营养不良性肺水肿大鼠肺泡液体清除机制的研究

目的探讨营养不良性肺水肿大鼠肺泡液体清除功能的变化及其机制。方法制备营养不良性肺水肿大鼠动物模型,分别于48h和120h测定大鼠肺泡液体清除率（AFC）、总肺水量（TLW）和肺血管外水量（EVLW）。将钠通道阻断剂氨氯毗眯、Na-K-ATP酶阻断剂畦巴因及β2受体激动剂特布他林分别灌注到正常及禁食120h大鼠的肺泡腔内,测定AFC的变化。结果大鼠禁食48hAFC（19．7±3．22％）与正常大鼠AFC（18．5±2．21％）比较没有明显变化;120h时AFC（9．50±2．19％）明显降低。氨氯吡咪、哇巴因明显降低营养不良性肺水肿大鼠AFC（P＜0．05）,特布他林对营养不良性肺水肿大鼠AFC的作用与对照组大鼠比较差异无显著性（P＞0．05）。结论营养不良性肺水肿与钠通道及Na-K-ATP酶及的活性被抑制,导致肺泡液体清除能力降低有关。     

急性冷暴露对大鼠肺组织中水通道蛋白AQP-1和AQP-5表达的影响

目的：探讨急性冷暴露后肺组织超微结构变化以及对水通道蛋白-1（AQP-1）和AQP-5表达的影响。方法：12只健康雄性Wistar大鼠随机分为室温（23℃±2℃）对照组和-25℃ 2 h冷暴露组（n=6）;记录冷暴露后大鼠直肠温度;透射电镜观察肺组织超微结构改变;RT-PCR法和Western blot法测定大鼠肺组织AQP-1和AQP-5基因和蛋白的表达水平。结果：急性冷暴露后大鼠的体心温度与对照组相比,明显降低（P<0.05）;肺组织超微结构亦发生改变,基底膜明显增厚,肺泡I上皮细胞（AT-I）核固缩,肺泡Ⅱ上皮细胞（AT-Ⅱ）胞浆空泡化增多;冷暴露后大鼠肺组织AQP-1的基因和蛋白表达未见明显变化,AQP-5的基因和蛋白表达均显著降低（P<0.05）。结论：急性冷暴露肺组织AQP-5基因和蛋白表达降低与寒冷暴露引发肺组织结构损伤可能存在一定因果关系。     

间歇性低氧防止缺血再灌注损伤引起的线粒体结构损伤和mtDNA片段缺失

本文用离体Ｌａｎｇｅｎｄｏｒｆｆ灌流大鼠心脏造成急性心肌缺血／再灌注损伤模型,观察间歇性低氧暴露保护心肌线粒体的作用。以聚合酶链式反应（ＰＣＲ）方法和电子显微镜技术,观察线粒体ＤＮＡ（ｍｔＤＮＡ＾４８３４）片段缺失和超微结构的变化。大鼠暴露于模拟海拔５０００米低氧环境（６ｈ／ｄ,２８ｄ）明显降低ｍｔＤＮＡ＾４８３４缺失的发生率（２８．５７％,ｖｓ常氧对照组８７．５％ Ｐ〈０．０５）;而且能够明显减     

经气管灌注血管紧张素Ⅱ导致凋亡性急性肺损伤(英文)

为研究外源性血管紧张素Ⅱ（angiotensinⅡ,ANG）在急性肺损伤和肺泡上皮细胞凋亡中的作用,经气管分别给雄性Wistar大鼠（175-200g）灌注ANG、ANG加caspase抑制剂ZVAD—fmk、ANG加ANG受体1阻断剂losartan和仅灌注磷酸盐缓冲溶液（PBS）。6或20h后在体灌洗动物肺脏,测定灌洗液中血红蛋白（hemoglobin,Hb）和荧光物质（BODIPY）标定的白蛋白含量（在灌洗前15min静脉注入BODIPY-白蛋白）。TUNEL测定显示,灌注ANG6h后,支气管和肺泡上皮细胞内标定的DNA片断显著增2H（P〈0．05）;ANG所致的DNA片断增加可被同时灌注ZVAD—fmk或losartan阻断。灌注ANG后免疫标定caspase3阳性细胞数量显著增多（P〈0．01）,ZVAD—fmk或losartan同样显著减少caspase3阳性细胞的数量。灌注ANG显著增加肺泡灌洗液中荧光标定的白蛋白（P〈0．01）和Hb的含量（P〈0．05）;ZVAD—fmk或losartan亦显著抑制荧光白蛋白和Hb含量的变化。结果表明,肺泡上皮细胞在体暴露于外源性ANG足以引起ANG受体1介导的上皮细胞凋亡和肺泡屏障损伤。     

