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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（transforminggrowth 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 type Ⅰ alpha 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对组（Con组）、1 mmol/L乳酸组（Lac1组）、10 mmol/L乳酸组（Lac10组）和20 mmol/L乳酸组（Lac20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Westen 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（Lac20组）和20 mmol/L乳酸+TGF‐β1受体抑制剂（SB431542）组（Lac20+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，Lac10组和Lac20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Lac20组比较，Lac20+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。
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Abstract: Objective To explore the mechanism of mechanical ventilation to promote lung fibroblast activation and pulmonaryfibrosis, and determine the role of the lactic acid‐transforming growth factor‐β1 (TGF‐β1) pathway in the process. Methods ①C57BL/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 groupwas mechanically ventilated over 2 h, with a tidal volume of 20 ml/kg and a ventilation frequency of 70 bpm. After 7 d, lung tissueswere collected. The injury and fibrosis of lung tissues were observed by hematoxylin‐eosin (H‐E) staining and Masson staining. The levelsof collagen type Ⅰ alpha 1 chain (COL1A1), α‐smooth muscle action (α‐SMA) and TGF‐β1 were dtected by Western blot. The concentrations of lactic acid in the bronchial alveolar lavage fluid (BALF) and serum were detected by colorimetry. ② Human 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 (Lac10) group and a 20 mmol/L lactic acid (Lac20) 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 of α‐SMA was observed by immunofluorescence. After exposure to lactic acid for 72 h, the levels of COL1A1 and α‐SMA in each group were detected by Western blot, and the content of procollagen type Ⅰ carboxyl peptide (PICP) in the supernatant was detected by ELISA. ③ MRC‐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 (Lac20) group, and a 20 mmol/L lactic acid+TGF‐β1 receptor inhibitor SB431542 (Lac20+SB) group. After treatment for 24 h, the levels of COL1A1 and α‐SMA in each group were detected by Western blot. Results ① Compared 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). ② Compared with the Con group, both the Lac10 and Lac20 groups produced increases in the content of TGF‐β1 and PICP in the supernatant (P<0.05) as well as the levels of COL1A1 and α‐SMA (P<0.05). Increased expression of α‐SMA was also observed by immunofluorescence (P>0.05). ③ Compared with the Lac20 group, the levels of COL1A1 and α‐SMA significantly decreased in the Lac20+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.
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