The Effect Of Ulinastatin On Lipopolysaccharide Induced Damage Of Endothelial Progenitor Cell By Activating TIE2 Signaling Pathway

ObjectiveTo observe the variation of the ulinastatin on lipopolysaccharide induced damage of early endothelial progenitor cell number and function and Tie2?AKT?eNOS protein express,and discuss the mechanism of ulinastatin im-proving endothelial progenitor cells function impaired by LPS in vitro.Methods1.we firstly isolated human peripheral blood-derived EPC by gradientcentrifug ation and characterized EPC by immunofluorescence and cellmorphol-ogy.2.After the different concentrations of LPS(0 ng/ml,5 ng/ml,10 ng/ml)sti-mulates endothelial progenitor cells 4 hours,we detected the functions of EPC and its Tie2 protein expression level via Western blot.3.After different concentrations of UTI(0 IU/ml,200 IU/ml,400 IU/ml,800IU/ml,1600 IU/ml)co-cultivated LPS stimulation EPC12 hours,we observed the functions of EPC again and detected the expression of Tie2 and AKT phosphory-lation level in EPC through Western blot.4.we divided the cell into the EPC group,EPC+LPS group,EPC+LPS+UTI group and respectively tested AKT and eNOS protein expression of phos-phorylation in endothelial progenitor cell after LY294002 and L-NAME respec-tively inhibition of PI3K and eNOS,and then further testing the adhesion?migration and proliferation activity of EPC.Results1.The 0-day MNC with small volume showed round.After 4 days culture,few cell colony can be found.The 7-days cultured EPC exhibited a spindle shape,and more cobblestone cells occurred in EPC of 14-days culture,similar to mor-phology of HUVEC.2.The 7-days cultured EPC could also devour Dil-ac-LDL and combined with FITC-lectin.3.After LPS treating EPC 4 hours,its adhesion(*P<0.05 vs 10ng/ml group;#P=0.056 vs 0ng/ml group,N=6),migration(*P<0.05 vs 10ng/ml group;#P=0.074 vs 0ng/ml group,N=6)and proliferation(*P<0.05 vs10ng/ml group;#P=0.588 vs 0ng/ml group,N=6)ability significantly des-cend when concentration of 10 ng/ml LPS,and its Tie2 protein expression also obviously fall(*P<0.05 vs 10ng/ml group;~#P=0.273 vs 0ng/ml group,N=6).4.After UTI co-cultivation damaged EPC 12 hours,its adhesion,migration and proliferation ability markedly improved when the concentration of 800 IU/ml UTI(*P<0.05 vs LPS+0IU/ml UTI group;#P<0.05 vs LPS+800IU/ml group,N=6),and its expression of Tie2 and AKT phosphorylation level obviously higher than control group(*P<0.05 vs.LPS+0IU UTI group,#P<0.05 vs.LPS+800IU UTI group,N=6).5.After LY294002 and L-NAME respectively inhibition of PI3K and eNOS,Western blot showed that the expression of phosphorylation of AKT and eNOS significantly decreased in endothelial progenitor cell(*P<0.05vs.EPC+LPS group,#P<0.05 vs.EPC+LPS+UTI group,N=6).Meanwhile,we also found that the adhesion,migration and proliferation of EPC were decreased ob-viously than EPC+LPS+UTI group(*P<0.05 vs.EPC+LPS group,#P<0.05vs.EPC+LPS+UTI group,N=6).ConclusionsUlinastatin can enhance the adhesion,migration and proliferation ability of endothelial progenitor cells stimulated by lipopolysaccharide in vitro,and its mechanism may be related to activate the Tie2 signal channel.