Simulated microgravity induces a NOX-sensitive oxidative response that is attenuated by resveratrol in human endothelial cells.

Spaceflight-associated microgravity perturbs endothelial physiology, at least in part, through oxidative stress and redox imbalance. Here, we investigated whether resveratrol protects human umbilical vein endothelial cells (HUVECs) exposed to simulated microgravity (µG) and examined whether an NADPH oxidase (NOX)-sensitive oxidative component contributes substantially to this response. Simulated µG significantly increased intracellular reactive oxygen species (ROS), enhanced protein carbonylation, disrupted glutathione homeostasis, and reduced cell viability. Pharmacological interrogation showed that diphenyleneiodonium (DPI), a broad inhibitor of flavin-dependent oxidases widely used to inhibit NOX activity, attenuated the oxidative response induced by µG. Resveratrol (1 µM) produced a comparable protective profile, reducing ROS accumulation, limiting oxidative protein damage, partially restoring the GSH/GSSG ratio, and preserving cell viability. In complementary assays, 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (MCLA) chemiluminescence, used here as a superoxide-associated readout, was increased under simulated µG and similarly reduced by both DPI and resveratrol. Given that NADPH oxidases (NOXs) are major endothelial sources of superoxide, the parallel inhibitory effects of DPI and resveratrol support the presence of a prominent NOX-sensitive, superoxide-associated component within the oxidative response induced by simulated µG. Collectively, these findings indicate that resveratrol attenuates simulated microgravity-induced oxidative stress and redox imbalance in endothelial cells and support its potential as a redox-active countermeasure under spaceflight-relevant conditions.