法舒地尔经Rho/ROCK通路影响尿道瘢痕成纤维细胞表型转化及细胞外基质合成  

Effects of Fasudil regulates the phenotypic transformation and extracellular matrix synthesis of urinary tract scar fibroblasts induced by TGF-βl through Rho/ROCK signaling pathway

作　　者：刘宇[1] 刘海玲 祖建成[1] 赵夭望[1] 胡建军 颜冲 陈毅夫[1] 何天衢 Liu Yu;Liu Hailing;Zu Jiancheng;Zhao Yaowang;Hu Jianjun;Yan Chong;Chen Yifu;He Tianqu(Department of Urolog,Hunan Children's Hospital,Changsha 410007,China;Department of Pharmacy,the People's Hospital of Hunan Province,Changsha 410005,China)

机构地区：[1]湖南省儿童医院泌尿外科,长沙410007 [2]湖南省人民医院药剂科,长沙410005

出　　处：《国际泌尿系统杂志》2022年第6期1106-1110,共5页International Journal of Urology and Nephrology

基　　金：湖南省卫健委课题项目(202104051896);国家临床重点专科建设项目(湘卫医发[2022]2号)。

摘　　要：目的探讨法舒地尔通过Rho/ROCK信号通路调控转化生长因子TGF-β1(TGF-β1)诱导的尿道瘢痕成纤维细胞表型转化及细胞外基质合成的机制。方法体外分离培养人尿道瘢痕组织来源成纤维细胞,将其分为对照组、TGF-β1诱导模型组及法舒地尔组;采用CCK-8测定各组细胞的增殖情况,采用ELISA法测定细胞分泌Ⅲ型胶原蛋白(ColⅢ)及纤维结合素蛋白(FN)的表达情况,采用Western blot法检测Rho相关蛋白激酶1、2(ROCK-1、ROCK-2)、α-平滑肌肌动蛋白(α-SMA)的表达情况。再用梯度浓度法舒地尔处理TGF-β1诱导的尿道瘢痕成纤维细胞模型,采用同样方法检测评估尿道瘢痕成纤维细胞表型转化及细胞外基质合成情况。结果TGF-β1诱导的细胞培养液随着TGF-β1浓度增加作用时间延长,其增殖活性增高,且5和10 ng/mL TGF-β1诱导的细胞增殖率均显著高于0 ng/mL(均P<0.01)。ColⅢ、FN含量随TGF-β1浓度和作用时间的增加而增高,与浓度、时间呈相关性(均P<0.01)。12.5、25.0、50.0μmol/L法舒地尔组的活细胞增殖减少。各组细胞的ColⅢ、FN含量随法舒地尔的浓度增加而降低,与浓度、时间有相关性(均P<0.01)。随着不同浓度法舒地尔梯度干预后,细胞中ROCK-1、ROCK-2、α-SMA表达逐步降低(均P<0.01)。结论TGF-β1可体外诱导人尿道瘢痕成纤维细胞表型转化及细胞外基质过度合成;法舒地尔可通过下调ROCK-1、ROCK-2、α-SMA的表达水平,从而抑制尿道瘢痕成纤维细胞的表型转化,降低细胞外基质ColⅢ及FN的分泌。Objective To investigate the effects of Fasudil regulates the phenotypic transformation and extracellular matrix synthesis of urinary tract scar fibroblasts induced by TCF-β1 through Rho/ROCK signaling pathway.Methods Fibroblasts from human urethral scar tissue were isolated and cultured in vitro and divided into normal control group,TGF-β1-induced model group and fasudil inhibiting TGF-β1-induced drug group.CCK-8 was used to measure the proliferation of cells in each group,and ELISA was used to measure the expression of collagen Il and fibronectin secreted by cells.The expressions of Rho-associated protein kinase 1/2(ROCK-1,ROCK-2)andα-smooth muscle actin(α-SMA)were detected by Western blot.The TCF-β1-induced urethral scar fibroblast model was then treated with fasudil at gradient concentration.The phenotypic transformation and extracellular matrix synthesis of urethral scar fibroblasts were evaluated by the same method.ResultsThe proliferation activity of TGF-β1 induced cell culture medium increased with the increase of TCF-β1 concentration,and the cell proliferation rate induced by 5 and 10 ng/mL TGF-β1 was significantly higher than that of O ng/mL(all P<0.01).The contents of Col II and FN increased with the increase of TCF-β1 concentration and action time,and were correlated with the concentration and action time(all P<0.O1).The proliferation of living cells in 12.5,25.0,50.0μmol/L fasudil groups decreased.The contents of Col Il and FN in each group decreased with the increase of fasudil concentration,which was related to the concentration and time(all P<0.01).The expressions of ROCK-1,ROCK-2 andα-SMA in the cells were gradually decreased with the intervention of fasudil at different concentrations(all P<O.01).Conclusions TGF-β1 can induce phenotypic transformation and extracellular matrix oversynthesis of human urinary tract scar fibroblasts in vitro.Fasudil inhibited phenotypic transformation and decreased the expression of Col Il and FN in extracellular matrix by down-regulating the expression

关 键 词：瘢痕 尿道 法舒地尔 成纤维细胞 细胞外基质 

分 类 号：R699[医药卫生—泌尿科学]