一氧化氮合成酶在不同压力氧气导致的肺型氧中毒中的表达变化

目的：临床上过量使用氧气可导致肺型氧中毒的发生,目前有文献认为常压氧和高压氧导致的肺型氧中毒具有不同的发病机制。本实验拟探讨一氧化氮合成酶在不同压力氧气导致的肺型氧中毒中的表达变化。方法：60只雄性SD大鼠,随机分为6组（n=10）,分别暴露于1绝对压（atmosphere absolute,ATA）、1．5ATA、2ATA、2．5ATA、3ATA,100％氧气中56、20、10、8、6h,暴露于空气组作为对照。出舱后测定各组大鼠肺组织湿干比、支气管肺泡灌洗液蛋白含量。收集肺组织,裂解提取蛋白。行Western blot检测内皮一氧化氮合酶（eNOS）、神经型一氧化氮合酶（eNOS）的表达变化。结果：相对于正常对照组,1．0ATA组肺湿干比、支气管肺泡灌洗液蛋白量表达明显增高。随着氧分压的增高,这种改变减弱。和1．0ATA组相比,高压氧组的肺湿干比、肺泡灌洗液蛋白含量显著降低。各个氧气压力暴露组大鼠肺组织中nNOS的含量没有明显改变。而eNOS含量则在氧气压力为2ATA时明显降低（P〈0．05）,氧气压力为2．5ATA及3ATA时明显增高（P〈0．05）。结论：eNOS在肺中的表达量随着氧气压力的变化而改变。     

BRL-37344对离体人肺脏肺泡液体清除的作用

目的探讨β1肾上腺素能受体激动剂BRL-37344对离体人肺脏肺泡液体清除率（AFC）的作用及其机制。方法首先将（10^-4-10^-8）mol／L的BRL-37344分别灌注到离体人肺脏肺泡腔内,测定AFC的变化。然后将10^-5moL／LBRL-37344分别与α受体阻滞剂酚妥拉明、13。受体阻滞剂阿替洛尔、β2受体阻滞剂ICI-118551、β3受体阻滞剂SR59230A、钠通道阻断剂氨氯吡咪、Na^＋-K^＋-ATP酶阻断剂哇巴因混合后灌注到离体人肺脏肺泡腔内,测定AFC的变化。结果10^-8mol／L的BRL-37344对离体人肺脏肺泡液体清除率没有影响,（10^-7-10^-4）mol／L的BRL-37344能够显著提高离体人肺脏肺泡液体清除率。BRL-37344增加离体人肺脏AFC的作用能够被SR59230A、氨氯吡咪和哇巴因抑制。结论BRL-37344主要通过调节Na^＋／K^＋-ATP酶的活性增加离体人肺脏肺泡液体清除能力,促进肺水肿液的吸收。     

