Effects of astragaloside-Ⅳ on human fibroblast apoptosis and myofibroblast transformation by regulating Wnt/β-catenin signaling pathway
Objective To simulate the pathological process of idiopathic pulmonary fibrosis(IPF)by stimulating human fibroblast(HFL-1)cells with transforming growth factor-β1(TGF-β1),and to explore the effects and mechanism of astragaloside-Ⅳ(AS-Ⅳ)on human fibroblast apoptosis and myofibroblast transformation.Methods HFL-1 cells were stimulated with 10 ng/mL TGF-β1 and then treated with different concentrations(0,1.25,2.5,5,10 μmol/L)of AS-Ⅳ solution for 24 h.The mRNA expression of fibronectin(FN)was determined by RT-PCR.Subsequently,the cells were divided into blank group,TGF-β1 group,TGF-β 1+AS-Ⅳgroup,and TGF-β1+XAV939 group.Flow cytometry was used to measure cell apoptosis rate,RT-PCR to examine the mRNA expressions of α-smooth muscle actin(α-SMA),FN,recombinant human collagen-1(collagen Ⅰ),B-cell lymphoma 2(Bcl-2),and Bcl-2 associated X protein(Bax),immunofluorescence to determine the protein expressions of α-SMA,E-Cadherin,collagen Ⅰ,FN,β-catenin,and Cleaved Caspase-3,and Western blot to check the protein expression levels of FN,β-catenin,and Cleaved Caspase-3.Results The optimal concentration of AS-Ⅳ for inhibiting FN expression was 5 μmol/L Compared with the blank group,the cell apoptosis rate in the TGF-β1 group increased(P＜0.05),the mRNA expressions of FN,collagen Ⅰ,α-SMA,and Bel-2 were elevated(P＜0.05),while Bax mRNA expression decreased(P＜0.05).Additionally,the protein expressions of α-SMA,collagen Ⅰ,FN,Cleaved Caspase-3,and β-catenin also increased(P＜0.05).Compared with the TGF-β1 group,both the TGF-β1+AS-Ⅳ group and TGF-β1+XAV939 group showed decreased cell apoptosis rates(P＜0.05),reduced mRNA expressions of FN,collagen Ⅰ,α-SMA.and Bcl-2(P＜0.05),increased Bax mRNA expression(P＜0.05),and decreased protein expressions of α-SMA,collagen Ⅰ,FN,Cleaved Caspase-3,and β-catenin(P＜0.05).Compared with the TGF-β1+XAV939 group,the cell apoptosis rate in the TGF-β1+AS-Ⅳ group decreased(P＜0.05),the mRNA expressions of FN,collagen Ⅰ,Bcl-2,and α-SMA decreased(P＜0.05),while that of Bax increased(P＜0.05).Conclusion AS-Ⅳ can inhibit TGF-β1-induced HFL-1 cell apoptosis and myofibroblast transformation,which may be related to the Wnt/β-catenin signaling pathway.
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