自噬对急性肺损伤小鼠肺泡上皮屏障功能的影响及机制探讨  

Influence of autophagy on alveolar epithelial barrier function in mice with acute lung injury

作　　者：邱玉环 贺天文 李国平 QIU Yuhuan;HE Tianwen;LI Guoping(The Affiliated Hospital of Southwest Medical University,Luzhou 646000,China)

机构地区：[1]西南医科大学附属医院,四川泸州646000 [2]西南医科大学

出　　处：《山东医药》2018年第22期28-31,共4页Shandong Medical Journal

基　　金：国家自然科学基金资助项目(81170032);泸州市科技厅基金项目(2016LZXNYD-Z01)

摘　　要：目的探讨自噬对急性肺损伤(ALI)小鼠肺泡上皮屏障功能的影响,并探讨其机制。方法将40只雄性BALB/c小鼠随机分为C组、LPS组、RAPA+LPS组、3-MA+LPS组,各10只。LPS组、RAPA+LPS组及3-MA+LPS组气管滴入脂多糖(LPS)制备小鼠ALI模型,C组滴入等量生理盐水;在气管滴入LPS前1 h,3-MA+LPS组和RAPA+LPS组分别腹腔预注射自噬抑制剂3-甲基腺嘌呤(3-MA,10 mg/kg)和自噬促进剂雷帕霉素(RAPA,5 mg/kg),C组、LPS组腹腔预注射等量生理盐水。造模18 h后处死小鼠,收集标本。HE染色观察各组小鼠肺组织病理学变化;测算肺组织干湿重比(W/D);测定支气管肺泡灌洗液(BALF)中总蛋白水平;RT-PCR法检测肺组织中紧密连接蛋白Zo-1、Occludin mRNA;Western blotting法检测肺组织中Zo-1、Occludin及自噬标志蛋白LC3Ⅱ/Ⅰ。结果LPS组和3-MA+LPS组肺损伤程度较C组明显加重,3-MA+LPS组较LPS组严重,RAPA+LPS组则较LPS组明显减轻。与C组相比,LPS组肺组织W/D、BALF中总蛋白水平、肺组织中LC3Ⅱ/Ⅰ蛋白表达高(P均<0.05),肺组织中Zo-1、Occludin mRNA和蛋白表达低(P均<0.05)。与LPS组相比,3-MA+LPS组肺组织W/D、BALF中总蛋白水平高,肺组织中Zo-1、Occludin mRNA和Zo-1、Occludin、LC3Ⅱ/Ⅰ蛋白表达低(P均<0.05);而RAPA+LPS组肺组织W/D、BALF中总蛋白水平低,肺组织中Zo-1、Occludin mRNA和Zo-1、Occludin、LC3Ⅱ/Ⅰ蛋白表达高(P均<0.05)。结论 LPS通过降低紧密连接蛋白Zo-1、Occludin的表达导致肺泡上皮屏障破坏,使肺泡上皮屏障功能受损;抑制自噬会进一步减少Zo-1、Occludin的表达,使肺泡上皮屏障功能受损更严重;而促进自噬,则明显增加Zo-1、Occludin的表达,明显改善肺泡上皮屏障功能,从而对肺脏起保护作用。Objective To investigate the influence of autophagy on alveolar epithelial barrier function in mice with acute lung injury and to explore its mechanism.Methods Forty male BALB/c mice were randomly divided into the group C,LPS group,RAPA+LPS group,and 3-MA+LPS group,with 10 mice in each group.Mice in the LPS group,3-MA+LPS group,and RAPA+LPS group were intratracheally given LPS to establish the ALI models,while mice in the group C received equal amount of saline.In the 3-MA+LPS group and RAPA+LPS group,mice were separately injected with autophagy inhibitor 3-MA(10 mg/Kg)and autophagy inducer RAPA(5 mg/Kg)intraperitoneally 1 h before LPS instillation,while mice in the group C and LPS group were injected intraperitoneally with equal amount of saline.At 18 h after LPS injection,the mice were sacrificed,then we collected the bronchoalveolar lavage fluid(BALF)and lung tissues.HE staining was used to observe the histopathological changes of lung tissues of each group.Detecting lung tissue dry/wet ratio and the total protein level of BALF.Moreover,the mRNA expression level of Zo-1 and Occludin in the lung tissues were examined by RT-PCR,the protein expression levels of Zo-1,Occludin and autophagy related protein LC3II/I were examined by Western blotting.Results The lung injury was more severe in the LPS group and 3-MA+LPS group than in group C,and more severe in the 3-MA+LPS group than in the LPS group,but much milder in the RAPA+LPS group than in the LPS group.Compared with the group C,the lung tissue W/D,total protein level in BALF and expression of LC3II/I protein in lung tissue increased,and the expression of ZO-1,Occludin mRNA and ZO-1,Occludin protein decreased in the LPS group(all P<0.05).Compared with the LPS group,the lung tissue W/D,total protein level in BALF increased,and the expression of ZO-1,Occludin mRNA and ZO-1,Occludin,and LC3II/I protein decreased in the 3-MA+LPS group(all P<0.05);however,the lung tissue W/D and total protein level in BALF decreased,and the expression of ZO-1,Occludin mRNA and ZO-1,O

关 键 词：急性肺损伤 自噬 肺泡上皮屏障 细胞紧密连接蛋白 小鼠 

分 类 号：R655.3[医药卫生—外科学]