目的：研究证明慢性酒精滥用是急性呼吸窘迫综合症(ARDS)的一个独立危险因子，并提示慢性酒精滥用导致肺泡上皮的还原型谷胱甘肽缺乏和肺泡上皮细胞(AEC)屏障功能损害。然而，慢性乙醇滥用导致损伤的具体分子并不清楚。以AEC屏障功能为主的AEC—内皮细胞双重屏障破坏是ARDS的病理生理学特征，而紧密连接(TJ)和粘附连接(AJ)是调节AEC屏障的关键。本研究通过观察大鼠肺泡上皮TJ特征性蛋白occludin，ZO-1和AJ蛋白E-cadherin的改变阐明慢性乙醇滥用对肺泡上皮屏障功能的损伤机制，并观察补充谷氨酰胺(Gln)对肺泡上皮屏障功能的保护作用，此外观察不同浓度乙醇暴露对人类肺泡上皮细胞株(A549细胞)单层的屏障功能影响及对TJ中occludin，ZO-1和AJ中E-cadherin的作用，并初步探讨不同浓度乙醇暴露对小G蛋白RhoA和Rho激酶(ROCK)信号转导途径的作用，为ARDS的临床治疗提供理论依据。 方法：体内部分实验选取60只雄性SD大鼠，随机分为对照组(control组)、慢性乙醇摄取组(ethanol组，10%(v/v)的乙醇为唯一饮料连续6周喂养)、内毒素处理组(LPS组，LPS 2㎎/㎏，静脉注射)、慢性乙醇摄取并内毒素处理组(ethanol+LPS组)、慢性乙醇摄取并Gln补充组(ethanol+ Gln， Gln 0.3g/㎏/d管饲)以及慢性乙醇摄取并内毒素处理Gln补充组(ethanol+LPS+Gln组)，每组均为10只。利用荧光示踪剂FD₄在血清中和支气管肺泡灌洗液中的荧光比值来测定每组的支气管肺泡渗透性、免疫荧光染色激光共聚焦显微镜下观察大鼠肺泡上皮occludin、ZO-1和E-cadherin蛋白分布及表达、分离培养各组大鼠Ⅱ型肺泡上皮细胞后运用免疫印迹和RT-PCR测定Ⅱ型肺泡上皮细胞中occludin、ZO-1和E-cadherin的蛋白表达及mRNA表达，并透射电镜、光镜下观察肺组织肺泡上皮的TJ结构及肺组织形态学变化。体外部分实验是培养A549细胞，当细胞在带微孔细胞隔膜的细胞培养小室(Cell insert)中形成完整细胞单层后，细胞下层培养基换为含不同浓度(0.1%、0.5%、1%以及2%)乙醇的培养基，细胞上层培养基换为含FD₄ (25 ㎎/ml)的培养基，乙醇暴露12小时后测定细胞上层和下层培养基中FD₄的荧光强度并计算FD₄的清除率以检测A549细胞单层的渗透性变化，以及运用免疫荧光、免疫印迹和RT-PCR观察不同浓度乙醇暴露对细胞单层中occludin、ZO-1和E-cadherin的定位、蛋白表达及mRNA表达的影响，并检测RT-PCR测定不同浓度乙醇暴露对细胞单层对RhoA、ROCK的mRNA表达的作用。 结果：体内实验结果显示ethanol组其支气管肺泡上皮渗透性明显较control组增高2倍左右(2.8887±1.1720 vs. 1.2063 ±0.3562，P＜0.05)；在LPS组同样观察到支气管肺泡上皮渗透性的增高(2.7127±1.5316 vs. 1.2063 ±0.3562， P＜0.05)；在ethancl+LPS组支气管肺泡渗透性进一步增高(6.0236±1.0945 vs. 1.2063±0.3562，P ＜0.01)；补充Gln可以明显改善由慢性乙醇摄取引起的支气管肺泡上皮渗透性增高(ethanol+Gln组 vs. ethanol组：1.2875± 0.5368 vs 2.8887 ± 1.1720，P＜0.05)；同样改善由慢性乙醇摄取并内毒素处理引起的支气管肺泡上皮渗透性显著增高(ethanol + LPS + Gln组 vs. ethanol + LPS组：3.854 ± 1.5933 vs.6.0236 ± 1.0945， P ＜0.01)。在正常对照大鼠中occludin、ZO-1和E-cadherin蛋白在肺泡上皮呈一连续均匀的胞膜及胞浆表达；慢性乙醇摄、内毒素导取导致occludin、ZO-1和E-cadherin蛋白在胞膜和胞浆的表达下降；并且肺泡上皮胞膜呈部分断裂不连续的表达(ethanol组和LPS组)；慢性乙醇摄取并内毒素处理导致肺泡上皮ocdudin、 ZO-1和E-cadherin蛋白表达显著下降；且胞膜表达呈明显的断裂甚至消失(ethanol + LPS组)；补充Gln可以明显改善由慢性乙醇摄取及慢性乙醇摄取并内毒素处理导致上述变化。免疫印迹结果显示在ethanol组和LPS组的Ⅱ型肺泡上皮细胞中occludin、ZO-1和E-cadherin的蛋白表达均有下降；RT-PCR结果显示这两组mRNA表达水平较control组的mRNA表达水平下降(P ＜0.05)；在Ethanol + LPS组中occludin、ZO-1和E-cadherin的蛋白表达下降最为明显，其mRNA表达水平较control组及其余各组的mRNA表达水平显著下降(P ＜0.01)；补充Gln可以明显改善由于慢性乙醇摄取并内毒素处理所引起occludin、ZO-1和E-cadherin的蛋白表达降低，同样也改善由于慢性乙醇摄取及慢性乙醇摄取并内毒素处理所引起occludin、ZO-1 和E-cadherin的mRNA表达降低〔(ethanol+Gln组 vs. ethanol组， P ＜0.05)、 (ethanol+LPS+Gln组 vs. ethanol+LP组，P ＜0.01)〕。在透射电镜下ethanol + LPS组可观察到肺泡上皮细胞间紧密连接出现明显的不规则増宽；在ethanol + LPS+Gln组其肺泡上皮细胞间紧密连接仅有少许的増宽。同时光镜下ethanol + LPS组观察到重度的肺组织急性炎症及出血坏死表现；ethanol组观察到部分小支气管周围纤维组织增生等慢性支气管炎的表现；在LPS组、ethanol + LPS + Gln组均可观察到肺组织的炎症及出血等损伤表现。 体外实验显示A549细胞单层在0.1%乙醇暴露12小时后其渗透性与对照组的相比较显著增高(286.1±95.63 vs. 145±43.82，P ＜ 0.01)；在0.5% 乙醇暴露后其渗透性继续增高至对照组的一倍多(356.7±64.27vs. 145±43.82， P＜0.01) ； A549细胞单层在1%及2%乙醇暴露12小时后其渗透性急剧增高至对照组的4倍多(672.3±128.45 vs. 145±43.82，P＜0.01)和5倍左右(809.5±115.29 vs. 145±43.82， P＜0.01)；并且不同浓度乙醇暴露组之间两两比较差异有显著性(P＜ 0.01)。正常A549细胞中occludin、ZO-1和E-cadherin蛋白呈一连续均匀的胞膜及胞浆表达；在0.1%乙醇暴露12小时后其胞膜及胞浆染色明显降低、胞膜上的染色不均匀；在0.5%乙醇暴露后的A549细胞的胞膜及胞浆染色进一步降低并且胞膜上的染色出现部分断裂；在1%乙醇暴露12小时后的A549细胞单层胞膜的染色基本上消失、部分细胞胞浆染色；在2%乙醇暴露12小时后A549细胞单层的胞膜表达完全消失、少量细胞胞浆染色。免疫印迹结果显不乙醇暴露导致occludin、ZO-1和E-cadherin蛋白表达的明显降低，并随着乙醇浓度的增加其表达水平进一步降低。RT-PCR结果亦显示乙醇暴露导致occludin、ZO-1和E-cadherin的mRNA表达的明显降低，并呈浓度依赖性降低(乙醇暴露组之间mRNA表达水平两两比较差异有显著性，P ＜0.05)；同时RhoA、ROCK的mRNA表达也随着乙醇暴露浓度增加而下降，不同组间两两比较亦有显著性差异(P＜0.05)。 结论：研究提示慢性乙醇摄取通过降低TJ分子occludin、ZO-1和AJ分子E-cadherin的mRNA、蛋白表达水平以及干扰各蛋白在胞膜上的定位导致肺泡上皮屏障功能受损，并增加了对内毒素诱导急性肺损伤的易感性，补充Gln对肺泡上皮屏障功能有保护作用。并且乙醇对occludin， Z0-1和E-cadherin的mRNA、蛋白表达水平的下调作用呈浓度依赖性，慢性乙醇对occludin，ZO-1和E-cadherin的这种作用可能是通过下调RhoA和ROCK的mRNA表达水平来实现的。 关键词：急性肺损伤；乙醇；occludin； ZO-1；谷氨酰胺；肺泡上皮屏障功能

