A Study on the Effect of IL-17A on Phenotypic Transformation of Fibroblasts in Bleomycin-induced Pulmonary Fibrosis in Mice and Its Mechanism

Abstract

Objective: In this study, lung fibroblasts were cultured and identified in mice lung fiber model with bleomycin. Under the induction of IL-17A, lung fibroblasts were gradually transformed into myofibroblasts in pulmonary fibrosis, and the specific induction effect of IL-17A in pulmonary fibrosis was analyzed, which could provide ideas for the prevention and treatment of clinical pulmonary fibrosis. Methods: To investigate the transcriptional expression of bleomycin-induced fractional pulmonary fibrosis in different pulmonary fibrosis processes. The 14-day mice model was taken as the research object, and the pulmonary fibrosis model was established by induction of myogenesis. After 14 days of modeling, lung tissue was removed, and after centrifugation and repeated adherent treatment, lung fibroblasts could be cultured at the origin. After three generations of culture, the morphological changes of lung fibroblasts could be observed under a microscope. Indirect immunofluorescence was used to establish the expression of vimentin, and IL-17 was used to stimulate primary cultured lung fibroblasts to detect the expression and specific localization of a-SMA in cells. Western blotting was used to stimulate the expression of lung fibroblast protein by IL-17A at different time points. Results: The typical characteristics of primary culture lung fibroblasts were obtained. After purification and culture, lung fibroblasts were obtained in morphology. The morphology of the 3rd and 4th generation cells was relatively uniform, showing long carboxyform. 1-2 nucleoli can be observed by microscope, which have distinct cell boundary and are lined up like fish schools. The results of indirect immunofluorescence showed that the vimentin staining in the third generation cells was positive, and the plasma was dark red. There were collagenous fibrous septa between the cells, which might make them develop into lung fibroblasts. A-SMA immunofluorescence results showed that in the absence of IL-17A induction, A-SMA signal was relatively weak in the lung fibroblasts of the control group and was in the cytoplasm, while after IL-17A induction, A-SMA signal was stronger in the lung fibroblasts of mice and the whole cells presented spindle structure. Western bletting showed that lung fibroblasts were stimulated by IL-17 in the 0h group. Compared with the 1h, 2h, and 4h groups, the expression of A-SMA in lung fibroblasts was significantly increased in the 1h, 2h, and 4h groups. The fibroblasts were very low in the 2h and 4h groups. There was no significant difference in the expression of AS MA signal. Compared with 0h, protein contents of p-IKB-a and p-p65 were higher in lung fibroblasts at 1h, 2h and 4h. Protein expressions of Acti, 1P6, IKB-a and P65 were different in lung fibroblasts, but there was no significant difference. However, there was no significant statistical difference in the expression of these proteins in lung fibroblasts at different times. Conclusion: By differential centrifugation and repeated adhesion, bleomycin-induced lung fibroblasts can be isolated and purified, and more cell production can be obtained. The staining vimentin was strongly positive after identification by indirect immunofluorescence. The stimulation of IL-17A could gradually transform non-fibroblasts into myofibroblasts and play an important role in pulmonary fibrosis. Therefore, through experimental studies, it was found that IL-17A stimulated F-kB signal and then increased the expression of P-IKB-a and P-P65 proteins, and transformed non-phosphorylated proteins into phosphorylated proteins, thus transforming lung fibroblasts into myofibroblasts and playing a role in pulmonary fibrosis.