FGF23对支气管上皮细胞生长、免疫平衡和上皮间充质转化的影响

Effects of fibroblast growth factor 23 on bronchial epithelial cell growth,immune balance and epithelial-mesenchymal transition

目的探究成纤维细胞生长因子23(FGF23)对支气管上皮细胞16HBE生长、免疫平衡和上皮间充质转化(EMT)的影响。方法使用Lipofectamine 3000转染试剂对16HBE细胞分别转染NC-shRNA或FGF23-shRNA。转染后,使用10 ng/mL TGF-β1处理细胞48 h。16HBE细胞分为对照组、NC-shRNA组、FGF23-shRNA组、TGF-β1组、NC-shRNA+TGF-β1组和FGF23-shRNA+TGF-β1组。通过CCK-8法检测细胞增殖。使用ELISA试剂盒检测16HBE细胞上清液中IFN-γ、IL-4、IL-17和IL-10的水平。通过qRT-PCR、Western blot或免疫荧光染色检测16HBE细胞中FGF23、Klotho、FGFR4、E-cadherin、α-SMA和N-cadherin的mRNA或蛋白表达水平。结果与TGF-β1组相比,FGF23-shRNA+TGF-β1组的OD 450nm值降低(P<0.05)。与TGF-β1组相比,FGF23-shRNA+TGF-β1组的IFN-γ和IL-10的水平升高,而IL-4和IL-17降低(P<0.05)。与TGF-β1组相比,FGF23-shRNA+TGF-β1组的E-cadherin蛋白表达水平升高,而α-SMA降低(P<0.05)。与TGF-β1组相比,FGF23-shRNA+TGF-β1组的E-cadherin相对荧光强度升高,而N-cadherin降低(P<0.05)。与TGF-β1组相比,FGF23-shRNA+TGF-β1组的FGF23和FGFR4的mRNA和蛋白表达水平均降低,而Klotho均升高(P<0.05)。结论下调FGF23抑制了TGF-β1诱导的16HBE细胞的生长和EMT过程,并纠正了Th1/Th2和Treg/Th17的失衡,FGF23-Klotho-FGFR4信号可能是哮喘治疗的一种分子靶标。

Objective To investigate the effect of fibroblast growth factor 23(FGF23)on the growth,immune balance and epithelial-mesenchymal transition(EMT)of bronchial epithelial cells(16HBE).Methods 16HBE cells were transfected with NC-shRNA or FGF23-shRNA using Lipofectamine 3000 transfection reagent.After transfection,cells were treated with 10 ng/mL of transforming growth factor-β1(TGF-β1)for 48 h.16HBE cells were grouped as follows:Control group,NC-shRNA group,FGF23-shRNA group,TGF-β1 group,NC-shRNA+TGF-β1 group and FGF23-shRNA+TGF-β1 group.Cell proliferation was detected by CCK-8 method.The levels of interferon-γ(IFN-γ),interleukin(IL)-4,IL-17 and IL-10 in the supernatant of 16HBE cells were detected using ELISA kits.The mRNA or protein expressions of FGF23,Klotho,FGFR4,E-cadherin,α-SMA and N-cadherin in 16HBE cells were detected by qRT-PCR,Western blot or immunofluorescence staining.Results Compared with TGF-β1 group,the OD_(450nm) value of FGF23-shRNA+TGF-β1 group decreased by 34.75%(P<0.05).Compared with TGF-β1 group,the levels of IFN-γand IL-10 in FGF23-shRNA+TGF-β1 group were increased,while the levels of IL-4 and IL-17 were decreased(P<0.05).Compared with TGF-β1 group,the expression level of E-cadherin protein in FGF23-shRNA+TGF-β1 group was increased,whileα-SMA was decreased(P<0.05).Compared with TGF-β1 group,the relative fluorescence intensity of E-cadherin in FGF23-shRNA+TGF-β1 group was increased,while the N-cadherin was decreased(P<0.05).Compared with TGF-β1 group,the mRNA and protein expression levels of FGF23 and FGFR4 in FGF23-shRNA+TGF-β1 group were decreased,while Klotho was increased(P<0.05).Conclusion Down-regulation of FGF23 inhibits TGF-β1-induced growth and EMT of 16HBE cells,and corrects the imbalance of Th1/Th2 and Treg/Th17.FGF23-Klotho-FGFR4 signaling may be a molecular target for asthma therapy.