法舒地尔通过抑制兔尿道成纤维细胞 Rho／ROCK通路激活减少尿道损伤后狭窄的发生

摘要

目的：研究法舒地尔对创伤后兔尿道狭窄发生的影响及机制，观察兔尿道成纤维细胞活力、迁移以及细胞外基质合成情况。方法：利用显微外科技术建立兔尿道创伤动物模型，分为5组：假手术组，手术组，不同剂量（3 mg／kg、10 mg／kg、30 mg／kg）法舒地尔组，术后3个月逆行尿道造影测量狭窄直径。从兔尿道瘢痕组织提取成纤维细胞进行传代培养，培养液中分别加入TGF-β1（10μg／L）和／或法舒地尔（12.5μmol／L，25μmol／L，50μmol／L）。 MTT比色法检测细胞活力；用Transwell小室实验检测细胞迁移能力；Western blot法检测各组Rho相关激酶（ ROCK）、α-平滑肌肌动蛋白（α-SMA）以及胶原蛋白I和III的表达情况。结果：法舒地尔显著减少尿道损伤后狭窄发生（P＜0.05）。随着法舒地尔浓度的增加，成纤维细胞的活力受到抑制，细胞迁移能力逐渐减弱，ROCK、α-SMA以及胶原蛋白I和III的表达受到抑制；法舒地尔对兔尿道成纤维细胞外基质及ROCK表达起抑制作用，并呈剂量依赖性（ P＜0.05）。结论：法舒地尔可以通过下调TGF-β1诱导的兔尿道成纤维细胞Rho／ROCK通路，抑制细胞外基质形成，减少尿道损伤后狭窄的发生。%AIM:To investigate the role of Rho-associated kinase ( ROCK) inhibitor fasudil in the formation of rabbit urethral stricture after injury and to observe the cell activity , migration and extracellular matrix synthesis in the rabbit urethra fibroblasts.METHODS:The rabbit model of urethral stricture was established by microsurgical techniques .The rabbits were divided into sham operation group , operation group and fasudil (3 mg/kg, 10 mg/kg, 30 mg/kg) groups.The diameter of the stenosis was measured by retrograde urethrography 3 months after surgery .The fibroblasts were isolated from urethral scar, and then incubated with fasudil (12.5 μmol/L, 25 μmol/L, 50 μmol/L) in the presence of transforming growth factor-β1 (TGF-β1, 10 μg/L).The untreated cells were used for control .The cell activity was measured by MTT assay.The cell migration ability was tested by the method of Transwell chambers .The protein expression of ROCK , α-smooth muscle actin (α-SMA) , collagen I and collagen III was determined by Western blot analysis .RESULTS:Fasudil significantly reduced formation of urethral stricture after injury (P<0.05).Cultured rabbit fibroblasts with different con-centrations of fasudil inhibited the cell activity and cell migration ability (P<0.05).The protein expression of ROCK,α-SMA, collagen I and collagen III was also inhibited by treatment with fasudil in a dose -dependent manner ( P<0.05 ) . CONCLUSION:Fasudil inhibits the formation of extracellular matrix and reduces the incidence of urethral stricture after injury by down-regulating TGF-β1-induced Rho/ROCK pathway activation in the rabbit urethra fibroblasts .