β-AR激活对脂多糖导致的肺微血管内皮屏障功能障碍的保护作用  

Protective Effect ofβ-AR Activation on Pulmonary Microvascular Endothelial Barrier Dysfunction Induced by Lipopolysaccharide

作　　者：孙欢[1] 蒋忠洋[1] 王关嵩 Sun Huan;Jiang Zhongyang;Wang Guansong(Intensive Care Unit,Mianyang Central Hospital,Mianyang,Sichuan 621000;Department of Respiratory Diseases,Xinqiao Hospital,Third Military Medical University,Chongqing 400037,China)

机构地区：[1]绵阳市中心医院重症医学科,四川绵阳621000 [2]陆军军医大学附属新桥医院呼吸科,重庆400037

出　　处：《四川医学》2021年第8期783-788,共6页Sichuan Medical Journal

基　　金：绵阳市卫生计生育委员会课题(编号:201624)。

摘　　要：目的研究β-AR在脂多糖导致的肺微血管内皮屏障功能障碍中的调控作用。方法LPS(10μg/ml)处理细胞2~8 h;分别激活β1-AR和β2-AR后再利用LPS处理6 h。采用Transwell小室法检测内皮屏障通透性,共聚焦显微镜观察细胞肌动蛋白(F-actin)的形态,蛋白质免疫印迹试验(Western Blot)检测细胞连接蛋白VE-cadherin和occludin的表达;质粒转染方法转染β2-AR-GFP,免疫荧光技术标记Rab5a,共聚焦显微镜观察Rab5a和β2-AR-GFP在细胞内的定位。结果LPS对肺微血管内皮屏障通透性的作用呈时间依赖性,随时间延长通透性增加,在6~8 h达到最高峰;LPS导致骨架蛋白F-actin形态学发生改变、应力纤维形成,下调VE-cadherin和occludin的蛋白表达水平;选择性激活β2-AR可部分逆转LPS对内皮屏障的损伤作用;Rab5a与β2-AR-GFP共定位于细胞膜,受体激活后主要共定位于细胞核周。结论β2-AR在肺微血管内皮细胞的激活可调控细胞骨架蛋白重构以及细胞间连接蛋白表达水平,对LPS导致的内皮屏障损伤有一定的保护作用。Objective To investigate the underlying protective effect ofβ-Adrenergic receptor activation on pulmonary microvascular endothelial barrier dysfunction induced by lipopolysaccharide.Methods Primary cultured rat pulmonary microvascular endothelial cells(PMECs)were stimulated by LPS(10μg/ml)for 2~8 hours.β1-AR andβ2-AR were activated separately,and then treated with LPS for 6 hours.The permeability of endothelial barrier was determined by Transwell assay.The morphological characteristics and distribution of F-actin was observed by laser confocal fluorescence microscope.VE-cadherin and occludin protein levels were detected by Western blotting.Cells were transfected with GFP-taggedβ2-AR,Rab5a was labeled by immunofluorescence technique,and then confocal microscopy was employed to observe the distribution ofβ2-AR-GFP and Rab5a in cells.Results The increased permeability of endothelial barrier was time-dependent and peaked at 6 to 8 hours.LPS changed F-actin morphology,obvious stress fiber,reduced VE-cadherin and occludin protein levels were observed.Selective activation ofβ2-AR could partially reverse the effect of LPS.Co-localization ofβ2-AR and Rab5a was observed on the cell membrane,and moved to perinuclear area after the activation of the receptor.Conclusions Activation ofβ2-AR could protect pulmonary microvascular endothelial barrier dysfunction from LPS stimulation via regulating cytoskeleton reorganization and VE-cadherin and occludin protein levels.

关 键 词：Β-AR 肺微血管内皮屏障 脂多糖 

分 类 号：R563.8[医药卫生—呼吸系统]