摘要
目的探讨机械通气促进肺成纤维细胞活化及肺纤维化的机制,明确乳酸-转化生长因子-β1(transforming growth factor-β1,TGF-β1)途径在该过程中的作用。方法①将C57BL/6小鼠采用随机数字表法分为假手术组(Sham组)和机械通气组(MV组),每组12只,其中Sham组仅行麻醉插管处理并保持自主呼吸,MV组采用20 ml/kg潮气量和70次/min通气频率行单次2 h机械通气,观察7 d后取材。采用H-E染色、Masson染色观察肺组织的损伤程度和纤维化程度,采用Western blot法检测肺组织Ⅰ型胶原蛋白α1链(collagen typeⅠalpha 1 chain,COL1A1)、α平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)和TGF-β1的蛋白表达情况,采用比色法检测小鼠肺泡灌洗液(bronchial alveolar lavage fluid,BALF)和血清的乳酸浓度。②将人胚肺成纤维细胞MRC-5采用随机数字表法分为4组(每组3个孔):PBS对照组(Con组)、1 mmol/L乳酸组(Lac1组)、10 mmol/L乳酸组(Lac_(10)组)和20 mmol/L乳酸组(Lac_(20)组),于乳酸刺激MRC-5细胞24 h后采用ELISA法检测细胞培养上清液中TGF-β1含量,同时采用细胞免疫荧光观察α-SMA的表达情况;于乳酸刺激MRC-5细胞72 h后采用Western blot法检测不同分组细胞COL1A1和α-SMA的表达情况,并通过ELISA法检测细胞培养上清液Ⅰ型前胶原羧基肽(procollagen type I carboxyl peptide,PICP)的含量变化。③另取MRC-5细胞采用随机数字表法分为3组(每组3个孔):PBS对照组(Con组)、20 mmol/L乳酸组(Lac_(20)组)和20 mmol/L乳酸+TGF-β1受体抑制剂(SB431542)组(Lac_(20)+SB组),处理24 h后采用Western blot法检测不同分组细胞COL1A1和α-SMA的表达情况。结果①与Sham组比较,MV组小鼠肺泡间隔增厚、胶原蛋白沉积、肺纤维化程度加重,伴有COL1A1、α-SMA和TGF-β1表达水平升高(P<0.05),血清和BALF乳酸浓度升高(P<0.05)。②与Con组比较,Lac_(10)组和Lac_(20)组细胞培养上清液中TGF-β1、PICP含量升高(P<0.05),细胞中COL1A1和α-SMA表达水平升高(P<0.05),免疫荧光观察到α-SMA在细胞内表达增多(P>0.05)。③与Lac_(20)组比较,Lac_(20)+SB组细胞COL1A1和α-SMA表达水平降低(P<0.05)。结论机械通气可以通过乳酸-TGF-β1途径活化肺成纤维细胞,引起胶原蛋白分泌,促进机械通气相关性肺纤维化进程。
Objective To explore the mechanism of mechanical ventilation to promote lung fibroblast activation and pulmonary fibrosis,and determine the role of the lactic acid-transforming growth factor-β1(TGF-β1)pathway in the process.Methods①C57BL/6 mice were divided into two groups according to the random number table method(n=12):a Sham group and a mechanical ventilation(MV)group.The Sham group only underwent anesthesia intubation and maintained spontaneous breathing,while the MV group was mechanically ventilated over 2 h,with a tidal volume of 20 ml/kg and a ventilation frequency of 70 bpm.After 7 d,lung tissues were collected.The injury and fibrosis of lung tissues were observed by hematoxylin-eosin(H-E)staining and Masson staining.The levels of collagen typeⅠalpha 1 chain(COL1A1),α-smooth muscle action(α-SMA)and TGF-β1 were detected by Western blot.The concentrations of lactic acid in the bronchial alveolar lavage fluid(BALF)and serum were detected by colorimetry.②Human embryonic lung fibroblasts MRC-5 cells were divided into four groups according to the random number table method(n=3):a PBS control(Con)group,a 1 mmol/L lactic acid(Lac1)group,a 10 mmol/L lactic acid(Lac_(10))group and a 20 mmol/L lactic acid(Lac_(20))group.After exposure to lactic acid for 24 h,the content of TGF-β1 in the supernatant was detected by enzyme-linked immunosorbent assay(ELISA),and the expression ofα-SMA was observed by immunofluorescence.After exposure to lactic acid for 72 h,the levels of COL1A1 andα-SMA in each group were detected by Western blot,and the content of procollagen typeⅠcarboxyl peptide(PICP)in the supernatant was detected by ELISA.③MRC-5 cells were divided into three groups according to the random number table method(n=3):a PBS control(Con)group,a 20 mmol/L lactic acid(Lac_(20))group,and a 20 mmol/L lactic acid+TGF-β1 receptor inhibitor SB431542(Lac_(20)+SB)group.After treatment for 24 h,the levels of COL1A1 andα-SMA in each group were detected by Western blot.Results①Compared with the Sham group,the MV group presented thickened alveolar septum,collagen deposition and aggravated pulmonary fibrosis,along with increases in the levels of COL1A1,α-SMA and TGF-β1(P<0.05)as well as the concentrations of lactic acid in the alveolar lavage fluid and serum(P<0.05).②Compared with the Con group,both the Lac_(10) and Lac_(20) groups produced increases in the content of TGF-β1 and PICP in the supernatant(P<0.05)as well as the levels of COL1A1 andα-SMA(P<0.05).Increased expression ofα-SMA was also observed by immunofluorescence(P>0.05).③Compared with the Lac_(20) group,the levels of COL1A1 andα-SMA significantly decreased in the Lac_(20)+SB group(P<0.05).Conclusions Mechanical ventilation can activate lung fibroblasts via the lactic acid-TGF-β1 pathway to cause the secretion of collagen,so as to accelerate the process of mechanical ventilator-associated pulmonary fibrosis.
作者
胡钺
梅舒雅
徐侨翌
胡晓婷
邢顺鹏
皋源
何征宇
李雯
Hu Yue;Mei Shuya;Xu Qiaoyi;Hu Xiaoting;Xing Shunpeng;Gao Yuan;He Zhengyu;Li Wen(Department of Critical Care Medicine,Renji Hospital,School of Medicine,Shanghai Jiao Tong University,Shanghai 200127,China)
出处
《国际麻醉学与复苏杂志》
CAS
2021年第3期227-233,共7页
International Journal of Anesthesiology and Resuscitation
基金
国家自然科学基金(81770060,81870052)
上海市闵行区自然科学研究课题(2019MHZ017)
2018年上海市"医苑新星"青年医学人才培养资助计划(SHWSRS(2019)-72)。
关键词
乳酸
转化生长因子-Β1
机械通气
肺成纤维细胞
肺纤维化
Lactic acid
Transforming growth factor-β1
Mechanical ventilation
Pulmonary fibroblasts
Pulmonary fibrosis
