分泌型钙激活Cl^-通道在ARDS小鼠肺组织表达的变化及规律

Changes and regularity of calcium-activated chloride channel in lung tissues of acute respiratory distress syndrome mice

目的观察小鼠分泌型钙激活Cl^-通道(mCLCA3)在急性呼吸窘迫综合征(ARDS)模型肺组织中的表达量及随时间的变化规律,探究其在急性肺损伤过程中的作用。方法18只雄性C57BL/6小鼠随机分为对照组和脂多糖(LPS)组ARDS小鼠(分为气道滴入LPS诱导6 h组、24 h组),每组6只。HE染色观察小鼠肺组织形态结构改变,并进行肺损伤评分;阿尔辛蓝-过碘酸雪夫(AB-PAS)染色观察小鼠气道上皮中杯状细胞的数量;反转录-聚合酶链反应(RT-PCR)法检测小鼠肺组织中mCLCA3的mRNA表达水平;蛋白质印迹法(Western blot)法检测mCLCA3蛋白在ARDS小鼠肺组织及支气管肺泡灌洗液(BALF)中的表达。结果HE染色结果,与对照组相比,LPS组ARDS小鼠肺部正常结构明显被破坏,肺泡上皮呈弥漫性损伤,肺泡内出血并伴有大量炎性细胞浸润,肺间质蛋白质沉积,有透明膜形成;与对照组小鼠相比较,LPS组ARDS小鼠的肺损伤评分(对照组:1.40±0.54;LPS组:7.40±0.54,P<0.05)显著升高;AB-PAS染色观察小鼠气道上皮中杯状细胞的数量;real-time RT-PCR(RT-qPCR)和Western blot结果显示:相比于对照组小鼠,LPS组ARDS小鼠的肺组织中mCLCA3的基因表达量明显下降(P<0.05);mCLCA3蛋白在ARDS小鼠的肺组织和BALF中均显著降低,且差异有统计学意义(P<0.05),且下降水平呈时间依赖性。结论LPS诱导的ARDS小鼠模型中气道杯状细胞数量明显增加,而其肺组织及BALF中mCLCA3的mRNA及蛋白表达水平(评分)均明显下降,并表现为时间依赖性。气道杯状细胞产生的分泌型CLCA表达水平与ARDS损伤程度呈负相关,提示前者可能在肺部炎症机制中扮演着负性调节作用。

Objective To observe the expression levels of murine calcium-activated chloride channel (mCLCA3) in lung tissue of acute respiratory distress syndrome (ARDS) mouse model and its changing rule with time, to explore its role in the process of lung tissue injury in the mice.Methods Eighteen male C57BL/6 mice were randomly divided into control group, lipopolysaccharide (LPS) group ARDS mouse (LPS tracheal instillation for 6 hours group and 24 hours group), with 6 mice in each group.HE staining was used to observe the morphological changes of lung tissue, and the lung injury score was calculated.Alcian blue-Periodic acid-Schiff (AB-PAS) staining was used to observe changes in the number of goblet cells in pulmonary of mice.Real-time RT-PCR (RT-qPCR) and Western blot were used to test the mRNA and the protein expression levels of mCLCA3 in lung tissue of mice.Results HE staining showed that compared with the control group, the normal tissue structure of alveolar and interstitial in ARDS mice were destroyed and filled with lots of inflammatory cells and red cells.The lung injury score of ARDS mouse in LPS group (7.40±0.54) was higher than that of the normal control group (1.40±0.54)(P<0.05). AB-PAS staining results showed that the numbers of bronchial epithelial goblet cells in LPS group were higher than those in normal control group (P<0.05). Real-time RT-PCR and Western blot results revealed that the mRNA expression levels of mCLCA3 were decreased significantly in lung tissue in ARDS mice (P<0.05). And the protein levels of mCLCA3 were decreased markedly in lung tissue and BALF in ARDS mice (P<0.05), its decline decreased more with time.Conclusions The number of the Goblet cell in the LPS-induced ARDS mice model is increase mildly, but mRNA expression and the protein levels of mCLCA3 are decreased significantly in its lung tissue and BALF lung tissue in ARDS mice, and its decline decreased more with time.The expression of CLCA produced by airway goblet cells is negatively correlated with the extent of ARDS damage, suggesting that the former may play an inhibitory role in the mechanism of lung inflammation.