Down‐regulation of miR‐21‐5p by pirfenidone to inhibit fibroblast proliferation in the treatment of acquired tracheal stenosis

Abstract Objective Treatment options for acquired tracheal stenosis (ATS) are limited due to a series of pathophysiological changes including inflammation and cell proliferation. Micro ribonucleic acid‐21‐5p (miR‐21‐5p) may promote the excessive proliferation of fibroblasts. However, various types of fibrosis can be prevented with pirfenidone (PFD). Currently, the effect of PFD on miR‐21‐5p and its biological function has not been clarified. In this study, PFD was evaluated as a potential treatment for ATS by inducing fibroblast proliferation in lipopolysaccharide (LPS)‐induced fibroblasts by targeting miR‐21‐5p. Methods For 48 h, 1 g/ml LPS was used to generate fibroblasts in vitro, followed by the separation of cells into four groups: control, PFD, mimic, and mimic + PFD. The Cell Counting Kit‐8 (CCK‐8) technique was adopted to measure the proliferation of fibroblasts. Real‐time quantitative polymerase chain reaction (RT‐qPCR) and Western blot (WB) were used to measure the relative expressions of tumor necrosis factor‐α (TNF‐α), transforming growth factor‐β1 (TGF‐β1), drosophila mothers against decapentaplegic 7 (Smad7) and collagen type I alpha 1(COL1A1) messenger RNA (mRNA) and proteins, respectively. Results (1) At 0, 24, 48, and 72 h, fibroblast growth was assessed using the CCK‐8 method. Compared with the control group, the mimic group showed the highest fibroblast viability, and the PFD group showed the lowest fibroblast viability. However, fibroblast viability increased in the mimic + PFD group but decreased in the mimic one. (2) RT‐qPCR and WB showed that the mimic group exhibited a significant up‐regulation in the relative expressions of TNF‐α, TGF‐β1, and COL1A1 mRNA and proteins but a down‐regulation in the relative expression of Smad7 mRNA and protein compared with the control one. In the PFD group, the results were the opposite. Nevertheless, the relative expressions of TNF‐α, TGF‐β1, and COL1A1 mRNA and proteins were increased, whereas that of Smad7 mRNA was decreased in the mimic + PFD group. The change was less in the mimic group. Conclusion PFD may have a preventive and curative effect on ATS by inhibiting fibroblast proliferation and the fibrotic process and possibly through down‐regulating miR‐21‐5p and up‐regulating Smad7 and its mediated fibrotic and inflammatory responses.