The Role Of Nuclear Factor-kappa B Activation And Fibroblast Phenotype Transdifferentiation In Pulmonary Fibrosis

ObjectiveInterstitial lung disease consists of more than 200 kinds of diseases.There are some common characteristics of the clinical manifestation,laboratory and pathological changes in them and respective characters in each one.Activation of fibroblasts,proliferation and accumulation of a great deal of extra-cell matrix are the primary pathological characteristic of pulmonary fibrosis.Multiple factors can cause pulmonary fibrosis,such as professional dust(SiO₂ et al),virus(coral virus et al),radiational injury and some medicine(bleomycin et al).Besides,one kind of pathogenesis—unclear pulmonary fibrosis—idiologic pulmonary fibrosis.Although those pathogenesis are different,the development and result of fibrosis are same basically.Present the detail mechanism is st(?)unclear and the dramatic advance on its early effective prevention and treatment have still not be gotten.Nuclear factor-κB(NF-κB)is one critical transcription factor required for maximal expression of many cytokines involved in the pathogenesis of fibrosis.The activation of NF-κB is associated with the generation of pulmonary fibrosis.Blocking the activation of NF-κB could be proved to be an important method to the therapy of pulmonary fibrosis.IκB,B is the inhibitor of NF-κB and may regulate the function of NF-κB. Fibroblasts differentiate to myofibroblasts which expressα-smooth muscle actin play an important role in the lung fibrosis pathological processes.This paper aims to study the effect of NF-κB activation to fibroblasts differentiate to myofibroblasts and study the regulation of IκB to NF-κB.Provide a theoretical basis for the prevention of pulmonary fibrosis.Methods1,Animal model establishing and experimental grouping12 female C57BL/6 mice(8 weeks old,weight:18-20g,maintained in specific pathogen-free conditions,Beijing Weitong Lihua experimental animal technology company)were randomly divided into two groups,six mice each group.Mice were anesthetized with 10%chloralhydrate by intraperitoneal ingestion and operated to expose the trachea in sterile condition.Model group:mice were treated with Bleomycin A5(BLM,5mg/kg,dissolved in 20μl sterile isotonic saline)by intertratracheal ingection.Control group:mice were treated with sterile isotonic saline(20μl)by intertratracheal ingection.2,Cell cultureIn the first and the third days after animal model establishing selected three mice from each group randomly.Fibroblasts were primary cultured by using tissue explants culture technique and were subcultured 4 passages.3,The identification of fibroblastsCell morphological changes were observed by inverted phase contrast microscope and the expressions of vimentin and keratin were identified by immunocytochemical staining.4,indicators and detecting methodsp65,IκB-αandα-SMA protein expression of Fb in primary and secondary cultures were detected by immunocytochemical staining.P65 and IκB-αmRNA expression of Fb in primary and secondary cultures were detected by in site hybridization staining. Metamorph image analysis system was used to quantitative analysis the results of immunocytochemistry and in site hybridization.Analysis the correlation between indicators,research the relationship betweenα-SMA expression and NF-κB of Fb. Result1,The cultured cells showed typical shape and feature of fibroblast cell,which were successfully dissociated and purified by subcultured.The cells of 3rd to 4th passage cultured in vitro were shuttle-shaped with one or two chromatospherite.The margin between cells was clear and arranged in fish or palisade.2,In immunocytochemical study,the vimentin was found strong positive in the primary passage cells and keratin was scattered and weak positive.The cells of 4th passage did not express keratin but vimentin.3,The expression of p65,IκB-αandα-SMA protein and p65,IκB-αmRNA significantly augmented in pathological process by blemycin-induced pulmonary fibrosis(P＜0.01).4,The expression of p65 protein is positively correlated with IκB-αprotein(r=0.801,P＜0.05),and is positively correlated withα-SMA protein (r=0.764,P＜0.05);p65 mRNA is positively correlated with IκB-αmRNA(r= 0.731,P＜0.05).Conclusion1,Explant culture technique is suitable for culturing the fibroblast cells.2,The phenotypic transformation of fibroblasts to myofibroblasts exist in animal model of pulmonary fibrosis,continue exist of myofibroblast is a key factor of pulmonary fibrosis.3,Nuclear factor-kappa B activation might promote the phenotypic transformation of fibroblasts to myofibroblasts and then play an important role in pulmonary fibrosis.4,IκB-αmight play an anti-fibrosis role in pulmonary fbrosis by inhibiting NF-κB activity.