不同时间睡眠剥夺对大鼠运动能力及谷氨酰胺的影响

目的：探索睡眠剥夺对大鼠运动能力及谷氨酰胺含量变化的影响,为睡眠剥夺后的运动训练等提供一定的实验依据。方法：将30只雄性SD大鼠按体重随机分安静对照组、0h睡眠剥夺力竭运动组（SDE）、24h SDE、48h SDE和72h SDE组（n=6）,采用轻柔刺激法建立睡眠剥夺模型和依据Bedford建立的大鼠运动模型。结果：24h SDE睡眠剥夺组与0h SDE组比较。大鼠后蹬跑时间明显长（P〈0．05）,48h SDE睡眠剥夺组和72h SDE睡眠剥夺与0h SDE睡眠剥夺组比较,后蹬跑时间显著性减少（P〈0．01）;24h SDE睡眠剥夺组与c组比较大鼠胸腺谷氨酰胺含量显著升高（P〈0．05）,48h SDE睡眠剥夺组和72h SDE睡眠剥夺组与C组比较大鼠胸腺谷氨酰胺含量降低（P〈0．01）;睡眠剥夺组与C组比较,血清谷氨酰胺含量的变化均具有高度显著性差异（P〈0．01）,睡眠剥夺24h后血清谷氨酰胺含量显著增多,却在睡眠剥夺48h、72h后血清谷氨酰胺含量明显下降。结论：①睡眠剥夺24h能提高大鼠运动能力,睡眠剥夺48h甚至是72h后大鼠运动能力开始降低。②睡眠剥夺24h后大鼠胸腺谷氨酰胺含量和血清谷氨酰胺含量升高,而睡眠剥夺48h后大鼠胸腺和血清的谷氨酰胺含量下降明显,睡眠剥夺72h后胸腺和血清谷氨酰胺含量显著性降低。     

硫化氢对大鼠内毒素性急性肺损伤肺泡表面活性物质的影响

目的：观察脂多糖（LPS）所致内毒素性急性肺损伤（ALI）大鼠肺泡表面活性物质（PS）的变化及硫化氢（H2S）对PS的影响,探讨H2S对肺脏的作用机制。方法：雄性SD大鼠共48只,随机分为6组（n=8）：空白对照组、LPS组、LPS＋NaHS低、中、高剂量组、LPS＋PPG组。空白对照组给予生理盐水,LPS组给予LPS,LPS＋NaHS低、中、高剂量组和LPS＋PPG组分别在给予LPS3h时腹腔注射低、中、高剂量氢硫化钠（NariS）或炔丙基甘氨酸（PPG）。各组均于给予生理盐水或LPS6h时电镜下观察肺泡Ⅱ型上皮细胞（AEC-Ⅱ）的形态改变,检测血浆中H2S含量、肺组织中胱硫醚-γ-裂解酶（CSE）活性、肺泡灌洗液（BALF）中总蛋白（1P）和总磷脂（TPL）含量、及肺组织中肺泡表面活性蛋白A、B、C（SP-A、B、C）mRNA表达的变化。结果：①与空白对照组比较,LPS组AEC-Ⅱ超微结构明显受损,血浆中H2S含量、肺组织中CSE活性、BALF中TPL的含量、及肺组织中SP-A、B、CmRNA表达均明显降低（P〈0．05,P〈0．01）,BALF中TP的含量明显增加（P〈0．01）;②与LPS组比较,LPS＋NaHS低、中、高剂量组,AEC-Ⅱ超微结构均有所恢复,血浆中H2S含量、肺组织中CSE海性、SP-AmRNA表达均明显升高（P〈0．05,P〈0．01）;LPS＋NaHS中、高剂量组BALF中吼含量明显增高,SP-BmRNA表达升高（P〈0．05）;LPS＋NaHS高剂量组BALF中,IP含量明显降低（P〈0．05）;LPS＋NaHS各剂量组SP-CznRNA表达无明显变化;③与LPS组比较,LPS＋PPG组AEC-Ⅱ超微结构仍损伤严重,血浆中H2S含量、肺组织中CSE活性、BALF中TPL的含量、及肺组织中SP-A、B、CmRNA表达均明显降低（P〈0．05）,BALF中TP的含量明显升高（P〈0．05）。结论：PS降低是内毒素性ALI的重要病理生理过程,H2S对LPS诱导的ALI有保护性作用,其机制可能与H2S对PS的调节有关。     

