肌球蛋白轻链激酶抑制剂对急性肺损伤的保护作用  

Protective effect of myosin light-chain kinase inhibitor on acute lung injury

作　　者：白建文[1] 邓伟吾[2] 章剑[1] 许淑敏[1] 张斗霞[1] 

机构地区：[1]同济大学附属东方医院急危重病科,上海200120 [2]上海交通大学附属瑞金医院呼吸科

出　　处：《中国危重病急救医学》2009年第4期215-218,I0002,共5页Chinese Critical Care Medicine

摘　　要：目的探讨肌球蛋白轻链激酶（MLCK）抑制剂ML-7对细菌脂多糖（LPS）诱导的人肺动脉内皮细胞（HPAEC）和急性肺损伤（ALI）的影响。方法体外培养HPAEC，待4～6代予ML-7（10μmol／L）孵育60min后，再给予LPS刺激60min，四甲基偶氮唑盐（MTT）比色法检测HPAEC活性；荧光显微镜下观察磷酸化肌球蛋白轻链激酶（p-MLCK）免疫反应细胞。20只雌性BALB／c小鼠随机分组，LPS组鼻内注入LPS（1μg／g）；ML-7组在LPS滴鼻前进行ML-7干预。观察小鼠肺湿／干重（W／D）比值、支气管肺泡灌洗液（BALF）蛋白含量、肺组织髓过氧化物酶（MPO）活性和病理学改变；用免疫组化法检测肺组织MLCK和CD11b阳性免疫反应细胞，逆转录-聚合酶链反应（RT-PCR）检测肺组织MLCK mRNA表达，蛋白质免疫印迹法（Western blotting）检测肺组织MLCK蛋白表达。结果与LPS组比较，HPAEC在ML-7孵育下吸光度（A）值增加（P〈0．01），P-MLCK免疫反应细胞明显减少（P〈0．05），肺W／D比值、肺组织MPO活性、BALF蛋白含量均明显降低（P〈0．05或P〈0．01）。病理结果显示，LPS组小鼠肺部炎症反应明显，以中性粒细胞浸润为主，肺泡充血、水肿；ML-7组肺部炎症及充血明显改善。免疫组化显示位于内皮的MLCK免疫反应细胞和位于炎性细胞的CD11b在ML-7组均较LPS组明显减少。ML-7组肺组织MLCK的mRNA和蛋白表达均较LPS组降低（P均〈0．05）。结论MLCK特异性抑制剂ML-7能提高LPS诱导的HPAEC生长活性，降低p-MLCK表达；减轻LPS诱导的中性粒细胞在肺内浸润、肺水肿及MLCK、CD11b蛋白和MLCK mRNA的表达。表明抑制MLCK活性可能通过减弱MLCK磷酸化而达到稳定血管屏障功能，防治ALI的作用。Objective To investigate the influence of inhibitor of myosin light-chain kinase （MLCK） on the human pulmonary arterial endothelial cell （HPAEC） challenged with lipopolysaccharide （LPS） and LPS induced of acute lung injury （ALI） in mice. Methods HPAECs were cultured in ECM medium and its passages 4 - 6 were used. After treatment with inhibitor of MLCK （ML-7） for 60 minutes, the HPAECs were incubated in LPS for another 60 minutes, and then cell viability was measured by the methyl thiazolyl tetrazolium （MTT） assay. Immunofluorescence microscope was used to detect phosphorylated-MLCK （p-MLCK） immunoreactive cells. Twenty female BALB/c mice were randomly divided into two groups. The mice of LPS group were exposed to LPS （1μg/g） through nasal instillation, and the mice of ML-7 group were pretreated with ML-7 before intranasal instillation of LPS. Wet/dry weight （W/D） ratio of lung, bronchoalveolar lavage fluid （BALF） protein content, myeloperoxidase （MPO） activity and histopathological changes of lung tissue were observed. Immunohistochemistry assays were used to determine the status of MLCK and CDllb immunoreactive cells in lung tissue, and expression of MLCK mRNA in lung tissue was assessed by reverse transcription-polymerase chain reaction （RT-PCR）. Expression of MLCK protein in lungs was assayed by Western blotting. Results Compared with LPS group, increased absorbance （A） value of HPAEC was found in ML-7 group （P〈0.01）. Immunoreactive cells of p-MLCK were more reduced in the ML-7 group （P〈0.05）, and W/D ratio of lung, MPO activity and BALF protein content of lung tissue were decresead in ML-7 group （P〈0. 05 or P〈0.01）. Histological examination showed that an extensive lung inflammation was seen in mice of LPS group, with an accumulation of a large number of neuyrophils, marked pulmonary edema and hemorrhage, but the inflammation and parenchymal hemorrhage was significantly alleviated in ML-7 group. Both MLCK immunoreactive cells located in

关 键 词：肌球蛋白轻链激酶抑制剂 肌球蛋白轻链激酶 急性肺损伤 

分 类 号：R686[医药卫生—骨科学]