NAH Ameliorates Endothelial Cell Injury In Lpsinduced Sepsis By Enhancing AngI-Tie2 Signal Pathway

Background and aims:Sepsis commonly progresses to disseminated intravascular coagulation and Endothelial barrier dysfunction is central to the pathogenesis of sepsis-associated complications.AngⅠ-Tie2 signaling pathway protect vascular endothelial cells that are crucial for the regulation of endothelial barrier function.non-anticoagulant heparin(NAH)possesses various biological activities,such as anti-inflammatory activity and endothelial barrier protection during sepsis.The present study aimed to evaluate the effect of NAH which is a non-anticoagulant heparin derivative,on improving sepsisinduced vascular endothelial cell injury by constructing a sepsis cell model using lipopolysaccharide(LPS).By comparing the cell survival rate,cell migration rate and the expression of inflammatory factors,the specific mechanism of non-anticoagulant heparin regulating the AngⅠ-Tie2 signaling pathway to protect vascular endothelial cells will be further studied,which will provide a basis for the prevention and treatment of vascular endothelial cell damage caused by sepsis in the future.The application of the method and its prevention and treatment drugs provide new scientific ideas.Materials and Method:Human umbilical vein endothelial cells were selected and randomly divided into 7 groups:control group(Control group),lipopolysaccharide group(Lipopolysaccharide,LPS group),non-anticoagulant heparin intervention group(NAH+LPS group),AngⅡgene knockout non-anticoagulant group Heparin intervention group(AngⅡ+LPS+NAH under SiRNA),non-anticoagulant heparin intervention group under SiRNA under Tie2 knockout(Tie2+LPS+NAH),lipopolysaccharide group under AngⅡ knockout(AngⅡ+LPS under SiRNA),Tie2 gene knockout under lipopolysaccharide group(SiRNA under Tie2+LPS).the LPS used to induce sepsis and then 1.5 ug of NAH were administered in those treated group and cells were incubated for 24 hr.before further analysis were done.Result:The results of qPCR showed that the mRNA expression of Ang2 and Tie2 in endothelial cells was significantly decreased compared to control group,while the negative control group had no significant decrease p<0.0001.NAH pretreatment significantly attenuated LPS-induced sepsis,and increased the endovascular integrity.Compared with the control group,the LPS group showed significantly reduced cell viability(P<0.001.In those LPS group that has been treated with NAH the cell viability is enhanced.We notice that the cell viability of the AngⅡ gene knockout non-anticoagulant heparin intervention group(SiRNA AngⅡ+LP S+NAH)was higher than that of the non-anticoagulant heparin intervention group(NAH+LPS group).On the other hand,when we do Tie2 gene knockout SiRNA,the cell viability of the nonanticoagulant heparin intervention group(Tie2+LPS+NAH)had little change compared with the non-anticoagulant heparin intervention group(NAH+LPS group).The cell viability of the LPS group undergoing AngⅡ knockout without NAH treatment(SiRNA AngⅡ+LPS)decreased compared to those with the NAH intervention group.Finally,the lipopolysaccharide group under Tie2 gene knockout(Tie2+LPS under SiRNA)shows the least cell viability(both P<0.05).The levels of TNF-α,IL-6,IL-1 and SDC-1 in lipopolysaccharide group were higher than those in control group(P<0.05).Furthermore,the levels of TNF-α,IL-6,IL-1,and SDC-1 in the AngⅡ knockout group were decreased when treated with NAH.Conclusion:1.The vascular endothelial cell injury caused by sepsis may lead to the decrease of HUVEC cell survival rate and cell mobilityLow and elevated levels of inflammatory cytokines.NAH pretreatment can improve cell survival rate and mobility level,and reduce the level of inflammatory factors.2.The decreased expression of AngⅡ is conducive to improving the survival rate and mobility of cells,and reducing the level of inflammatory factors.3.These results indicate that NAH can alleviate the damage of vascular endothelial cells caused by sepsis and reduce the inflammatory process,which may be caused by the activation of the activity of AngⅠ-Tie2 pathway and the reduction of endoglycemic calyx shedding.NAH may protect endocalyx by activating AngⅠ-Tie2 pathway,reduce the level of inflammatory factors and alleviate the damage of vascular endothelial cells caused by sepsis.