特布他林短时间作用促进油酸急性肺损伤大鼠肺泡液清除的机制与αENaC的相关性
|
摘要
急性肺损伤/急性呼吸窘迫综合症(Acute Lung Injury/Acute RestpiratoryDistress Syndrome,ALI/ARDS)是在严重感染、休克、创伤及烧伤等非心源性疾病过程中,肺毛细血管内皮细胞和肺泡上皮细胞损伤造成弥漫性肺间质及肺泡水肿,导致的急性低氧性呼吸功能不全或衰竭。流行病学调查显示,ALI/ARDS是临床常见危重症。根据1994年欧美联席会议提出的ALI/ARDS诊断标准,ALI发病率为每年18/10万,ARDS为每年13～23/10万。2005年的研究显示ALI/ARDS发病率分别在每年79/10万和59/10万提示ALI/ARDS发病率显著增高,明显增加了社会和经济负担,这甚至可与胸部肿瘤、AIDS、哮喘或心肌梗死等相提并论。虽然不同研究对ARDS病死率的报道差异较大,总体来说,目前ARDS的病死率仍较高。对1967年～1994年国际正式发表的ARDS临床研究进行荟萃分析,3264例ARDS患者的病死率在50%左右。2001年3月至2002年3月上海市15家成人ICU中ARDS病死率也高达68.5%。
ALI/ARDS的基本病理生理改变是多种炎症细胞(巨噬细胞、中性粒细胞和淋巴细胞)参与的肺脏局部炎症反应和(或)系统性炎症反应失控所致的肺泡毛细血管膜损伤、通透性增加所致的富含蛋白的非心源性肺水肿。由于肺水肿肺水含量增加,肺泡水肿导致肺泡表面活性物质层破坏,降低了肺的顺应性;肺泡水肿增加弥散距离,减少散面积及其后形成的肺泡透明膜致弥散障碍;肺血管痉挛和肺微小血栓形成加重了通气/血流比例失调,产生严重的低氧血症。严重者,因呼吸机疲劳,通气减少,致Ⅱ型呼吸衰竭。肺血管痉挛和肺微小血栓形成可引发肺动脉高压。肺水肿在ALI/ARDS的病理生理中起着重要作用。
ALI/ARDS早期的病理特征为肺毛细血管内皮细胞与肺泡上皮细胞屏障的通透性增高,肺泡与肺间质内积聚大量的水肿液,其中富含蛋白及以中性粒细胞为主的多种炎症细胞。所以ALI/ARDS患者早期抗炎治疗的同时若能及时减轻,甚至治愈肺水肿,有助于逆转其病理生理过程,改善预后。不少临床研究已表明肺泡液体转功能的正常维持能降低ALI患者的死亡率。
肺泡内液体的主动吸收机制发现以前仅以Starling机制来解释,即由于肺泡内外存在流体静力压和渗透压差异而导致水自肺泡吸收。自Matthay等发现了肺泡内液体的主动转运机制后,为更好地治疗肺水肿提供新的可能方法。目前认为肺水肿的主动重吸收为:Na~+肺泡上皮细胞(包括肺泡Ⅰ型上皮细胞和肺泡Ⅱ型上皮细胞)顶膜的上皮钠离子通道(ENaC)重吸收入细胞质,再经其基底膜侧的Na~+,K~+-ATPase泵入肺间质,Cl~-通过肺泡上皮细胞CFTR,K~+通过K~+通道跨细胞转运,从而形成跨肺泡上皮的渗透压差,使肺泡内的水通过单纯扩散或肺泡上皮膜的水通道进入肺间质。其中ENaC和Na~+,K~+-ATPase是两个重要的环节和调节位点。
已发现多种调节肺泡液体主动转运的机制:CAMP介导的信号通路,多巴胺能通路,腺苷通路,激素通路,生长因子通路,丝氨酸蛋白酶通路等,其中CAMP介导的信号通路中的β_2肾上腺能受体(β_2AR)激动剂研究的最多并已进入不少临床试验中。β_2AR分布于肺的气道平滑肌和肺泡上皮,分布密度随气道级数增加而增加,90%以上分布于肺泡上皮。目前认为β_2AR激动剂短时间通过β_2AR激动剂-β_2AR-GPCR-CAMP-PKA-细胞骨架蛋白,促进细胞内的ENaC转运至细胞膜上增加肺泡上皮细胞膜ENaC数量;直接激活βENaC、γENaC;长时间作用增加αENaCmRNA促进αENaC的表达,增加ENaC数量,促进Na~+的跨肺泡上皮转运促进肺泡液体的重吸收。
ENaC由三个蛋白质亚基(αENaC、βENaC、γENaC)组成。分布于从鼻至肺泡的呼吸道上皮细胞膜。三个亚基以不同的方式组合形成了具有不同生物学特点和不同调节方式的钠离子通道。不同钠离子通道都包涵有αENaC,αENaC基因缺失的小鼠,很快死于肺内过多液而致的呼吸窘迫,γENaC基因缺失小鼠肺泡内液清除速度减慢,未死于呼吸系统并发症,βENaC、γENaC基因缺失小鼠患有假性醛固酮减少症,因失钠和高钾血症而死亡。由此可见αENaC对肺泡液体重吸收很重要。
为此我们产生了β_2AR激动剂是否通过增加αENaC的基因表达增加肺泡上皮细胞ENaC的数量促进肺泡液体的重吸收设想,为此我们以αENaC为研究对象,选择经典的油酸ALI大鼠模型,以肺组织的肺水含量为指标观察特布他林这一临床中应用多年的β_2AR激动剂治疗早期(1小时)ALI大鼠肺水肿的效果,检测正常组大鼠,ALI/ARDS状态组大鼠和β_2AR激动剂组大鼠远端肺组织αENaCmRNA和αENaC蛋白表达,研究特布他林短时间作用增强油酸急性肺损伤大鼠肺泡液清除的机制与αENaC的相关性,为探索β_2AR激动剂治疗ALI的机制,为ALI的治疗选择可行、有效方法提供参考。
材料和方法
1,实验分组:按随机数字法将24只200～220g的SPF级雄性SD大鼠(南方医科大学实验动物中心提供)分成正常组,ALI组和特布他林治疗组共3组,n=8。
2,油酸ALI大鼠模型的复制:经腹腔动脉抽血测血气分析后再,注射高纯度油酸0.08ml/kg,可观察大鼠呼吸急促、颜面部皮肤粘膜发绀,1h后再抽腹腔动脉血,测血气分析,并放血活杀,取材HE染色,根据PaO_2氧合指数,HE染色结果判断模型是否复制成功。
3,肺水肿吸收效果的测定:特布他林治疗组经气管插管滴入硫酸酸特布他林气雾剂4×10~(-4)mol/L 1ml/kg,放血活杀后,取相同部位肺组织测定湿重,再在80℃烤箱内放置72hs,再称肺组织干重,肺水含量=肺湿重-肺干重/肺湿重×100%。
4,远端肺组织总α-ENaC mRNA表达测定:按Trizol(invitrogen TRIZOL~(?)Reagent)说明书提肺组织总RNA,选OD260/OD280比值在1.7～1.9的,再跑1%琼脂糖电泳,得到明亮的28S、18S和暗淡的5S三条带后,作合格标准,选来自合格总RNA的RT反应后的产物进qRT-PCR反应。采用ΔΔCt相对定量法定量分析各组间α-ENaCmRNA表达差异。
