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Oxidative stress play a pivotal role in the development of cardiovascular diseases. ATP-binding cassette transporter G1 (ABCG1), a regulator of cellular lipid metabolism, has been found to reverse the high glucose-induced oxidative stress in previous our study. The present study was aimed to investigate the possible regulation of ABCG1 on oxidative stress production induced by TNF-a in endothelial cells and its possible mechanism of imbalances between prooxidants and antioxidants.
Human umbilical vein endothelial cells (HUVECs) were treated with 10ng/mL TNF-α with or without liver X receptor (LXR) agonist T0901317 for 0, 12 and 24h. Furthermore, HUVECs were transfected with specific ABCG1 siRNA or ABCG1 overexpression plasmid to obtain different expression of ABCG1. Intracellular reactive oxygen species (ROS) was measured using dihydroethidium (DHE) fluorescence. Real time PCR and Western blot were employed to measure the expression of prooxidant Nox4, one of NADPH oxidase subunit and the expression of antioxidant SOD1 and SOD2 as well as ABCG1. Furthermore, NADPH oxidase activity was measured using lucigenin chemiluminescence assay and translocation of cytosolic p47phox to the membrane.
ABCG1 mRNA and protein expressions were inhibited by 40% when incubation of HUVECs with TNF-α for 24h. Intracellular ROS generation was significantly induced by TNF-α accompanied with increased NADPH oxidase activity by 55% and a strong membrane translocation of p47phox. Furthermore, Nox4 protein expression was also upregulated by TNF-α. In contrast, there was a decreased SOD1 or SOD2 mRNA and protein levels were found in HUVECs treated by TNF-α. However, upregulation of ABCG1 by transfection of ABCG1 expression plasmid or pre-incubation of LRX agonist T0901317 significantly blunted TNF-α-induced the activity of NADPH oxidase by 47% and by 35% respectively and attenuated the expression of Nox4 protein by 53% and 47%. While the expression of SOD1 and SOD2 was promoted with the upregulation of ABCG1. Conversely, downregulation of ABCG1 by ABCG1 siRNA increased the NADPH oxidase activity and Nox4 expression and inhibited the SOD enzyme expression in TNF-α-treated HUVECs.
The results demonstrated that ABCG1 attenuated TNF-α-induced oxidative stress in endothelial cells, which may involve decreased NADPH oxidase activity and expression of NADPH oxidase subunits and increased antioxidant SOD1 and SOD2 enzyme via ABCG1 pathway.