The Effect And Mechanism Of C3a-c3aR Complement Axis In Sepsis-induced Acute Lung Injury Of Mice

Background:Acute lung injury secondary to sepsis is one of the leading causes of death in sepsis.ALI is characterized by diffuse injury to the lung endothelial and epithelial cells,which leads to an increase in alveolar-capillary permeability and alveolar pulmonary edema.Recent studies have shown that C3a-C3a R promotes the extracellular release of ATP through extracellular signal-regulated kinase 1/2（ERK1/2）,and further promotes secretion of the inflammatory factor IL-1βand activation of inflammasome NLRP3 through the ATP-sensitive receptor P2X7.In addition,C3a can also activate Casapse-11 through the Cpb1-C3a-C3a R pathway,aggravating bacterial endotoxin-induced sepsis.However,the role of C3a-C3a R activation in sepsis relate lung injury remains unclear.Obstructive:To explore the role and mechanism of C3a-C3a R in acute lung injury in mice model of sepsis.Methods:1)The mice model with sepsis was established with cecal ligation and puncture（CLP）.Mice were randomly divided into Sham group,CLP group,CLP+PBS group,CLP+C3a Ra（C3a R antagonist,SB290157）group,and GSDMD^-/-group.In C3a Ra group,C3a R inhibitor was injected intraperitoneally at 12,24,and 48 h after surgery.2)The spleen,lung and plasma were collected at 72h after the operation.After processing,immunology experiments were performed to compare the severity of tissue damage,levels of inflammatory cytokines,and levels of pyroptosis-associated protein.3)In addition,a 72-hour survival analysis was performed to evaluate the effect of C3a R inhibitors on the prognosis of sepsis.4)In vitro experiments,pulmonary vascular endothelial cells were randomly divided into LPS+ATP group,LPS+ATP+PBS group,LPS+ATP+C3a Ra group.After stimulation the total cell proteins was collected for western blotting to compare the expression of pyroptosis-associated proteins.Results:1)Levels of C3/C3a in lung and spleen of septic mice were higher than that in normal mice.2)Degree of lung injury of the C3a Ra group was lower than that of the CLP and GSDMD^-/-group.Levels of IL-1βand IL-18 in the plasma and tissues of the C3a Ra group was lower than that of the CLP group.The survival ratio of mice in C3a Ra group was significantly better than that in CLP group and GSDMD^-/-group.3)The levels of pyroptosis associated proteins in the total proteins of lung of mice in C3a Ra group was decreased.4)Mice pulmonary vascular endothelial cells were treated with C3a R inhibitor,and the level of pyroptosis associated proteins was significantly lower than that in the LPS+ATP group.Conclusion:The level of C3/C3a was increased in septic mice.Inhibition of C3a-C3a R complement axis can inhibit pyroptosis of pulmonary vascular endothelial cells,relieve acute lung injury in sepsis,and reduce inflammatory cytokines level.