Effects Of HIPK2 Expression Changes On Lung Fibroblasts And Pulmonary Fibrosis Of Bleomycin Mice By Wnt-β/catenin Pathway

Objective: To identify the effects of homologous domain interaction protein kinase 2(HIPK2)expression changes on mouse fibroblasts.To elucidate the effects of HIPK2 on Wnt / β-catenin signaling pathway and the possible mechanism of regulating pulmonary fiber process.the expression of HIPK2 and the activation of the mouse lung fibroblast(MLFs)and Wnt-β/catenin.To explore the effects of overexpression of HIPK2 on the degree of fibrosis and activation of Wnt / β-catenin signaling pathway in mice induced by bleomycin.Methods:1.Construction of adenoviral vectors containing HIPK2 with green fluorescent protein(GFP)(Ad-HIPK2)and specificity ShRNA adenoviral vector(Ad-sh-HIPK2).2.Infect the mouse lung fibroblasts(MLF)with recombinant adenovirus.Then detecte the cell proliferation,invation and apoptosis by CCK8 assay,Transwell and Flow cytometry.Detect the mRNA levels of HIPK2,β-catenin,CollaI,CollaIII,α-SMA by RT-Qpcr and Western blot.3.To construct a mouse model of pulmonary fibrosis induced by bleomycin,and inject recombinant adenovirus into mice through the tail vein.The mice were sacrificed on days7,14,and 28 respectively,and the mouse lung tissue specimens were retained for further testing.Using HE staining,Masson staining,qPCR,Western blot,immunohistochemistry techniques to study the molecular mechanism of HIPK2 in the development of mouse lung fibrosis.4.Statistical analysis of experimental data using SPSS 25.0 statistical software.The data of each group is indicated by,and the comparison between the two groups adopts independent sample t test.One-way analysis of variance and Dunnet ex-post comparison were used for comparison between the three groups.The difference was statistically significant at P <0.05.Results:1.In the in vitro cell culture experiment,the proliferation rate,invation,synthesis and secretion of HIPK2 silencing rexpression group increased and the apoptosis decreased.The HIPK2 overexpression group otherwise.2.The expression of β-catenin increased in HIPK2 silencing rexpression group,and the synthesis and secretion of HIPK2 silencing rexpression group decreased after added in XAV939.3.HE staining,Masson staining 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;while the degree of fibrosis in Ad-HIPK2 group lighten.HIPK2 was fond to be decreased in mouse models of bleomycin-induced pulmonary fibrosis.In Ad-HIPK2 group of mice,the expression of Colla Ⅰ,Colla Ⅲ and α-SMA was decreased,while the expression of Bc1-2、caspase3 increased.The expression of β-catenin increased in mouse models of bleomycin-induced pulmonary fibrosis and decreased in Ad-HIPK2 group.Conclusions:1.Down-regulating HIPK2 may promote the occurrence of lung fibrosis and up-regulating HIPK2 is possible to inhibate the form of pulmonary fibrosis.2.There is the activation of wnt-β/catenin pathway in the process of lung fibrosis.3.The down-regulated HIPK2 may involve in lung fibrosis by activating the wnt-β/catenin pathway,and block this pathway could lighten the degree of fibrosis.