The Expression Of Aquaporin 5 In Acute Pancreatitis Associated Lung Injury In Rats

Acute pancreatitis (AP) is a common clinical disorder presenting as an acute abdomen, with sudden onset, many complications and high mortality. It causes damages not only to pancreas, but also to distant organs, such as lung, liver, kidney, intestines etc. Among them, acute lung injury (ALI) is the most common complication of AP. The pathogenesis of lung injury in AP is not very clear. The activity of trypsin, cytokines and inflammatory mediators, oxidative stress, microcirculation disturbance may play important roles in this process.Aquaporins (AQPs) are a group of membrane transport proteins with relations to water-permeability. So far, 13 members of AQPs have been found in mammalian (AQP0 ~ AQP12), 6 of which are distributed in the lung tissue (AQP1, AQP3, AQP4, AQP5, AQP8, AQP9). Studies have shown that AQP5 is involved in the pathogenesis of freshwater drowning, hyperoxia-induced lung injury and acute respiratory distress syndrome, and it may affect the bronchial contraction and participate in the occurrence of asthma and glandular body secretion.One of the main pathological changes of acute lung injury is pulmonary edema, there are many studies on the relationship between lung injury and AQPs. On the basis of current research findings, the AQP1 expression of the lung tissue was significantly decreased in the mRNA level and protein level in AP associated lung injury. This indicated that the decreased expression of AQP1 played an important role in pulmonary edema of AP. Dexamethasone (DEX) can improve AP associated lung injury by inhibiting inflammatory mediators and increasing AQP1 expression. However, there is no relevant research on the relationship between the expression of AQP5 and AP associated lung injury.Objective: To investigate the pathological changes of lung tissue, wet / dry weight ratio, PaO2 and AQP5 expression in the lung tissue of AP rat,and by admistrating DEX therapy, to explore the effect of DEX on AQP5 expression of the AP associated lung injury.Method:1 Animal model: The experiment was performed in male Wistar rats under anesthesia injected intra-peritoneally with 10% chloral hydrate. Then, the abdomen median incision was performed. Used non-invasive arterial clip to block pancreatic duct near the hepatic portal before pancreatic duct was injected antidromicly with 5% sodium taurocholate (1 ml / kg) at the speed of 0.2ml/min by microinjector. Then pressed the pancreatic duct entering duodenum. 3 minutes later, removed the artery clip and sutured abdomen.2 Animal groups: All male Wistar rats (n=54) were divided into three groups at random: sham-operation group(n=18), AP group(n=18), and DEX group(n=18). Then every group was divided into 3 subgroups(n=6) at different time points: 3 h,6 h,12 h. In DEX group,the rats were injected DEX intraperitoneally immediately after making the animal model.3 Blood gas analysis: The levels of PaO2 were determined by blood gas analyzer.4 The wet/dry weight ratio of lung: The surface fluid of the left lobe of lung was dried up with absorbent papers, then weighed the lung lobe and put it into the 60°C baker for 72 h, then weighed it again and caculated the wet/dry weight ratio.5 Determination of serum amylase: The level of serum amylase was determined by automatic biochemical analyzer.6 Pancreas and lung histological analysis: Paraffin section was stained by hematoxylin-eosin (HE).7 Immunohistochemical staining of AQP5: After dehydration and antigen heat plerosis, the sections were incubated with 3% hydrogen peroxide for 20 min. The sections were dripped with goat serum for 30 min, then incubated with first antibody at 4°C overnight. In negative control group, PBS replaced first antibody. Dripping successively the second antibody and the third antibody, which were kept 30 min respectively. The results were visualized by using 3, 3-diaminobenzidine (DAB) as chromogen. At last, the sections were redyed with hematoxylin.8 Western blot: A piece of lung tissue about 250 mg was homogenized thoroughly by Glass homogenizer, then 1ml lysis buffer was added to it for extraction of the protein (the process according to the instruction of kit), the protein about 100μg was mixed with loading buffer, then it was performed SDS denaturing polycrylamide gel electrophoresis, successively it was thansferred to PVDF film. Then it was incubated with first antibody at 4°C overnight, successively the second antibody at room temperature for 2 h.Results:1 Histological findings of pancreas: It was seen by naked eyes and light microscope that pancreatic gland and its surrounding tissue was approximatly normal in sham-operation group; edema, hemorrhage, necrosis and inflammatory cells infiltration of pancreatic tissue were more and more severe, and the ascites volume was growing in AP group and DEX group.2 Histological findings of lung: It was seen by naked eyes and light microscope that lung tissue was approximatly normal in sham-operation group; pulmonary edema, hemorrhage, and inflammatory cells infiltration were becoming more and more severe in AP group; in DEX group, the injury of lung tissue was lighter than in AP group.3 Serum amylase: There was no significant difference in sham-operation group at different time points. Compared with sham-operation group, the levels of serum amylase were increased in AP group and DEX group (P<0.05). Compared with AP group, the levels of amylase were significantly decreased at 6h and 12h time points in DEX group (P<0.05), but there was no significant difference between the two groups at 3h time point.4 The wet/dry weight ratio: There was no significant difference in sham-operation group at different time points. Compared with sham-operation group, the wet/dry weight ratios were increased in AP group and DEX group (P<0.05).Compared with AP group, the wet/dry weight ratios in DEX group were declined at different time points (P<0.05).5 PaO2: There was no significant difference in sham-operation group at different time points. Compared with sham-operation group, PaO2 were significantly lower at all time points in AP group and DEX group (P<0.05). Compared with AP group, PaO2 were significantly increased at all time points in DEX group (P<0.05).6 Immunohistochemical staining of AQP5: In sham-operation group, we found an overexpression of AQP5 in alveolar epithelial cells and thin bronchial surface epithelial cells. Compared with sham-operation group, the expression of AQP5 in AP group was significantly reduced (P<0.05). The expression of AQP5 in DEX group was significantly increased than that in AP group (P<0.05).7 Western blot of AQP5: There were no significant differences in sham-operation group at different time points. Compared with sham-operation group, it showed that the expression of AQP5 was decreased at all time points in AP group and DEX group (P<0.05).Compared with AP group, the expression of AQP5 was increased at all time points in DEX group (P<0.05).Conclusions:1 On the basis of the increasing of serum amylase and lung wet /dry weight ratio, the decreasing of PaO2, pancreatic histological findings of edema, hemorrhage and necrosis,and lung tissue edema, congestion, hemorrhage and flammatory infiltration, we concluded that AP associated lung injury model has been successfully manufactured by pancreatic duct injecting antidromicly with 5% sodium taurocholate.2 Compared with sham-operation group, the expression of AQP5 was decreased in the lung tissue of AP group. This indicates that AQP5 plays a role in the water-liquid equilibrium of lung and AQP5 may be a protective protein in AP rats.3 In DEX group, the expression of AQP5 was increased and lung injury was reduced. This indicates that DEX upregulates AQP5 expression to improve lung injury.