机械通气诱导内质网应激促进机械通气相关性肺纤维化的机制研究  

Research on the mechanism of mechanical ventilation induced endoplasmic reticulum stress promoting mechanical ventilation-induced pulmonary fibrosis

作　　者：汤日 冯金华 梅舒雅 徐侨翌 周洋 邢顺鹏[1] 皋源[1] 何征宇[1] 张志贇 Tang Ri;Feng Jinhua;Mei Shuya;Xu Qiaoyi;Zhou Yang;Xing Shunpeng;Gao Yuan;He Zhengyu;Zhang Zhiyun(Department of Critical Care Medicine,Renji Hospital,Shanghai Jiaotong University School of Medicine,Shanghai 200127,China)

机构地区：[1]上海交通大学医学院附属仁济医院重症医学科,上海200127

出　　处：《中华危重病急救医学》2023年第11期1171-1176,共6页Chinese Critical Care Medicine

基　　金：国家自然科学基金(82172150)。

摘　　要：目的通过动物实验探讨机械通气(MV)诱导内质网应激(ERS)促进机械通气相关性肺纤维化(MVPF)的机制,明确血管紧张素受体1(AT1R)通路在该过程中的重要作用。方法将C57BL/6小鼠按随机数字表法分为对照组(Sham组)、MV组、AT1R敲减组(AT1R-shRNA组)、AT1R敲减+MV组(MV+AT1R-shRNA组),每组6只。MV组和MV+AT1R-shRNA组小鼠气管插管后MV 2 h(参数设置:呼吸频率70次/min,潮气量20 mL/kg,吸入氧浓度0.21)以建立MVPF动物模型;Sham组和AT1R-shRNA组小鼠气管插管后自主呼吸。AT1R-shRNA组和MV+AT1R-shRNA组小鼠于制模前1个月经气管注射AT1R-shRNA-腺相关病毒悬液以抑制肺组织内AT1R基因表达。分别用免疫荧光和蛋白质免疫印迹试验(Western blotting)检测肺组织AT1R、ERS标志性蛋白免疫球蛋白重链结合蛋白(BIP)、蛋白质二硫键异构酶(PDI),以及纤维化标志性蛋白Ⅰ型胶原蛋白(COL1A1)、α-平滑肌肌动蛋白(α-SMA)的表达情况;苏木素-伊红(HE)染色评估肺损伤情况,Masson染色评估肺纤维化程度。结果与Sham组相比,MV组小鼠肺纤维化程度和肺损伤情况显著,肺组织AT1R、BIP、PDI、COL1A1、α-SMA蛋白表达水平明显升高(AT1R/β-actin:1.40±0.02比1,BIP/β-actin:2.79±0.07比1,PDI/β-actin:2.07±0.02比1,COL1A1/α-Tubulin:2.60±0.15比1,α-SMA/α-Tubulin:2.80±0.25比1,均P<0.01),肺组织内E-cad^(+)/AT1R+和E-cad^(+)/BIP^(+)细胞数量增加,COL1A1和α-SMA荧光强度增强。与MV组相比,MV+AT1R-shRNA组小鼠肺纤维化程度和肺损伤情况显著缓解,肺组织AT1R、BIP、PDI、COL1A1、α-SMA蛋白表达水平明显下降(AT1R/β-actin:0.53±0.03比1.40±0.02,BIP/β-actin:1.73±0.15比2.79±0.07,PDI/β-actin:1.04±0.07比2.07±0.02,COL1A1/α-Tubulin:1.29±0.11比2.60±0.15,α-SMA/α-Tubulin:1.27±0.10比2.80±0.25,均P<0.01),肺组织内E-cad^(+)/AT1R^(+)和E-cad^(+)/BIP^(+)细胞数量减少,COL1A1和α-SMA荧光强度减低。AT1R-shRNA组各指标与Sham组比较差异均无统计学意义。结�ObjectiveTo demonstrate the mechanism of mechanical ventilation(MV)induced endoplasmic reticulum stress(ERS)promoting mechanical ventilation-induced pulmonary fibrosis(MVPF),and to clarify the role of angiotensin receptor 1(AT1R)during the process.MethodsThe C57BL/6 mice were randomly divided into four groups:Sham group,MV group,AT1R-shRNA group and MV+AT1R-shRNA group,with 6 mice in each group.The MV group and MV+AT1R-shRNA group mechanically ventilated for 2 hours after endotracheal intubation to establish MVPF animal model(parameter settings:respiratory rate 70 times/minutes,tidal volume 20 mL/kg,inhated oxygen concentration 0.21).The Sham group and AT1R-shRNA group only underwent intubation after anesthesia and maintained spontaneous breathing.AT1R-shRNA group and MV+AT1R-shRNA group were airway injected with the adeno-associated virus one month before modeling to inhibit AT1R gene expression in lung tissue.The expressions of AT1R,ERS signature proteins[immunoglobulin heavy chain-binding protein(BIP),protein disulfide isomerase(PDI)],fibrosis signature proteins[collagenⅠ(COL1A1),α-smooth muscle actin(α-SMA)]in lung tissues were detected by immunofluorescence and Western blotting.Hematoxylin-eosin(HE)staining was used to evaluate lung injury and Masson staining was used to evaluate pulmonary fibrosis.ResultsCompared with the Sham group,the degree of pulmonary fibrosis and lung injury were more significant in the MV group.In the MV group,the protein expressions of AT1R,BIP,PDI,COL1A1 andα-SMA were increased(AT1R/β-actin:1.40±0.02 vs.1,BIP/β-actin:2.79±0.07 vs.1,PDI/β-actin:2.07±0.02 vs.1,COL1A1/α-Tubulin:2.60±0.15 vs.1,α-SMA/α-Tubulin:2.80±0.25 vs.1,all P<0.01).The number of E-cad^(+)/AT1R^(+)/and E-cad^(+)//BIP^(+)/cells in lung tissue increased,and the fluorescence intensity of COL1A1 andα-SMA increased.Compared with the MV group,the degree of pulmonary fibrosis and lung injury were significantly relieved in the MV+AT1R-shRNA group.In the MV+AT1R-shRNA group,the protein expressions of AT1R,BI

关 键 词：机械通气 肺纤维化 内质网应激 血管紧张素受体1 

分 类 号：R459.7[医药卫生—急诊医学] R563[医药卫生—治疗学]