PD-1 Regulates The Role And Mechanism Of Alveolar Macrophage Polarization In Lung Ischemia Reperfusion Injury

Objective To investigate the role and mechanism of programmed death factor-1(PD-1)in the regulation of macrophage polarization during the occurrence of lung ischemia-reperfusion injury(LIRI).Methods 1.24 healthy and clean male SD rats were divided into blank control group(Control group),PD-1 inhibitor group(BMS202 group),and lung ischemia-reperfusion group(I/Group R),pulmonary ischemia-reperfusion+BMS202 group(I/R+BMS202 group),6 animals in each group.The left lung hilum was clamped with a vascular clip for 1h and then reperfused for 2h to establish a rat LIRI model;rats in the I/R+BMS202 group were given BMS202(5mg/kg)1 hour before surgery by gavage;BMS202 group was given the same amount;The group does nothing.Rats were sacrificed 2 hours after reperfusion in each group,and the left lung tissue was taken to determine the lung wet/dry weight ratio(W/D).After hematoxylin-eosin(HE)staining,the pathological changes of the lung tissue were observed under light microscope and the degree of injury was evaluated.Observe the ultrastructural changes of lung tissue under electron microscope.The expression of CD86 and CD206 were detected by flow cytometry.Using ELISA to detect the expression of TNF-α and IL-1β,,IL-6 in lung tissue.Divide rat alveolar macrophages into 5 groups:blank control group(Control group),negative lentivirus transfection control group(NC group),PD-1 short hairpin RNA lentivirus transfection group(shRNA-PD 1 group),oxygen and glucose deprivation reperfusion group(OGD/R group),oxygen and glucose deprivation reperfusion+PD-1 short hairpin RNA lentivirus infection group(OGD/R+shRNA-PD 1 group).Cell oxygen and glucose were deprived for 1h,and then restored to supply 2h to establish the cell OGD/R model.The expressions of PD-1,and hypoxia-inducible factor 1α(HIF-1α)were detected by WB,the expression of M86 macrophage surface marker CD86 and M2 macrophage surface marker CD206 were detected by flow cytometry,and TNF was detected by ELISA-α,IL-1β,IL-6 levels.2.To further explore the mechanism by which PD-1 regulates the polarization of alveolar macrophages during lung ischemia-reperfusion injury,we used PD-1 inhibitors(BMS202)and transfected lentiviral vectors(shRNA-PD 1)to inhibit PD-1,WB detection of the expression of PI3K/AKT signaling pathway in animal models and cell models of lung ischemia-reperfusion to clarify that PD-1 participates in lung ischemia-reperfusion injury by mediating activation of PI3K/AKT signaling pathway;then,further application AKT inhibitor(LY294002)was used to detect macrophage polarization after lung ischemia-reperfusion to confirm the regulation of PI3K/AKT signaling pathway on macrophage polarization.24 healthy and clean male SD rats were divided into blank control group(Control group),PI3K/AKT inhibitor group(LY294002 group),lung ischemia-reperfusion group(I/R group),lung In the ischemia-reperfusion+PI3K/AKT inhibitor group(I/R+LY294002 group),6 animals in each group.Except for the LY294002 group and the I/R+LY294002 group,the other groups were modeled and intervened according to experiment 1.The I/R+LY294002 group was injected with LY294002(0.3 mg/kg)via the tail vein 5 minutes before surgery,and the LY294002 group was injected with the same amount Agent.Flow cytometry was used to detect the expression,about CD86 and CD206.Results 1.Compared with the Control group,the lung tissue morphology and ultrastructure of the I/R group were significantly changed,and the lung pathology score,lung W/D ratio,and TNF-α,IL-1β,and IL-6 levels were significantly increased.High,the expression of M86 type macrophage polarization marker CD86 increased significantly.Compared with the I/R group,the lung tissue morphology and ultrastructural changes were significantly improved,the lung pathology score was reduced,and the lung W/D ratio and TNF-α,IL-1β,and IL-6 levels were significantly reduced after BMS202 pretreatment.The expression of polarization marker CD86 of M1 type macrophages was significantly decreased,and the differences were statistically significant(both P<0.05).There was no statistically significant difference in CD206 among the groups.There was no statistically significant difference between the Control group and the BMS202 group.2.Compared with Control group and NC group,we could foud that the expression of PD-1 in shRNA-PD 1 group decreased significantly,and the virus was successfully transfected.Compared with the Control group,the levels of TNF-α,IL-1β,and IL-6 in the OGD/R group cells were significantly increased,the expression of M86 type macrophage polarization marker CD86 was significantly increased,and the expression of HIF-la was significantly increased.Compared with the OGD group,the levels of TNF-α,IL-1β,and IL-6 in the OGD/R+shRNA-PD 1 group were significantly decreased,the expression of M86 type macrophage polarization marker CD86 was significantly decreased.The differences were statistically significant(all P<0.05).There was no statistically significant difference in CD206 among the groups.2.WB detection showed that the phosphorylation level of AKT decreased after I/R,and the phosphorylation level increased after intervention with PD-1 inhibitors,and similar results were obtained in vitro.Compared with the I/R group:the expresion of TNF-α,IL-1βand IL-6 were significantly increased,in the I/R+LY294002 group,and the expression of the polarization marker CD86 of M1 type macrophages was significantly increased.The differences were statistically significant(all P<0.05).Conclusion PD-1 may stimulate the release of inflammatory factors by regulating macrophage polarization to M1,leading to inflammation and tissue damage in the lung.Its mechanism of action is related to PD-1 inhibiting the activation of PI3K/AKT signaling pathway.