急性低氧和复氧对牛主动脉内皮细胞损伤的研究

本实验通过一个自制的用于低氧研究的装置,将细胞暴露于严重低氧条件下（ＰＯ２＝５．３ＫＰａ）观察不同时间低氧和复氧时内皮细胞超微结构和存活率的变化。研究显示随低氧时间延长细胞存活率逐步降低,急性低氧后再复氧细胞存活率进一步降低。细胞超微结构损伤也随低氧时间延长逐步加重,低氧后再复氧超结构损伤程度进一步加重。实验观察到在急性低氧和复氧条件下,培养的牛主动脉内皮细胞超微结构损伤以脂质体和空泡的增加为其最明显特点,而不是线粒体和内质网等细胞器的肿胀和破裂。用ＳＯＤ孵育（１５０μ／ｍｌ）能减少复氧引起的牛主动脉内皮细胞死亡（Ｐ＜０．０５）,提示复氧时可能有超氧自由基产生,并在细胞损伤中起重要作用。     

急进高原大鼠肠黏膜机械屏障损伤后细胞自噬变化的初步研究

目的探讨急进高原大鼠肠黏膜机械屏障损伤后细胞自噬的变化情况。方法50只Wistar大鼠随机分为5组：平原组,急进高原6h组、12h组、24h组、48h组,每组各10只。通过低压低氧动物实验舱模拟急进海拔4767m建立急进高原大鼠模型。观察各组大鼠肠黏膜机械屏障损伤情况,用透射电镜观察肠上皮细胞中自噬体;用免疫组织化学法检测肠上皮细胞中自噬相关蛋白Beclinl及LC3B表达。结果与平原组比较,急进高原组可使其肠黏膜机械屏障发生损伤,并且在急进高原肠黏膜损伤6h后可观测到自噬体,24h后检测到自噬相关蛋白Beclinl及LC3B表达显著增高（P〈0．01）。结论急进高原组大鼠肠黏膜机械屏障损伤后自噬相关蛋白Beclinl及LC3B表达明显增加,表明急进高原大鼠肠黏膜机械屏障损伤后细胞自噬活性上调。     

Sepsis impairs alveolar epithelial function by downregulating Na-K-ATPase pump

Widespread vascular endothelial injury is the major mechanism for multiorgan dysfunction in sepsis. Following this process, the permeability of the alveolar capillaries is augmented with subsequent increase in water content and acute respiratory distress syndrome (ARDS). Nevertheless, the role of alveolar epithelium is less known. Therefore, we examined alveolar fluid clearance (AFC) using isolated perfused rat lung model in septic rats without ARDS. Sepsis was induced by ligating and puncturing the cecum with a 21-gauge needle. AFC was examined 24 and 48 h later. The expression of Na-K-ATPase proteins was examined in type II alveolar epithelial cells (ATII) and basolateral membrane (BLM). The rate of AFC in control rats was 0.51 ± 0.02 ml/h (means ± SE) and decreased to 0.3 ± 0.02 and 0.33 ± 0.03 ml/h in 24 and 48 h after sepsis induction, respectively (P < 0.0001). Amiloride, significantly decreased AFC in sepsis; conversely, isoproterenol reversed the inhibitory effect of sepsis. The alveolar-capillary barrier in septic rats was intact; therefore the finding of increased extravascular lung water in early sepsis could be attributed to accumulation of protein-poor fluid. The expression of epithelial sodium channel and Na-K-ATPase proteins in whole ATII cells was not different in both cecal ligation and puncture and control groups; however, the abundance of Na-K-ATPase proteins was significantly decreased in BLMs of ATII cells in sepsis. Early decrease in AFC in remote sepsis is probably related to endocytosis of the Na-K-ATPase proteins from the cell plasma membrane into intracellular pools, with resultant inhibition of active sodium transport in ATII cells.     

Alveolar epithelial fluid transport can be simultaneously upregulated by both KGF and beta-agonist therapy.

