The Role And Mechanism Of EI24 Delays Pulmonary Fibrosis In Mouse By Promoting Autophagy Through Regulating AMPK/mTOR Pathway

Background:Pulmonary fibrosis(PF)is a common end-stage pathological change in interstitial pulmonary diseases,with poor prognosis and high mortality.At present,the molecular mechanism that causes the occurrence and development of PF is not fully understood,and there is a lack of effective treatment in clinical practice.To further clarify the pathogenesis of pulmonary fibrosis is of great significance for the prevention and treatment of pulmonary fibrosis.Excessive deposition of extracellular matrix(ECM)is a major factor in the occurrence and development of PF,and myofibroblasts,as effector cells of this link,have always been the source of concern.Alveolar epithelial cell is one of the source cells of myofibroblasts,which can promote the continuous progress of pulmonary fibrosis after EMT transformation.It has been reported that autophagy deficiency is an important factor that triggers EMT transformation in alveolar epithelial cells.Abnormal AMPK/mTOR axis may be one of the important reasons for autophagy deficiency.In the process of pulmonary fibrosis,AMPK activation is reduced,mTOR is abnormally activated,and the AMPK/mTOR axis is in an abnormal state,but the specific mechanism is not clear.Etoposide-induced 2.4 protein(EI24)is expressed in a variety of tissues,and its deletion leads to the emergence of the corresponding disease phenotypes.However,current studies on EI24 mainly focus on tumors.How does EI24 change in pulmonary fibrosis? What role does it play? No research has been reported.Studies have shown that EI24 is an important protein involved in autophagy.The current studies mainly found that EI24 can promote the degradation of autophagy lysosome.Whether it can participate in the regulation of autophagy through other pathways is not clear.Literature has suggested that EI24 is closely related to the activation of AMPK and mTOR,respectively.However,whether EI24 can promote autophagy through the AMPK/mTOR axis and its role in pulmonary fibrosis remains to be confirmed.Therefore,this study aims to investigate the role and mechanism of EI24 in pulmonary fibrosis mice and alveolar epithelial cells.Meanwhile,molecular biological methods were used to determine whether EI24 enhances autophagy level through AMPK/mTOR pathway and its role in pulmonary fibrosis.To further elucidate the pathogenesis of pulmonary fibrosis and to explore new effective targets for the prevention and treatment of pulmonary fibrosis.Objective:1.To observe the expression of EI24 and the changes of the corresponding fibrosis indexes in lung tissue from pulmonary fibrosis model and in mouse alveolar epithelial cells(MLE-12)treated by bleomycin(BLM).2.To further investigate the effects of up-regulation of EI24 on EMT,ECM deposition and fibrosis indexes in lung tissues and MLE-12 cells,and to clarify the role of EI24 in pulmonary fibrosis.3.To investigate whether EI24 promotes autophagy through the AMPK/mTOR axis,and to explore the role of EI24 in pulmonary fibrosis and in the EMT transformation of alveolar epithelial cells.Methods:1.The pulmonary fibrosis model of mice was created by nasal feeding bleomycin and the alveolar epithelial cells were interfered with bleomycin.The expression of EI24 and the changes of the corresponding fibrosis indexes in lung tissues and cells in each group were observed:(1)In vivo experiment: C57BL6 mouse was taken as the research object and randomly divided into normal control group and pulmonary fibrosis model group.The pulmonary fibrosis model was established through the administration of bleomycin by nasal feeding,and all mouse were sacrificed 21 days after model replication.HE and Masson staining were used to observe the morphological changes of lung tissue.Immunohistochemical staining and Western blot were used to detect EI24,E-cadherin,α-SMA,Collagen-Ⅰand Fibronectin levels;EI24 m RNA was detected by q PCR.(2)In vitro experiments: Mouse alveolar epithelial cells(MLE-12)was taken as the research object.MTT was used to detect the activity of alveolar epithelial cells after the treatment of bleomycin.The changes of cell morphology were observed under microscope.Western blot were used to detect EI24,E-cadherin,α-SMA,Collagen-Ⅰ and Fibronectin expression level in MLE-12 after the stimulation by different concentration of bleomycin;the EI24 m RNA level was detected by q PCR.2.Mice were injected with overexpressed plasmid EI24 through tail vein and cells were transfected with overexpressed plasmid EI24,and the effects of upregulation of EI24 on EMT,ECM deposition and fibrosis indexes were observed at animal and cell levels:(1)In vitro experiments: MLE-12 cells were used as the research object.