Study on Effect of Astragaloside Ⅳ on Mesenchymal Transition of Human Fibroblasts Based on Notch and Wnt/β-catenin Pathway
Objective To investigate the mechanism of astragaloside Ⅳ(AS-Ⅳ)in an in vitro model of rheumatoid arthritis-associated interstitial lung disease(RA-ILD)based on Notch and Wnt/β-catenin pathways.Methods Inflammatory factors were detected in the bronchoalveolar lavage fluid of RA-ILD model rats by Luminex assay to screen out key inflammatory factors.Two cell lines,THP-1 and NHLF,were co-cultured,and inflammatory factors were added to simulate RA-ILD in vitro model with different interventions.The promoter activity and mRNA expressions of Snail,ZEB-1 and E-Cadherin,expres-sions of key proteins in Notch pathway,nuclear translocation level of β-catenin and the proliferation of NHLF cells,the secre-tion levels of Col Ⅰ,Col Ⅲ and Fibronectin,and the interaction of β-catenin with NICD,β-catenin with LEF,and NICD with CSL were observed by RT-PCR,Western blot,COIP,EdU,ELISA and dual luciferase reporter gene activity assays.Result-s After NHLF cells were treated with inflammatory factors,the promoter activity and mRNA expressions of Snail and ZEB-1 were significantly increased,the expression levels of key proteins in the Notch pathway were significantly up-regulated,the nuclear translocation of β-catenin was significantly increased,and EdU-positive cells were significantly increased.The secretion levels of Col Ⅰ,Col Ⅲ and Fibronectin in cell supernatants were significantly up-regulated,while the protein binding of β-catenin with NICD,β-catenin with LEF and NICD with CSL was significantly increased.The results were the opposite when using the Notch inhibitors DAPT and Jagged1 shRNA.AS-Ⅳ was able to down-regulate the overactivation of Notch and β-catenin sig-naling pathways by inhibiting Jagged1 and thus AS-Ⅳ was able to inhibit the excessive activation of Notch and β-catenin sig-naling pathways,inhibit the protein binding of β-catenin with NICD,β-catenin with LEF and NICD with CSL,dose-depend-ently inhibit Snail and ZEB-1 and up-regulate the promoter activity,mRNA and protein expression levels of E-Cadherin,in-hibit the excessive proliferation of NHLF cells and the secretion of Col Ⅰ,Col Ⅲ and Fibronectin.Conclusion AS-Ⅳ may inhibit NHLF cell proliferation and reduce excessive ECM deposition by suppressing Notch and Wnt/β-catenin signaling pathways,thereby slowing the progression of RA-ILD interstitial lung fibrosis.
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