5,远端肺组织总α-ENaC蛋白表达测定:用RIPA强裂解液提取远端肺组织总蛋白,以β-actin为内参western blot测定各组大鼠远端肺组织α-ENaC蛋白相对表达量。
6,统计学方法:采用SPSS13.0统计软件分析,Microsoft Office Excel 2007作统计表。各组样本量正态性检验用对偏度和峰度来检验;各组间方差齐性检验用levene方差齐性检验,Pa0_2、Pa02/FiO_2油酸处理1 h前后对比用配对样本t检验,其余各组间差别检验用两独立样本t检验或Satterthwaite近似t检验,P≤0.05有统计学差异。3组均数比较则行one-way ANOVA方差分析,P≥0.1无统计学差异。
结果
1、ALI大鼠模型复制:
1.1:各组大鼠临床表现:正常组大鼠呼吸无变化(呼吸频率(R):60～90次/分),无气管插分泌物,唇鼻部皮肤仍为粉红色;ALI组大鼠在下腔静脉注射油酸后,很快(2min)内出现持续呼吸急促(R:130～200次/分),多数在30min后有叹气样呼吸,胸腹予盾运动,气管插管内泡沫状红色分泌物,大鼠唇鼻部皮肤发绀。
1.2:ALI组油酸注射前及油酸注射后1h的PaO_2为:12.37±0.14、6.73±0.10;氧合指数为:441.87±5.18、240.38±3.52两指标两样配对t检验结果P值为别0.000和0.000,都小于0.05。
1.3:肺组织病理学:大体观:正常组肺呈粉红色,肺形态正常,肺缘锐利;ALI组肺深红近紫色,肿胀,发亮,以双下肺为重,气道分泌物多;HE染色:正常组大鼠肺组织切片可见呼吸性支气管,肺泡管,肺泡等肺组形态正常清晰,肺泡腔不肿大,内无红染,肺泡壁、间质薄。ALI组的肺泡等组织形态欠清晰,肺泡腔扩大,内有红染(蛋白质性肺水肿),正在形成透明膜;肺泡壁及间质明显增宽、充血,粒细胞显著增多、局灶性肺不张、坏死。
2、特布他林促进ALI大鼠肺泡液体重吸收效果的观察:ALI组大鼠和特布他林治疗组大鼠的PaO_2:6.73±0.10、9.58±0.12;氧合指数:240.38±3.52、342.00±4.28;肺水含量:0.855±0.001、0.828±0.002经两样本t检验,P值分别为:0.000、0.000、0.000都小于0.05。
HE染色:ALI组的肺组织形态欠清晰,肺泡腔扩大,内有红染(蛋白质性肺水肿),正在形成透明膜;肺泡壁、间质明显增宽,充血,粒细胞显著增多,局灶性肺不张。特布他林治疗组的肺组织形态尚可,间质增宽,充血,但腔内大都清晰无红染,浸润粒细胞较ALI组的明显减少。
3、各组大鼠远端肺组织α-ENaC mRNA的表达差异:正常组、ALI组和特布他林治疗组3组大鼠远端肺组织αENaC mRNA的相对表达量为别0.97±0.23、1.06±0.13和0.90±0.19,one-way ANOVA方差分析3者的差异:P=0.825＞0.1;各组大鼠远端肺组织α-ENaC蛋白的表达差异:3组αENaC蛋白相对表达量为0.217±0.003、0.213±0.002和0.217±0.002,P=0.344＞0.1。
结论
1、静脉注射0.08ml/kg高纯度油酸制备ALI大鼠模型成功;
2、β_2AR激动剂特布他林能促进早期ALI大鼠的肺水肿重吸收;
3、β_2AR激动剂特布他林能促进早期ALI大鼠的肺水肿重吸收不是通过增强α-ENaC基因表达实现。
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 typeⅡrespiratory 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 TⅠand TⅡcells via ENaC(HSC and NSC channels) and via CNG channels in TⅠcells.Electroneutrality is conserved with Cl-movement through CFTR in TⅠand TⅡcells(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 TⅠor TⅡcells.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.β_2AR,a cAMP-mediated pathways is the best prospective and has entered a number of clinical trials.β_2AR is distributed in the epithelium and smooth muscle of airway,and the density of it is increasing along airway extention,more than 90%β_2AR in the alveolar epithelium.is now it is considered thatβ_2ARagonist-β_2AR-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,β_2AR agonist,terbutaline,pulmonary water content of lung tissue as outcome measure of the effectsβ_2AR agonist treatment of early(1 hour) ALI rats of pulmonary edema;detectαENaCmRNA andαENaC protein of distal lung tissue from normal rats,ALI/ARDS rats,β_2AR agonist rats,to study and explore the mechanism ofβ_2AR 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~(-4)mol/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.
