Fasudil reduces the recurrence of urethral stricture treated by laser internal urethrotomy in rabbits

Abstract

Objective To establish a stable laser internal urethrotomy model in New Zealand rabbits with post-traumatic urethral stricture and to explore the role of Rho-associated protein kinase (ROCK) inhibitor fasudil in the recurrence of urethral stricture treated by laser internal urethrotomy. Methods Post-traumatic urethral stricture was established by microsurgical techniques. Thereafter, the rabbits underwent laser internal urethrotomy. The rabbits were allocated into 5 groups: the sham operation group, the operation with fasudil injection (3, 10, 30 mg/kg) groups, and the operation with normal saline group. After 3 months, the diameter of the urethra was evaluated by urethrography. The fibroblasts were isolated from urethral scar, and then incubated with fasudil (0, 12.5, 25.0, 50.0 μmol/L), or co-incubated with fasudil (0, 12.5, 25.0, 50.0 μmol/L) in the presence of transforming growth factor-β1 (TGF-β1, 10 μg/L). The cell activity was measured by methyl thiazol tetrazolium (MTT) assay. The protein expression of collagen Ⅰ, collagen Ⅲ and ROCK was detected by Western blotting. Results Fasudil significantly reduced the recurrence of urethral stricture treated by laser internal urethrotomy, the diameter of the urethra was (2.26±1.21), (5.57±1.13), (6.61±1.03), (7.59±1.39), (8.18±1.22) mm, respectively (F=49.207, P=0.000; F=7.010, P=0.001). Van Gieson stain showed that the content of collagen fiber was decreased as the dose of fasudil increased, the fiber density was 50.67±6.73, 48.51±6.58, 42.11±8.10, 38.52±4.98, 33.41±7.10, respectively (F=10.172, P=0.000; F=9.221, P=0.000). As the dose of fasudil increasing, the cell activity was decreased. In non-TGF-β1-treated group, after 24 h treatment of fasudil, the inhibitory rate was 0, 0.208 8±0.012 5, 0.364 0±0.012 4, 0.520 3±0.015 6, respectively (F=47.117, P=0.000); after 72 h treatment of fasudil, the inhibitory rate was 0, 0.244 2±0.013 4, 0.396 5±0.010 9, 0.556 8±0.011 1, respectively (F=53.069, P=0.000). In TGF-β1-treated group, after 24 h treatment of fasudil, the inhibitory rate was 0, 0.117 6±0.013 8, 0.294 1±0.014 5, 0.431 4±0.013 7, respectively (F=55.341, P=0.000); after 72 h treatment of fasudil, the inhibitory rate was 0, 0.150 2±0.013 5, 0.322 9±0.010 3, 0.459 6±0.010 9, respectively (F=61.217, P=0.001). And the synthesis of collagen Ⅰ and Ⅲ, and ROCK was also decreased as the dose of fasudil increased. The expression of ROCK was 0.398±0.011, 0.317±0.017, 0.254±0.032, 0.178±0.057, 0.549±0.024, 0.469±0.033, 0.381±0.027, 0.264±0.014, respectively (F=11.318, P=0.000); the expression of collagen Ⅰ was 0.509±0.036, 0.415±0.025, 0.337±0.046, 0.227±0.037, 0.872±0.044, 0.704±0.021, 0.582±0.019, 0.425±0.023, respectively (F=9.715, P=0.000); the expression of collagen Ⅲ was 0.387±0.019, 0.301±0.010, 0.230±0.033, 0.116±0.015, 0.507±0.042, 0.404±0.023, 0.339±0.036, 0.240±0.026, respectively (F=13.657, P=0.000). Conclusion Fasudil could be efficacious in preventing the recurrence of urethral stricture treated by laser internal urethrotomy and the formation of urethral scar, and the proliferation of fibroblasts by downregulating TGF-β1-induced Rho/ROCK pathway activation. Key words: Fasudil; Urethral trauma; Urethral stricture; Laser therapy