Effects of Diazepam on Oxidative Stress, Apoptosis, and Vascular Reactivity in Human Umbilical Arteries Under Experimental Hyperhomocysteinemia

In obstetrics, diazepam is commonly used to treat anxiety, insomnia and as a second‐line treatment for seizures in preeclampsia and eclampsia. However, the effects of diazepam on the oxidative balance and vascular function of the umbilical circulation are still insufficiently studied. In this study, we investigated the effects of suprapharmacological concentrations of diazepam in an in vitro model of human umbilical artery (HUA) injury, induced by incubation with 1 mM homocysteine, representing experimental conditions that mimic hyperhomocysteinemia. HUAs were divided into four groups: control (C), diazepam (D) alone (100 μmol/L), homocysteine (Hcy) alone (1 mM), and combined treatment (HcyD). For oxidative stress assays, data were obtained from 8–10 individual HUA samples per group, with lipid peroxidation, nitrite (NO2 −) levels, and SOD, CAT, and GSH activities determined spectrophotometrically. Histological and TUNEL analyses were performed in triplicate (n = 3 per group), whereas vascular reactivity experiments, using an organ bath system, included six individual vessel samples per group. Statistical analysis was performed using the paired Student′s t‐test (p < 0.05). Diazepam alone significantly reduced superoxide dismutase (SOD) activity, indicating an apparent impairment of antioxidant defenses. In contrast, concomitant treatment under hyperhomocysteinemia lowered the levels of thiobarbituric acid reactive substances (TBARSs) and NO2 −, suggesting partial protection against lipid peroxidation and nitrosative stress. Histologically, diazepam preserved arterial structure with minor subendothelial changes, whereas cotreatment reduced homocysteine‐induced endothelial disruption, smooth muscle disarray, and medial connective tissue loss. Apoptotic index was significantly reduced with cotreatment comparing to hyperhomocysteinemia, whereas diazepam alone showed values similar to control. Functionally, diazepam significantly reduced serotonin‐induced contraction of HUAs, but this vascular effect was attenuated under hyperhomocysteinemic conditions. Overall, concomitant treatment with diazepam partially attenuated oxidative and structural changes induced by hyperhomocysteinemia in our experimental setting, in a preclinical model mimicking selected pathophysiological aspects of preeclampsia.