Thymoquinone reverses homocysteine-induced endothelial dysfunction via inhibition of ER-stress induced oxidative stress pathway

Abstract

Abstract Hyperhomocysteinemia has been linked to an increased risk of cardiovascular diseases. High levels of homocysteine (Hcy) promote endoplasmic reticulum (ER) stress that can increase reactive oxygen species (ROS), leading to endothelial dysfunction. Thymoquinone (TQ) is the major active ingredient in Nigella sativa seeds volatile oil and is shown to have a cardioprotective effect. However, no study evaluated the effect of TQ against Hcy-induced endothelial dysfunction. Thus, this study aims to investigate the effects and mechanisms of TQ in reversing Hcy-induced endothelial dysfunction. Isolated aorta from male Sprague-Dawley (SD) rats incubated with Hcy (500 µM) and co-treated with or without TQ (0.1 µM, 1 µM, and 10 µM), 20 µM TUDCA, 100 µM Apocynin or 1 mM Tempol in organ bath to study the vascular function. Additionally, human umbilical vein endothelial cells (HUVECs) were incubated with Hcy (10 mM) and various concentrations of TQ (1 and 10 𝜇M), Tempol (100 𝜇M), Apocynin (100 𝜇M), TUDCA (100 𝜇M) or H2O2 (0.25 mM) to evaluate the cell viability by using a phase contrast microscope and dye exclusion assay. Involvement of ER stress pathway, ROS and NO bioavailability were accessed via immunoassay and fluorescent staining respectively. Molecular docking was performed to evaluate the binding affinity of TQ to GRP78. Our results revealed that Hcy impaired endothelium-dependant relaxation in isolated aorta and induced apoptosis in HUVECs. These effects were reversed by TQ, TUDCA, tempol and apocynin. Treatment with TQ (10𝜇M) also reduced ROS level, improved NO bioavailability as well reduced GRP78 and NOX4 protein in HUVECs. Result from the molecular docking study showed that TQ could bind well to GRP78 through hydrogen bond and hydrophobic interaction with the amino acid at GRP78 ATP binding pocket. Taken together, the present results suggest that TQ preserved endothelial function in rat aorta and reduced apoptosis of HUVECs induced by Hcy through the inhibition of ER stress-mediated ROS and eNOS uncoupling.