| Part I Changes of PD-L1~+neutrophil ratio in sepsis miceObjective:Make a mouse model of sepsis,and determine the trend of PD-L1~+neutrophil ratio during the onset of sepsis.Methods:?C57BL/6 male mice(8-10 weeks old,weighing 20-25g)were anesthetized intraperitoneally with 0.3%sodium pentobarbital,and then a mouse model of sepsis was made by cecal ligation and puncture(CLP).?Blood samples were taken from the heart under anesthesia at 24 hours,48hours,72 hours,and 96 hours after modeling.Leukocytes were stained with Anti-Mouse Ly-6G and Anti-Mouse CD274,and then were detected by flow cytometry.After the voltage and fluorescence channel compensation of the flow cytometer was performed,the double positive cells of Ly-6G~+and CD274~+were defined as PD-L1~+neutrophils.?The bone marrow was taken from the femur,and the method of detecting the ratio of PD-L1~+cells in the bone marrow neutrophils was the same as that of blood flow cytometry.Results:?25 mice in the CLP group were operated,of which 2 mice were unsuccessfully awakened after the operation,and the remaining 23 mice were awakened successfully after the operation and had manifestations of sepsis.15mice in the Sham group were operated and all recovered smoothly after operation.?In the blood of CLP mice,the proportion of PD-L1~+neutrophils was4.433±0.77%,9.583±2.222%,16.3±3.716%and 12.933±1.877%at 24 hours,48 hours,72 hours and 96 hours,respectively.while the proportion of PD-L1~+neutrophils in the Sham group at 24 hours,48 hours,72 hours,and 96 hours was2.647±1.117%,3.867±1.22%,7.003±0.512%,and 4.387±2.531%.Through statistical analysis,it was found that the ratio of blood PD-L1~+neutrophils in the CLP group was significantly higher than that in the sham group at 48 hours,72hours and 96 hours(P<0.05).?In bone marrow,the proportion of PD-L1~+neutrophils in the CLP group at24 hours,48 hours,72 hours,and 96 hours was 10.867±0.493%,14.7±1.353%,25.367±6.037%,and 17.433±2.616%,respectively.And the proportion of PD-L1~+neutrophils in Sham group at 24 hours,48 hours,72 hours and 96 hours was 6.197±1.127%,5.2±2.05%,9.8±1.928%and 9.417±2.029%,respectively.Through statistical analysis,it was found that the proportion of bone marrow PD-L1~+neutrophils in the CLP group was significantly higher than that in the Sham group at 24h,48h,72h,and 96h(P<0.05).Conclusion:?The cecal ligation and perforation method is an ideal method for making a mouse model of sepsis.?After the onset of sepsis,the proportion of PD-L1~+neutrophils in the blood and bone marrow of mice gradually increased,reaching a peak at about 72hours.Part II Analysis of the efficacy of atezolizumab in sepsis miceObjective:To evaluate the efficacy of atezolizumab in sepsis mice by endotoxin levels,ileal permeability,ileal histopathological score detection,ileal tight junction protein and survival analysis.Methods:?The experimental group was divided into three groups:Sham group,CLP group and Atezolizumab group.Atezolizumab group mice were sepsis mice treated with atezolizumab.There were 15 mice in the Sham group,27 mice in the CLP group,and 27 mice in the Atezolizumab group.In each of the three groups,15 mice were used to detect endotoxin levels,intestinal permeability,ileal histopathology scores,and ileal tight junction protein expression.The other12 CLP mice and 12 Atezolizumab mice were used for survival analysis.?Blood was taken from the heart under anesthesia at 24 hours,48 hours,and72 hours after modeling,and the endotoxin level was measured by Limulus test kit;?Fluorescently labeled dextran 40S(FD40S)was administered to the stomach5 hours before blood collection,Blood was taken from the heart under anesthesia at 24 hours,48 hours,and 72 hours after septic mice modeling.A fluorescence spectrophotometer was used to detect the fluorescence value of each sample,and this fluorescence value was converted to a concentration value according to the standard curve.The concentration of FD40S was used to reflect intestinal permeability.?At 72 hours after modeling,a section of the ileum was cut under anesthesia,and Hematoxylin-eosin(HE)staining was performed,and the ileal pathological