Authors: A1 Isabella Fiandra. A2 Stephanie Whittam. A3 Andrew English

Affiliations: A1, Science department, School of Health & Life Sciences, Teesside University, Middlesbrough, TS1 3BX, United Kingdom. A2, Science department, School of Health & Life Sciences, Teesside University, Middlesbrough, TS1 3BX, United Kingdom. A3, Science department, School of Health & Life Sciences, Teesside University, Middlesbrough, TS1 3BX, United Kingdom

Background/ Introduction: Endothelial dysfunction driven by oxidative stress and reduced nitric oxide bioavailability is a critical early event in cardiovascular disease. This study investigated the effects of the dietary flavonoid quercetin on oxidative stress and endothelial NO signalling in human EA.hy926 endothelial cells, and defined the concentration and time dependency of these effects.

Material & Methods: EA.hy926 cells (passages 7–9) were exposed to hydrogen peroxide (H₂O₂; 100 µM) to induce oxidative stress and treated with quercetin. Intracellular reactive oxygen species (ROS) were quantified using DCFH DA fluorescence, while NO bioavailability was assessed by nitrite accumulation using the Griess assay. Acute responses were assessed after 2 h with quercetin (100µM), followed by extended dose–response (5–500 µM) and time course (1–24 h) analyses. All experiments were conducted in triplicate and analysed using one way ANOVA with Tukey post hoc testing

Results: H₂O₂ exposure (100 µM, 2 h) significantly increased ROS levels by 1.3 fold, from 30,500 ± 5,000 to 51,000 ± 4,050 RFU (p<0.001), and markedly reduced NO bioavailability from 56±8 to 35±6 µM nitrite (p<0.0001), confirming oxidative endothelial dysfunction. Quercetin co treatment (100µM) significantly attenuated ROS accumulation (21,000±2,080 RFU; p<0.01 vs H₂O₂*) and partially restored NO levels (*51±7 µM nitrite; p < 0.01). Quercetin alone significantly increased basal NO bioavailability (*62±9 µM nitrite; p<0.05). Dose–response analysis demonstrated maximal protection at 150 µM quercetin, with ROS reduced to 18,000 ± 2,100 RFU and NO increased to 55±4 µM nitrite. Higher concentrations (200–500µM) conferred no additional benefit. Time course analysis revealed significant ROS reduction within 1–2 h (p<0.01), followed by sustained preservation of NO bioavailability at 4–24 h (p<0.01).

Conclusion: Quercetin protects endothelial cells from oxidative damage by reducing ROS and maintaining NO availability, making it a redox-modulating dietary bioactive. Future work will explore its synergy with other dietary bioactives to further improve endothelial redox balance and NO bioavailability.