 Objective: Previous studies show that chronic alcohol abuse is an independent risk factor for acute respiratory distress syndrome (ARDS) and impairs alveolar epithelial barrier function through glutathione depletion. However, the precise molecular structures that are damaged by chronic ethanol ingestion have not been identified. Acute respiratory distress syndrome is characterized by disruption of the alveolar epithelial barrier, allowing the abnormal passage of water, proteins, and sodium ions into the alveolar space. To test whether chronic ethanol ingestion impairs the alveolar epithelium barrier by tight junction proteins deterioration and predisposes to ALI, this study determined the alterations in tight junction proteins occludin, zonula occludens (ZO)-1 and adherens junction protein E-cadherin in alveolar epithelium of rats and observed the protective effect of glutamine (Gln) supplementation. This study also investigated the effects of exposure to different concentrations ethanol on the barrier function of human alvealor epithelial cells (A549 cells), alterations of occludin, ZO-1 and E-cadherin in A549 cells monolayers and expressions of small small guanosine triphosphatases RhoA and ROCK, which can offer theory ground for the clinical treatment of ARDS. Methods: In vivo, 60 rats were assigned to the six groups as follows (10 rats per group). Rats received a control diet with standard chow and water (control group), the six weeks ethanol diet (ethanol group), a control diet and an LPS administration (2㎎/㎏, i.v.) 12 h before measurement (LPS group), the ethanol diet and LPS administration (ethanol + LPS group), the ethanol diet and a Gln supplementation (0.3 g/㎏, gavage ) once daily to prevent glutathione depletion (ethanol + Gln group), or the ethanol diet and Gln supplementation and LPS administration (ethanol + Gln + LPS group). The bronchoalveolar epithelial permeability was detected by the bronchoalveolar lavage fluid (BALF): serum FITC-dextran (FD₄) fluorescence ratio. Protein localization and expression of occludin, ZO-1 and E-cadherin in alveolar epithelium of rats lung were examined by immunofluorescence. The alveolar type Ⅱ Cells were isolated and protein, messenger RNA expression of occludin, ZO-1 and E-cadherin in the isolated alveolar type Ⅱ cells were examined by immunoblotting and reverse transcription-polymerase chain reaction (RT-PCR). Finally, morphological alterations of tight junction of alveolar epithelium and acute lung injury in rats were observed by transmission electron microscopy and microscopy. In vitro, A549 cells were seeded onto microporous cells inserts. Monolayers growing on Transwell inserts were used 12 days after seeding. The medium bathing the basolateral surface was replaced with DMEM/F12 supplemented with ethanol in different concentration (0.1%, 0.5%, 1% and 2%). The medium bathing the apical surface of the monolayers was replaced with DMEM/F12 containing FD₄ at 25 ㎎/ml. The cells were then incubated for 12 h. The concentrations of FD₄ in the apical