(1)The alveolar epithelial cells were treated with bleomycin and EI24 overexpression plasmid simultaneously.The cells were divided into normal control group,bleomycin intervention group,bleomycin and vector group,bleomycin and EI24 overexpression plasmid intervention group.Western blot was used to detect EI24,E-cadherin,α-SMA,Collagen-Ⅰ and Fibronectin level;the m RNA level of EI24 was detected by q PCR.(2)The alveolar epithelial cells were treated with TGF-β1 and EI24 overexpression plasmid simultaneously.Cells were divided into: normal control group,the TGF-β1intervention group,the TGF-β1 and vector plasmid intervention group,TGF-β1 and EI24 overexpression plasmid intervention group.Western blot was used to detect EI24,E-cadherin,α-SMA,Collagen-Ⅰ and Fibronectin level;the m RNA level of EI24 was detected by q PCR.(2)In vivo experiment: C57BL6 mouse were randomly divided into normal control group,PF model group,PF model and vector plasmid injection group,PF model and EI24 overexpression plasmid injection group.EI24 overexpressing plasmid or vectors was injected through the mouse tail vein at 14 days after the administration of BLM.The lungs in the mouse were harvested at 7 days after the administration of EI24-overexpressing plasmid.HE and Masson staining were used to observe the morphological changes of lung tissue.Western blot was used to detect EI24,E-cadherin,α-SMA,Collagen-Ⅰ,and Fibronectin level;EI24 m RNA was detected by q PCR.Immunohistochemical staining was used to detect Collagen-Ⅲexpression;the expression of E-cadherin andα-SMA were also detected by immunofluorescence staining.3.To observe the effects of EI24 on the level of autophagy and the activation of AMPK/mTOR during pulmonary fibrosis;By intervening cells with autophagy inhibitors,AMPK inhibitors and agonists,the changes in the effects of autophagy or AMPK inhibition on EI24 were observed:(1)In vivo experiment:(1)(1)C57BL6 mouse were divided into normal control group and pulmonary fibrosis model group.The changes of LC3 and p62 were detected by Western blot.(2)C57BL6 mouses were divided into normal control group,pulmonary fibrosis model group,PF model and vector plasmid injection group,PF model and EI24 overexpression plasmid injection group.The changes of LC3,p62,p-mTOR,mTOR,p-AMPK and AMPK were detected by Western blot,and the expression of p-AMPK was also detected by immunohistochemical staining.(2)In vitro experiments:(1)MLE-12 were cultured,then divided into normal control group and autophagy inhibitor 3-MA intervention group,Western blot was used to detect LC3,p62,E-cadherin,α-SMA,Collagen-Ⅰ,and Fibronectin in cells;(2)MLE-12 were divided into normal control group,BLM intervention group,BLM intervention and vector plasmid transfection group,BLM intervention and EI24 overexpression plasmid transfection group.The changes of LC3 and p62 were detected by Western blot.(3)MLE-12 were cultured and divided into normal control group,BLM intervention group,BLM intervention and EI24 overexpression plasmid transfection group,BLM intervention and EI24 overexpression plasmid transfection group and autophagy inhibitor 3-MA intervention group.Western blot was used to detect EI24,LC3,p62,E-cadherin,α-SMA,Collagen-Ⅰ,and Fibronectin levels.(4)MLE-12 were cultured and divided into normal control group,BLM intervention group,BLM intervention and EI24 overexpression plasmid transfection group,BLM intervention and AMPK agonist intervention group,BLM intervention and EI24overexpression plasmid transfection and AMPK inhibitor intervention group.Western blot was used to detect EI24,p-AMPK,AMPK,p-mTOR,mTOR,LC3,p62,E-cadherin,α-SMA,Collagen-Ⅰ,and Fibronectin levels.Results:1.The expression of EI24 decreased during pulmonary fibrosis:The model group showed fibrosis in the lung tissue,and the model replication was successful;compared with NC group,EI24 m RNA and protein level were reduced,while E-cadherin decreased,α-SMA,Collagen-Ⅰ and Fibronectin level increased in PF group.With the increased concentration of BLM intervention,survival rate of cell decreased gradually.Moreover,the administration of BLM resulted in a morphological change of MLE-12 cells,similar to the scattered morphology of mesenchymal cells.And with the increased concentration of bleomycin intervention,EI24 m RNA and protein level gradually reduced,accompanied by E-cadherin level gradually reduced,α-SMA,Collagen-Ⅰ and Fibronectin level gradually increased.2.Upregulation of EI24 delayed the occurrence of pulmonary fibrosis:(1)In vitro,Compared with bleomycin group,after transfection of EI24 overexpression plasmid,EI24 m RNA and protein increased,E-cadherin expression increased,whileα-SMA,Collagen-Ⅰ and Fibronectin level decreased.These results suggest that EI24 can inhibit bleomycin-induced EMT transformation in alveolar epithelial cells.