The Relevance Between The Mechanism Of Terbutaline Enhancing Oleic Acid-induced Acute Lung Injury Adult Rat Pulmonary Alveolar Edema Clearance In Short Time And αENaC Subunit

Background:Acute lung injury/acute respiratory distress syndrome are syndromes of respiratory dysfunction/failure,which is resulted from diffuse pulmonary interstitial and alveolar edema caused by alveolar epithelial and endothelial taking place during serious infection shock trauma burns and other non-cardiogenic disease.Most epidemiological survey show ALI/ARDS are common clinical critical illness. Acording to the definition recommended by American-European Consensus Conference Committee in 1994,the morbility of the ALI is 18/0.1million each year,and 13～18/0.1million for ARDS.ALI/ARDS morbility shown by study in 2005 have reached 79/0.1 million and 59/0.1 million each,are significantly higher than before,markedly increased social and economic burden,which can compare with the chest tumor,AIDS,asthma or myocardial infarction and et.al.Although the difference of the mortalities of lots of studies reported are very big,general speaking, the current fatality rate of ARDS remains high.The motality of 3264 ARDS patients reported by a clinical meta-analysis collecting ARDS clinical researchs from international publications between 1967 and 1994 is about 50%;another one reached 68.5%,which come from 15 ICUs of Shanghai hospitals between 2001.03 and 2002.03.The basic physiopathological change of ALI/ARDS is non-cardiogenic pulmonary edema caused by increased permeability of alveolar epithelium and pulmonary capillary endothelium.That interstitial and alveolar edema increasing pulmonary water content and alveolar edema can damage alveolar surfactant layer, reduce pulmonary compliance,increase work of breathing;that alveolar well edema and alveolar edema extend the O2' diffusion distance and alveolar edema reduce diffusion area,decrease the diffusing capacity of lung.regional atelectasis caused by alveolar edema,regional atelectasis,regional thromboembolism aggravate ventilation/perfusion ratio imbalance.In severe cases,because of respiratory muscle fatigue,hypercapnia can occur,that is typeâ…¡respiratory failure.Early pathological features of ALI/ARDS is hyperpermeability of pulmonary capillary endothelium and alveolar epithelial barrier,pulmonary interstitium and and alvoelar filling with edema fluid,which is rich in protein and neutrophil consisting mainly of a variety of inflammatory cells.so ALI/ARDS patients with early anti-inflammatory and primary disease treatment receive pulmonary edema clearance therapy can reverse the pathophysiological course so some extent.Many clinical study[2]have shown that maintaining of normal alveolar fluid transfer function in patients with ALI/ARDS can improve their prognosis.Lots of research manifest normal normal alveolar fluid transfer function can reduce ALI/ARDS patients mortality.The mechanism of alvoelar edema clearance was previously explained by Starling mechanism:alveolar edema fluid was reabsorbed by difference hydrostatic pressure and osmotic pressure between inside and outside alveolar epithelium.Since the matthay[3],etc have discovered alvoelar active fluid transport,a new therapy of pulmonary edema will be created.Under conditions of net absorption of Na+,Na+ is absorbed from the apical surfaces of both Tâ… and Tâ…¡cells via ENaC(HSC and NSC channels) and via CNG channels in Tâ… cells.Electroneutrality is conserved with Cl-movement through CFTR in Tâ… and Tâ…¡cells(and possibly other anion channels) and/or paracellularly through tight junctions.Na+ is transported from the basal surface of both cell types into the interstitial space by Na+,K+-ATPase.K+ may be transported from alveolar epithelial cells via K+ channels located on the apical surface of Tâ… or Tâ…¡cells.If the directionality of net ion transport is from the apical surface to the interstitium,an osmotic gradient would be created,which would in turn direct water transport in the same direction,either through aquaporins or by diffusion. Conversely,in the formation of alveolar liquid,there may be net Cl- secretion mediated by CFTR(and possibly other Cl- channels).Under these conditions,there would be net secretion of Na+ by unidentified pathways,.Both the ENAC and NA+,K- ATPase are two important regulatory sites.A variety mechanisms of regulating alveolar fluid transport have been found: cAMP-mediated pathways,Dopaminergic pathways,Adenosine,Hormonal effects, Growth factors,Serine proteases,et al.β₂AR,a cAMP-mediated pathways is the best prospective and has entered a number of clinical trials.β₂AR is distributed in the epithelium and smooth muscle of airway,and the density of it is increasing along airway extention,more than 90%β₂AR in the alveolar epithelium.is now it is considered thatβ₂ARagonist-β₂AR-GPCR -CAMP-PKA-skeleton protein,promoting that cytoplasm ENAC insert to membrane;makeβENaC,γENaC in the membrane active;increasingαENaCmRNA andαENaC protein in order to increase the number of ENaC to promote alveolar fluid transport. ENaC consists of three(αENaC,βENaC,γENaC)subunits,is distributed in the epithelium membrane from transnasal to alvoelar of airway.Different ENaCs with different biological characteristics are combined by different combination among three subunits,Mice deficient forαENaC die of respiratory distress because of an inability to clear lung liquid.To this end,we chooseαENaC as the research object,select the classic rat model:oleic acid rat ALI,β₂AR agonist,terbutaline,pulmonary water content of lung tissue as outcome measure of the effectsβ₂AR agonist treatment of early(1 hour) ALI rats of pulmonary edema;detectαENaCmRNA andαENaC protein of distal lung tissue from normal rats,ALI/ARDS rats,β₂AR agonist rats,to study and explore the mechanism ofβ₂AR agonist ehnacing pulomanry edema reabsortion in ALI/ARDS rats,for the ALI