Although keratinocyte growth factor (KGF) protects against experimental acute lung injury, the mechanisms for the protective effect are incompletely understood. Therefore, the time-dependent effects of KGF on alveolar epithelial fluid transport were studied in rats 48-240 h after intratracheal administration of KGF (5 mg/kg). There was a marked proliferative response to KGF, measured both by in vivo bromodeoxyuridine staining and by staining with an antibody to a type II cell antigen. In controls, alveolar liquid clearance (ALC) was 23 +/- 3%/h. After KGF pretreatment, ALC was significantly increased to 30 +/- 2%/h at 48 h, to 39 +/- 2%/h at 72 h, and to 36 +/- 3%/h at 120 h compared with controls (P < 0.05). By 240 h, ALC had returned to near-control levels (26 +/- 2%/h). The increase in ALC was explained primarily by the proliferation of alveolar type II cells, since there was a good correlation between the number of alveolar type II cells and the increase in ALC (r = 0.92, P = 0.02). The fraction of ALC inhibited by amiloride was similar in control rats (33%) as in 72-h KGF-pretreated rats (38%), indicating that there was probably no major change in the apical pathways for Na uptake in the KGF-pretreated rats at this time point. However, more rapid ALC at 120 h, compared with 48 h after KGF treatment, may be explained by greater maturation of alpha-epithelial Na channel, since its expression was greater at 120 than at 48 h, whereas the number of type II cells was the same at these two time points. beta-Adrenergic stimulation with terbutaline 72 h after KGF pretreatment further increased ALC to 50 +/- 7%/h (P < 0.5). In summary, KGF induced a sustained increase over 120 h in the fluid transport capacity of the alveolar epithelium. This impressive upregulation in fluid transport was further enhanced with beta-adrenergic agonist therapy, thus providing evidence that two different treatments can simultaneously increase the fluid transport capacity of the alveolar epithelium.     

不同浓度TGF-β1对肺泡II型细胞系MLE-12表型及功能的影响

探讨不同浓度及不同时间点TGF-β1对肺泡II型上皮细胞（AECII）表型及功能的影响。小鼠肺泡II型细胞系MLE-12,随机分为：对照组（0ng／mL）、低浓度组（0．1ng／mL）、中浓度组（1ng／mL）和高浓度组（10ng／mL）。应用细胞免疫荧光双标法及荧光定量PCR法观察各组12,24,48,72h细胞形态变化、AECII标记（肺表面活性物质蛋白B,SP—B）及成纤维细胞标记（成纤维细胞特异性蛋白1,FSP—1）蛋白及mRNA的表达情况。结果表明,随着TGF—β1干预时间的延长及浓度的升高,AECII逐渐由鹅卵石样变成纺锤体形状,获得成纤维细胞样外观。蛋白水平,AECII标记SP-B表达逐渐减弱,成纤维细胞标记FSP-1表达逐渐增强,48h中浓度组及24h高浓度组两者可见明显的共表达,同时,其SP-BmRNA表达较同时间对照组下调,而FSP1 mRNA表达较同时间对照组上调。低浓度组各时间点上述表现不明显。TGF—β1促使AECII向成纤维细胞转化（EMT）,且具有时间及浓度依赖性。     

Hypoxia and beta 2-agonists regulate cell surface expression of the epithelial sodium channel in native alveolar epithelial cells

Alveolar hypoxia may impair sodium-dependent alveolar fluid transport and induce pulmonary edema in rat and human lung, an effect that can be prevented by the inhalation of beta(2)-agonists. To investigate the mechanism of beta(2)-agonist-mediated stimulation of sodium transport under conditions of moderate hypoxia, we examined the effect of terbutaline on epithelial sodium channel (ENaC) expression and activity in cultured rat alveolar epithelial type II cells exposed to 3% O(2) for 24 h. Hypoxia reduced transepithelial sodium current and amiloride-sensitive sodium channel activity without decreasing ENaC subunit mRNA or protein levels. The functional decrease was associated with reduced abundance of ENaC subunits (especially beta and gamma) in the apical membrane of hypoxic cells, as quantified by biotinylation. cAMP stimulation with terbutaline reversed the hypoxia-induced decrease in transepithelial sodium transport by stimulating sodium channel activity and markedly increased the abundance of beta-and gamma-ENaC in the plasma membrane of hypoxic cells. The effect of terbutaline was prevented by brefeldin A, a blocker of anterograde transport. These novel results establish that hypoxia-induced inhibition of amiloride-sensitive sodium channel activity is mediated by decreased apical expression of ENaC subunits and that beta(2)-agonists reverse this effect by enhancing the insertion of ENaC subunits into the membrane of hypoxic alveolar epithelial cells.     