and basolateral media were determined spectrofluorometrically and clearances of FD₄ were counted. The permeability of the monolayers was expressed as a clearance of FD₄. Protein localization and expression of occludin, ZO-1 and E-cadherin in monolayers of A549 were examined by immunofluorescence. The protein, messenger RNA expression of occludin, ZO-1 and E-cadherin in the A549 cells were examined by immunoblotting and RT-PCR. The messenger RNA expression of RhoA and ROCK were also determined by RT-PCR to investigate the effect of ethanol exposure on monolayers. Results: The BALF:serum fluorescence ratios in the ethanol group were approximately 2-fold higher than those in the control group (2.8887 ± 1.1720 vs. 1.2063± 0.3562, P ＜0.05). A similar increase was observed in the LPS group that had ALI (2.7127±1.5316 vs. 1.2063 ±0.3562, P＜0.05). The BAJLF:serum ratios in the ethanol + LPS group were increased further (6.0236 ± 1.0945 vs. 1.2063± 0.3562, P < 0.01). The protective effects of Gln supplementation on the bronchoalveolar epithelial permeability were examined in the ethanol+ Gln group and ethanol +LPS+ Gln group. Gln supplementation significantly attenuated the enhanced permeability induced by ethanol (ethanol + Gln group vs. ethanol group: 1.2875± 0.5368 vs. 2.8887 ±1.1720, P＜ 0.05) and ethanol + LPS (ethanol + LPS + Gln group vs. ethanol + LPS group : 3.854±1.5933 vs.6.0236± 1.0945, P＜0.01). A continuous along the membrane and diffuse in the cytoplasm staining of occludin, ZO-1 and E-cadherin was shown in the alveolar epithelium of control rats. Ethanol feeding induced partial breakdown of membrane staining and decreased cytoplasm staining of occludin,ZO-1 and E-cadherin in the alveolar epithelium. Similar changes were observed in the LPS group. Treatment with ethanol plus LPS induced fragmented staining pattern on membrane and mostly disappeared cytoplasm staining pattern of occludin,ZO-1 and E-cadherin. Glutamine supplementation markedly ameliorated these changes of occludin,ZO-1 and E-cadherin elicited by ethanol and ethanol plus LPS. Immunoblotting shows that both chronic ethanol ingestion and LPS significantly depressed the protein levels of occludin, ZO-1, and E-cadherin in isolated alveolar epithelial cells. Treatment with ethanol plus LPS further depressed the protein levels of occludin, ZO-1, and E-cadherin in isolated alveolar epithelial cells. Glutamine supplementation markedly ameliorated those downregulated protein levels induced by ethanol and ethanol plus LPS. The changes of occludin, ZO-1 and E-cadherin protein expression and messenger RNA expression in isolated alveolar epithelial cells detected by immunoblotting and RT-PCR were similar. RT-PCR shows that messenger RNA levels of occludin, ZO-1, and E-cadherin in ethanol group and LPS group are low (ethanol group vs. control group, P＜ 0.05; LPS group vs. control group, P＜0.05). Treatment with ethanol plus LPS had the greatest depressive effect on these messenger RNA levels (ethanol + LPS group vs. the other groups, P＜0.01). Glutamine supplementation markedly ameliorated those downregulated messenger RNA levels induced by ethanol (ethanol + Gln group vs. ethanol group, P＜0.05) and ethanol plus LPS (ethanol + LPS + Gln group vs. ethanol + LPS