Expression And Its Significance Of ET-1,VEGF In Trypsin-induced Acute Lung Injury

Objective: To establish a model of acute lung injury by continuous pumping trypsin(TPS)into caudal vein,which is simulated in patients with severe acute pancreatitis(SAP)when the pancreatin is released into blood,observing the expressions of endothelin-1(ET-1),vascular endothelial growth factor(VEGF)in the lung tissue and detect the capillary permeability of lung tissue,and to explore the mechanism and significance of VEGF and ET-1 in trypsin-induced acute lung injury.Mythods: A total of 72 healthy adult male Sprague-Dawley(SD)rats weighing 230-250 g were randomly selected and divided into three groups: model group(trypsin injection group,TPS group),drug inhibition group(trypsin+ulinastatin(UTI)injection group,UTI group),and normal control group(normal saline injection group,CON group).There were 24 rats in each group,which was randomly divided into four time point sub-groups,and there were 6 rats in each time point sub-group,respectively named 3h sub-group,6h sub-group,12 h sub-group and 24 h sub-group.The TPS group was injected with trypsin(15000U / Kg / h)for 2 hours into rat caudal vein to make the model of acute lung injury.The CON group was treated with equal volume of normal saline for 2 hours in rat caudal vein to make the control.The UTI group was treated the same as the TPS Group,then ulinastatin 50,000 units were injected subcutaneous.After that we observed the animals' general situation of three groups.Evans blue(EB)(0.8 ml / kg)was injected into the femoral vein 30 minites before the animals were sacrificed.The rats were sacrificed at 3h,6h,12 h and 24 h the time point after drawing the fundusvenous blood,and then we collected specimens.We draw the fundus venous blood 1.5ml to detect the changes of ET-1 and VEGF in plasma by enzyme-linked immunosorbent assay(ELISA).After sacrificing the rats at each time point,we took some lung tissue to measure the EB content for evaluating the capillary permeability.Took a certain amount of lung tissue to measure wet weight and dry weight and calculate the wet / dry weight ratio(W/D)of lung tissue.Took a certain amount of lung tissue which were fixed with 4% paraformaldehyde and 3% glutaraldehyde separately and observed the pathomorphology of the tissues by light microscopy and transmission electron microscopy;Detected the expression of ET-1 protein and VEGF protein in lung tissue by ELISA method,and the expression of ET-1 m RNA and VEGF m RNA in lung tissue by real-time quantitative PCR detecting system(QPCR).Results:1.The survival rate of TPS group,CON group and UTI group were 100%.The W / D value of TPS group was significantly higher than that of CON group and UTI group(P <0.05),and the W / D value increased with the time of TPS group.At the each time point,gross and microscopic pathology of the lung tissue of the CON group was normal basically,and the UTI group had different degrees of injury,which was lighter than the same time point of the TPS group.The lung injury of TPS group was significantly increased than the other two groups: lung tissue began to appear dot flake dark red bleeding,local edema,a small amount of non-bloody exudation at 3h,and with the time prolonging the lesions showed a significant increasing trend,some developed into "lobar pneumonia",accompanied by blood exudation;Light microscope pathology: the size and distribution of alveolar was disorderly.Pulmonary interstitial was significantly widened,and the capillaries were expansion and congested.Pulmonary interstitial was congested and edema and the exudation of alveolar cavity increased.Around bronchial andalveolar,there were a large number of inflammatory cells,and the degree of injury was gradually increased with the time prolonging.Electron microscopic pathology: lung tissue capillary and alveolar epithelial cells began to damage at 3h,appeared organelle degeneration,basement membrane edema and degeneration.With the time prolonging the damage of capillary and alveolar epithelial cells was gradually increased,the basement membrane degeneration aggravated,and appeared local fracture,capillary lumen stenosis,blood stasis.The cell membrane ruptured,the mitochondria,endoplasmic reticulum and other organelles were severe degeneration,the nucleus was fixed and dissolved.2.The levels of ET-1 and VEGF protein in plasma detected by ELISA method showed that: CON group had no significant difference at each time point;Compared with CON group,the expression of ET-1 and VEGF protein in TPS group was significantly higher,which began to increase from 3h,reached a peak at 12h(P < 0.01);Compared with TPS group,the expression level of ET-1 and VEGF protein of UTI group decreased(P < 0.01).3.The levels of ET-1 and VEGF protein in lung tissue detected by ELISA method showed that: CON group had no significant difference at each time point;Compared with CON group,the expression of ET-1 and VEGF protein in TPS group was significantly higher,which began to increase from 3h,reached a peak at 12h(P < 0.01);Compared with TPS group,the expression level of ET-1 and VEGF protein of UTI group decreased(P < 0.01).4.The levels of ET-1 m RNA and VEGF m RNA in lung tissues detected by QPCR method showed that: CON group had no significant difference at each time point;Compared with CON group,the expression of of ET-1 m RNA and VEGF m RNA in TPS group was significantly higher,which began to increase from 3h,reached a peak at 12h(P < 0.01);Compared with TPS group,the expression level of ET-1 m RNA and VEGF m RNA of UTI group decreased(P < 0.01).5.The capillary permeability of lung tissue evaluated by Evans blue leakage rate method showed that: CON group had no significant difference at each time point;Compared with CON group,the capillary permeability of lung tissue in TPS group significantly increased(P<0.01);Compared with TPS group,the capillary permeability in UTI group decreased(12h group,24 group P < 0.05).Conclusion:1.Trypsin can cause the acute lung injury,which is one of the important causes of external pancreatic organ injuries in severe acute pancreatitis.2.The high expression of ET-1 and VEGF in lung tissue and the increasing permeability in early stage of trypsin-induced acute lung injury show that there exists vascular endothelial injury and capillary endothelial barrier disruption,which may be one of the main mechanisms of acute lung injury induced by trypsin.The expression trend of ET-1 and VEGF factor in plasma is similar to that in the lung tissue,but the value is higher than that of lung tissue,which indicates that ET-1 and VEGF were highly expressed in other tissues and organs except lung tissue.3.Ulinastatin has a protective effect on acute lung injury and microcirculation in SAP.