The effect of verapamil on transforming growth factor-.BETA.1 induced alpha-smooth muscle actin and extracellular matrix of fibroblasts in cultured derived urethral scars

Abstract

Objective To investigate the effect of verapamil on transforming growth factor-β1 (TGF-β1) induced alpha-smooth muscle actin (α-SMA) and extracellular matrix of fibroblasts in cultured derived urethral scars,and the molecular mechanisms.Methods Fibroblasts were isolated from urethral scar and cultured in vitro.Cells were divided into five groups,cultured by cell medium containing 5 μg/L TGF-β1 and different doses of verapamil (0,10,50 and 100 μmol/L).After 72 hours incuation,the levels of α-SMA mRNA and Smad7 mRNA expression were assayed by semiquantitative real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) technique.The productions of collagenI,collagen Ⅲ,fibronectin and larninin were examined by enzyme linked immunosorbent assay (ELISA).Results TGF-β1 can significantly induce α-SMA mRNA expression.The α-SMA mRNA in each group were 0.497 ± 0.085,1.460 ± 0.062,1.070 ±0.066,0.780 ± 0.060,0.293 ± 0.067,respectively.With the increase of the verapamil concentration,the expression of α-SMA mRNA significantly was suppressed.The Smad7 mRNA in each group were 0.477 ± 0.065,0.223 ± 0.055,0.680 ± 0.053,0.833 ± 0.032,1.077 ± 0.060,respectively.Verapamil increased the expression of Smad7 mRNA in a dose-dependent manner (P ＜ 0.05).Verapamil decreased expression of the extracellular matrix in a dose-dependent manner (P ＜ 0.05).Conclusion Verapamil can inhibit the excessive synthesis of TGF-β1 induced α-SMA mRNA and extracellular matrix of fibroblasts in cultured derived urethral scars.The mechanisms may regulate the way of TGF-β/Smads,increasing the expression of Smad7. Key words: Urethral scar tissue; Fibroblasts ; Verapamil ; Transforming growth factor; Extracellular matrix