大黄䗪虫丸对博来霉素致肺纤维化大鼠TGF-β1/Smad3信号通路的影响

Effects of Dahuang Zhechong pills on TGF-β1/Smad3 signaling pathway of pulmonary fibrosis rats induced by bleomycin

目的基于“肺络干血”假说,探究大黄䗪虫丸对博来霉素致肺纤维化大鼠TGF-β1/Smad3信号通路的影响。方法将60只健康雄性SD大鼠随机分为空白组(A)、模型组(B)、吡非尼酮组(C)、大黄虫丸大剂量组(D)、大黄䗪虫丸中剂量组(E)、大黄䗪虫丸小剂量组(F),每组10只。除A组外各组采用气管内滴入博来霉素(5 mg/kg)复制大鼠肺纤维化模型。连续给药28天后处死大鼠,取肺组织行Masson染色观察病理变化,实时PCR检测转化生长因子-β1(transforming growth factor-β1,TGF-β1)、I型胶原(Collagen I)及Smad3蛋白的mRNA表达;Western blot法检测Smad3、p-smad3的蛋白表达水平。结果病理:B组纤维化改变明显,F组纤维化程度为各用药组中最轻。PCR:较A组相比,B^E组大鼠TGF-β1、Smad3及Collagen I的基因表达均显著升高(P<0.01),F组Smad3、Collagen I的基因表达显著升高(P<0.01),TGF-β1的基因表达明显升高(P<0.05);较B组相比,F组TGF-β1、Smad3的基因表达显著降低(P<0.01),Collagen I的基因表达明显降低(P<0.05);较C组相比,F组TGF-β1的基因表达显著降低(P<0.01),Smad3、Collagen I的基因表达明显降低(P<0.05)。Western blot:与A组相比,B组Smad3蛋白表达明显升高(P<0.05)、B^D组p-smad3蛋白表达均显著升高(P<0.01),E组p-smad3蛋白表达明显升高(P<0.05);与B组相比,E组Smad3、D组p-smad3蛋白表达明显降低(P<0.05),E、F组p-smad3蛋白表达均显著降低(P<0.01);与C组相比,F组p-smad3蛋白表达显著降低(P<0.01)。结论大黄䗪虫丸有抗纤维化的作用,可能与抑制TGF-β1/Smad3信号通路,减少细胞外基质沉积有关,小剂量使用效果最佳。

Objective To explore the effects of Dahuang Zhechong pills on TGF-β1/Smad3 signaling pathway of pulmonary fibrosis rats induced by bleomycin,based on the hypothesis of‘dried blood in lung collateral’.Methods 60 healthy male SD rats were randomly divided into blank group(group A),model group(group B),pirfenidone group(group C),high,medium and low dose of Dahuang Zhechong pills group(group D,E and F),10 rats in each group.In addition to group A,the rat model with pulmonary fibrosis was established by dripping bleomycin(5 mg/kg)into trachea in other groups.Rats were sacrificed after 28 days continuous administration.The lung tissue was taken to observe the pathological changes by Masson staining.The mRNA expression of transforming growth factor-β1(TGF-β1)and Collagen I and Smad3 were detected by real-time PCR.The protein expression of Smad3 and p-smad3 were detected by Western blot.Results Pathology:The fibrosis changes in group B was obvious,and the fibrosis degree in group F was the least in group among all medicine groups.PCR:Compared with group A,the gene expression of TGF-β1,Smad3 and Collagen I in group B,C,D and E increased significantly(P<0.01),and the gene expression of Smad3 and Collagen I in group F increased significantly(P<0.01),and the gene expression of TGF-β1 increased significantly(P<0.05);Compared with group B,the gene expression of TGF-β1 and Smad3 in group F decreased significantly(P<0.01),and the gene expression of Collagen I decreased significantly(P<0.05);Compared with group C,gene expression of TGF-β1 in group F decreased significantly(P<0.01),and gene expression of Smad3 and Collagen I decreased significantly(P<0.05).Western blot:Compared with group A,the expression of Smad3 protein in group B increased significantly(P<0.05),the expression of p-smad3 protein in group B,C and D increased significantly(P<0.01),and the expression of p-smad3 protein in group E increased obviously(P<0.05);Compared with group B,the expression of p-smad3 protein in group E and Smad3 protein in group D both decreased significantly(P<0.05),and the expression of p-smad3 protein in group E and F was significantly reduced(P<0.01);Compared with group C,the expression of p-smad3 protein in group F and E was both decreased significantly(P<0.01).Conclusion Dahuang Zhechong pills has anti-fibrotic effect,which may be related to inhibition of TGF-β1/Smad3 signaling pathway and reduction of extracellular matrix deposition,and low dose administration shows the best improvement effect.