Fig. 3

Induction of NETs Following TCb Stimulation, Demonstrating NETs-Mediated Damage in Human Umbilical Vein Endothelial Cells (HUVECs). A Schematic representation of the experimental setup, illustrating the in vitro application of the TCb chemotherapeutic agent to induce NETs formation in neutrophils and generate conditioned media containing NETs for treating HUVECs, B neutrophils extracted from healthy volunteers were treated with TCb for 4, 8, and 24 h in vitro. An increase in NETs dsDNA concentration in the cell culture supernatant was observed starting 8 h post-treatment, C After 8 h of TCb treatment, significant elevations in MPO and histone H3 concentrations were observed in the cell culture supernatant of the TCb-treated group compared to the untreated control, D, E immunofluorescence staining revealed a more pronounced co-expression of MPO and histone H3 in TCb-treated human neutrophils compared to untreated controls, F cell viability of HUVECs treated with TCb-induced NETs was assessed using the CCK-8 assay, revealing a significant decrease compared to untreated controls, G, H western blot analysis revealed significant downregulation of VE-cadherin, CD31, and Syndecan-4, and upregulation of Bax in HUVECs treated with TCb-induced NETs, indicating endothelial cell injury and apoptosis. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001