Mechanism Of PD-L1 Delaying PMN Apoptosis In Sepsis

Objective:To investigate the regulatory role and possible mechanism of Programmed death ligand 1(PD-L1)on the delayed apoptosis of sepsis neutrophil(PMN).Methods:(1)Venous peripheral blood of 18 sepsis patients and the same number of healthy volunteers were collected,and Ficoll density gradient separation method was used to obtain the neutrophil(PMN).(2)After obtaining PMN,the purity and vitality of PMN were detected by trypan blue and white-giemsa mixed staining.(3)PMN cells were cultured in vitro for 5h,10h and 20h,and the apoptosis rate of PMN at different time periods was detected by flow cytometry.(4)The chemokines of PMN were detected by agar-chemokines experiment.(5)Culture the PMN of 18 patients with sepsis,PI staining was used to detect the apoptotic rate and the expression of PD-L1 by Western Blot,gray value analysis was performed,and the ratio of PD-L1/β-actingray value was calculated.(6)Under normal PMN conditions,interfero-γ(IFN-γ)and interferon-γ/Lipopolysaccharide(LPS)were used to promote the expression of PD-L1on PMN in vitro,and PD-L1 siRNA was used to silence the expression of PD-L1.Flow cytometry and Western blot were used to detect the apoptosis rate of PMN cells and the expression level of PD-L1 protein,and the relationship between the apoptosis rate of PMN cells and PD-L1 after the expression level of PD-L1 was detected after the stimulation and silencing of PMN.(7)PD-L1 siRNA was used to silence the expression of PD-L1 in PMN sepsis.Flow cytometry and Western blot were used to detect the apoptosis rate of PMN cells and the expression level of PD-L1 protein,and the relationship between the apoptosis rate of PMN cells and PD-L1 was detected after the expression level of PD-L1 was silenced by PMN.Results:The staining results showed that the purity and activity of PMN in the sepsis group and the normal control group met the requirements of further experiments within a range of more than95%.Using flow cytometry to detect the apoptosis of PMN cells,it was found that the number of PMN cells in peripheral blood of patients in the sepsis group at 5h,10h and 20h was significantly lower than that in the normal control group,with statistically significant difference(P<0.05).The results of agarosaccharide chemotaxis test showed that the chemotaxis distance of PMN in sepsis patients was significantly lower than that in the blank control group(P<0.01).In the experiment of PMN when IFN-γtreatment interfered with normal state,Western blot and flow cytometry showed that the apoptosis rate of PMN after IFN-γtreatment and IFN-γ/LPS stimulation was significantly lower than that of the normal control group(P<0.01),and the expression level of PD-L1 was also higher than that of the normal control group.The results of PD-L1siRNA interference experiment suggested that the apoptosis rate of PMN increased significantly after the silencing of PD-L1(P<0.05).Western blot assay showed that PD-L1 expression level in PMN of sepsis patients was negatively correlated with apoptosis rate,R~2=0.4718.Conclusion:1)The expression level of PD-L1 on PMN was negatively correlated with its apoptosis,and the large amount of PD-L1 expression could significantly delay the life of PMN.2)In sepsis,the chemotactic function of PMN was significantly inhibited.3)PD-L1 is an important molecule in the regulation of delayed apoptosis of sepsis PMN.