Results
1、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,aβ_2AR 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.
ALI/ARDS的基本病理生理改变是多种炎症细胞(巨噬细胞、中性粒细胞和淋巴细胞)参与的肺脏局部炎症反应和(或)系统性炎症反应失控所致的肺泡毛细血管膜损伤、通透性增加所致的富含蛋白的非心源性肺水肿。由于肺水肿肺水含量增加,肺泡水肿导致肺泡表面活性物质层破坏,降低了肺的顺应性;肺泡水肿增加弥散距离,减少散面积及其后形成的肺泡透明膜致弥散障碍;肺血管痉挛和肺微小血栓形成加重了通气/血流比例失调,产生严重的低氧血症。严重者,因呼吸机疲劳,通气减少,致Ⅱ型呼吸衰竭。肺血管痉挛和肺微小血栓形成可引发肺动脉高压。肺水肿在ALI/ARDS的病理生理中起着重要作用。
ALI/ARDS早期的病理特征为肺毛细血管内皮细胞与肺泡上皮细胞屏障的通透性增高,肺泡与肺间质内积聚大量的水肿液,其中富含蛋白及以中性粒细胞为主的多种炎症细胞。所以ALI/ARDS患者早期抗炎治疗的同时若能及时减轻,甚至治愈肺水肿,有助于逆转其病理生理过程,改善预后。不少临床研究已表明肺泡液体转功能的正常维持能降低ALI患者的死亡率。
肺泡内液体的主动吸收机制发现以前仅以Starling机制来解释,即由于肺泡内外存在流体静力压和渗透压差异而导致水自肺泡吸收。自Matthay等发现了肺泡内液体的主动转运机制后,为更好地治疗肺水肿提供新的可能方法。目前认为肺水肿的主动重吸收为:Na~+肺泡上皮细胞(包括肺泡Ⅰ型上皮细胞和肺泡Ⅱ型上皮细胞)顶膜的上皮钠离子通道(ENaC)重吸收入细胞质,再经其基底膜侧的Na~+,K~+-ATPase泵入肺间质,Cl~-通过肺泡上皮细胞CFTR,K~+通过K~+通道跨细胞转运,从而形成跨肺泡上皮的渗透压差,使肺泡内的水通过单纯扩散或肺泡上皮膜的水通道进入肺间质。其中ENaC和Na~+,K~+-ATPase是两个重要的环节和调节位点。
已发现多种调节肺泡液体主动转运的机制:CAMP介导的信号通路,多巴胺能通路,腺苷通路,激素通路,生长因子通路,丝氨酸蛋白酶通路等,其中CAMP介导的信号通路中的β_2肾上腺能受体(β_2AR)激动剂研究的最多并已进入不少临床试验中。β_2AR分布于肺的气道平滑肌和肺泡上皮,分布密度随气道级数增加而增加,90%以上分布于肺泡上皮。目前认为β_2AR激动剂短时间通过β_2AR激动剂-β_2AR-GPCR-CAMP-PKA-细胞骨架蛋白,促进细胞内的ENaC转运至细胞膜上增加肺泡上皮细胞膜ENaC数量;直接激活βENaC、γENaC;长时间作用增加αENaCmRNA促进αENaC的表达,增加ENaC数量,促进Na~+的跨肺泡上皮转运促进肺泡液体的重吸收。
ENaC由三个蛋白质亚基(αENaC、βENaC、γENaC)组成。分布于从鼻至肺泡的呼吸道上皮细胞膜。三个亚基以不同的方式组合形成了具有不同生物学特点和不同调节方式的钠离子通道。不同钠离子通道都包涵有αENaC,αENaC基因缺失的小鼠,很快死于肺内过多液而致的呼吸窘迫,γENaC基因缺失小鼠肺泡内液清除速度减慢,未死于呼吸系统并发症,βENaC、γENaC基因缺失小鼠患有假性醛固酮减少症,因失钠和高钾血症而死亡。由此可见αENaC对肺泡液体重吸收很重要。
为此我们产生了β_2AR激动剂是否通过增加αENaC的基因表达增加肺泡上皮细胞ENaC的数量促进肺泡液体的重吸收设想,为此我们以αENaC为研究对象,选择经典的油酸ALI大鼠模型,以肺组织的肺水含量为指标观察特布他林这一临床中应用多年的β_2AR激动剂治疗早期(1小时)ALI大鼠肺水肿的效果,检测正常组大鼠,ALI/ARDS状态组大鼠和β_2AR激动剂组大鼠远端肺组织αENaCmRNA和αENaC蛋白表达,研究特布他林短时间作用增强油酸急性肺损伤大鼠肺泡液清除的机制与αENaC的相关性,为探索β_2AR激动剂治疗ALI的机制,为ALI的治疗选择可行、有效方法提供参考。
材料和方法
1,实验分组:按随机数字法将24只200～220g的SPF级雄性SD大鼠(南方医科大学实验动物中心提供)分成正常组,ALI组和特布他林治疗组共3组,n=8。
2,油酸ALI大鼠模型的复制:经腹腔动脉抽血测血气分析后再,注射高纯度油酸0.08ml/kg,可观察大鼠呼吸急促、颜面部皮肤粘膜发绀,1h后再抽腹腔动脉血,测血气分析,并放血活杀,取材HE染色,根据PaO_2氧合指数,HE染色结果判断模型是否复制成功。
3,肺水肿吸收效果的测定:特布他林治疗组经气管插管滴入硫酸酸特布他林气雾剂4×10~(-4)mol/L 1ml/kg,放血活杀后,取相同部位肺组织测定湿重,再在80℃烤箱内放置72hs,再称肺组织干重,肺水含量=肺湿重-肺干重/肺湿重×100%。
4,远端肺组织总α-ENaC mRNA表达测定:按Trizol(invitrogen TRIZOL~(?)Reagent)说明书提肺组织总RNA,选OD260/OD280比值在1.7～1.9的,再跑1%琼脂糖电泳,得到明亮的28S、18S和暗淡的5S三条带后,作合格标准,选来自合格总RNA的RT反应后的产物进qRT-PCR反应。采用ΔΔCt相对定量法定量分析各组间α-ENaCmRNA表达差异。
5,远端肺组织总α-ENaC蛋白表达测定:用RIPA强裂解液提取远端肺组织总蛋白,以β-actin为内参western blot测定各组大鼠远端肺组织α-ENaC蛋白相对表达量。
