Ⅱ型肺泡上皮细胞间质转分化中的Notch-1/Twist-1信号通路机制

Effects of Notch-1/Twist-1 axis in the process of epithelial-mesenchymal transition of type II alveolar epithelial cell and its mechanism

目的:通过研究Notch-1/Twist-1信号通路参与Ⅱ型肺泡上皮细胞间质转分化(EMT)作用,为阐明肺纤维化(PF)发病机制提供理论依据。方法:动物实验设对照组和博莱霉素(BLM)组(n=15)。气管注射BLM(7500 U/kg)诱导肺纤维化大鼠模型,造模后28 d取左肺下叶固定于10%福尔马林中,进行HE、Masson及转化生长因子-β1(TGF-β1)免疫组化染色。体外培养Ⅱ型肺泡上皮细胞(RLE-6TN),细胞实验设对照(Control)组、TGF-β1(5 ng/ml)组、TGF-β1+Notch-1 siRNA阴性对照组(NC siRNA,100 pmol/L)和TGF-β1+Notch-1 siRNA干扰组(Notch-1 siRNA,100 pmol/L),每组设9个复孔。细胞先用NC siRNA或Notch-1 siRNA预处理24 h,再用TGF-β1处理48 h。检测肺组织和(或)II型肺泡上皮细胞内TGF-β1、I型胶原(collagen I)、III型胶原(collagen III)、E-钙粘蛋白(E-Cadherin)、紧密连接蛋白-1(ZO-1)、波形蛋白(Vimentin)、N-钙粘蛋白(N-Cadherin)、Notch-1、Notch-1胞内域(NICD)、Hes-1和Twist-1 mRNA和(或)蛋白表达。结果:动物实验结果显示,与对照组相比,BLM组肺泡萎缩、塌陷并发生融合,肺泡间隔明显增宽,可见大量炎性细胞的浸润。肺间质胶原纤维沉积明显增多,collagen I和collagen III的表达明显增加(P<0.01);另外,肺BLM组织中E-cadherin和ZO-1表达明显下降而Vimentin和N-cadherin的表达明显增加(P<0.01);同时,BLM组肺组织TGF-β1、Notch-1、NICD、Hes-1和Twist-1的表达也明显上调(P<0.01)。细胞实验结果显示,与Control相比,TGF-β1组Notch-1、NICD、Hes-1、Twist-1、collagen I和collagen III的表达明显升高,同时E-Cadherin和ZO-1的表达明显降低而Vimentin和N-cadherin的表达明显升高(P<0.01)。与TGF-β1组相比,Notch-1 siRNA能够明显降低TGF-β1诱导Notch-1、NICD、Hes-1和Twist-1的表达(P<0.05或P<0.01),同时E-Cadherin和ZO-1的表达明显升高而Vimentin和N-cadherin的表达明显降低(P<0.05或P<0.01)。另外Notch-1 siRNA还能够明显降低TGF-β1诱导的collagen I和collagen III的表达(P<0.05或P<0.01)。结论:Notch-1/Twist-1信号通路参与了Ⅱ型肺泡上皮细胞EMT,可能参与了肺纤维化的发生发展。

Objective:To study the role of Notch-1/Twist-1 axis in the process of epithelial-mesenchymal transition(EMT)of type II alveolar epithelial cells in pulmonary fibrosis(PF)and hope to provide a new theoretical basis for the pathogenesis of PF.Methods:Thirty rats were randomly divided into control group and bleomycin(BLM)group,15 rats in each group.The PF rat model was induced by intratracheal injection of BLM(7500 U/kg).Excised inferior lobe of left lung was fixed in 10%formalin for HE staining,Masson staining and transforming growth factor-beta 1(TGF-β1)immunohistochemistry staining after BLM injection for 28 days.The cultured type II alveolar epithelial cells(RLE-6TN)were divided into 4 groups(Control group,transforming growth factor-beta 1(TGF-β1)group,Notch-1 negative control siRNA(NC siRNA,100 pmol/L)group and Notch-1 siRNA(100 pmol/L)group),each group was established nine holes.The cells were treated with TGF-β1(5.0 ng/ml)for 24 h following NC siRNA or Notch-1 siRNA for 48 h.The mRNA and(or)proteins levels of TGF-β1,collagen I,collagen III,E-Cadherin,zonula occludens-1(ZO-1),Vimentin,E-Cadherin,Notch-1,Notch-1 intracellular domain(NICD),Hes-1 and Twist-1 were detected in lung tissue and type II alveolar epithelial cells.Results:In vivo,compared with the control group,the alveolar atrophy,collapse and fusion occurred,alveolar septum widened significantly,and a large number of inflammatory cells infiltrated in the pulmonary interstitial of the rats in the BLM group.And compared with control group,BLM obviously increased collagen deposition and collagen I and collagen III expressions,while the expressions of ZO-1 and E-cadherin were decreased,and the expressions of Vimentin and N-cadherin were increased,and concomitantly with increasing Notch-1,NICD,Hes-1 and Twist-1 expression in lung tissues of rats(P<0.01).In vitro,compared with control group,TGF-β1 treatment obviously induced collagen I,collagen III,Notch-1,NICD,Hes-1 and Twist-1 expressions,and the expressions of E-cadherin and ZO-1 were decreased and the expressions of Vimentin and N-cadherin were increased(P<0.01).Compared with TGF-β1 group,Notch-1 siRNA treatment significantly inhibited the expressions of Notch-1,NICD,Hes-1 and Twist-1,and the expressions of E-cadherin and ZO-1 were increased and the expressions of Vimentin and N-cadherin were decreased,and also obviously reduced the expressions of collagen I and collagen III induced by TGF-β1(P<0.05 or P<0.01).Conclusion:Notch-1/Twist-1 axis is involved in the EMT process of type II alveolar epithelial cells,suggesting that Notch-1/Twist-1 signaling may be involved in the development of pulmonary fibrosis.