Resveratrol Ameliorates Low Shear Stress-induced Oxidative Stress In Human Umbilical Endothelial Cells

Objective Atherosclerosis has been reported to preferentially settle at the bifurcations, bends or stenosis of the artery where shear stress is low. Shear stress, a dragging force generated by blood flow, exerts a variety of effects on endothelial function and contributes a lot to the focal location of atherosclerotic lesions. The main process of AS involves the deposition of lipid, formation of firbous cap and then the development into AS. In which, endothelium dysfunction is the initial of AS. Resveratrol (RSV), a nature polypobe, can be found in various plants such as grapes, and nuts. RSV has been found to possess various functions such as antiflammation, antioxidant, influence cell proliferation and cell apoptosis. The current study aim to investigate the antioxidant effect of RSV on low shear stress (LSS) treated cells and the possible mechanisms involved.Methods A parallel-plate flow chamber that can apply LSS of2dynes/cm2was used. At first, MTT assay was applied to determine a proper concentration of RSV in this study. After flow treatment, the levels of reactive oxygen species (ROS) and nitric oxide (NO) were measured by chemiluminescence. Apoptotic cells were determined by TUNEL staining. eNOS and its three well known sites:Ser1177, Thr495and Ser633were tested by western blot to confirm the regulation of LSS on eNOS. After that, different inhibitors of MAPK were used to pre-treat cells to study the upstream regulator of eNOS. Intracellular SOD activity was assayed to measure cellular antioxidant defenses, and LDH was tested to measure cell permeability. At last Real-time PCR was applied to assess the level of endothelial nitric oxide synthase (eNOS) mRNA in LSS-treated ECs.Results For our time course study, oxidative damage such as enhanced ROS, decreased NO and increased cell apoptosis was found in LSS-treated cells. eNOS-Thr495and eNOS-Serl177were activated by LSS in a time-dependent manner and reached a maximal level at30min. eNOS-Ser633remained unchanged in a short time LSS stimulus. ERK inhibition increased intracellular SOD activity and suppressed LSS-activated eNOS-Thr495. LSS activated pi-ERK which peaked at15min and was prior to the time points at which eNOS-Thr495reached its maximal level. RSV exhibited cellular protected effect by suppressing LSS-induced oxidative damage:inhibited ROS accumulation and attenuated cell apoptosis. RSV suppressed ERK activation and deactivated eNOS-Thr495. The expression of eNOS mRNA was in independent of RSV or LSS in our short time flow study.Conclusion In our time course study:1) LSS evokes oxidative stress via activating ERK/eNOS-Thr495, and2) RSV attenuates LSS-induced oxidative damage, at least in part due to the suppression of ERK/eNOS-Thr495.