6,统计学方法:采用SPSS13.0统计软件分析,Microsoft Office Excel 2007作统计表。各组样本量正态性检验用对偏度和峰度来检验;各组间方差齐性检验用levene方差齐性检验,Pa0_2、Pa02/FiO_2油酸处理1 h前后对比用配对样本t检验,其余各组间差别检验用两独立样本t检验或Satterthwaite近似t检验,P≤0.05有统计学差异。3组均数比较则行one-way ANOVA方差分析,P≥0.1无统计学差异。
结果
1、ALI大鼠模型复制:
1.1:各组大鼠临床表现:正常组大鼠呼吸无变化(呼吸频率(R):60～90次/分),无气管插分泌物,唇鼻部皮肤仍为粉红色;ALI组大鼠在下腔静脉注射油酸后,很快(2min)内出现持续呼吸急促(R:130～200次/分),多数在30min后有叹气样呼吸,胸腹予盾运动,气管插管内泡沫状红色分泌物,大鼠唇鼻部皮肤发绀。
1.2:ALI组油酸注射前及油酸注射后1h的PaO_2为:12.37±0.14、6.73±0.10;氧合指数为:441.87±5.18、240.38±3.52两指标两样配对t检验结果P值为别0.000和0.000,都小于0.05。
1.3:肺组织病理学:大体观:正常组肺呈粉红色,肺形态正常,肺缘锐利;ALI组肺深红近紫色,肿胀,发亮,以双下肺为重,气道分泌物多;HE染色:正常组大鼠肺组织切片可见呼吸性支气管,肺泡管,肺泡等肺组形态正常清晰,肺泡腔不肿大,内无红染,肺泡壁、间质薄。ALI组的肺泡等组织形态欠清晰,肺泡腔扩大,内有红染(蛋白质性肺水肿),正在形成透明膜;肺泡壁及间质明显增宽、充血,粒细胞显著增多、局灶性肺不张、坏死。
2、特布他林促进ALI大鼠肺泡液体重吸收效果的观察:ALI组大鼠和特布他林治疗组大鼠的PaO_2:6.73±0.10、9.58±0.12;氧合指数:240.38±3.52、342.00±4.28;肺水含量:0.855±0.001、0.828±0.002经两样本t检验,P值分别为:0.000、0.000、0.000都小于0.05。
HE染色:ALI组的肺组织形态欠清晰,肺泡腔扩大,内有红染(蛋白质性肺水肿),正在形成透明膜;肺泡壁、间质明显增宽,充血,粒细胞显著增多,局灶性肺不张。特布他林治疗组的肺组织形态尚可,间质增宽,充血,但腔内大都清晰无红染,浸润粒细胞较ALI组的明显减少。
3、各组大鼠远端肺组织α-ENaC mRNA的表达差异:正常组、ALI组和特布他林治疗组3组大鼠远端肺组织αENaC mRNA的相对表达量为别0.97±0.23、1.06±0.13和0.90±0.19,one-way ANOVA方差分析3者的差异:P=0.825＞0.1;各组大鼠远端肺组织α-ENaC蛋白的表达差异:3组αENaC蛋白相对表达量为0.217±0.003、0.213±0.002和0.217±0.002,P=0.344＞0.1。
结论
1、静脉注射0.08ml/kg高纯度油酸制备ALI大鼠模型成功;
2、β_2AR激动剂特布他林能促进早期ALI大鼠的肺水肿重吸收;
3、β_2AR激动剂特布他林能促进早期ALI大鼠的肺水肿重吸收不是通过增强α-ENaC基因表达实现。
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 typeⅡrespiratory 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 TⅠand TⅡcells via ENaC(HSC and NSC channels) and via CNG channels in TⅠcells.Electroneutrality is conserved with Cl-movement through CFTR in TⅠand TⅡcells(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 TⅠor TⅡcells.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.β_2AR,a cAMP-mediated pathways is the best prospective and has entered a number of clinical trials.β_2AR is distributed in the epithelium and smooth muscle of airway,and the density of it is increasing along airway extention,more than 90%β_2AR in the alveolar epithelium.is now it is considered thatβ_2ARagonist-β_2AR-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,β_2AR agonist,terbutaline,pulmonary water content of lung tissue as outcome measure of the effectsβ_2AR agonist treatment of early(1 hour) ALI rats of pulmonary edema;detectαENaCmRNA andαENaC protein of distal lung tissue from normal rats,ALI/ARDS rats,β_2AR agonist rats,to study and explore the mechanism ofβ_2AR 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~(-4)mol/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.
