A Preliminary Study On The Mechanism Of Neutrophil Function Changes In Sepsis

Objectives:1.To study the changes of neutrophil function in peripheral blood of burn sepsis patients during severe infection,including the number of neutrophils,the chemotactic function of neutrophils,and the changes of neutrophil apoptosis.To reveal the phenotypic changes of neutrophils in patients with sepsis during severe infection period.2.CLP sepsis mice model were established.Bone marrow neutrophils were stimulated by LPS in vitro.The functions on chemotaxis and apoptosis,phenotype of bone marrow neutrophils in CLP sepsis mice were detected.Bone marrow neutrophils were stimulated by LPS in vitro.Using P38 inhibitor to stimulate these neutrophils,the regulatory effect of P38 MAPK signaling pathway on neutrophil phenotype was preliminarily revealed,which provided a theoretical basis for the role of neutrophils in the pathogenesis of sepsis.It might provide a new idea for the treatment in clinical sepsis.Methods:1.Clinical trial: Blood samples were collected from 5 patients in burn sepsis and 5 healthy young volunteers as control group.Neutrophils were isolated and purified by Ficoll density gradient centrifugation and their activity was identified by trypan blue staining.The purity was identified by Wright staining and CD66 b staining by the flow cytometry detection,and the chemotactic distance was detected by agarose gel chemotaxis test.The apoptosis ratio of neutrophils was detected by the flow cytometry,and the changes of neutrophil surface molecule PD-L1(CD274)were detected at the same time.The correlation analysis was carried out,and the above data were statistically analyzed.2.Animal model: Twenty male C57/B6 mice were randomly divided into sham operation group(Sham group,n=10)and cecal ligation and perforation group(CLP group,n=10).The survival rate was analyzed after 72 hours of continuous observation.Ten male C57/B6 mice were also randomly divided into sham operation group(Sham group,n=5)and cecal ligation perforation group(CLP group,n=5).The phenotype of bone marrow neutrophils PD-L1(CD274)in mice was detected.At the same time,bone marrow neutrophils were extracted by Percoll density gradient centrifugation in Sham group and CLP group,and these neutrophils were identified by trypan blue staining,and their purity was identified by Wright staining and Ly6 G staining by flow cytometry.Agarose gel chemotactic assay was used to detect the chemotactic function of bone marrow neutrophils in mice,and flow cytometry was used to detect the proportion of apoptosis.3.In vitro: Bone marrow neutrophils of C57/B6 normal mice were purified and divided into normal control group,lipopolysaccharide(LPS)stimulation group,P38 inhibitor SB 203580 group and P38 inhibitor SB 203580 + lipopolysaccharide(LPS)group.After 12 hours of LPS stimulation,the phenotypic changes PD-L1(CD274)of neutrophils in each group were detected by flow cytometry.Results:1.The peripheral blood neutrophils purified by Ficoll density gradient centrifugation showed lobulated or horseshoe-shaped nuclei and reddish cytoplasm by Wright staining.The purity of CD66 b was detected by flow cytometry and the activity of neutrophils was detected by trypan blue.The purity and activity were more than 95%.2.Agarose gel chemotaxis test showed that the distance of neutrophil chemotaxis in sepsis patients was lower than that in normal control group,and apoptosis was delayed in sepsis group.The higher expression of PD-L1(CD274)on peripheral blood neutrophils in sepsis patients was detected.3.In the animal experiment,the survival rate of CLP model group was significantly lower than that of Sham group.The expression of PD-L1(CD274)on the surface of bone marrow neutrophils in CLP group was higher than that in Sham group.The purity activity of bone marrow neutrophils extracted by Percoll density gradient centrifugation in Sham group and CLP group was more than 95% and the agarose gel chemotaxis of bone marrow neutrophils in CLP group was lower than that in Sham group.Apoptosis in CLP group was delayed and the proportion of apoptosis was lower than that in Sham group.4.In vitro,the positive expression rate of PD-L1(CD274)in bone marrow neutrophils stimulated by lipopolysaccharide was significantly higher than that in normal group.However,after using P38 MAPK inhibitor SB 203580,bone marrow neutrophils stimulated by lipopolysaccharide(LPS),the positive rate of neutrophil PDL1(CD274)was significantly lower than that of lipopolysaccharide group.Conclusions:1.The chemotactic function of neutrophils in burn sepsis patients and CLP model mice was inhibited,and at the same time apoptosis was delayed.In addition,the expression of neutrophil surface molecule PD-L1(CD274)in sepsis was higher.2.The expression of PD-L1(CD274)on neutrophils was regulated by P38 signaling pathway and the P38 inhibitor might decrease the expression of PD-L1(CD274)in neutrophils during sepsis.