Role Of Pulmonary Endothelial Cell-barrier Dysfunction Mediated By P38 MAPK In Lung Ischemia-reperfusion Injury

Objective:The chief purpose of our study was to examine whether p38 MAPK plays an important role in the development of LIRI by mediating barrier dysfunction of pulmonary endothelial cell.Methods:1.Twenty-four adult male SD rats were divided into four groups at random:control group,p38 MAPK inhibitor group(SB203580 group),ischemia-reperfusion group(I/R group),ischemia-reperfusion+p38 MAPK inhibitor group(I/R+SB203580 group).I/R group:In I/R group,the rats subjected with thoracotomy.Then the left hilus pulmonis were clamped by a vascular clamp for 1h,and then accepted reperfusion for 4h.The rats in I/R+SB203580 group received SB203580 lOmg/kg by intraperitoneal injection 30 min before an operation begins.Degree of lung injury induced by ischemia-reperfusion(I/R)was assessed by pathohistological lung injury score,the changes of morphology and ultrastructure.Endothelial barrier permeability was detected by the lung tissues wet/dry(W/D)ratio,the total proteins in bronchoalveolar lavage fluid(BALF)and the leakage of evans blue.The contents of IL-6 and IL-1? in the lung tissues were tested to evaluate inflammatory reaction.The protein expression levels of aquaporin 1(AQP1),intercellular adhesion molecule 1(ICAM-1)and zonulae occludente 1(ZO-1),vascular endothelial cadherin(VE-cadherin),p-p38 MAPK in lung tissues were detected by western blot.Protein expression of ZO-1 and VE-cadherin were estimated by Immunohistochemistry.2.The rat pulmonary microvascular endothelial cells(rPMVECs)were divided into five roups:Control group,NC group(rPMVECs were infected with a negative control lentiviruses),shRNA-p38 MAPK group(rPMVECs were infected with the short hairpin-RNA(shRNA)of p38 MAPK lentiviruses),OGD/R group(rPMVECs subjected with OGD/R),OGD/R+shRNA-p38 MAPK group(shRNA-p38 MAPK group was treated with OGD/R).OGD/R group:The cells were placed in the special cell culture chamber,which was flushed with 1%O2,5%CO2 and 94%N2 at 37? for 1 h then continued to be cultured in glucose-containing medium in 5%CO2,95%air at 370C for 4 h.Cell viability and apoptosis were measured to evaluate the degree of cell injury.The assay of transwell to detect permeability of pulmonary microvascular endothelial cell barrier.The protein expression levels of aquaporin 1(AQP1),intercellular adhesion molecule 1(ICAM-1)and zonulae occludente 1(ZO-1),vascular endothelial cadherin(VE-cadherin),p-p38 MAPK in lung tissues were detected by western blot.Protein expression of ZO-1 and VE-cadherin were estimated by immunofluorescence.Results:Compared with the control group and SB203580 group,the lung tissues inflammatory changes were marked and the ultrastructure of lung was severely damaged in I/R group.After pretreatment with SB203580,the inflammatory changes of lung tissue and the degree of lung injury were significantly reduced.The W/D ratio of lung,the protein content in BALF and the leakage of Evans blue in I/R group increased significantly,which indicated that the permeability of lung endothelial barrier increased.After pretreatment with SB203580,the permeability of endothelial barrier decreased significantly.In cell experiments,we found that decreasing the expression of p38 MAPK could alleviate the OGD/R-induced cell damage and reduce the permeability of pulmonary microvascular endothelial cell barrier.Inhibiting the expression of p38 MAPK can also increase the pulmonary ischemia-reperfusion-induced low expression of AQP1,ZO-1 and VE-cadherin,and decrease the pulmonary ischemia-reperfusion-induced overexpression of ICAM-1 in vitro and vivo.Conclusions:inhibition of p38 MAPK could alleviate lung ischemia-reperfusion injury(LIRI)by decreasing pulmonary blood-air permeability.