Results
1、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,aβ_2AR 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.
引文
[1]中华医学会重症医学分会.急性肺损伤/急性呼吸窘迫综合征诊断和治疗指南.[J].医学与哲学(临床决策论坛版)2007;28(2):19-28.
[2]Mutlu G.M.Factor P.Alveolar Epithelial b2-Adrenergic Receptors.Am J Respir Cell Mol Biol 2008,38:127-134.
[3]Matthay MA,Landolt CC,Staub NC.Differential liquid and protein clearance from the alveolar of anesthetized sheep.J Appl Physiol,1982.
[4]Johnson,M.D.,Bao,H.F.,Helms,M.N.,et al.Functional ion channels in pulmonary alveolar type Ⅰ cells support a role for type Ⅰ cells in lung ion transport.Proc.Natl.Acad.Sci.2006.103:4964-4969.
[5]Dobbs L.G.,Johnson M.D..Alveolar epithelial transport in the adult lung.Respiratory Physiology & Neurobiology 2007,159:283-300.
[6]李新甫,汪建新.急性肺损伤动物模型研究进展.[J].国外医学呼吸分册,2005(7):506-508.
[7]熊俊,吴旭王,武军.急性肺损伤大鼠肺血管通透性的变化规律.[J].实用医学杂志,2006,22(12):1370-1371.
[8]陆月明.油酸致急性肺损伤动物模型析评.[J].中华急诊医学杂志,2005;14(1):81-83
[9]李新甫,汪建新.急性肺损伤动物模型进展.[J].国外医学呼吸分册.2005:25(7):506-511.
[10]陈智伟,白春学,方晓惠.特布他林对急性肺损伤大鼠肺泡液体清除功能影响的实验研究.[J].中国危重病急救医学.2000,12(9):552-555.
[11]Wang H.M.,Bodenstein M.,Markstaller K.Overview of the Pathology of Three Widely Used Animal Models ofAcute Lung Injury.[J]:Eur Surg Res 2008;40:305-316
[12]陆再英,钟南山.内科学,第七版,北京:人民卫生出版社,2008
[13]毛宝龄,钱桂生,陈正堂.急性呼吸窘迫综合征.北京:人民卫生出版社,2002:231-241.
[14]彭朝胜,段蕴铀.β_2肾上腺素受体激动剂在ARDS中的应用进展.[J]海军总医院学报2006;19(4):224-227.
[15]孙振涛;韩雪萍;苗丽君等.脑死亡状态巴马小型猪肺水含量及肺超微结构变化.[J].中国组织工程研究与临床康复,2007;11(8):1428-1430.
[16]宗酉明,曾因明.兔内毒素性急性肺损伤时总肺水含量和肺组织Na+-K+-ATPase活性的变化.[J].中华麻醉学杂志,2003;23(11):846-847.
[17]乔莉娟,王立贤,颜华.猪油酸-急性肺损伤模型的建立.[J].中国畜牧兽医 2008(10):82-85.
[18]裴凌;傅文;王俊科等.氯胺酮对内毒素休克鼠动脉血浆TNF-α浓度、氧分压及肺水含量的影响.[J].中华外科杂志.1999;37(6):363.
[19]Chollet Martin S1 Polymorphonuclear neut rophil activation during the acute respiratory dist ress syndrome[J].Intensive Care Med,2000,26(10):1575-1577.
[20]樊毫军,张健鹏,刘又宁.钠离子通道与急性肺损伤关系研究进展.国际呼吸杂志.2006,26(3):203-205;
[21]Perkins G.D.,McAuley D.F.,Richter A.et al.β2 -agonists and acute respiratory syndrome.[J].Critical Care 2004,8:25-32
[22]Spurzam J R,Gupta J,Yeys T,et al1Activation of protein kinase A accelerates bovine bronchial epithelial cell migration[J].Am J Physiol Lung Cell Mol Physiol 2002,82(5):L1108-L1116.
[23]Eliat C,Lassel L,Guillou YM.Intravenous beta-2-adrenergic agonists for tocolytic therapy in pre-eclampsia:two cases of acute pulmonary edema [J].Ann Fr Anesth Reanim,2002,21(9):737-740.
[24]Matthay MA,Fukuda N,Frank J,et,al.Alveolar epithelial barrier.Role in lung fliud balance in clinical lung injury [J].Clin Chest Med,2000,21(3):477-90
[25]Berthiaume Y , Matthay M.A.Alveolar edema fluid clearance and acute lung injury.[J].Respiratory Physiology & Neurobiology 2007,159: 350-359.
[26]Dobbs LG, Johnson MD.Alveolar epithelial transport in the adult lung.[J].Respiratory Physiology & Neurobiology.2007,159:283-300.
[27]Pitkanen,O.M., Smith, D., 0'brodovich,H.,et al.Expression ofα 、β、andy-hENaC mRNA in the Human Nasal, Bronchial, and Distal Lung Epithelium Am J Respir Crit Care Med 2001,163.pp 273-276.
