Heparin reduces endothelial cell damage induced by neutrophil extracellular traps / 中华危重病急救医学

ABSTRACT

Objective Clarify the effect of neutrophil extracellular traps (NETs) on endothelial cell injury, and investigate whether the heparin can exert a protective effect on endothelial cells by reducing the endothelial cell injury induced by NETs.Methods Neutrophils (PMN) were obtained from healthy human peripheral blood by Percoll-Histopaque density gradient centrifugation, and was stimulated with phorbol ester (PMA) to induce NETs. The qualitative and quantitative analysis of NETs was detected by immunofluorescence staining combined with fluorescence detector. The NETs were used to induce human umbilical vein endothelial cells (HUVEC)in vitro. Recombinant DNA hydrolytic enzymes (rhDNase) and heparin intervention were added respectively. The activity of HUVEC was measured by methyl thiazolyl tetrazolium (MTT) method after 6 hours.Results PMA can stimulate PMN to produce NETs. Immunofluorescence staining showed the formation of reticular formation around the PMN. The concentration of cell-free DNA in the supernatant of PMN stimulated by PMA was significant increased compared with the control group through the detection of PicoGreen fluorescent labeling instrument (2 hours 119.62±14.83 vs. 24.27±0.67, 4 hours 146.67±21.24 vs. 28.35±2.98, bothP < 0.05). Application of NETs to stimulate the HUVEC, cell damage was dose dependent and inhibition rate increased gradually. The endothelial cell inhibition induced by NETs can be antagonized after adding rhDNase [10μg/L NETs (8.65±0.51)% vs. (10.99±0.35)%, 20μg/L NETs(14.85±0.43)% vs. (16.85±0.49)%, 30μg/L NETs (26.06±3.51)% vs. (27.54±0.62)%, allP < 0.05]. Heparin with different concentrations were added into the experimental group (0.01, 0.1, 1, 10 kU/L). We found that the endothelial cell inhibition rate decreased compared with control group [10μg/L NETs (8.96±0.70)%, (5.32±1.36)%, (0.70±0.30)%, (0.75±0.20)% vs. (10.99±0.35)%; 20μg/L NETs (15.57±0.62)%, (13.28±0.65)%, (6.91±0.15)%, (5.86±0.17)% vs. (16.85±0.49)%; 30μg/L NETs (30.49±0.74)%, (29.41±1.41)%, (23.45±0.75)%, (21.72±1.52)% vs. (27.54±0.62)%, allP < 0.05].Conclusions NETs can induce endothelial cell injury, and the injury degree was increased with the concentration of NETs. Heparin can reduce endothelial cell injury induced by NETs, which may be a potential mechanism for the protective effect of heparin on sepsis.