group, P＜0.01). Morphological alterations of tight junction were observed by transmission electron microscopy. Obviously, after treatment with ethanol plus LPS, the tight junction was irregularly widened. When supplementing Gln to the same treatment, the tight junction also widened but to a less extent. Morphology of lung tissue showed severe lung injury and necrosis in ethanol +LPS group and chronic inflammation such as part of bronchia fibroplasias in ethanol group. The lung injury and hemorrhage were observed in LPS group and ethanol + LPS + Gln group. The study of in vitro shows the permeability of A549 cell monolayers increased when the cells monolayers exposure to 0.1%ethanol (286.1±95.63 vs. 145±43.82, P ＜0.01) and to 0.5% ethanol (356.7±64.27 vs. 145±43.82, P＜0.01) . A549 cells monolayers increased further when the cell monolayers exposure to 1%ethanol (672.3±128.45 vs. 145±43.82, P＜ 0.01) and 2% ethanol (809.5±115.29 vs. 145±43.82, P＜0.01) . The ethanol exposure increased the monolayers permeability in a concentration dependent manner. Immunofluorescence of occludin, ZO-1 and E-cadherin in normal A549 cell monolayer was even and continuous along the membrane and diffuse in the cytoplasm. The stainings of membrane and cytoplasm were decreased and stainings of membrane were nonuniform in A549 cell monolayer after 0.1%ethanol exposure. The stainings of membrane and cytoplasm were decreased further and stainings of membrane were disrupted in A549 cell monolayer after 0.5% ethanol exposure. The stainings of membrane were almost disappeared and partial cytoplasm stainings were seen in A549 cell monolayer after 1% ethanol exposure. The stainings of membrane were totally disappeared and small amounts cytoplasm stainings were seen in A549 cell monolayer after 2% ethanol exposure. Immunoblotting shows that ethanol exposure induced the down-regulation of protein expression of occludin, ZO-1 and E-cadherin in A549 cell monolayers. It shows that the more high concentration of ethanol exposure the more down-regulation of protein expression of occludin, ZO-1 and E-cadherin. RT-PCR shows that ethanol exposure induced down-regulation of messenger RNA levels of occludin, ZO-1, and E-cadherin in A549 cell monolayers. The effects of ethanol were concentration dependent ( etanol exposure group compare with the other etanol exposure group, P＜0.05) . The messenger RNA expression of RhoA and ROCK were down-regulation by ethanol exposure in concentration dependent (etanol exposure group compare with the other etanol exposure group, P ＜0.05) Conclusion: These data suggest that chronic ethanol ingestion impairs the alveolar epithelial barrier function through down-regulation the messenger RNA expression and disruption of membrane localization of occludin, ZO-1 and E-caoherin. Those deteriorations of occludin, ZO-1 and E-cadherin predisposes to ALI induced by LPS. Gln supplementation has protective effect. The ethanol induced the down-regulation of protein expression and messenger RNA expression of occludin, ZO-1 and E-cadherin in concentration dependent. The effect of ethanol may via down-regulation of messenger RNA expression level of RhoA and ROCK. Key words: acute lung injury; alcohol; occludin; ZO-1; glutamine; alveolar epithelial barrier function