LIMK1 耗竭增强了 fasudil 依赖型对尿道成纤维细胞增殖和迁移的抑制作用。

LIMK1 depletion enhances fasudil-dependent inhibition of urethral fibroblast proliferation and migration.

机构信息

Departments of Urology, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China.

出版信息

J Cell Biochem. 2019 Aug;120(8):12977-12988. doi: 10.1002/jcb.28569. Epub 2019 Mar 12.

Abstract

LIM kinase 1 (LIMK1) is an important regulator of the cell cytoskeleton. This study aimed to examine the role of LIMK1 in mediating the effects of the Rho kinase (ROCK) inhibitor fasudil. In vitro cultures of urethral fibroblasts were divided into LIMK1 knockdown (LIMK1 KD) and LIMK1 control (LIMK1 NC) experimental groups. Each group was incubated with fasudil (50 μmol/L) with or without transforming growth factor β1 (10 ng/mL) for 24 hours. Wound healing and Transwell assays were performed to determine cell migration. Flow cytometry was used to determine apoptosis. LIMK1, collagen I, collagen III, phospho-myosin light chain (p-MLC), alpha smooth muscle actin (α-SMA), and phospho-Cofilin (p-Cofilin) expression was examined by Western blot analysis. The expression of LIMK1 was further validated in human urethral scar tissues. Transwell and wound healing assays revealed that the cells of the LIMK1 KD group exhibited significantly attenuated migration, when compared with those of the LIMK1 NC group ( P < 0.05). Cell migration was also attenuated in the LIMK1 KD group treated with fasudil ( P < 0.05). Flow cytometry analysis revealed that apoptosis was higher in the LIMK1 KD group than that in LIMK1 NC group ( P < 0.05). Apoptosis was also enhanced in the LIMK1 KD group treated with fasudil ( P < 0.05). Western blot analysis demonstrated that LIMK1, collagen I, collagen III, p-MLC, α-SMA, and p-Cofilin expression was significantly attenuated in both the fasudil-treated and untreated LIMK1 KD groups ( P < 0.05). LIMK1 was positively expressed in human urethral scar tissues while it was negatively expressed in normal urethra tissues. In conclusion, loss of LIMK1 expression inhibits the Rho/ROCK pathway-dependent proliferation and migration via downregulation of collagen I, collagen III, p-Cofilin, and α-SMA. LIMK1 loss can also enhance the inhibitory effects of fasudil on the proliferation and migration of urethral fibroblasts.

摘要

LIM 激酶 1（LIMK1）是细胞细胞骨架的重要调节因子。本研究旨在探讨 LIMK1 在介导 Rho 激酶（ROCK）抑制剂法舒地尔作用中的作用。体外培养尿道成纤维细胞分为 LIMK1 敲低（LIMK1 KD）和 LIMK1 对照（LIMK1 NC）实验组。每组用或不用转化生长因子β1（10ng/ml）孵育 24 小时，用 fasudil（50μmol/L）处理。划痕实验和 Transwell 实验用于测定细胞迁移，流式细胞术用于测定细胞凋亡。Western blot 分析检测 LIMK1、胶原 I、胶原 III、磷酸化肌球蛋白轻链（p-MLC）、α平滑肌肌动蛋白（α-SMA）和磷酸化原肌球蛋白结合蛋白（p-Cofilin）的表达。进一步在人尿道瘢痕组织中验证 LIMK1 的表达。Transwell 和划痕实验显示，与 LIMK1 NC 组相比，LIMK1 KD 组细胞迁移明显减弱（P<0.05）。LIMK1 KD 组用 fasudil 处理后细胞迁移也减弱（P<0.05）。流式细胞术分析显示，LIMK1 KD 组细胞凋亡高于 LIMK1 NC 组（P<0.05）。LIMK1 KD 组用 fasudil 处理后细胞凋亡也增强（P<0.05）。Western blot 分析显示，fasudil 处理和未处理的 LIMK1 KD 组 LIMK1、胶原 I、胶原 III、p-MLC、α-SMA 和 p-Cofilin 的表达均明显减弱（P<0.05）。LIMK1 在人尿道瘢痕组织中阳性表达，在正常尿道组织中阴性表达。结论：LIMK1 表达缺失通过下调胶原 I、胶原 III、p-Cofilin 和α-SMA 抑制 Rho/ROCK 通路依赖性增殖和迁移。LIMK1 缺失还可以增强 fasudil 对尿道成纤维细胞增殖和迁移的抑制作用。