Research Of ERK5 Promoting Phenotypic Transformation Of Fibroblasts And Its Role In Pulmonary Fibrosis In Mice

Objective: The purpose of this study was to investigate whether extracellular signal-regulated protein kinase-5(ERK5)is involved in the pathogenesis of pulmonary fibrosis both in vitro and in vivo,and to further investigate the effects of ERK5 on autophagy,apoptosis and proliferation of human lung fibroblasts cultured in vitro and the influences of ERK5 inhibitor on bleomycin-induced pulmonary fibrosis in mice.The research provides a new idea of the theoretical basis for the treatment of pulmonary fibrosis.The first part is to study the regulation of ERK5 on autophagy,apoptosis and proliferation in human lung fibroblasts and the affection of ERK5 on the transformation of human lung fibroblasts to myofibroblasts(phenotypic transformation).Methods:(1)Human lung fibroblasts(HLFs)were treated with TGF-?1 at a dose of 10ng/ml for 24 hours.And then to detect the protein expression levels of Fibronectin and ?-SMA by Western Blot and cellular immunofluorescence respectively.(2)HLFs were treated with si RNA(si-ERK5)that knocked down ERK5 gene and plasmid(over-ERK5)that overexpressed ERK5 gene respectively before treated with TGF-?1.The intracellular ?-SMA protein expression level was detected by Western Blot.And then treated with autophagy inhibitor and autophagy activator respectively,the expression levels of ?-SMA,LC3 II,Beclin-1 and p62 were detected by Western Blot.(3)HLFs were treated with BIX02189(ERK5inhibitor),si-ERK5 and over-ERK5 respectively before treated with TGF-?1.And Western Blot was used to detect intracellular ?-SMA protein expression level,apoptosis related protein Bcl-2?cleaved-caspase-3 expression levels,and proliferation related protein PCNA expression level.Apoptosis rate of HLFs was analyzed by flow cytometry and proliferation rate was analyzed by CCK8.Results:(1)The protein expression levels of Fibronectin and ?-SMA in HLFs treated with TGF-?1 were higher than those without treated with TGF-?1.(2)After treated with TGF-?1,the expression level of ?-SMA protein in the knock-down group was lower than that in blank control group.And the protein expression levels of ?-SMA and p62 in knock-down and autophagy inhibitor group were higher than that in knock-down group while the protein expression levels of LC3 II and Beclin-1 were lower.After treated with TGF-?1,the expression level of ?-SMA protein in over-expression group was higher than that in blank control group.And the protein expression levels of ?-SMA and p62 in over-expression and autophagy activator group were lower than that in over-expression group while the protein expression levels of LC3 II and Beclin-1 were higher.(3)After the treatment with TGF-?1,compared with the blank control group,the expression of cleavedcaspase3 of HLFs was increased in BIX02189(ERK5 inhibitor)group and siERK5 group,and the expression of Bcl-2 and PCNA was decreased,and the cell proliferation was inhibited detected by CCk8 while the apoptosis rate increased detected by flow cytometry.And the over-ERK5 group compared with the blank control group,the expression of cleaved-caspase3 wasdecreased and the expression of Bcl-2 and PCNA was increased,and the cell proliferation rate was increased detected by CCK8.The second part is to study the effects of ERK5 inhibitors on the expression of ?-SMA and LC3 II of lung tissue in pulmonary fibrosis mice.Methods: 24 male C57BL/6 mice were randomly divided into saline blank control group(NS group),bleomycin-administered experimental group(BLM group),BIX02189 treatment group(BT group)and BIX02189 blank control group(BC group).There were 6 mice in each group.NS group and BLM group were i.p.inoculated with 100?L saline while BT group and BC group were i.p.inoculated with 100?l of BIX02189(10mg/kg).Two hours later,50?L of saline was i.t.inoculated into NS group and BC groups while50?L of bleomycin(3.5mg/kg)was i.t.inoculated into BLM group and BT group.The general state of the mice was daily observed.28 days later,the mice were sacrificed,and the left lung tissue was taken for Masson staining and HE staining and the right lung tissue was taken for Western Blot to detect protein expression of ?-SMA and LC3 II.Results:(1)Compared with mice in NS group and BC group,the general state in BLM group and BT group were worse,they had sparse hair,no gloss,breathe hard and stayed inactive.Their food intake and water intake reduced and they were lighter than the other two groups.But the general state in BT group mice was slightly better than that in BLM group.(2)Dissected the mice after anesthesia and found that the lungs of mice in NS group and BC group were ruddy in color and soft in texture.The lungs of mice in BLM group and BT group were pale in color and hard in texture.(3)After bleeding from the abdominal aorta,the left lungs of mice were taken for pathological sections for the next Masson staining and HE staining.The lung tissue structure of mice in NS group and BC group wasintact,while a large amount of collagen fibers were deposited in BLM group and a small amount of collagen fibers were deposited in BT group.(4)After bleeding from the abdominal aorta,the right lungs of the mice were taken for Western Blot.The protein expression level of ?-SMA in BLM group was obviously higher than that in NS group.And the expression of ?-SMA in BT group was obviously lower than that in BLM group.The protein expression level of LC3 II in BLM group was lower than that in NS group.And the expression of LC3 II in BT group was higher than that in BLM group.Conclusions:(1)ERK5 takes an important role in TGF-?1-reduced phenotypic transformation of HLFs by inhibiting cell autophagy and apoptosis and promoting proliferation.(2)ERK5 promotes the phenotypic transformation of lung fibroblasts and participates in the occurrence and development of pulmonary fibrosis in vitro and in vivo.(3)ERK5 inhibitor BIX02189 can alleviate the pathogenesis of bleomycin-induced pulmonary fibrosis in mice.