[28]Hummler, E., Barker, P., Gatzy, J.et al.Early death due to defective neonatal lung liquid clearance in alpha-ENaC-deficient mice.Nat.Genet.12:325-328.
[29]Barker, P.M., Nguyen, M.S., Gatzy, J.T.,et al.Role of gammaENaC subunit in lung liquid clearance and electrolyte balance in newborn mice.Insights into perinatal adaptation and pseudohypoaldosteronism.J.Clin.Invest.102:1634-1640.
[30]Liebler, J.M., Borok, Z., Li, X.,et al.Alveolar epithelial type I cells express beta2-adrenergic receptors and G-protein receptor kinase 2.J.Histochem.Cytochem.2004,52:759-767.
[31]MINAKATA Y, SUZUKI S, GRYGORCZYK C, Impact of b-adrenergic agonist Na+ channel and Na+-K+-ATPase expression in alveolar type Ⅱ cells.Am J Physiol Lung Cell Mol Physiol, 1998,275:414-422.
[1]彭朝胜,段蕴铀.β_2肾上腺素受体激动剂在ARDS中的应用进展.海军总医院学报,2006,19(4):224-227.
[2]Johnson MD,Bao HF,Helms MN,et al.Functional ion channels in pulmonary alveolar type Ⅰ cells support a role for type Ⅰ cells in lung ion transport.Proc Natl Acad SciUSA,2006,103(13):4964-4969.
[3]Dobbs LG,Johnson MD.Alveolar epithelial transport in the adult lung.Respir Physiol Neurobiol,2007,159(3):283-300.
[4]Liebler,JM.,Borok,Z,Li,X,et al.Alveolar epithelial type Ⅰ cells express beta2-adrenergic rceptors and G-protein receptor kinase 2.Histochem.Cytochem,2004,52:759-767.
[5]Berthiaume Y.Long-term stimulation of alveolar epithelial cells by beta-adrenergic agonists: increased Na+ transport and modulation of cell growth?Am J Physiol Lung Cell Mol Physiol, 2003, 285: L798-L801.
[6]Jiang X, Ingbar DH, O' Grady SM.Adrenergic stimulation of Na~+ transport across alveolar epithelial cells involves activation of apical Cl~- channels.Am J Physiol ,1998,275:C1610-C1620.
[7]Chalfant ML, Coupaye-Gerard B, Kleyman TR.Distinct regulation of Na+ reabsorption and CI" secretion by arginine vasopressin in the amphibian cell line A6.Am J Physiol, 1993,264: C1480-C1488.
[8]Fang X, Fukuda N, Barbry P, et al.Novel role for CFTR in fluid absorption from the distal airspaces of the lung.Gen Physiol.2002,119: 199-208.
[9]Mutlu GM, Dumasius V, Burhop J, et al.Interdependency of beta-adrenergic receptors and CFTR in regulation of alveolar active Na+ transport.Circ Res,2005,96(9):999-1005.
[10]Yue G, Shoemaker RL, Matalon S.Regulation of low-amiloride-affinity sodium channels in alveolar type Ⅱ cells.Am J Physiol 1994;267:L94-L100.
[11]Chen XJ, Eaton DC, Jain L.Beta-adrenergic regulation of amiloride sensitive lung sodium channels.Am J Physiol Lung Cell Mol Physiol 2002;282:L609-L620.
[12]Maron MB, Folkesson HG, Stader SM, et al.PKA delivery to the distal lung air spaces increases alveolar liquid clearance after isoproterenol-induced alveolar epithelial PKA desensitization.Am J Physiol Lung Cell Mol Physiol ,2005, 289:L349-L354.
[13]Smith PR, Saccomani G, Joe EH, et al.Amiloridesensitive sodium channel is linked to the cytoskeleton in renal epithelial cells.Proc Natl Acad Sci USA ,1991,88:6971-6975.
[14]Snyder PM .Minireview: Regulation of epithelial Na~+ channel trafficking.Endocrinology, 2005,146: 5079-5085.
[15]Senyk O, Ismailov Ⅱ, Bradford AL, et al.Reconstitution of immunopurified alveolar type Ⅱ cell Na~+ channel protein into planar lipid bilayers.AmJ Physiol ,1995,268:C1148-C1156.
[16]Jain L, Chen XJ, Ramosevac S, et al.Expression of highly selective sodium channels in alveolar type Ⅱ cells is determined by culture conditions.Am J Physiol Lung Cell Mol Physiol, 2001,280:L646-L658.
[17]Suzuki S, Zuege D, and Berthiaume Y.Sodium-independent modulation of Na~+-K~+-ATPase activity by P-adrenergic agonist in alveolar type Ⅱ cells.Am J Physiol Lung Cell Mol Physiol, 1995, 268:L983-L990.
[18]Lecuona E, Ridge K, Pesce L, et al.The GTP-binding protein RhoA mediates Na~+,K~+-ATPase exocytosis in alveolar epithelial cells.Mol Biol Cell,2003,14:3888-3897.
[19]Maron MB, Folkesson HG, Stader SM, et al.Impaired alveolar liquid clearance after 48h isoproterenol infusion spontaneously recovers by 96 h of continuous infusion.Am J Physiol Lung Cell Mol Physiol, 2006,291: L252-L256.
[20]Minakata Y, Suzuki S, Grygorczyk C, et al.Impact of P-adrenergic agonist on Na~+ channel and Na~+-K~+-ATPase expression in alveolar type Ⅱ cells.Am J Physiol Lung Cell Mol Physiol, 1998,275:414-422.
