The Role Of PD-1 In Lung Ischemia-reperfusion Injury In Rats

OBJECTIVE: To investigate the role of PD-1 in lung ischemia-reperfusion(I/R)injury of rat model.METHODS: Thirty Sprague-Dawley rats were randomly divided into five groups: Control group,2 hours post I/R group(I/R 2h),6 hours post I/R group(I/R 6h),24 hours post I/R group(I/R 24h),72 hours post I/R group(I/R 72h),six animals in each.Rats were anesthetized by intraperitoneal injection of 10% chloral hydrate.Except the control group,the rats in the other experimental groups opened the chest after successful tracheal intubation.Establishment of a model of lung ischemia-reperfusion by clamping the left hilar with non-invasive microvascular clamp.Release the vessel clamp after 60 minutes of ischemia and close the chest cavity.Reperfusion for 2 h,6 h,24 h,72 h.After the set time,the rats were sacrificed by cardiac bloodletting,and the left lung tissue samples of each group were collected for examination.The wet and dry weights were calculated separately,and the wet-dry weight ratio(W/D)of lung tissues was calculated.The pathological changes of lung tissues were observed by HE staining,and the lung injury scores were evaluated.Ultrastructural changes in lung tissue were observed by transmission electron microscopy(TEM).Detection of total protein concentration in bronchoalveolar lavage fluid by BCA.The expression of interleukin-1?(IL-1?),interleukin-4(IL-4),interleukin-6(IL-6)and interleukin-10(IL-10)in lung tissue was detected by enzyme-linked immunosorbent assay(ELISA).The expression of PD-1 protein in lung tissue was detected by Western blot.The expression of CD 11 c,CD 16,CD 206 and Arg 1 in lung tissue was detected by immunohistochemistry.RESULTS: Successful establishment of a model of lung ischemia-reperfusion injury by clamping the left hilar.Compared with the control group,the W/D values of the ischemia-reperfusion group increased,and increased significantly in the I/R 6h group.The difference was statistically significant(P<0.05).Compared with the control group,the BALF protein content increased significantly in the 6h reperfusion group,the difference was statistically significant(P<0.05).The HE results showed that there was no significant change in the morphology of the lung tissue of the control group.Lung injury began to appear in the 2h after ischemia-reperfusion,and the lung injury score increased.The morphology of the lung tissue showed destruction of the alveolar septum,thickening of the interstitial lung,and exudation of inflammatory substances.The lung injury was the most serious in I/R 6h group,and there was obvious edema,pulmonary interstitial thickening,and a large number of inflammatory cell infiltration.The lung injury score was the highest,the difference was statistically significant(P<0.05).The microstructure of lung tissue was observed by electron microscopy.It was found that the ultrastructure of lung tissue in the control group is normal,in I/R 2h group began to change and the microvilli decreased.The injury in I/R 6h group was the mostserious,the microvilli decreased sharply,the lamellar bodies were sparse,the distribution was uneven,then the degree of injury gradually decreased.The results of ELISA showed that compared with the control group,the expression levels of IL-1?,IL-4,IL-6 and IL-10 in the lung tissue of each experimental group increased,the difference was statistically significant(P<0.05).Among them,IL-1?,IL-6,IL-10 began to increase in I/R 2h group,reached the peak in group and then decreased in expression;IL-4 began to increase in I/R 2h group,and reduce expression after reaching the peak in I/R 24 h group.Western blot showed that compared with the control group,the expression of PD-1 increased in each experimental group,the difference was statistically significant(P<0.05).The expression of the group was the highest in I/R 6h group.Immunohistochemistry showed that compared with the control group,the expression of CD11 c,CD16,CD206 and Arg1 in the lung tissue increased significantly in I/R 6h group(P<0.05).Compared with the I/R 6h group,the expression in I/R 72 h group decreased.CONCLUSION: Changes of PD-1 might be closely related to lung ischemia-reperfusion injury in rats,and its mechanism may be related to PD-1 mediated inflammation induced by pulmonary macrophage polarization.