Studies On The Influence Of HIPK2 Expression Changes On Pulmonary Fibrosis Through The P53 Signaling Pathway

Objective: To investigate the expression of p53,Bax and Bcl-2 in lung fibroblasts when HIPK2 expression was changed and its effect on lung fibroblast function in vitro.Further,vivo experiments were conducted to verify the effect of HIPK2 on bleomycin induced pulmonary fibrosis in mice and its mechanism.Methods: The expression of HIPK2 was up-regulated and down-regulated by constructing recombinant adenovirus vectors.In vitro,mouse lung fibroblasts were first divided into four groups,namely Ad-HIPK2 group and control Ad-GFP group,Ad-HIPK2-shRNA group and control Ad-GFP-shRNA group.After addition of p53 inhibitor PFT-α,they were divided into Ad-GFP group,Ad-HIPK2 group,and Ad-HIPK2+PFT-α group.Western Blot and RT-PCR were used to detect the expression of HIPK2,apoptotic related factors p53,Bax,Bcl-2 and the main extracellular matrix including CollaI,CollaⅢ proteins and mRNA in lung fibroblasts,respectively.Besides,the apoptosis,proliferation and migration of lung fibroblasts were detected by flow cytometry,CCK-8 and transwell chamber assay,respectively.Further,in vivo,54 8-week-old male C57BL/6 mice were randomly divided into 3 groups,namely control group,pulmonary fibrosis group,and pulmonary fibrosis+Ad-HIPK2 group.First of all,the pulmonary fibrosis model in mice was constructed by intratracheal injection of bleomycin,and the control group was injected with equal volume of aseptic saline,and the corresponding adenovirus was injected into the tail vein of mice on the next day after modeling.On the 7th,14 th and 28 th days after modeling,5 mice in each group were sacrificed,and some lung tissues were taken for HE and Masson staining and pulmonary fibrosis Ashcroft score.The expressions of HIPK2,p53,Bax,Bcl-2,CollaⅢ,α-SMA proteins and mRNA in lung tissues were detected by Western Blot and RT-PCR,respectively.Results: The up-regulated and down-regulated expression of HIPK2 was successfully achieved by adenovirus.When the expression of HIPK2 was up-regulated,the expression of HIPK2,p53 and Bax in lung fibroblasts increased,the expression of Bcl-2,CollaI and CollaⅢ decreased,the cell apoptosis was enhanced,and the ability of proliferation and migration was weakened(all P values < 0.05).On the contrary,When HIPK2 expression was down-regulated,the results were reversed.After the addition of p53 inhibitor,the expression of p53 and Bax decreased in the inhibitor group,while the expression of Bcl-2,CollaI and CollaⅢ increased,the apoptosis decreased,and the proliferation and migration ability were all enhanced(all P values < 0.05).HE and Masson staining indicated that the pulmonary fibrosis mouse induced by bleomycin was successfully constructed.When HIPK2 was overexpressed,the expression of p53 and Bax in the lung tissue increased,and the expression of Bcl-2,CollaⅢ and α-SMA decreased(all P values < 0.05),and the degree of pulmonary fibrosis in mice decreased.Conclusion: HIPK2 may promote cell apoptosis by activating the p53/Bax/Bcl-2 apoptotic signaling pathway in lung fibroblasts,inhibit cell proliferation,migration,and reduce the production of extracellular matrix,thereby reducing pulmonary fibrosis in mice and delaying the progression of pulmonary fibrosis.