[21]Pesce L, Guerrero C, Cornelias A, et al.L-Agonists regulate Na,K-ATPase via novel MAPK/ERK andrapamycin-sensitive pathways.FEBS Letters, 2000,486:310-314.
[22]Pesce L, Cornelias A, and Sznajder JI.P-Adrenergic agonists regulate Na~+,K~+-ATPase via p70~(S6k) .Am J Physiol Lung Cell Mol Physiol, 2003, 285:L802-L807.
[2]Mutlu G.M.Factor P.Alveolar Epithelial b2-Adrenergic Receptors.Am J Respir Cell Mol Biol 2008,38:127-134.
[3]Matthay MA,Landolt CC,Staub NC.Differential liquid and protein clearance from the alveolar of anesthetized sheep.J Appl Physiol,1982.
[4]Johnson,M.D.,Bao,H.F.,Helms,M.N.,et al.Functional ion channels in pulmonary alveolar type Ⅰ cells support a role for type Ⅰ cells in lung ion transport.Proc.Natl.Acad.Sci.2006.103:4964-4969.
[5]Dobbs L.G.,Johnson M.D..Alveolar epithelial transport in the adult lung.Respiratory Physiology & Neurobiology 2007,159:283-300.
[6]李新甫,汪建新.急性肺损伤动物模型研究进展.[J].国外医学呼吸分册,2005(7):506-508.
[7]熊俊,吴旭王,武军.急性肺损伤大鼠肺血管通透性的变化规律.[J].实用医学杂志,2006,22(12):1370-1371.
[8]陆月明.油酸致急性肺损伤动物模型析评.[J].中华急诊医学杂志,2005;14(1):81-83
[9]李新甫,汪建新.急性肺损伤动物模型进展.[J].国外医学呼吸分册.2005:25(7):506-511.
[10]陈智伟,白春学,方晓惠.特布他林对急性肺损伤大鼠肺泡液体清除功能影响的实验研究.[J].中国危重病急救医学.2000,12(9):552-555.
[11]Wang H.M.,Bodenstein M.,Markstaller K.Overview of the Pathology of Three Widely Used Animal Models ofAcute Lung Injury.[J]:Eur Surg Res 2008;40:305-316
[12]陆再英,钟南山.内科学,第七版,北京:人民卫生出版社,2008
[13]毛宝龄,钱桂生,陈正堂.急性呼吸窘迫综合征.北京:人民卫生出版社,2002:231-241.
[14]彭朝胜,段蕴铀.β_2肾上腺素受体激动剂在ARDS中的应用进展.[J]海军总医院学报2006;19(4):224-227.
[15]孙振涛;韩雪萍;苗丽君等.脑死亡状态巴马小型猪肺水含量及肺超微结构变化.[J].中国组织工程研究与临床康复,2007;11(8):1428-1430.
[16]宗酉明,曾因明.兔内毒素性急性肺损伤时总肺水含量和肺组织Na+-K+-ATPase活性的变化.[J].中华麻醉学杂志,2003;23(11):846-847.
[17]乔莉娟,王立贤,颜华.猪油酸-急性肺损伤模型的建立.[J].中国畜牧兽医 2008(10):82-85.
[18]裴凌;傅文;王俊科等.氯胺酮对内毒素休克鼠动脉血浆TNF-α浓度、氧分压及肺水含量的影响.[J].中华外科杂志.1999;37(6):363.
[19]Chollet Martin S1 Polymorphonuclear neut rophil activation during the acute respiratory dist ress syndrome[J].Intensive Care Med,2000,26(10):1575-1577.
[20]樊毫军,张健鹏,刘又宁.钠离子通道与急性肺损伤关系研究进展.国际呼吸杂志.2006,26(3):203-205;
[21]Perkins G.D.,McAuley D.F.,Richter A.et al.β2 -agonists and acute respiratory syndrome.[J].Critical Care 2004,8:25-32
[22]Spurzam J R,Gupta J,Yeys T,et al1Activation of protein kinase A accelerates bovine bronchial epithelial cell migration[J].Am J Physiol Lung Cell Mol Physiol 2002,82(5):L1108-L1116.
[23]Eliat C,Lassel L,Guillou YM.Intravenous beta-2-adrenergic agonists for tocolytic therapy in pre-eclampsia:two cases of acute pulmonary edema [J].Ann Fr Anesth Reanim,2002,21(9):737-740.
[24]Matthay MA,Fukuda N,Frank J,et,al.Alveolar epithelial barrier.Role in lung fliud balance in clinical lung injury [J].Clin Chest Med,2000,21(3):477-90
[25]Berthiaume Y , Matthay M.A.Alveolar edema fluid clearance and acute lung injury.[J].Respiratory Physiology & Neurobiology 2007,159: 350-359.
[26]Dobbs LG, Johnson MD.Alveolar epithelial transport in the adult lung.[J].Respiratory Physiology & Neurobiology.2007,159:283-300.
[27]Pitkanen,O.M., Smith, D., 0'brodovich,H.,et al.Expression ofα 、β、andy-hENaC mRNA in the Human Nasal, Bronchial, and Distal Lung Epithelium Am J Respir Crit Care Med 2001,163.pp 273-276.
[28]Hummler, E., Barker, P., Gatzy, J.et al.Early death due to defective neonatal lung liquid clearance in alpha-ENaC-deficient mice.Nat.Genet.12:325-328.
[29]Barker, P.M., Nguyen, M.S., Gatzy, J.T.,et al.Role of gammaENaC subunit in lung liquid clearance and electrolyte balance in newborn mice.Insights into perinatal adaptation and pseudohypoaldosteronism.J.Clin.Invest.102:1634-1640.
