Effects Of Urinary Trypsin Inhibitor On Sepsis-induced Endothelial Cell Injury By Lipopolysaccharide-activated Platelets

Objective:To explore the effects of urinary trypsin inhibitor (UTI) on endothelial cell injury by lipopolysaccharide(LPS)-activated platelets in viro.Methods:1. Platelets were isolated from healthy volunteers’blood and were randomly divided into four groups:group control; group LPS; group UTI; group platelets TLR4 monoclonal antibodies. Group LPS:platelets were incubated with LPS and final concentrations were 0.5,1,5,10 ug/ml. Group UTI:platelets were incubated with LPS and UTI, the final concentrations of UTI were 5000,10000,15000,20000 U/ml. The expressions of TLR4 on platelets were detected by flow cytometry. Choose the concentration of LPS and UTI have the most obvious effects on the expressions of TLR4 on platelets.2. Neutrophil cells and platelets were isolated from healthy volunteers’blood, co-cultured with human umbilical vein endothelial cells (HUVECs). They were randomly divided into six groups: group control; group LPS; group UTI; group platelets TLR4 monoclonal antibodies; group no platelets; group UTI+no platelets. The neutrophil extracellular traps (NETs) were detected by PicoGreen dsDNA Quantitation Kits in each group at 3/6/18 hour. The vascular cell adhesion molecule-1 (VCAM-1) and endothelial cell specific molecule-1(endocan/ESM-1) were detected by enzyme linked immunosorbent assay in each group at 3/6/18 hour. The association of NETs between endocan, VCAM-1 were analyzed by Pearson or Spearman correlation analysis.Results:1.The effects of LPS on the expression of TLR4 on platelets:the expression of TLR4 on platelets in group control were 40.99±1.22%. The expression of TLR4 on platelets were obviously up-regulated with the concentration of LPS to 5ug/ml.2. The effects of UTI on the expression of TLR4 on platelets:The expression of TLR4 on platelets were down-regulated with increasing concentration of UTI treatment. Comparing with group control, platelets TLR4 expression treated with LPS were obviously down-regulated with the concentration of UTI to 15000U/ml.3. The expression of NETs in co-cultured HUVECs group:①Compared with group control, the expression of NETs in group LPS were significant higher at 3/6/18h (P<0.01).② Compared with group LPS, the expression of NETs in group no platelets were significant lower at 3/6/18h (P<0.05); the expression of NETs in group platelets TLR4 monoclonal antibodies were significant lower at 18h (P<0.05),while the other time point was not statistically significant; the expression of NETs in group UTI were significant lower at 3/6/18h (P<0.05).③Compared with group UTI, the expression of NETs in group UTI+no platelets were significant lower at 3/18h (P<0.05),while the other time point was not statistically significant.4. The expression of endocan in co-cultured HUVECs group: ①Compared with group control, the expression of endocan in group LPS were significant higher at 3/6/18h (P<0.01) ② Compared with group LPS, the expression of endocan in group UTI were significant lower at 18h (P<0.05),while the other time point was not statistically significant. ③Compared with group LPS, the expression of endocan in group platelets TLR4 monoclonal antibodies were significant lower at 3/6/18h (P<0.05).④Compared with group LPS, the expression of endocan in group no platelets were significant lower at 3/6/18h (P<0.05).⑤ Compared with group UTI, the expression of endocan in group UTI+ no platelets were significant lower at 3h (P<0.01), while the other time point was not statistically significant.5. The expression of VCAM-lin co-cultured HUVECs group:①Compared with group control, the expression of VCAM-1 in group LPS were significant higher at 3/6/18h (P<0.01) ②Compared with group LPS, the expression of VCAM-1 in group UTI were significant lower at 3/6/18h (P<0.05).③Compared with group LPS, the expression of VCAM-1 in group platelets TLR4 monoclonal antibodies were significant lower at 3/6/18h (P<0.05).④ Compared with group LPS, the expression of VCAM-1 in group no platelets were significant lower at 3/6/18h (P<0.05). ⑤Compared with group UTI, the expression of VCAM-1 in group UTI+ no platelets were significant lower at 3/6h (P<0.01), while the other time point was not statistically significant.6. The association of NETs between endocan, VCAM-1:The level of NETs was positively correlated with endocan, VCAM-1 (r=0.871,P<0.001; r=0.747, P=0.005)。Conclusions:(1) endothelial cell injury from LPS were enhanced by LPS mediated increasing expression of platelets TLR4 and NETs.(2)UTI may acted on platelets TLR4 to decrease endothelial cell injury and NETs expression.(3)NETs induced endothelial cell injury from LPS.