滇龙胆草调控TGF-β1/Smad2/3信号通路抑制肺纤维化小鼠肺上皮间质转化

Longdancao(龙胆草)From Yunnan Suppresses Epithelial Mesenchymal Transition in Mice with Pulmonary Fibrosis by Regulating TGF-β1/Smad2/3 Signaling Pathway

目的:研究滇龙胆草对肺纤维化小鼠转化生长因子β1及其信号转导蛋白Smad(TGF-β1/Smad)信号通路和肺上皮间质转化的影响。方法:健康雄性昆明小鼠随机分为正常对照组、模型对照组、吡非尼酮50 mg/kg组、滇龙胆草50、100、200 mg/kg组。气管滴注博莱霉素5 mg/kg制备小鼠肺纤维化模型,造模同时各组灌胃相应药物或CMC混悬液,建模第28 d,摘眼球取血,取肺组织进行检测;HE染色检测小鼠肺组织病理变化;Masson染色检测肺组织胶原沉积;羟脯氨酸试剂盒测定小鼠肺组织中的羟脯氨酸含量;ELISA检测小鼠血清中转化生长因子(TGF-β1)含量;Western blot检测肺组织中TGF-β1、Smad2/3、E-钙黏蛋白(E-cadherin)表达;免疫组织化学检测小鼠肺组织α-平滑肌肌动蛋白(α-SMA)、E-Cadherin表达。结果:与正常对照组比较,模型对照组小鼠肺组织结构破坏,肺泡壁增厚,支气管周围可见大量纤维组织增生、胶原沉积,肺泡炎评分和肺纤维化评分增加,肺组织羟脯氨酸含量增多,血清TGF-β1含量增多,肺组织中TGF-β1、Smad2/3、α-SMA蛋白表达显著上调,E-Cadherin蛋白表达显著下调(P<0.01);与模型对照组比较,滇龙胆草各剂量组及吡非尼酮50 mg/kg组胶原沉积减少,肺组织病变改善,肺泡炎评分和肺纤维化评分降低,肺组织羟脯氨酸含量减少,TGF-β1、Smad2/3、α-SMA蛋白表达下调,E-Cadherin蛋白表达上调,血清TGF-β1含量降低(P<0.05或P<0.01)。结论:滇龙胆草对博莱霉素诱导小鼠肺纤维化具有保护作用,其机制与调控TGF-β1/Smad2/3信号通路抑制肺上皮-间质转化有关。

Objective:To study the effects of Longdancao(龙胆草)from Yunnan on transforming growth factorβ1 and signal transduction protein(TGF-β1/Smad)signaling pathway and epithelial mesenchymal transition(EMT)in mice with pulmonary fibrosis.Methods:Healthy male KM mice were randomly divided into six groups,namely the normal control group,model group,50 mg/kg pirfenidone group,and low-,medium-,and high-dose(50,100,and 200 mg/kg)Longdancao(龙胆草)groups.Bleomycin(5 mg/kg)was instilled into the trachea of mice for inducing pulmonary fibrosis.During modeling,all the mice were gavaged with the corresponding drugs or CMC suspension.On the 28 th day of modeling,the blood was sampled from eyeball,and the lung tissues were taken for detection.The pathological changes in lung tissue were observed by HE staining,followed by the determination of collagen deposition in lung tissue by Masson staining.The hydroxyproline assay kit was used to determine the content of hydroxyproline.The content of TGF-β1 in mouse serum was detected by ELISA.The expression levels of TGF-β1,Smad2/3,and E-cadherin in lung tissue were measured by Western blotting,and the expression ofα-smooth muscle actin(α-SMA)and E-Cadherin in lung tissue by immunohistochemistry.Results:Compared with the normal control group,the model group exhibited destroyed lung tissue structure,thickened alveolar walls,a large amount of fibrous tissue proliferation and collagen deposition around the bronchi,increased score of alveolitis and pulmonary fibrosis,elevated hydroxyproline content in lung tissue and TGF-β1 in the serum,up-regulated TGF-β1、Smad2/3、α-SMA protein expression,down-regulated E-Cadherin protein expression(P<0.01).Compared with the model group,Longdancao(龙胆草)at each dose and 50 mg/kg pirfenidone reduced the collagen deposition,improved the pathological changes in lung tissue,lowered the score of alveolitis and pulmonary fibrosis,the content of hydroxyproline in lung tissue,and TGF-β1 content in the serum,down-regulated the protein expression levels of TGF-β1,Smad2/3,andα-SMA,and up-regulated E-Cadherin protein expression(P<0.05 or P<0.01).Conclusion:Longdancao(龙胆草)has a protective effect against bleomycin-induced pulmonary fibrosis in mice,which may be related to its regulation of TGF-β1/Smad2/3 signaling pathway and its inhibition of lung EMT.