[30]Liebler, J.M., Borok, Z., Li, X.,et al.Alveolar epithelial type I cells express beta2-adrenergic receptors and G-protein receptor kinase 2.J.Histochem.Cytochem.2004,52:759-767.
[31]MINAKATA Y, SUZUKI S, GRYGORCZYK C, Impact of b-adrenergic agonist Na+ channel and Na+-K+-ATPase expression in alveolar type Ⅱ cells.Am J Physiol Lung Cell Mol Physiol, 1998,275:414-422.
[1]彭朝胜,段蕴铀.β_2肾上腺素受体激动剂在ARDS中的应用进展.海军总医院学报,2006,19(4):224-227.
[2]Johnson MD,Bao HF,Helms MN,et al.Functional ion channels in pulmonary alveolar type Ⅰ cells support a role for type Ⅰ cells in lung ion transport.Proc Natl Acad SciUSA,2006,103(13):4964-4969.
[3]Dobbs LG,Johnson MD.Alveolar epithelial transport in the adult lung.Respir Physiol Neurobiol,2007,159(3):283-300.
[4]Liebler,JM.,Borok,Z,Li,X,et al.Alveolar epithelial type Ⅰ cells express beta2-adrenergic rceptors and G-protein receptor kinase 2.Histochem.Cytochem,2004,52:759-767.
[5]Berthiaume Y.Long-term stimulation of alveolar epithelial cells by beta-adrenergic agonists: increased Na+ transport and modulation of cell growth?Am J Physiol Lung Cell Mol Physiol, 2003, 285: L798-L801.
[6]Jiang X, Ingbar DH, O' Grady SM.Adrenergic stimulation of Na~+ transport across alveolar epithelial cells involves activation of apical Cl~- channels.Am J Physiol ,1998,275:C1610-C1620.
[7]Chalfant ML, Coupaye-Gerard B, Kleyman TR.Distinct regulation of Na+ reabsorption and CI" secretion by arginine vasopressin in the amphibian cell line A6.Am J Physiol, 1993,264: C1480-C1488.
[8]Fang X, Fukuda N, Barbry P, et al.Novel role for CFTR in fluid absorption from the distal airspaces of the lung.Gen Physiol.2002,119: 199-208.
[9]Mutlu GM, Dumasius V, Burhop J, et al.Interdependency of beta-adrenergic receptors and CFTR in regulation of alveolar active Na+ transport.Circ Res,2005,96(9):999-1005.
[10]Yue G, Shoemaker RL, Matalon S.Regulation of low-amiloride-affinity sodium channels in alveolar type Ⅱ cells.Am J Physiol 1994;267:L94-L100.
[11]Chen XJ, Eaton DC, Jain L.Beta-adrenergic regulation of amiloride sensitive lung sodium channels.Am J Physiol Lung Cell Mol Physiol 2002;282:L609-L620.
[12]Maron MB, Folkesson HG, Stader SM, et al.PKA delivery to the distal lung air spaces increases alveolar liquid clearance after isoproterenol-induced alveolar epithelial PKA desensitization.Am J Physiol Lung Cell Mol Physiol ,2005, 289:L349-L354.
[13]Smith PR, Saccomani G, Joe EH, et al.Amiloridesensitive sodium channel is linked to the cytoskeleton in renal epithelial cells.Proc Natl Acad Sci USA ,1991,88:6971-6975.
[14]Snyder PM .Minireview: Regulation of epithelial Na~+ channel trafficking.Endocrinology, 2005,146: 5079-5085.
[15]Senyk O, Ismailov Ⅱ, Bradford AL, et al.Reconstitution of immunopurified alveolar type Ⅱ cell Na~+ channel protein into planar lipid bilayers.AmJ Physiol ,1995,268:C1148-C1156.
[16]Jain L, Chen XJ, Ramosevac S, et al.Expression of highly selective sodium channels in alveolar type Ⅱ cells is determined by culture conditions.Am J Physiol Lung Cell Mol Physiol, 2001,280:L646-L658.
[17]Suzuki S, Zuege D, and Berthiaume Y.Sodium-independent modulation of Na~+-K~+-ATPase activity by P-adrenergic agonist in alveolar type Ⅱ cells.Am J Physiol Lung Cell Mol Physiol, 1995, 268:L983-L990.
[18]Lecuona E, Ridge K, Pesce L, et al.The GTP-binding protein RhoA mediates Na~+,K~+-ATPase exocytosis in alveolar epithelial cells.Mol Biol Cell,2003,14:3888-3897.
[19]Maron MB, Folkesson HG, Stader SM, et al.Impaired alveolar liquid clearance after 48h isoproterenol infusion spontaneously recovers by 96 h of continuous infusion.Am J Physiol Lung Cell Mol Physiol, 2006,291: L252-L256.
[20]Minakata Y, Suzuki S, Grygorczyk C, et al.Impact of P-adrenergic agonist on Na~+ channel and Na~+-K~+-ATPase expression in alveolar type Ⅱ cells.Am J Physiol Lung Cell Mol Physiol, 1998,275:414-422.
[21]Pesce L, Guerrero C, Cornelias A, et al.L-Agonists regulate Na,K-ATPase via novel MAPK/ERK andrapamycin-sensitive pathways.FEBS Letters, 2000,486:310-314.
[22]Pesce L, Cornelias A, and Sznajder JI.P-Adrenergic agonists regulate Na~+,K~+-ATPase via p70~(S6k) .Am J Physiol Lung Cell Mol Physiol, 2003, 285:L802-L807.