尼达尼布减轻脂多糖诱导的小鼠急性呼吸窘迫综合征后肺纤维化

Nintedanib ameliorates lipoplysaccharide-induced lung fibrosis in acute respiratory distress syndrome

目的：探讨尼达尼布对脂多糖（lipopolysaccharide,LPS）所诱导的小鼠急性呼吸窘迫综合征（acute respiratory distress syndrome,ARDS）后肺纤维化的作用。方法：体外实验中,以LPS（O55：B5）刺激C57BL/6小鼠肺成纤维细胞转化为肌成纤维细胞,以不同浓度尼达尼布干预,半定量PCR法检测转化生长因子-β1（transforming growth factor-β1,TGF-β1）、α-平滑肌激动蛋白（α-smooth muscle actin,α-SMA）、成纤维细胞生长因子-2（fibroblast growth factor-2,FGF-2）、血小板衍生生长因子（platelet-derived growth factor,PDGF）和血管内皮生长因子（vascular endothelial growth factor,VEGF）mRNA的表达水平;ELISA法检测细胞上清液中TGF-β1蛋白的含量。体内实验中,将SPF级6~8周龄雄性C57BL/6小鼠随机分为0.9%Na Cl溶液对照组、LPS（O111：B4）模型组（实验d 1气管内滴入LPS,5 mg·kg-1）、LPS＋尼达尼布早期干预组（尼达尼布于实验d 7~d 21,50 mg·kg-1·d-1,ig）、LPS＋尼达尼布晚期干预组（尼达尼布于实验d 14~d 28,50 mg·kg-1·d-1,ig）。实验d 28处死各组小鼠,收集小鼠支气管肺泡灌洗液（brochoalveolar lavage fluid,BALF）,计数炎性细胞总数并分类。HE染色及Masson染色观察肺组织炎性改变及肺纤维化变化;碱性水解法检测肺组织中羟脯氨酸（hydroxyproline,Hyp）含量;Western blotting检测肺组织中α-SMA和I型胶原的表达。结果：体外实验中,尼达尼布可抑制TGF-β1,α-SMA,FGF-2,PDGF和VEGF mRNA及TGF-β1蛋白的表达。体内实验中,尼达尼布干预能显著减轻小鼠BALF中炎性细胞总数及各种炎性细胞数量。HE染色及Masson染色结果显示,LPS可诱导小鼠ARDS后肺纤维化形成,而尼达尼布早期和晚期干预均能不同程度减轻小鼠肺纤维化程度,均可降低肺组织中Hyp,α-SMA和I型胶原的含量,且早期干预效果要优于晚期干预效果。结论：尼达尼布可抑制LPS诱导的小鼠肺成纤维细胞向肌成纤维细胞转化;尼达尼布可减轻LPS导致的小鼠ARDS后肺纤维化。

Objective： To investigate the effect of nintedanib on lung fibrosis after lipopolysaccharide（ LPS） induced acute respiratory distress syndrome（ ARDS） in mice. Methods： Mouse C57BL/6 lung fibroblasts were induced by LPS（ O55： B5） to trasform into myofibroblasts,and the trasformation process was inhibited by nintedanib at various concentrations. The mRNAexpressions of transforming growth factor-β1（ TGF-β1）,α-smooth muscle actin（ α-SMA）,fibroblast growth factor-2（ FGF-2）,platelet-derived growth factor（ PDGF）,and vascular endothelial growth factor（ VEGF） were detected by semi-quantitative PCR or ELISA. Male SPF C57 BL /6 mice,aged 6 ~ 8 weeks,were randomly divided into groups,and treated with saline（ control）,intratracheal instillation of LPS O111： B4（ 5 mg·kg- 1·d- 1on day 1）,LPS ＋ early nintedanbib treatment（ 50 mg·kg- 1·d- 1,ig,from day 7to day 21）,LPS ＋ late nintedanbib treatment（ 50 mg·kg- 1·d- 1,ig,from day 14 to day 28）. All mice were sacrificed in day 28 and the bronchoalveolar lavage fluid（ BALF） was collected and characterized. Mouse lung tissue sections were processed and analyzed after Hematoxylin-Eosin（ HE） and Masson staining. Lung hydroxyproline（ Hyp） contents were detected by alkaline hydrolysis method,and the expressions of α-SMA and type I collagen were detected by Western blotting. Results： Nintedanib inhibits LPS-induced expressions of TGF-β1,α-SMA,FGF-2,PDGF,and VEGF mRNAs. Both early and late nintedanib treatments decreased the levels of inflammatory cells in BAL fluid. LPS stimulation resulted in lung fibrosis after ARDS. Both early and late nintedanib treatments decreased Hyp content,and α-SMA and type I collagen expression in C57 BL /6 mice. Meanwhile,early nintedanib treatment was more effective than the late treatment. Conclusion： Nintedanib can inhibit LPS-induced transformation of fibroblastsd into myofibroblasts in vitro,and may ameliorate LPS-induced lung fibrosis in vivo after ARDS in mice.