent inhibitory effect on the ROS production and ROS-activated signaling pathway in the TNF-alpha stimulated endothelial cells, mainly through the inhibition of NADPH oxidase.