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中国应用生理学杂志 ›› 2021, Vol. 37 ›› Issue (6): 577-583.doi: 10.12047/j.cjap.6124.2021.048

• 研究论文 •    下一篇

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*

汤娟1+, 柯洁2+, 付裕3, 何晓伟1, 李先伟1△   

  1. 1.皖南医学院药学院病理学教研室, 安徽 芜湖 241002;
    2.皖南医学院医学影像学院, 安徽 芜湖 241002;
    3.皖南医学院临床医学院, 安徽 芜湖 241002
  • 收稿日期:2020-07-12 修回日期:2020-12-29 出版日期:2021-11-28 发布日期:2021-11-25
  • 通讯作者: Tel: 13866384544; E-mail: wnmclixianwei69@163.com.
  • 作者简介:: 为共同第一作者
  • 基金资助:
    *安徽省高校自然科学研究重点项目(2018A0254、KJ2019A0419);安徽省卫生健康委科研重点项目(AHWJ2021a033);2019、2020年国家级大学生创新创业训练计划项目(201910368002、202010368046)

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

TANG Juan1+, KE Jie2+, FU Yu3, HE Xiao-wei1, LI Xian-wei1△   

  1. 1. Department of Pharmacology, Wannan Medical College, Wuhu 241002, China;
    2. School of Medical Image, Wannan Medical College, Wuhu 241002, China;
    3. School of Clinical Medicine, Wannan Medical College, Wuhu 241002, China
  • Received:2020-07-12 Revised:2020-12-29 Online:2021-11-28 Published:2021-11-25

摘要: 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(7 500 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。

关键词: 肺纤维化, 上皮细胞间质转分化, Notch-1, Twist-1, 大鼠

Abstract: 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 (7 500 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.

Key words: pulmonary fibrosis, epithelial-mesenchymal transition, Notch-1, Twist-1, rat

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