histology score was performed according to the Chiu score method.?At 72 hours after modeling,a section of the ileum was cut under anesthesia,and the expression of three tight junction proteins was detected by immunohistochemistry.The expression differences of claudin-1 protein,occludin protein,and zonula occludens-1(ZO-1)protein were compared in each group.?Recorded the death time of the mice in the CLP group and the Atezolizumab group,drew a survival curve,then compared the difference in survival time between the two groups by log-rank statistical test.Results:?The blood endotoxin levels measured in the Sham group at 24 hours,48hours,and 72 hours after surgery were 0.224±0.008 EU/ml,0.241±0.128EU/ml,and 0.23±0.064 EU/ml;The endotoxin levels in the CLP group were0.496±0.102 EU/ml,0.742±0.153 EU/ml,and 0.956±0.162 EU/ml,while the endotoxin levels in the Atezolizumab group were 0.477±0.069 EU/ml,0.488±0.145 EU/ml,and 0.564±0.128 EU/ml.By comparison,the endotoxin levels in the CLP group were significantly higher than those in the Sham group at 24 hours,48 hours,and 72 hours(P<0.05).Compared with the CLP group,the endotoxin levels in the Atezolizumab group were significantly reduced at 24,48 and 72 hours(P<0.05).?The concentrations of FD40S at 24 hours,48 hours,and 72 hours after surgery in the Sham group were 1.754±0.575 ug/ml,2.758±0.636 ug/ml,and3.473±1.434 ug/ml,respectively.In the CLP group,48-hour and 72-hour FD40S concentrations were 8.919±2.681 ug/ml,25.024±3.68 ug/ml,and32.283±3.456 ug/ml,while the concentrations of FD40S in Atezolizumab group at 24,48,and 72 hours were 8.519±2.335 ug/ml,13.1±4.098 ug/ml and17.981±3.211 ug/ml,respectively.By comparison,the concentrations of FD40S at 24 hours,48 hours,and 72 hours in the CLP group were significantly higher than that in the Sham group(P<0.05).Compared with CLP group,the concentrations of FD40S in the Atezolizumab group were significantly lower at48 hours and 72 hours(P<0.05).?The histopathological score of ileum in the Sham group was 0.875±0.835,the histopathological score of ileum in the CLP group was 3.5±1.069,and the histopathological score of ileum in the Atezolizumab group was 2.25±1.035.The comparison results indicated that the histopathological score of ileum in the CLP group was higher than that in the Sham group(P<0.05),while the histopathological score of ileum in the Atezolizumab group was significantly lower than that in the CLP group(P<0.05).?In terms of ileum tight junction protein expression,the expression of claudin-1 protein in the Sham group,CLP group and Atezolizumab group was1.03±0.061,0.652±0.17,and 0.846±0.127,respectively.The expression of claudin-1 protein in the CLP group was significantly lower than that in the Sham group(P<0.05),while the expression of claudin-1 protein in the Atezolizumab group was higher than that in the CLP group(P<0.05).The expression of occludin protein in Sham group,CLP group and Atezolizumab group was1.02±0.125,0.68±0.194 and 0.888±0.122,respectively.Compared with Sham group,the expression of occludin protein in the CLP group was significantly lower(P<0.05),while the expression of the occludin protein in the Atezolizumab group was significantly higher than that in the CLP group(P<0.05).The expression of ZO-1 protein in Sham group,CLP group and Atezolizumab group was 1.01±0.137,0.794±0.124 and 0.896±0.092,respectively.The ZO-1 protein expression in CLP group was significantly lower than that in the Sham group(P<0.05).The ZO-1 protein expression in the Atezolizumab group was higher than that in the CLP group,but the difference between the two groups was not statistically significant(P>0.05).?Sepsis mice treated with atezolizumab had a longer survival time(P<0.05).Within 90 hours,about 50%of the mice in the CLP group died,while only about25%of the mice in the Atezolizumab group died.Conclusion:?During sepsis,the level of endotoxin in the blood is increased,the intestinal epithelial cells are damaged,and the expression of tight junction protein is decreased,which leads to the damage of the intestinal mucosal barrier and an increase in intestinal permeability.The