Effects Of HIPK2 Expression On Pulmonary Fibroblasts And Pulmonary Fibrosis In Mice Through JNK Pathway

Objective:1.To clarify the role of homologous domain interaction protein kinase 2(HIPK2)in mouse fibroblast apoptosis and its possible mechanism.2.To determine the effect of exogenous regulation of HIPK2 on JNK apoptosis signal pathway,and to explore the possible regulatory mechanism between them.3.To investigate the effects of HIPK2 overexpression on bleomycin(Bleomycin,BLM)-induced pulmonary fibrosis and JNK pathway in C57BL/6 mice.Research methods: The HIPK2 adenovirus vector(Ad-HIPK2)with green fluorescent protein(GFP)tracer gene and the specific shRNA adenovirus vector targeting HIPK2(Ad-sh-HIPK2)were purchased.Mouse lung fibroblasts(MLF),were cultured in vitro and divided into 7 groups:(1)adenovirus vector infection group: Ad-GFP group,Ad-HIPK2 group,Ad-sh-control group and Ad-sh-HIPK2 group;(2)After adding JNK inhibitor SP60025,the rats were divided into three groups: Ad-GFP group,Ad-HIPK2 group and Ad-HIPK2+SP600125 group.Then CCK8,flow cytometry and Transwell were used to detect the ability of proliferation,apoptosis and invasion.Real-time quantitative real-time PCR(RT-PCR)was used to detect the mRNA expression of HIPK2,JNK,Daxx,Bax,Bc1-2,caspase3,CollaⅠ / Ⅲ,and α-SMA in each group of cells.Westernblot was used to detect the expression of HIPK2,JNK,Daxx,Bax,Bc1-2,caspase3,CollaⅠ / Ⅲ and α-SMA in each group of cells.Mice were randomly divided into 3 groups: Mock group,Ad-control-GFP group,and Ad-HIPK2 group,and a mouse lung fibrosis model was constructed.The mice were sacrificed on day 7,14 and 28 respectively.Take peripheral blood and lung tissue for backup.The lung tissues of mice in each group were treated with hematoxylin-eosin(HE)staining and Masson staining.The degree of pulmonary fibrosis was observed under microscope,and the degree of fibrosis was graded and the score was calculated according to the pulmonary fibrosis grading method proposed by Ashcroft.The types and expression of positive cells in lung tissues of mice in each group were observed by microscope after immunohistochemistry.Westernblot was used to detect the expression of HIPK2,JNK,Daxx,Bax,Bc1-2,caspase3,CollaⅠ / Ⅲ,and α-SMA in the lung tissue of each group of mice.RT-PCR was performed on the lung tissue of each group of mice to detect mRNA expressions of HIPK2,JNK,Daxx,Bax,Bc1-2,caspase3,CollaⅠ / Ⅲ,and α-SMA.Results: 1.The results of Westernblot detection showed that:(1)compared with Ad-GFP group,the expression of HIPK2,JNK,Daxx,Bax,and caspase3 in Ad-HIPK2 group increased(t=-23.872,-66.446,-35.216,t=-18.682,-33.121,P<0.05),while the expression ofα-SMA,Colla Ⅰ / Ⅲ and Bcl-2 decreased in Ad-HIPK2group(t=13.807,26.961,33.033,80.682,P < 0.05).Compared with Ad-sh-control group,the expression of HIPK2,JNK,Daxx,Bax and caspase3 in Ad-sh-HIPK2 group decreased(t=27.092,81.004,44.752,22.381,9.164,P < 0.05),and the expression ofα-SMA,Colla Ⅰ / Ⅲ and Bcl-2 increased(t=-49.693,-11.699,-51.133,-22.107,P<0.05).(2)after adding JNK inhibitor SP60025,compared with Ad-HIPK2 group,the decrease ofα-SMA,Colla Ⅰ / Ⅲ and Bcl-2 expression was reversed(t=-8.935,-25.368,-42.521,-41.955,P<0.05)and the increase of JNK,Daxx,Bax and caspase3 expression was reversed in Ad-HIPK2+sp600125 group(t=38.243,36.927,25.003,26.964,P < 0.05).