| Objective:To identify the expression of CXXC5 and the activation of Wnt-β/catenin pathway in bleomycin-induced pulmonary fibrosis mice.To investigate the effect of exogenously regulated CXXC5 overexpression on lung fibrosis and wnt-β/catenin pathway in bleomycin mice.To investigate the possible mechanism of CXXC5 involvement in pulmonary interstitial fibrosis and explore new therapeutic targets for pulmonary interstitial fibrosis.Methods: Construction of over-expressed CXXC5 adenoviral vectors(Ad-CXXC5)with a green fluorescent protein(GFP)as a tracer gene.This experiment was devided into six groups: normal saline group(Mock group),fibrotic group(BLM group),saline + GFP infection group(Mock + Ad-GFP group),physiological saline + Ad-CXXC5 infection group(Mock + Ad-CXXC5 group),fiber In the +GFP infection group(BLM+Ad-GFP group),fibrosis+Ad-CXXC5 infection group(BLM+ Ad-CXXC5 group).In this experiment,normal saline and bleomycin were dripped into the trachea,and the mice were injected with adenovirus on the tail vein of the next day after modeling.A batch of experimental mice was treated on the 7th,14 th,and 28 th days respectively.The collection of mouse lung tissue specimens was further tested.HE staining,Masson staining,hydroxyproline assay of lung tissue,and Ashcroft score were used to evaluate the degree of fibrosis of lung tissue in each group at different time points.Cell sorting and counting of BALF fluid in each group.Western Blot was used to detect the expression of CXXC5,β-catenin,Dvl,α-SMA,E-cadherin,Vimentin,Colla I,Colla III in lung tissue of each group.RT-q PCR measured the expression of CXXC5,α-SMA,pro Colla I m RNA and pro CollaⅢ m RNA in lung tissue of each group of mice at each time point.ELISA measured the expression of Colla I and Colla III in lung tissue of each group.Results:CXXC5-overexpressing adenovirus vector Ad-CXXC5 was successfully constructed,and mouse models of bleomycin-induced pulmonary fibrosis was constructed.HE,Masson staining,hydroxyproline assay and fibrosis score in mouse lung tissue revealed that the degree of lung fibrosis in BLM group was significantly worse than that in Mock group;lung tissue in BLM group and BLM+ Ad-GFP group mice,there was no statistically significant difference in the degree of fibrosis,suggesting that the adenoviral vector itself has no effect on the degree of lung fibrosis in mice;the degree of fibrosis in the BLM+ Ad-CXXC5 group was significantly lower than that in the BLM group and the BLM+ Ad-GFP group.The cell sorting and counting of BALF fluids in each group at three time points revealed that there was no significant difference in total cell number,the percentage of macrophages,and the percentage of neutrophils between Mock group and Mock+Ad-GFP group,Mock+Ad-CXXC5 group at three time points.The total cell number and the percentage of neutrophils in the BLM group was significantly higher than that in the Mock group,the percentage of macrophages was lower than that in the Mock group.And the total number of cells and the percentage of neutrophils in the BLM+Ad-CXXC5 group was lower than that of BLM group and BLM+Ad-GFP group,and the percentage of macrophages was significantly higher than that of the latter two groups.Western Blot measured the expression of CXXC5,β-catenin,Dvl,α-SMA,E-cadherin,Vimentin,Colla I,Colla III protein in each group of lung tissues at each time point.Compared with Mock group,the expression of CXXC5 in BLM group was significantly decreased,but the activity of wnt-β/catenin pathway-related proteins β-catenin and Dvl was significantly increased,and the expression of E-cadherin,an epithelial cell marker,was down-regulated,while that of mesothelial cells and collagen markers,α-SMA,Vimentin,Colla I,Colla III was up-regulated.The expression of CXXC5 and E-cadherin in BLM+ Ad-CXXC5 group was significantly higher than that in BLM+ Ad-GFP group,while the expression of β-catenin,Dvl,α-SMA,Vimentin,Colla I,and Colla III were all decreased,suggesting that CXXC5 was overexpressed.The activity of the wnt-β/catenin pathway decreases and the degree of fibrosis decreases.The expression of CXXC5,α-SMA,pro CollaⅢ m RNAand pro Collal I m RNA was detected by RT-q PCR.The expression of CXXC5 m RNA in BLM group was significantly lower than that in Mock group,but the expression of α-SMA m RNA,pro CollaⅢ m RNAand pro Collal I m RNA was increased.The expression of CXXC5 m RNA in BLM+Ad-CXXC5 group was higher than that in BLM+Ad-GFP group,while the m RNA levels ofα-SMA,pro CollaⅢ m RNA and pro Collal I were lower than those in BLM+Ad-GFP group.The expression of Colla I and Colla III in lung tissue of each group was detected by ELISA.The content of Colla I and Colla III in lung tissue of BLM group was higher than that of Mock group,and it gradually increased with time.Comparing Mock and Mock+Ad-GFP groups,BLM group and BLM+Ad-GFP group respectively,found that there was no significant difference in the contents of Colla I and Colla III,while the content of BLM+Ad-CXXC5 group was significantly lower than that of BLM group and BLM+Ad-GFP group.Conclusions:1.The expression of CXXC5 was down-regulated in the bleomycin-induced pulmonary fibrosis mouse model.2.The wnt-β/catenin pathway was activated in the lungs of bleomycin mice.3.Over-expression of CXXC5 could inhibit the expression of key proteins of Wnt/β-catenin pathway,the expression of EMT and fibroblast transdifferentiation-related proteins,cx xreduced the degree of pulmonary fibrosis in mice.4.CXXC5 may regulate the Wnt/β-catenin pathway by negative feedback,inhibit EMT and the transdifferentiation of fibroblasts,participate in the pathogenesis and progression of pulmonary fibrosis. |
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