Water Metabolism Of AQP-4 In Rats With Oleic Acid Induced Acute Lung Injury During The Initial Stage

ObjectivesAcute Lung Injury /Acute respiratory distress syndrome(ALI/ARDS) is a clinical condition of severe acute respiratory failure,resulting from disruption of the alveolar-capillary barrier membrane and subsequent influx of protein-rich edema into the alveolar spaces.One study discovered lungs of 40%patient with ALI can partly absorbe edema,to cue alveolar epithelial cells have the function of eliminating fluid in 30%-40%,as to estimate alveolar epithelium Integrity and functions.It also obviously correlated to survival rate of ARDS.So it is significance that we study pellating of alveolar epithelial cells in earlier period of ALI/ARDS for recovering.Alveolar typeâ…¡cells(ATâ…¡),which is only 5%surface area of alveoli pulmonum,but amount to 60%.The main functions are transporting fluid,excreting surface active substance and inversion typeâ… cells in ALI.The cells play important role to repair alveolar epithelium.Na⁺ is transported to interstitium by Na⁺,K⁺-ATPase which is on the alveolar epithelial cell's basolateral surface;osmotic pressure effect water into cells or intercellular substance through aquaporins.The key to repair injury of lung is how Alveolar typeâ…¡cells absorb water in earlier period.The aquaporins(AQPs) are a family of water-selective channels membrane proteins whose function is to increase plasma membrane water permeability and thus provide a route of rapid fluid movement.Nowadays,it has no clear mechanism to explain AQPs effection in early acute lung injury.It is known at present that there are aquaporin 1,aquaporin 3,aquaporin 4,aquaporin 5,aquaporin 8,and aquaporin 9 in lung.To identify how AQPs work,we plan to study ATâ…¡which act as "alveolar stem cell".Alveolar typeâ…¡cells are the stem cells of alveolar epithelium;they are multifunctional pneumocytes and play an important role in lung fluid transport. However,no studies have addressed the regulation of AQPs in alveolar typeâ…¡cells on ARDS.The purposes of my study is to determine whether AQP4 of rat is expressed in the alveolar typeâ…¡cells,furthermore we investigate the alteration of AQP4 in ARDS rats induced by oleic acid on the base of setting up rat ARDS models of oleic acid style,isolating and puritying alveolar typeâ…¡cells.It will provid new experimental and theoretic evidence in the study of ARDS' pathogenesis.We have already proved AQP1,5 on the ATâ…¡.Now we study the mechanism which depends on terbutaline uptake by beta-adrenergic receptor on the apical membrane of alveolar typeâ…¡cells followed by extrusion of water on the basolateral surface by the AQP4. That pharmacological treatment with beta-adrenergic agonists may induce a more sustained stimulation of alveolar fluid reabsorption and in turn facilitate recovery from experimental pulmonary edema.Similar results have been achieved experimentally by extravascular lung water content of the abundance of water transports in the alveolar epithelium.It will provide new experimental direction and evidence in the study of pathogenesis,and consummate the theory about mechanisms of lung edema in ARDS.MethodsFor all experiments,adult Sprague-Dawley rats weighing 180-220g were used. We divided 3 groups concluding control group,oleic acid induce ALI/ARDS group,terbutaline therapy group.In the first part,we want to find an effective way to purify alveolar typeâ…¡cells in both normal and ARDS rats;in the second part,the content of extravascular lung water in rat lung was observed in each groups;in the last part,the expression of aquaporin4 in rat alveolar typeâ…¡cells in all rats was studied. In the first part of our experiment,ALI/ARDS rat models were set up using injection oleic acid.Oleic acid was one of saturated fatty acids possessed strong toxicity.If it enters into bodies occasionally,it will bring complicated effects to bodies,especially to lung tissues where it can place a premium on and enlarge lung injury by oxidation.The models caused by oleic acid were recognized to be better models.In this study,rat ALI/ARDS models were made by intravenous injection of oleic acid.The dosage of oleic acid was 0.1ml/kg.Isolating,purifying and identifying of ATâ…¡cells:ATâ…¡cells were isolated by enzymatic digestion of lung tissue from adult Sprague-Dawley rats using previously described methods with a little amelioration.The purification of cells was based on differential adherence of cells to dishes coated with rat IgG.The