Effects of hypoxia on alveolar fluid transport capacity in rat lungs.

There is little information regarding the effect of hypoxia on alveolar fluid clearance capacity. We measured alveolar fluid clearance, lung water volume, plasma catecholamine concentrations, and serum osmolality in rats exposed to 10% oxygen for up to 120 h and explored the mechanisms responsible for the increase in alveolar fluid clearance. The principal results were 1) alveolar fluid clearance did not change for 48 h and then increased between 72 and 120 h of exposure to hypoxia; 2) although nutritional impairment during hypoxia decreased basal alveolar fluid clearance, endogenous norepinephrine increased net alveolar fluid clearance; 3) the changes of lung water volume and serum osmolality were not associated with those of alveolar fluid clearance; 4) an administration of beta-adrenergic agonists further increased alveolar fluid clearance; and 5) alveolar fluid clearance returned to normal within 24 h of reoxygenation after hypoxia. In conclusion, alveolar epithelial fluid transport capacity increases in rats exposed to hypoxia. It is likely that a combination of endogenous norepinephrine and nutritional impairment regulates alveolar fluid clearance under hypoxic conditions.     

Impairment of transalveolar fluid transport and lung Na(+)-K(+)-ATPase function by hypoxia in rats.

We examined whether hypoxic exposure in vivo would influence transalveolar fluid transport in rats. We found a significant decrease in alveolar fluid clearance of the rats exposed to 10% oxygen for 48 h. Terbutaline did not stimulate alveolar fluid clearance, and alveolar fluid cAMP levels were lower than those determined in normoxia experiment. Hypoxia did not influence the alveolar fluid lactate dehydrogenase levels, Evans blue dye fluid-to-serum concentration ratio, or lung wet-to-dry weight ratio, indicating no significant change in the permeability of alveolar-capillary barrier. Histological examination showed no significant fluid accumulation into the interstitium and the alveolar space. Hypoxia did not reduce lung ATP content; however, we found significant decrease in Na(+)-K(+)-ATPase hydrolytic activity in lung tissue preparations and isolated alveolar type II cells. Our data indicate that hypoxic exposure in vivo impairs transalveolar fluid transport, and this impairment is related to the decrease in alveolar epithelial Na(+)-K(+)-ATPase hydrolytic activity but is not secondary to the alteration of cellular energy source.     

Effects of hypoxia and hypercapnia on surfactant protein expression proliferation and apoptosis in A549 alveolar epithelial cells

During lung injury alveolar epithelial cells are directly exposed to changes in PO(2) and PCO(2). Integrity of alveolar epithelial type II cells (AECII) is critical in lung injury but the effect of hypoxia and hypercapnia on AECII function, viability and proliferation has not been clearly investigated. Aim of the present work was to determine the direct effect of hypoxia and hypercapnia on surfactant protein expression, proliferation and apoptosis of lung epithelial cells in vitro. A549 alveolar epithelia cells were subjected to hypoxia (1%O(2)-5% CO(2)) or hypercapnia (21% O(2-) 15% CO(2)) and expression of surfactant protein C was measured and compared to normal conditions (21% O(2)- 5% CO(2)). Cell cycle progression and apoptosis were measured by flow cytometric analysis. RESULTS: A549 alveolar epithelial cells produce surfactant proteins, including surfactant protein C, when cultured under normal conditions, which is reduced under hypoxic conditions. Specifically, pro-SpC expression is moderately decreased after 8 h of culture in hypoxia, and is completely attenuated after 48 h. Hypercapnia decreases pro-SpC expression only after 48 h of exposure. Stimulation with TNF-alpha partly reverses pSPC decrease observed under hypoxic and hypercapnic conditions. Hypoxic culture of A549 cells results in progressive arrest of cells in the G1 phase of the cell cycle and increased apoptosis first observed 4 h following exposure and peaking at 24 h. In contrast hypercapnia has no significant effect on alveolar epithelial cell proliferation or apoptosis. CONCLUSIONS: Taken together we can conclude that hypoxia rapidly and severely affects AECII function and viability while hypercapnia has an inhibitory effect on pro-SpC production only after prolonged exposure.     