treatment providing a viable,effective way in future.Methods:1,experimental grouping:24 SPF male adult Sprague-Dawley rats(from experimental animal center of Suthern Medical University) weighing 200-220g were divided into normal,ALI and Terbutaline treatment groups,by random number method,n=8 per group.2,Replication of ALI rat model:After absorbing artery blood for gas analysis,we inject 0.08ml/kg highly purified oleic acid through inferior vena cava in abdomen,soon we can observe distress breath,cyanosis in rats' face and lips.1 hour later we absorb artery blood for gas analysis once again,and then bloodletting to kill rats,choose right median loble for HE staining.we judge whether the replication is successful or not,according to results of artery gas blood analysis and HE staining.3,Measurement of pulmonary water content:Terbutaline sulphate aerosol was instilled through tracheal intubation at 4×10^-4mol/L 1ml/kg dose.1h later,choose right lower lobes to check their wet weight.and then grilled them in baker at 80℃for 72hs,after that,check their dry weigh,pulmonary water content=wet weight-dry weight/wet weight×100%.4,The diterminationof total expression ofα-ENaC mRNA in distal lung tissue:to extract total RNA of distal lung tissue according to protocol of Trizol(invitrogen TRIZOL(?) Reagent).choose qualified total RNA sample for RT reaction,the qualify criterion is OD260/OD280 ratio range from1.7 to 1.9;Occuring two bright bands(28S,18S) and a light band(5S)after running 1%agarose gel electrophoresis.and then make qPCR,choose△△Ct relative quantitation method to analysis the difference among the three groups rats'α-ENaC mRNA expression.5,The determination ofα-ENaC protein expression in distal lung tissue:to extract the distal lung tissue total protein by RIPA,and chooseβ-actin as internal reference to make Western blot to determinateα-ENaC protein relative expression in sample.6,Statistical Methods:All the data in the experimental were analysis by SPSS 13.0.Normality test of data all the groups were checked with skewness and kurtosis,each group homogeneity of variance were tested by Levene homogeneity of variance test.PaO2,PaO2/FiO2 of data before and after 1h of oleic acid treatment were compared by Paired Sample T Test,other difference between groups were compaired by Independent-Sample T Test or Satterthwaite approximate T Test P≤0.05 for the difference statistical significance,mean difference of all three groups were compared by One-Way ANOVA.P≥0.1 for difference no statistical significance.Results1,Reproduction of ALI rat model and observation of the effect of terbutaline for rat pulmonary edema clearance:1.1:Clinical performance of rats in each group:ALI rats following intravenous injection of oleic acid cavity soon after(2min) accur a persistent short breath with high frequency(130～180 times / min),after 30min later the majority has sighing respiration,thoracoabdominal paradoxical motion,red secretion in tracheal intubation,cyanosis in rats lip nasal skin.Terbutaline rats degree of shortness of breath and tracheal secretion less than that in ALI rats significantly,the majority have no paradoxical motion,cyanosis in rats lip nasal skin is extenuated markedly.Normal rats no change in breathing(60～90 times / min),no tracheal intubation secretions, nasal labial skin is still pink.1.2,HE staining of lung tissue:general view:ALI lung crimson near purple,swollen especial for lower part of lung.Airway full of secretion;terbutaline group was bright red lungs,swelling of the ALI group lighter than that of ALI rats;normal lung is pink,normal lung morphology,pulmonary sharp edge.HE staining:normal lung tissue can be clearly seen respiratory bronchiole,alveolar tubes,elveolar,such normal lung tissue morphosis,alveolar space does not swell,no red dye,alveolar wall,interstitial thin.ALI group,such as alveolar morphology unclear,expanded alveolar space(edema),which has red dye(protein),is the transient formation of a transparent membrane;alveolar wall,interstitium significantly widened,edema, significant increase of granulocytes,some regional atelectasis.Terbutaline treatment group organization such as the alveolar form is near normal,but there is still expanding the alveolar space(edema),congestion,but the cavity without a clear red staining,non-transparent film forming,interstitium widening relieved,increased neutrophil infiltrating reduced significantly than that of ALI.2,The effect of terbutaline enhancing ALI rat alveolar liquid clearance:PaO2 of ALI group rats' lung and terbutaline treatment group rats' lung:6.73±0.10,9.58±0.12;PaO2/FiO2 of ALI group rats' lung and terbutaline treatment group rats' lung:240.38±3.52,342.00±4.28;fraction of pulmonary water of ALI group rats' lung and terbutaline treatment group rats' lung:0.855±0.001,0.828±0.002.by two-sample t test,P values are as follows:less than 0.05 are 0.000,0.000,0.000.3,the difference ofα-ENaC mRNA expression among the three group rats' distal lung tissue is no statistic significance:relative expression of normal group,ALI group and terbutaline treatment group ratsαENaC mRNA in distal lung tissue are 0.97±0.23,1.06±0.13 and 0.90±0.19,by one-way ANOVA analysis P = 0.825＞0.1;4,the difference ofα-ENaC protein expression among the three group rats' distal lung tissue is no statistic significance:relative expression of normal group,ALI group and terbutaline treatment group ratsαENaC protein in distal lung tissue are 0.217±0.003,0.213±0.002 and 0.217±0.002,by one-way ANOVA analysis P = 0.344＞0.1.Conclusion:1,The ALI rat model in the experiment are successful by oleic acid intravenous injection administration at 0.08ml/kg;2,Terbutaline,aβ₂AR agonist can promote the ALI rats pulmonary edema reaborsorption at a early stage;3,The mechanism of terbutaline enhancing ALI rats pulmonary edema reaborsorption at early stage of the disease is not through theαENaC subunit gene expression.