viability of cells was identified by 4%Trypan blue solution.And purity was identified with a tannic acid stain and electron microscope.The electron microscope also was used to observe the ultrastructure of these ATâ…¡cells.Extravascular lung water content(EVLW) were examined at 3 groups respectively after infusion by gravity.Liquid extrude alveolar space by hydrostatic pressure and osmotic pressure,then subtract liquid from absorbent vessels to vessels.For AQP4 immunohistochemical studies in rat lung.Reverse transcriptase polymerase chain reaction:After the rats were killed,the total RNA was extracted from freshly dissected sensory retinas using the TRIzol(?) Total RNA Isolation System. The total RNA was quantified by ultraviolet spectrophoto- -metry by measuring OD₂₆₀.The images were quantitated using the strom 860 soft system.The ratio of produce AQP4 andβ-actin is result of AQP4 express.Results1.It was the precondition and basis in this study that setting up rat ARDS models triumphantly with oleic acid and isolating,purifying and identifying of ATâ…¡cells successfully.Rat ARDS models were made by injection of oleic acid through coelio-vena,the rate of success was well-pleasing.After 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.After HE coloration,it was shown that the alveoli and interstitial became dropsical and hemorrhagic;capillary vessel been overspread,engorgement and leukocytic infiltrate;and there are a lot of liquid like thin blood permeated into the alveoli.There were more difficulties existed for us to isolate ATâ…¡cells in ARDS models than in control group.To judge the damage score of lung the adnephrinβ2-acceptor excitomotor therapy is between of ALI/ARDS and normal group.2.The 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.The s graphy of electron microscope3.EVLW were markedly increased,with ensuing severe hypoxemia.Compared with injured rats,terbutaline significantly decreased EVLW,and improved hypoxemia.Terbutaline can reduce edema accumulation and improve hypoxia in acute lung injury,and may have therapeutical effect on pulmonary edema after acute lung injury.Injury degree of lung tissues in ALI group was worser than those of control group under microscope and blood gas analysis.Smith lung injury score and EVLW in ALI group were higher than those in the control group(all P＜0.05).4.AQP4 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 ARDS rat alveolar cells.AQP4 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(Figure 25-30).Through RT-PCR of amplification,the absorbance integral proportion which is between AQP4 product and the product ofβ-actin can judge the m-RNA expression levels of AQP4.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 (Figure 31,32).Conclusions1.We had successfully isolated and purified ATâ…¡cells from normal and ARDS rats.It is simple and feasible that using 4%Trypan blue solution to confirm viability of cells and staining for tannic acid to identify purity of cells.Electron microscope is the best way to authenticate these cells.2.The practice indicated that setting up rat ARDS model by injection of oleic acid through coeliac vena was an easy and efficient method.In this model,the rat experienced a decrease of PaO₂,an increase of PaCO₂,Smith lung injury score and EVLW in ALl group was worser than those of control group under microscope and blood gas analysis,accompanied with notable ultrastructure of ATâ…¡cells.The rats were treatmented with terbutaline could lessen symptoms,blood gas analysis and lung injury degreens.3.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.Pulmonary vascular permeability is declined, reducing interstitial edema.Terbutaline maybe effect Alveolar epithelial cells to strengthen the active reabsorption.The extravascular lung water decreased significantly reflect that terbutaline may has a therapeutic effect in early lung injury.4.AQP4 is present at the membranes of isolated rat typeâ…¡cells.When rats in ALI get therapy of terbutaline the expression of AQP4 in typeâ…¡cells is significantly increased as compared with.control and lung injury group.Terbutaline induce cAMP up-regulated to effect of transporting and synthesis in AQP4.The change is studied on the expression of AQP4 in ARDS rats'typeâ…¡cells in three different stratifications and level including constitution,cell and protein molecule.The increased expression of AQP4 in injury lungs suggests that AQP4 may play a important role in fluid transportation.It is significative that EVLW reduced significantly.