Effect Of CXXC5 Expression On Pulmonary Fibrosis In Bleomycin-induced Mice Via CD40/CD40L Pathway

Objective:1.To clarify the expression of CXXC5 in lung tissue of mice with pulmonary fibrosis induced by bleomycin(BLM)and its expression in CD4/CD40 L pathway.To investigate the effect of CXXC5 overexpression on BLM-induced pulmonary fibrosis and CD4/CD40 L pathway in C57BL/6 mice.3.To investigate whether CXXC5 inhibits epithelial-mesenchymal transition(EMT)via BMD-induced pulmonary fibrosis in mice to inhibit pulmonary fibrosis.Research methods: 1.Construct a CXXC5 overexpressing adenovirus expression vector containing green fluorescent protein(GFP).2.Grouping: C57BL/6 mice were randomly divided into 6 groups: saline group(N group),fibrosis group(group B),saline + Ad-GFP group(N+Ad-GFP group),normal saline.+Ad-CXXC5 group(N+ Ad-CXXC5 group),fibrosis+Ad-GFP group(B+ Ad-GFP group),fibrosis+Ad-CXXC5 group(B+ Ad-CXXC5group),construct a mouse model of pulmonary fibrosis The mice were sacrificed by cervical dislocation on the 7th,14 th and 28 th day after modeling,and lung tissue specimens were taken for experimental use.3.The lung tissues were subjected to hematoxylin-eosin staining(HE staining)and horsesson staining(Masson staining).The degree of fibrosis was observed under a microscope,and the Ashcroft fibrosis score was further measured and the hydroxyproline content was determined.RT-qPCR was performed on the lung tissues of each group to detect the expression of CXXC5 mRNA,CollaI mRNA and CollaIII mRNA.The expression of CXXC5,CD40,CD40 L,Fibronectin,Keratin-14,CollaI and CollaIII protein was detected by Western Blot in the lung tissues of each group.The expression levels of CollaI and CollaIII in lung tissue of each group were determined by ELISA.The results were as follows:1.An adenoviral expression vector overexpressing CXXC5 was constructed and a mouse model of pulmonary fibrosis induced by BLM was constructed.2.HE staining,Masson staining and fibrosis scoring of lung tissue were performed to determine the content of hydroxyproline.We found that the degree of pulmonary fibrosis was significantly aggravated in group B compared with group N;N+Ad-GFP,B+Ad Compared with N group and B group,there was no significant change in the degree of pulmonary fibrosis,indicating that green fluorescent protein had no effect on pulmonary fibrosis;group B,B+Ad-GFP group and B+Ad-CXXC5 group had lung The degree of fibrosis is significantly aggravated,indicating that overexpression of CXXC5 can inhibit the progression of pulmonary fibrosis.3.Comparing the changes in total number of cells,percentage of macrophages(MACR%)and percentage of neutrophils(NEUT%)in BALF ateach time point in each group of mice,we found that on days 7,14,and 28 Compared with the N group and the N+Ad-GFP group,there was no significant difference in the N+Ad-CXXC5 group.Compared with the N group,the total number of cells and NEUT% in the alveolar lavage fluid were significantly increased,while the MACR was increased.% was significantly lower than N group;B+Ad-CXXC5 group compared with group B and B+Ad-GFP group,the total number of cells and NEUT% were significantly decreased,while MACR% was significantly higher than the two groups.Western blot was used to detect the expression of CXXC5 and CXXC5 related proteins in lung tissue of mice.We found that the expression of mesenchymal cell marker Fibronectin and collagen components Cola1 and CollaIII in group B was significantly higher than that in group N at three time points,and the expression of epithelial cell marker Keratin-14 was significantly decreased.CXXC5 was significantly decreased,and the expression levels of CD40 and CD40 L related to CD40/CD40 L signaling pathway were significantly increased.Compared with the N+Ad-GFP group,the B group and the B+Ad-GFP group were compared at three time points,and the expression levels of CXXC5,Fibronectin,CollaI and CollaIII in the lung tissue were not significantly changed.Compared with the B+Ad-CXXC5 group,the expression of Fibronectin,CollaI and CollaIII was significantly increased,CXXC5 was significantly decreased,and the expression of CD40/CD40 L signaling pathway-related protein CD40/CD40 L was significantly increased in group B,B+ Ad-GFP and B+Ad-CXXC5.increase.The expression of Fibronectin mRNA,CollaI mRNA and CollaIII mRNA in group B was significantly higher than that in group N by RT-qPCR.Compared with the N+Ad-GFP group,the B group and the B+Ad-GFP group were compared at three time points,and the expression levels of CollaI mRNA and CollaIII mRNA in the lung tissue were not significantly changed.The expression of CollaI mRNA and CollaIII mRNA was significantly increased in group B,B+ Ad-GFP and B+Ad-CXXC5 at three time points.The expression of CollaI and CollaIII in lung tissue of each group was detected by ELISA.The expression of CollaI and CollaIII in group B was significantly higher than that in N group.The N group and the N+Ad-GFP group,the B group and the B+Ad-GFP group were compared at three time points,and the expression levels of CollaI and CollaIII in the lung tissue were not significantly different.The expression of CollaI and CollaIII was significantly increased in group B,BLM+ Ad-GFP group and BLM+Ad-CXXC5 group at three time points.Conclusions: 1.The expression of CXXC5 in lung tissue of C57 BL mice induced by bleomycin was decreased.2.Bleomycin-induced pulmonary fibrosis Increased expression of CD4/CD40 L pathway in lung tissue of C57 BL mice.3.CXXC5 overexpression inhibits the occurrence of EMT in bleomycin mice by inhibiting CD4/CD40 L pathway,which in turn affects the progression of fibrosis.4.CXXC5 overexpression inhibits the progression of fibrosis and may provide a new target for the treatment of pulmonary fibrosis.