The Mechanism Of Pirfenidone Inhibiting Gli2-mediated HAS2 Expression Affecting Fibroblast Activation And Alleviating Urethral Stricture Fibrosis In Rats

Objective:To investigate the effect and mechanism of pirfenidone on fibrosis of Urethral stricture in rats.Method:Rat urethral stricture model was established in vivo.HE,Masson and immunohistochemical staining were used to detect negative control group,positive control group and pirfenidone group,respectively group),Transforming growth factor-β 1(a-SMA and TGF-β 1)protein expression levels in urethra.The inhibitory effects of pirfenidone on cell proliferation,apoptosis,migration and invasion were detected by cck-8,flow cytometry,scratch and Transwell methods in vitro.Gene sequencing was used to detect the expression of cytokines in fibroblasts treated with pirfenidone.The expression of Hyaluronan Synthase2(HAS2)and the transcription factor Gli2 were significantly down-regulated.qRt-PCR,Western Blot,immunofluorescence and immunohistochemistry were used to verify the effect of pirfenidone on HAS2 in cells and urethra tissue.The influence of Gli2 gene protein expression level.The effect of pirfenidone on the expression levels of Glil,SMO and SHH proteins in fibroblasts was detected by Western Blot.Subsequently,the influence of small interference and overexpressed plasmid on cell proliferation,migration and invasion function was observed by transfection of small interference and overexpressed plasmid.The changes of cell functional phenotype were observed with or without the addition of pirfenidone at the same time of transfection of overexpressed plasmid.Finally,the regulatory relationships among pirfenidone,Gli2,HAS2 and TGF-β 1/Smad3 signaling pathways were verified by Western Blot.Results:Pirfenidone could inhibit the degree of urethral stricture fibrosis and the functional phenotype of fibroblasts in rats.Pirfenidone could significantly inhibit the expression of HAS2 and Gli2 in vivo and in vitro.Silenced HAS2 and Gli2 could inhibit the functional phenotype of fibroblasts,but overexpression could inhibit the functional phenotype of fibroblasts.In addition,pirfenidone inhibited Gli2 expression in a SHH-independent manner,and silenced Gli2 decreased HAS2 expression by inhibiting TGF-β 1/Smads signaling pathway.Conclusion:Pirfenidone can inhibit the expression of HAS2 and the functional phenotype of fibroblasts by regulating Gli2/TGF-β 1/Smads pathway,thus alleviating urethral stricture and fibrosis in rats.