damage of the intestinal mucosal barrier in turn aggravates the sepsis and forms a vicious circle.?In sepsis mice,atezolizumab treatment can reduce blood endotoxin levels,reduce intestinal epithelial cell damage and increase intestinal tight junction protein expression,protect the intestinal mucosal barrier and extend survival time.Part III Exploring the mechanism of atezolizumab in the treatment of sepsis from the perspective of bioinformaticsObjective:To understand the gene expression changes of sepsis mice before and after treatment with atezolizumab,and explore the molecular biological mechanism of atezolizumab treatment of sepsis.Methods:The experiment grouping was the same described as above.72 hours after modeling,the blood of Sham group,CLP group and Atezolizumab group mice was collected from the heart under anesthesia.There were 5 mice in each group.Neutrophils were isolated from the blood,then RNA was extracted and expression profiling was performed.DESeq software was used to screen differentially expressed genes,and the differentially expressed genes screened were used for PPI(Protein-protein interaction)network construction,GO(Gene ontology)annotation analysis,KEGG(Kyoto encyclopedia of genes and genomes)signal path analysis.Results:There were 375 up-regulated and 275 down-regulated genes in the CLP group mice compared with the Sham group mice,and 94 up-regulated and 27down-regulated genes in the Atezolizumab group mice compared with the CLP group mice.The most significantly up-regulated pathway in the CLP group mice compared with the Sham group mice was TNF signaling pathway.The most significantly up-regulated pathway in the Atezolizumab group mice compared to the CLP group mice was Leukocyte transendothelial migration.Conclusions:?Gene expression is in dynamic changes,and differences in gene expression often determine differences in phenotype.?The most significantly upregulated signal pathway in septic mice is the TNF signal pathway,which indicates that an obvious inflammatory response occurs during sepsis.?The most significantly upregulated signaling pathway in septic mice treated with atezolizumab is leukocyte transendothelial migration,indicating that atezolizumab can improve the mice's immune defense ability.Part IV In vitro cell experiments to explore the mechanism of the treatment atezolizumab during sepsisObjective:Through in vitro experiments to prove that atezolizumab can resist T lymphocyte apoptosis induced by neutrophils during sepsis and explore its mechanism.Methods:?Neutrophils extracted from mice were co-cultured with T lymphocyte line at a 1:1 ratio for 24 hours.The experimental group was divided into three groups,each group has 8 mice.The Sham group was neutrophils of sham-operated mice+T lymphocyte line+isotype Ig G2a(10ug/ml),and the CLP group was neutrophils of sepsis mice+T lymphocyte line+isotype Ig G2a(10ug/ml),Atezolizumab group was neutrophils of sepsis mice+T lymphocyte line+atezolizumab(10ug/ml)in sepsis mice.After Annexin V-FITC,PI,Anti-CD3-APC staining,flow cytometry was used to detect apoptosis rate of T lymphocytes.?Five blood samples of sepsis mice were collected.Neutrophils were extracted and co-cultured with T lymphocytes at a 1:1 ratio for 24 hours,respectively.After Anti-PD-1 staining,the proportion of T lymphocytes expressing PD-1 was detected.Results:?The apoptosis rates of T lymphocytes in the Sham,CLP and Atezolizumab group were 8±1.638%,27±2.855%,and 12.93±2.15%,respectively.After statistical analysis,the apoptosis rates of T lymphocytes in the CLP group were significantly higher than that in the Sham group(P<0.05),while the apoptosis rates of T lymphocytes in the Atezolizumab group were significantly lower than that in the CLP group(P<0.05).?All 5 co-cultured T lymphocytes could express PD-1,and the median ratio of expressing PD-1 T lymphocytes was 21.9%.Conclusion:?The apoptosis of T lymphocytes induced by neutrophils increases during sepsis.?The PD-L1 antibody atezolizumab can reduce the apoptosis rate of T lymphocytes during sepsis,which may work by blocking PD-1/PD-L1 signal pathway. |
PDF Full Text Request