益生菌对胆汁淤积性黄疸大鼠小肠上皮细胞紧密连接蛋白的影响

目的探讨双歧三联活菌(培菲康)对胆汁淤积性大鼠小肠上皮细胞紧密连接蛋白ZO-1(zonula oc-cludens-1)和Occludin表达的调控机制。方法雄性3周龄SD大鼠随机分为对照组、模型组和培菲康组,模型组和培菲康组均给予α-异硫氰酸萘酯(ANIT)50 mg/kg一次性灌胃,建立急性肝内胆汁淤积动物模型,培菲康组于造模前4 d开始给予培菲康4.2×107个活菌数/(kg.d)灌胃大鼠。分别于造模后24、48和72 h三个时间点处死大鼠,取末端回肠黏膜组织,采用免疫组化和Western blots免疫印迹法检测紧密连接蛋白ZO-1、闭锁蛋白(Occludin)的分布和表达,并利用图像分析系统对Western blots图像结果进行定量分析。结果ZO-1和Occludin蛋白主要沿大鼠小肠黏膜上皮细胞膜的顶端呈线状分布,模型组大鼠24 h时ZO-1和Occludin的阳性染色较对照组减少,48 h减少最为明显,72 h阳性染色有所恢复,而培菲康组大鼠各时间点ZO-1和Occludin的阳性染色和模型组相比均明显增多。Western blots结果与免疫组织化学结果相一致,模型组24 h已经开始下降(ZO-1 0.1294±0.0481)、(Oc-cludin 0.1950±0.0441),48 h达到最低(ZO-1 0.0395±0.0095)、(Occludin 0.0137±0.0092),72 h开始恢复(ZO-10.2024±0.0498)、(Occludin 0.1494±0.0355),各时间点与对照组(ZO-1 0.2887±0.0237)、(Occludin 0.4266±0.0670)相比差异有统计学意义(P0.01);而培菲康组各时间点蛋白表达分别为24 h(ZO-1 0.2110±0.0367)、(Occludin 0.3056±0.0572),48 h(ZO-1 0.1173±0.0423)、(Occludin 0.0521±0.0123),72 h(ZO-1 0.2601±0.0191)、(Occludin 0.2050±0.0721),与模型组相应时间点数据相比差异有统计学意义(P0.05)。结论双歧三联活菌能够影响胆汁淤积性大鼠小肠黏膜上皮紧密连接蛋白的分布和表达,可以恢复肠黏膜上皮屏障的完整性。     

Low expression of the beta-ENaC subunit impairs lung fluid clearance in the mouse

Transepithelial alveolar sodium (Na+) transport mediated by the amiloride-sensitive epithelial sodium channel (ENaC) constitutes the driving force for removal of fluid from the alveolar space. To define the role of the beta-ENaC subunit in vivo in the mature lung, we studied a previously established mouse strain harboring a disruption of the beta-ENaC gene locus resulting in low levels of beta-ENaC mRNA expression. Real-time RT-PCR experiments confirmed that beta-ENaC mRNA levels were decreased by >90% in alveolar epithelial cells from homozygous mutant (m/m) mice. beta-ENaC protein was undetected in lung homogenates from m/m mice by Western blotting, but alpha- and gamma-ENaC proteins were increased by 83% and 45%, respectively, compared with wild-type (WT) mice. At baseline, Na+-driven alveolar fluid clearance (AFC) was significantly reduced by 32% in m/m mice. Amiloride at the concentration 1 mM inhibited AFC by 75% and 34% in WT and m/m mice, respectively, whereas a higher concentration (5 mM) induced a 75% inhibition of AFC in both groups. The beta2-agonist terbutaline significantly increased AFC in WT but not in m/m mice. These results show that despite the compensatory increase in alpha- and gamma-ENaC protein expression observed in mutant mouse lung, low expression of beta-ENaC results in a moderate impairment of baseline AFC and in decreased AFC sensitivity to amiloride, suggesting a possible change in the stoichiometry of ENaC channels. Finally, adequate beta-ENaC expression appears to be required for AFC stimulation by beta2-agonists.