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Objectives and questions: Chronic wounds like diabetic foot ulcers are characterized by persistent inflammation. In this process neutrophils release so-called neutrophil extracellular traps (NETs) to eliminate invading pathogens. However, excessive neutrophil activation and overproduction of NETs may also contribute to tissue damage and delayed wound healing. Reactive oxygen species (ROS) are key drivers of NET formation, promoting oxidative damage and pro-inflammatory signaling. Vanillic acid (VA), a phenolic compound with known antioxidative properties, has been suggested to modulate oxidative stress, but its effects on neutrophil activation and NET formation remain unclear. This study aimed to investigate whether VA affects ROS generation and NET formation in human neutrophils and to explore potential underlying antioxidative signaling mechanisms.

Material and methods: Primary neutrophils were isolated from EDTA-blood of healthy and diabetic donors using density gradient centrifugation (Ethical Vote: 844/2020BO2). Neutrophils were pretreated with VA (0.5–500 µM) for 3 hours and then stimulated with phorbol 12-myristate 13-acetate (PMA, 100 nM) to induce ROS production and NET formation. Cell viability was assessed by Calcein-AM assay. Intracellular ROS generation was quantified by DCFH-DA and dihydrorhodamine 123 (DHR-123) assays, while NET formation was measured fluorometrically using Sytox Green and visualized by immunofluorescence staining of neutrophil elastase and DNA. Statistical analyses were performed using the Kruskal–Wallis test followed by Dunn’s post hoc test.

Results: VA did not significantly affect neutrophil viability at concentrations ranging from 0.5 to 500 µM as determined by Calcein-AM assay. VA pretreatment resulted in a dose-dependent reduction of PMA-induced intracellular ROS levels, with a reduction of approximately 65% at 50 µM and 75% at 500 µM compared to PMA-stimulated controls. Correspondingly, NET formation was significantly decreased, showing reductions of approximately 30% and 40% at 50 µM and 500 µM VA, respectively. Immunofluorescence analysis demonstrated that VA treatment was associated with reduced extracellular DNA release and a retention of neutrophil elastase close to the cell membrane following PMA stimulation.

Discussion and conclusions: Vanillic acid effectively reduced ROS production and ROS-dependent NET formation in human neutrophils without compromising cell viability. These findings indicate that VA attenuates neutrophil-driven oxidative stress and NET release, potentially via modulation of antioxidative signaling pathways. VA may therefore represent a promising therapeutic candidate for limiting excessive neutrophil activation in chronic inflammatory conditions.