Effects Of IL-17A On Fibro Blast Phenotype Transdifferentiation In Pulmonary Fibrosis And Its Possible Mechanism

Objective:Lung fibroblasts were cultured in vitro and identified in the pulmonary fibrosis mice induced by Bleomycin,to study the effects of IL-17Aon the transdifferentiation of fibroblasts to myofibroblasts in pulmonary fibrosis and its possible mechanism to discuss its possible role in the pathogenesis of pulmonary fibrosis so as to provide a new theoretical basis for the prevention of pulmonary fibrosis.Methods:According to the initial study of IL-17RAmRNA expressive tendency at different stages in the development of pulmonary fibrosis of mice with bleomycin-induced pulmonary fibrosis,14-day Model mice whose IL-17RAmRNA reach the highest expression were used as this experimental research object,modeling method and process in the pulmonary fibrosis mice induced by Bleomycin consistent with the previous experiments.Taking lung tissues of14-day model mice used for subsequent experiments.Primary murine lung fibroblasts were purified with high purity by different centrifugal force and different adherence and were subcultured3Passages.Cell morphological changes were observed by inverted phase contrast microscope and the expressions of vimentin were identified by indirect immunofluorescence.a-SMA expression and localization of mice lung fibroblasts in primary culture and passing the third generation with IL-17A stimulated was detected by indirect immunofluorescence.α-SMA,Act1,p65,P-p65,IκB-α and P-IκB-α protein expression of mice lung fibroblasts with IL-17A stimulated at different time points(0h,lh,2h,4h) were detected by Western blotting. Results:1, The cultured cells showed typical shape and feature of fibroblast cell,which were successfully dissociated and purified by subcultured.The cells of3rd to4th 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, The indirectly fluorescent immunocytochemistry results:under the laser scanning confocal microscope,the cells of3rd passage in which the vimentin was found strong positive were observed,their cytoplasms were dark red and there were numerous collagen fibers being apart between them Above could prove that they were lung fibrob lasts.3,The immune fluorescence performance of a-SMA in the lung fibroblasts:under the laser scanning confocal microscope,in the control group,a few of lung fibrob lasts without IL-17A stimulated expressed the fluorescent signal of a-SMA,and the signal was weak and mainly located in the cytoplasm of the cells.On the contrary,in the lung fibrob lasts with IL-17A stimulated expression, the quantity and strength of the expression of the fluorescent signal of a-SMA were significantly stronger than the lung fibroblasts'without IL-17A stimulated,and these cells present the typical change of spindle.4, Western blotting shows that,with the stimulation of IL-17A(50ng/ml) to lung fibroblasts.at0hours group,there were weaker expression of a-SMA;In the1st,2nd and4th hours groups,there were more expression of a-SMA which are significantly higher than the0hours group's(P<0.05); Compared with the1st hours group's,in the2nd and4th hours gourps,there were significantly more expression of a-SMA(P<0.05);In the2nd and4th hours groups,there were no significant difference between the two groups of the a-SMA's expression level of lung fibroblasts(P<0.05).In the lst,2nd and4th hours groups,the expression of protein P-p65and P-IκB-a were significantly higher than the0hours group's(P<0.05);Compared with the1st hours group's,the expression of protein P-p65and P-IκB-a in the2nd and4th hours groups were significantly lower(P<0.05);In the2nd and4th hours groups,there were no significant difference between the two groups of the P-p65's and P-IκB-a's expression level of lung fibroblasts(P<0.05).At different time points(Oh,lh,2h,4h),there all were the expression of protein p65,IKB-a and Actl in the lung fibroblasts.But in these time points,the compares of the expression of protein p65,IκB-a and Actl of each two in the lung fibroblasts were no significant difference.Conclusion:1,The different centrifugal force and different adherence are a kind of reliable technology which successfully allows lung fibroblasts of the pulmonary fibrosis mice induced by Bleomycin,to be isolated,purified,and cultivated,and greatly improves the production of cells.Through the indirect immunofluorescence,the result of analysis of lung fibroblasts of mice point out that the vimentin immunofluorescence staining are positive.lt is suitable for the cells of3rd passage to study lung fibroblast for the experimental research object.2,IL-17A can participate in the formation of pulmonary fibrosis by promoting the transdifferentiation of fibroblasts to myofibroblasts.3,By activating the NF-κB signal transduction pathways,IL-17A may raise the expression of protein P-p65and P-lκB-a,and activate the dephosphorylated proteins into phosphorylated proteins to promote the transdifferentiation of fibroblasts to myofibroblasts.These eventually lead to the occurrence and development of pulmonary fibrosis.