(2)Compared with normal control group,EI24 m RNA and protein expression reduced after TGF-β1 stimulation,accompanied by E-cadherin reduced,α-SMA,Collagen-Ⅰand Fibronectin increased;compared with the TGF-β1 stimulation group,after transfection EI24 overexpression plasmid,EI24 m RNA and protein expression increased,with E-cadherin level increased,while α-SMA,Collagen-Ⅰ and Fibronectin level decreased.These results suggested that EI24 could inhibit TGF-β1-induced EMT transformation in alveolar epithelial cells.(3)compared with pulmonary fibrosis model group,the EI24 level increased significantly in mouse lungs after injecting EI24-overexpressing plasmid.While E-cadherin level increased,α-SMA,Collagen-Ⅰ,Collagen-Ⅲand Fibronectin level decreased,and the fibrotic lesions were alleviated after the upregulation of EI24.3.Autophagy deficiency is closely related to EMT transformation and fibrosis:(1)In the lung tissue of mice and pulmonary fibrosis model bleomycin intervention in alveolar epithelial cells,the level of autophagy was suppressed,showed by the LC3-Ⅱ/LC3-Ⅰ ratio decreases,p62 protein level increased;(2)Use autophagy inhibitor processing cell,cell autophagy was significantly inhibition,EMT occured,the performance of E-cadherin protein expression level decreased,α-SMA,Collagen-Ⅰ and Fibronectin expression are increased;4.EI24 inhibits the occurrence of EMT by enhancing autophagy:(1)The autophagy level was enhanced in lung tissue from mouses treated with overexpressed plasmid EI24 through tail vein injection and in cells transfected by overexpression of EI24 plasmid.(2)The expression of EI24 decreased after BLM stimulation,accompanied by inhibition of autophagy and occurrence of EMT.BLM intervention was accompanied by transfection of overexpression of EI24 plasmid,with the increase of EI24 expression,the level of autophagy was enhanced,and the cell EMT transformation was decreased.When autophagy specific inhibitor 3-MA was present in cells,although the expression of EI24 was increased,the anti-fibrosis effect of EI24 was weakened due to autophagy inhibition.5.EI24 plays an anti-fibrosis effect by mediating AMPK/mTOR to promote autophagy:(1)AMPK was inhibited and mTOR was in abnormal activation both in mouse lung tissue of pulmonary fibrosis model and cells treated by bleomycin,showing a significant decrease in p-AMPK/AMPK ratio and a significant increase in p-mTOR/mTOR ratio.After intervention with exogenous plasmid,with the increase of EI24 expression,AMPK was activated in lung tissues and alveolar epithelial cells,and mTOR was significantly inhibited.It is suggested that EI24 can promote the activation of AMPK and inhibit the activation of mTOR.(2)After BLM intervention,the expression of EI24 decreased,along with the inhibition of AMPK activation,mTOR was abnormally activated,autophagy was inhibited,and EMT occurred.With the increase of EI24,AMPK was activated,mTOR activation was inhibited,autophagy level was enhanced and the occurrence of EMT was reduced.With the addition of AMPK agonist in bleomycin intervention,EI24 level did not change,but due to the significant activation of AMPK,mTOR activation was still inhibited,autophagy level was enhanced,and the occurrence of EMT was inhibited.When AMPK inhibitor was present in cells,although EI24 expression was increased,mTOR was in an abnormal activated state due to the significant inhibition of AMPK,and autophagy level was not enhanced,accompanied with the occurrence of cell EMT,suggesting that the anti-fibrosis effect of EI24 is weakened.Conclusion:(1)In the process of pulmonary fibrosis,the expression of EI24 is decreased,accompanied by the occurrence of EMT and excessive deposition of ECM.As a potential anti-fibrosis factor,EI24 may delay the progression of pulmonary fibrosis by inhibiting EMT transformation and reducing the production and deposition of extracellular matrix proteins.(2)The abnormal AMPK-mTOR axis leads to insufficient autophagy in lung tissue cells,which will promote the occurrence and development of pulmonary fibrosis.EI24 can enhance autophagy level by mediating the AMPK-mTOR axis,and thus exert anti-pulmonary fibrosis effect.