Core Fucosylation Modification Regulates Alveolar Epithelial Cells Senescence In Pulmonary Interstitial Fibrosis

Background: Idiopathic pulmonary fibrosis(IPF),a chronic and fatal disorder of unknown etiology associated with aging,has a terrible prognosis.To date,no novel pharmacological or other therapies have received strong recommendations or widely used for the treatment of pulmonary fibrosis.Cell senescence has been found to be a cell state triggered by various physiological processes and associated with multiple age-related diseases,characterized by an irreversible cell-cycle arrest.Alveolar epithelial cells senescence causes interstitial cells to secrete large amounts of inflammatory factors that exacerbate alveolar epithelial cells injury,while it also promotes the synthesis of extracellular matrix and activates a variety of fibrotic genes to promote fibrosis progression.Therefore,it is hypothesized that alveolar epithelial cells senescence is an important node in causing pulmonary interstitial fibrosis.Glycosylation is a common post-translational modification of proteins,andα1,6-fucosyltransferase(FUT8)is the only enzyme that catalyzes core fucosylation(CF)in mammals.Recent studies have demonstrated that CF is involved in the regulation of cell growth,cell signaling,tumorigenesis and other biological processes under physiological and pathological conditions.In addition,the pro-fibrotic effect of CF has been found in fibrotic diseases such as renal interstitial fibrosis and peritoneal fibrosis.However,there are no studies on whether CF modification may regulate the mechanism of alveolar epithelial cells senescence leading to pulmonary interstitial fibrosis.Objective:(1)To clarify the characteristic alterations of CF modification in alveolar epithelial cells senescence causing pulmonary interstitial fibrosis.(2)To elucidate the role of CF modification induced alveolar epithelial cells senescence in the progression of pulmonary interstitial fibrosis and its molecular mechanism.Methods: In vivo,we use C57/BL6 male mice or(and)male alveolar epithelial cell-specific FUT8 conditional knockout(CKO)mice to construct a bleomycin(BLM)-induced pulmonary fibrosis mice model.HE and Masson pathological staining were performed to observe the alveolar structure and extent of pulmonary interstitial fibrosis 21 days after BLM injecting.Immunofluorescence and western blotting were used to detect senescence markers p16 and p21,fibrosis indexes E-cadherin,collagenⅠand collagenⅢ,changes in CF expression levels in alveolar epithelial cells and related pathway protein expression in lung tissue.Immunoprecipitation was performed to observe the correlation between CF modifications and key proteins of the pathway.In vitro,MLE12 cell line was selected and stimulated with BLM for 72 h to construct an alveolar epithelial cells injury model.After transfection with si RNA that knockdown FUT8 gene or(and)infection with adenoviral vector that overexpressed FUT8,MLE12 cells were treated with or without BLM stimulation for 48 h.q RT-PCR was performed to detect the efficiency of cell knockdown or overexpression of FUT8.Senescence associated beta-galactosidase(SA-β-gal)staining was performed to observe the degree of alveolar epithelial cells senescence.Western blotting and immunofluorescence were performed to observe the changes in the expression of FUT8,p16,p21,E-cadherin,collagen Ⅰ,collagen Ⅲ and other proteins as well as the changes in CF expression levels and related pathway protein expression in alveolar epithelial cells.Immunoprecipitation was performed to detect the extent of CF modification associated with key proteins of the pathway.Results: In vivo: In the lung tissue of BLM-induced mice,HE and Masson staining exhibited that destruction of the alveolar structure and accumulation of collagen fibers.Immunofluorescence and western blotting suggested that BLM treatment increased the expression of senescence indicators p16,p21,and fibrosis markers collagenⅠ,collagenⅢ,while decreased expression of E-cadherin,and significantly increased expression of FUT8 compared with the control group.However,after induction of CKO mice with BLM,FUT8 expression was significantly decreased,and senescence markers p16,p21 protein expression was also decreased,while fibrosis indicators collagenⅠand collagenⅢ protein expression was decreased,and E-cadherin expression was increased compared with BLM-induced control mice.Immunoprecipitation showed that TGF-βRⅠ protein could be regulated by CF modification.Immunofluorescence and western blotting suggested that induction of CKO mice with BLM significantly reduced the BLM-induced increase in TGF-β pathway downstream signal Smad2/3 protein phosphorylation(p-Smad2/3)expression in lung tissue of normal mice.In vitro: BLM stimulation increased the number of SA-β-gal positive cells in MLE12 cells,and western blotting showed a significant increase in FUT8,p16 and p21 protein expression.Immunofluorescence double staining suggested that the expression of senescence indicators p16,p21 showed the same trend as the expression of FUT8 or lens culinaris agglutin(LCA).Western blotting and immunofluorescence results indicated that compared with MLE12 cells transfected with non-targeted(NT)si RNA and induced with BLM,MLE12 cells transfected with FUT8 si RNA and then induced with BLM attenuates BLM-stimulated increase in the expression of FUT8,p16,p21,collagenⅠ,collagen Ⅲ proteins and decrease in E-cadherin expression.Meanwhile,Immunoprecipitation showed that TGF-βRⅠ protein was regulated by CF modification.Western blotting and immunofluorescence suggested that transfection with FUT8 si RNA before stimulation of MLE12 cells with BLM inhibiting CF modification significantly reduced p-Smad2/3 protein expression compared to transfection with NT si RNA.Application of an adenovirus vector that overexpressed FUT8 to infect MLE12 cells showed a significant increase in FUT8,p16,p21 and p-Smad2/3 protein expression by western blotting,while treatment with the TGF-βR inhibitor EW-7197 resulted in a decrease in p-Smad2/3 protein expression,senescence indicators p16 and p21 expression and SA-β-gal positive cells.Conclusion: Through in vitro and in vivo experiments,we found that CF expression levels were elevated during alveolar epithelial cells senescence induced pulmonary interstitial fibrosis.Inhibition of CF modification played a key role in reducing alveolar epithelial cells senescence and attenuating pulmonary interstitial fibrosis.Furthermore,CF regulated alveolar epithelial cells senescence causing pulmonary interstitial fibrosis through activation of TGF-β signaling pathway.It is indicated that CF may be an important potential intervention target for the treatment of pulmonary interstitial fibrosis.