保肺化纤方调控TGF-β1/smad2/3、Wnt1/β-catenin信号通路对BLM/PM2.5诱导的特发性肺纤维化大鼠肺组织胶原沉积的影响

Baofei Huaxian Formula(保肺化纤方)Improves Collagen Deposition in IPF Rats Induced by BLM/PM2.5 via Regulating TGF-β1/smad2/3 and Wnt1/β-catenin Signaling Pathways

目的探讨保肺化纤方经转化生长因子(transforming growth factor,TGF)-β1/smad2/3、Wnt1/β-连环蛋白(catenin)信号通路减轻博来霉素(bleomycin,BLM)/PM2.5暴露诱导的特发性肺纤维化(idiopathic pulmonary fibrosis,IPF)大鼠肺组织胶原沉积的作用机制。方法SD大鼠随机分为对照(Control)组、BLM+PM2.5(Model)组、保肺化纤方(BHF)组、吡非尼酮(pirfenidone,PFD)组、XAV-939组、BHF+PFD组、BHF+XAV-939组。采用一次性气管内雾化BLM法制备IPF大鼠模型,大气PM2.5实时浓缩全身暴露和给予相应药物干预。检测大鼠肺功能,观察肺组织病理和胶原沉积;采用免疫组化技术检测肺组织Ⅰ、Ⅳ型胶原(collagen,COL)、TGF-β1、smad2/3、β-catenin蛋白水平;采用qPCR技术检测肺组织TGF-β1、smad2/3、Wnt1、β-catenin mRNA水平。结果与Control组比较,Model组大鼠肺功能显著降低(P<0.01),肺组织可见大量炎症细胞浸润,肺泡壁断裂、增厚,胶原沉积等肺纤维化特征性病理改变,肺组织胶原容积分数(collagen volume fraction,CVF)、COLⅠ、COLⅣ蛋白及TGF-β1、smad2/3、β-catenin基因与蛋白表达均显著升高(P<0.01);与Model组比较,各治疗组均有效改善大鼠肺组织病理改变,显著降低肺组织CVF及COLⅠ、COLⅣ、TGF-β1、smad2/3、β-catenin蛋白表达水平(P<0.05,P<0.01),同时BHF组、BHF+PFD组、BHF+XAV-939组均能显著提高大鼠肺功能(P<0.05,P<0.01),明显下调肺组织TGF-β1、smad2/3、β-catenin基因表达水平(P<0.01);与PFD组比较,BHF+PFD组大鼠肺组织COLⅠ、TGF-β1蛋白及smad2、β-catenin基因表达均显著降低(P<0.05,P<0.01);与XAV-939组比较,BHF+XAV-939组smad3基因表达显著降低(P<0.01)。结论保肺化纤方可改善BLM/PM2.5诱导的IPF大鼠肺组织病理损伤,减少胶原沉积,提高肺功能,改善肺纤维化,其机制可能与下调TGF-β1/smad2/3、Wnt1/β-catenin通路有关。

Objective To investigate the mechanism of Baofei Huaxian Formula(保肺化纤方)via transforming growth factor(TGF)-β1/smad2/3 and Wnt1/β-catenin signaling pathway to reduce collagen deposition in lung tissue of rats with idiopathic pulmonary fibrosis(IPF)induced by bleomycin(BLM)/PM2.5.Methods SD rats were randomly divided into control(Control)group,BLM+PM2.5(Model)group,Baofei Huaxian Formula(BHF)group,pirfenidone(PFD)group,XAV-939 group,BHF+PFD group and BHF+XAV-939 group.The IPF rat model was prepared by disposable endotracheal nebulization bleomycin method,and atmospheric PM2.5 real-time concentrated animal exposure and corresponding drug intervention were given.The lung function of rats was detected,and lung histopathology and collagen deposition were observed.The protein levels of I andⅣcollagen(COL),TGF-β1,smad2/3 andβ-catenin in lung tissue were detected by immunohistochemistry.The mRNA levels of TGF-β1,smad2/3,Wnt1 andβ-catenin in lung tissue were detected by qPCR.Results Compared with those of the Control group,the lung function of rats in the Model group was significantly reduced(P<0.01),and the lung tissue was seen to be infiltrated by a large number of inflammatory cells,alveolar wall fracture,thickening,collagen deposition and other characteristic pathological changes of pulmonary fibrosis,and collagen volume fraction(CVF),gene and protein expressions of COLⅠ,COLⅣ,TGF-β1,smad2/3 andβ-catenin in lung tissue were significantly increased(P<0.01).Compared with the Model group,all treatment groups effectively improved the lung histopathological changes in rats,and significantly reduced CVF and the protein expression levels of COLⅠ,COLⅣ,TGF-β1,smad2/3 andβ-catenin in lung tissue(P<0.05,P<0.01),while the BHF group,BHF+PFD group and BHF+XAV-939 group significantly improved the lung function of rats(P<0.05,P<0.01)and significantly down-regulated the expression levels of TGF-β1,smad2/3 andβ-catenin genes in lung tissues(P<0.01).Compared with the PFD group,the expressions of COLⅠ,TGF-β1 protein and smad2 andβ-catenin genes in lung tissues of rats in the BHF+PFD group were significantly lower than those in the PFD group.The catenin gene expressions were significantly lower in the BHF+XAV-939 group compared with those in the PFD group(P<0.05,P<0.01)and smad3 gene expression was significantly lower in the BHF+XAV-939 group compared with that in the XAV-939 group(P<0.01).Conclusion The mechanism of Baofei Huaxian Formula to improve BLM/PM2.5-induced lung histopathological damage and lung function and reduce collagen deposition in IPF rats may be related to the down-regulation of TGF-β1/smad2/3 and Wnt1/β-catenin pathways.