Expression Of AQP-4 In Rats With Oleic Acid Induced Acute Lung Injury And Influence Of Terbutaline On The Expression

ObjectivesTo study an acute lung injury/acute respiratory distress syndrome(ALI/ ARDS)animal model by oleic acid in rats with stability and repeatability used to investigate lung tissue pathology,alveolar typeâ…¡epithelial cell morphology and water content of extravascular lung water(EVLW)Early changes in lung tissue to evaluate the extent of injury determined by AQP4 in lung tissue and alveolar typeâ…¡cells and then observe the tracheal instillation of terbutaline therapy,analysis of the pathological changes of lung tissue,EVLW determine changes in pulmonary edema formation and clearance liquid volume of poor lung tissue and alveolar typeâ…¡cells,AQP-4 expression differences,and thus analysis of terbutaline in acute lung injury is how it affects the mechanism of pulmonary edema to study theβ2 receptor to stimulate the cAMP pathway on the water channel protein impact and provide a theoretical basisMethods1.Establish the model of lung injury in rats by oleic acidThirty male Sprague-Dawley rats of clean grade were used to experiment after one week of adaptive feeding in our laboratory,weighing 220±20 g. According to the serial number,We divided 3 groups concluding control group,oleic acid induce ALI group,terbutaline therapy group.Three groups underwent tracheal intubation,model group and the terbutaline group of intravenous injection of oleic acid by intraperitoneal vein 0.1 ml/kg after the injection of normal saline control group.15 minutes after terbutaline instillation with endotracheal intubation group terbutaline 2.5mg/kg,more than the equivalent of normal saline instillation group.30 minutes after the abdominal aortic artery pumping 0.3ml blood gas analysis to determine the extent of lung injury.2.Assess the rats' lung injury after injection of oleic acidIntravenous injection of oleic acid,30 minutes after the blood gas analysis; after 30 minutes the bloodletting through abdominal aorta.HE staining through the use of Smith pathological score on pulmonary edema,alveolar and interstitial inflammation,hemorrhage,atelectasis and hyaline membrane formation were analysis of from 0 to 4(11),arterial blood gas analysis to determine lung injury in rats the extent of the establishment of the model.3.Isolating,purifying and identifying of alveolar typeâ…¡cells in ratsmarked lung tissue of choice,using trypsin digestion method improved the separation of typeâ…¡alveolar epithelial cells,using rat IgG purified cell immune adherence,Trypan blue cell viability test,improved tannin staining and transmission electron microscopy to identify cell purity.4.AQP-4 express in lung tissue and ATII cells of ratsKit immunohistochemistry for AQP-4 in lung tissue by immunohistochemical staining;Trizol extraction of alveolar typeâ…¡cells total RNA,UV Spectrophotometer content OD260 detected.Reverse transcription-polymerase chain reaction(RT-PCR)detected the expression of AQP-4mRNA.Strom 860 using image analysis system scanning line density to calculate the product of AQP4 andβ-actin product of the ratio of absorbance points,as the expression level of AQP4m-RNA.5.To test Extravascular lung water content(EVLW) EVLW were examined at 3 groups respectively after infusion by gravity. Lung tissue removed bronchial,by detecting the distribution of pulmonary blood vessels in the outer liquid,Liquid extrude alveolar space by hydrostatic pressure and osmotic pressure.EVLW contents in accordance with formula.Results1.Assessment of the oleic acid rat modelsAfter injection of oleic acid for more than 30s,most experimental animals appeared obviously symptoms such as short of breath,skin of face and legs became purple.PaO₂ and PH were significantly decreased and PaCO₂ was increased obviously apply to diagnostic criteria of ALI/ARDS.The pathological changes of lungs seem to be engorgement,edema,bleeding,etc.2.To assesse the methods of isolation and purification ATâ…¡cellsThe improved methods we used to isolate and purify ATâ…¡cells were feasible.Using these methods,we were able to obtain an ATâ…¡cell population with 84.3±3.21%confirmed by 4%Trypan blue solution,and confirmed by staining for tannic acid,we got 83.7±2.96%purity of oleic acid group.Decreasing the concentration of digestive enzyme and centrifugal rate could raise the cellular vitality.It could be found the lamellar bodies with a tannic acid stain and electron microscope.3.Expression of AQP4 in Alveolar organization and alveolar typeâ…¡cellsAQP4 was present at the membranes of isolated rat alveolus lung by immunohistochemical staining.The distribution of aquaporin-4(AQP-4)on the cellular membrane was observed and it also stained strongly in ALI rat alveolar cells.4.To analysis blood gas analysis,pathology,and EVLW after terbutaline treatmentAfter Terbutaline therapy blood gas analysis does not match the criteria for ALI diagnosis,PO2 increased;Smith pathology score than the model group decreased,with statistical significance;EVLW decreased significantly reflect that terbutaline may has a therapeutic effect in early lung injury,markedly improved alveolar epithelial fluid clearance.5.Terbutaline affect AQP-4 on alveolar and alveolar typeâ…¡epithelial cellsAQP4 in the therapy group is expressed mostly in three groups.It is demonsteated that AQP4 was up-regulated at the membranes of rat alveolar cells in injuried and therapy stage.Through RT-PCR of amplification,the terbutaline group is significantly increased,followed by the model,it could explain that AQP4 of m-RNA is increasing in the nucleus of ATâ…¡and terbutaline may stimulate the expression.ConclusionsIn this model,the rat Blood gas analysis,Smith lung injury score in ALI group was worser than those of control group under microscope and blood gas analysis.The practice indicated that setting up rat ALI model by injection of oleic acid through coeliac vena was an easy and efficient method.We had successfully isolated and purified ATâ…¡cells from normal and ARDS rats.It is simple and feasible that staining for tannic acid to identify purity of cells.Electron microscope is the best way to authenticate these cells.Alveolar typeâ…¡under the microscope the number of epithelial cells than the control group is no significant decrease,cells have ultrastructure injury in the electron microscope.By immunohistochemistry to prove that AQP4 is present at the membranes of alveolar epithelial cells and isolated rat typeâ…¡cells.RT-PCR showed that the model group after ATâ…¡cell damage,AQP4 expression increased,its still on the water diversion channel protein with transport function.Terbutaline administration by air tube,the results which draw from observation of the lungs, blood gas analysis,pathology,alveolar typeâ…¡epithelial cells are less than the standard lung injury.When rats in ALI get therapy of terbutaline the expression of AQP4 in typeâ…¡cells is significantly increased as compared with control group.AQP4 was strongly positive immunohistochemical staining shows that AQP4 transfer from the cytoplasm to the membrane and an increase in cell RTPCR proved to increase synthesis of AQP4,AQP4 could play to promote and alveolar space fluid re-absorption may be the promotion of early lung injury pulmonary edema re-absorption of compensatory mechanisms.