(3)compared with Ad-control group,the expression of HIPK2,JNK,Daxx,Bax and caspase3 in Ad-HIKP2 group was significantly increased(F=207.399,164.085,52.908,28.319,409.572,27.594,110.366,81.654,51.857,147.352,868.200,584.09,592.618,101.180,102.021,P < 0.05),while the expression of Bcl-2,Colla Ⅰ / Ⅲ and α-SMA was decreased in Ad-HIKP2group(F=231.765,634.970,665.99,50.553,203.060,126.400,758.839,499.804,997.980,63.884,7.516,111.564,P < 0.05).2.The results of qPCR detection showed that:(1)Compared with Ad-GFP group,the mRNA expression of HIPK2,JNK,Daxx,Bax and caspase3 in Ad-HIPK2 group increased(t=-25.682,-102.237,-75.136,-37.712,-58.409,P< 0.05),while the mRNA expression of α-SMA,Colla Ⅰ / Ⅲ and Bcl-2decreased(t=88.236,120.736,82.103,82.918,P<0.05).Compared with Ad-sh-control group,the mRNA expression of HIPK2,JNK,Daxx,Bax and caspase3 in Ad-sh-HIPK2 group decreased(t=107.293,55.432,81.967,81.412,134.987,P < 0.05),while the mRNA expression of α-SMA,Colla Ⅰ / Ⅲ and Bcl-2 increased(t=-47.065,-30.113,-47.609,-65.331,P<0.05).(2)After adding JNK inhibitor SP60025,compared with the Ad-HIPK2 group,the m RNA expression of α-SMA,CollaⅠ / Ⅲ,and Bcl-2 in the Ad-HIPK2 + sp600125 group was reversed(t=2.876,-21.515,-22.850,-10.138,P<0.05),and the mRNA expression of HIPK2,JNK,Daxx,Bax and caspase3 expression was reversed(t=18.840,40.464,17.536,18.925,27.360,P < 0.05).(3)Compared with the Ad-control group,the mRNA expression of HIPK2,JNK,Daxx,Bax and caspase3 in the Ad-HIKP2 group was significantly increased in mouse lung tissues(F=498.286,7726.904,20487.425,947.675,961.972,1179.546,289.350,368.329,486.140,581.471,303.795,594.657,323.379,356.309,403.481,P < 0.05),and the mRNA of Bcl-2,CollaⅠ / Ⅲ,and α-SMA Reduced expression(F=400.861,219.497,985.603,176.682,394.411,1971.132,459.149,952.629,3063.192,201.780,257.067,254.260,P < 0.05).3.Transwell,CCK-8 and flow cytometry test results showed that:(1)compared with the Ad-GFP group,the number of cells passing through the cell in the Ad-HIPK2 group was reduced(t = 12.369,P <0.05),24 h,48h and 72 h decreased proliferation(t = 10.988,17.163,22.269,P <0.05),decreased the number of cells passing through the cell(P <0.05),and increased the MLF apoptosis rate(t =-89.628,P <0.05).Compared with the Ad-sh-control group,Ad-sh-HIPK2 increased the number of cells passing through the cell(t = 23.401,P <0.05),and increased proliferation at 24 h,48h,and 72h(t =-6.245,-33.459,-37.844,P <0.05),the MLF apoptosis rate decreased(t = 17.835,P <0.05).(2)After adding the JNK inhibitor SP60025,compared with the Ad-GFP group,the number of cells passing through the cell in the Ad-HIPK2 group was reduced(t = 10.335,P <0.05),and the proliferation was reduced at 24 h,48h,and 72h(t = 15.563,13.025,45.481,P <0.05),the MLF apoptosis rate increased(t =-24.165,P <0.05);compared with the Ad-HIPK2 group,the number of cells passing through the cell in the Ad-HIPK2-sp600125 group increased(t =-5.326,P <0.05),proliferation increased at 24 h,48h,and 72h(t =-15.052,-8.030,-30.905,P <0.05),and the MLF apoptosis rate decreased(t = 5.147,P <0.05).4.Bleomycin-induced mouse pulmonary fibrosis model HE,Masson staining and fibrosis scores,Ashcroft score results show that compared with the Mock group,fibrosis scores in the Ad-control-GFP group on days 7,14,28 Increased(t =-7.604,-62.000,-176.317,P<0.05).At 7,14,and 28 days,compared with the Ad-control-GFP group,the fibrosis score of the Ad-HIPK2 group was reduced(t = 3.170,12.004,40.880,P <0.05).5.The results of immunohistochemistry showed that the expression of HIPK2 protein was significantly increased in lung tissue of Ad-HIPK2 group.Conclusion: 1.Overexpression of HIPK2 may inhibit MLF proliferation and promote its apoptosis,reduce collagen deposition and extracellular matrix(ECM)expression,and alleviate pulmonary fibrosis.The result was opposite when the expression of HIPK2 was down-regulated.2.Overexpression of HIPK2 may promote the apoptosis of fibroblasts in bleomycin mice and reduce pulmonary fibrosis in mice.3.HIPK2 may lead to the imbalance of fibroblast proliferation / apoptosis and alleviate pulmonary fibrosis by affecting JNK signal pathway.