ABSTRACT
Objective To investigate the influence of heparin pretreatment on serum and lung tissue level of neutrophil extracellular traps (NETs) in septic mice model and its molecular mechanism.Methods Ninety male C57BL/6J mice were randomly divided into control group (n = 30), lipopolysaccharides (LPS) group (n = 30, 30 mg/kg LPS in 100μL normal saline was intraperitoneally injected) and LPS+heparin group (n = 30, 8 U of heparin in 20μL normal saline was subcutaneously injected 30 minutes before the injection of LPS). Six hours later of LPS injection, blood was collected and lung tissue was harvested. Enzyme linked immunosorbent assay (ELISA) was used to assess the concentration of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and histones 2AX (H2AX), neutrophil elastase (NE), which reflected NETs concentration. PicoGreen fluorescent dyes was used to detect serum circulating free DNA (cf-DNA/NETs) concentration. The protein expression levels of H2AX and NE in lung tissue were examined by Western Blot.Results The serum concentrations of TNF-α, IL-6, H2AX, NE, cf-DNA/NETs, and the protein expression levels of H2AX and NE in lung tissue of septic mice were significantly higher than those of control group [TNF-α (ng/L): 133.0±14.1 vs. 2.7±1.0, IL-6 (ng/L): 3911.2±189.2 vs. 298.9±52.5, H2AX (ng/L): 545.5±40.0 vs. 21.9±8.3, NE (μg/L): 6.48±0.12 vs. 0.47±0.15, cf-DNA/NETs (μg/L): 846.3±137.5 vs. 152.7±36.4, H2AX protein (gray value): 1.14±0.09 vs. 0.68±0.04, NE protein (gray value): 0.56±0.03 vs. 0.32±0.04, allP 0.05).Conclusion Heparin pretreatment could significantly decrease the level of NETs in serum and lung tissue, and can be the potential mechanism of its organ protection in sepsis.
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.