The Effect Of TRB3 Expression On Bleomycin Induced Pulmonary Fibrosis In Mice Through Wnt/β-catenin Signaling Pathway

Objective: To elucidate the mechanism of Tribbles homologue 3(TRB3)overexpression and underexpression on bleomycin induced pulmonary fibrosis in C57BL/6 mice and the exogenous regulation of TRB3 in Wnt / β-catenin signaling pathway on bleomycin induced pulmonary fibrosis in C57BL/6 mice.Methods: Experimental group: 60 C57 black mice were randomly divided into the Mock group,the fibrosis group(F group),the F+ ad-GFP infection group(F+GFP group),the F+ ad-trb3 infection group(F+TRB3 group),and the F+ad-trb3-shrna infection group(F+ sh-trb3 group),with 12 mice in each group.Modeling: the mouse pulmonary fibrosis model was constructed by slowly injecting bleomycin solution into the trachea.On the second day of modeling,mice were injected with corresponding adenovirus through tail vein,and the same amount of normal saline was injected into the normal saline group and the fibrotic group.A group of experimental mice were sacrificed at three time points,namely,day 7,day 14 and day 28 after modeling,and alveolar lavage fluid(BALF)and lung tissue samples were collected for experimental use.The changes of pulmonary inflammation and degree of fibrosis in the experimental mice at different time points were determined: HE staining and Masson staining were used to observe the changes of lung tissue morphology and score the fibrosis,hydroxyproline was used to determine the collagen content,and alveolar lavage fluid was stained.The expression of TRB3,EMT-related proteins E-cadherin,Vimentin,Fibronectin and α-SMA and β-cateninin proteins in mouse lung tissues were detected by immunohistochemical assay.Western blot was used to detect the expression of TRB3,E-cadherin,Vimentin,Fibronectin,α-SMA and β-catenin in lung tissues of each group.RT-q PCR detection TRB3 in lung tissue,pro CollaⅠ/Ⅲ m RNA expression.ELISA to detect each lung tissue Col1aⅠ/Ⅲ content.Results: The model of bleomycin induced pulmonary fibrosis in mice was successfully constructed.HE staining,Masson staining,fibrosis score,hydroxyproline assay,and BALF staining showed that the over-expression of TRB3 increased the degree of bleomycin-induced lung tissue inflammation and fibrosis in mice,while the results of the low-expression group of TRB3 were opposite.Immunohistochemical results showed that the positive expression of EMT-related proteins vimentin,α-SMA and Fibronectin in the TRB3 over expression group was significantly higher than that in the simple fibrosis group,while the positive expression of E-cadherin was opposite.Western blot showed that the over expression of TRB3 increased the expression of EMT-related proteins Vimentin,Fibronectin,α-SMA and β-catenin and decreased the expression of E-cadherin in pulmonary fibrosis mice,while the expression of TRB3 decreased in the group.RT-q PCR detection hint at m RNA level,TRB3 express group pro Colla Ⅰ / Ⅲ expression,TRB3 low expression group was opposite results.ELISA test shows: TRB3 expression make each lung tissue Col1aⅠ/Ⅲ content increased significantly,TRB3 cut set expression result instead.Conclusion: Changes in TRB3 expression can affect the expression of emt-related proteins and collagen m RNA,suggesting that exogenous regulation of TRB3 expression may influence the EMT process of bleomycin induced pulmonary fibrosis in mice,and thus participate in the formation of pulmonary fibrosis.The overexpression of TRB3 increased the expression of the key protein of the Wnt/ he-catenin signaling pathway,suggesting that TRB3 could participate in the process of bleomycin induced pulmonary fibrosis in mice through the Wnt/he-catenin signaling pathway.