Hsieh Shu-Chung, Wu Chun-Chi, Hsu Shih-Lan, Feng Chin-Hsing, Yen Jung-Hsing
Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan; Department of Education & Research, Taichung Veterans General Hospital, Taichung, Taiwan.
Institute of Medicine, Chung-Shan Medical University, Taichung, Taiwan; Department of Medical Research, Chung Shan Medical University Hospital, Taichung, Taiwan.
Life Sci. 2016 Sep 15;161:19-26. doi: 10.1016/j.lfs.2016.07.011. Epub 2016 Jul 22.
To examine the effect and molecular mechanism of gallic acid (GA) on transforming growth factor-β1 (TGF-β1)-stimulated hypertrophic scar fibroblast (HSF) contraction.
A fibroblast-populated collagen lattice (FPCL) was developed to examine the effect of GA on TGF-β1-enhanced HSF contraction. The changes in crucial factors related to cell contraction including α-smooth muscle actin (α-SMA), F-actin, and the phosphorylation level of myosin light chain (MLC) were evaluated using western blot and immunostaining. The activation and expression of RhoA/ROCK after the TGF-β1 challenge and GA insult were evaluated using RhoA-G-LISA and RhoA-ELISA kit while the phosphorylation level of MYPT1 and the expression of ROCK1 and ROCK2 were examined by western blot, respectively.
GA significantly suppressed TGF-β1-stimulated HSF contraction in a dose- and time-dependent manner. Moreover, the TGF-β1-enhanced α-SMA expression, F-actin formation, and MLC phosphorylation were obviously attenuated by GA. TGF-β1 significantly stimulated RhoA activation but did not alter the expression of RhoA in the HSFs. However, both the activation and expression of RhoA decreased obviously with GA pretreatment followed by TGF-β1 stimulation. Furthermore, GA inhibited ROCK activity but did not affect its expression after TGF-β1 stimulation.
These results suggest that GA exhibited the potential to prevent HSF contraction after TGF-β1 stimulation by down regulating the RhoA/ROCK signal cascade, followed by the inhibition of the expression of α-SMA, F-actin formation, and phosphorylation of MLC.
研究没食子酸（GA）对转化生长因子-β1（TGF-β1）刺激的增生性瘢痕成纤维细胞（HSF）收缩的影响及其分子机制。
构建成纤维细胞填充胶原晶格（FPCL）以检测GA对TGF-β1增强的HSF收缩的影响。使用蛋白质免疫印迹法和免疫染色评估与细胞收缩相关的关键因子α-平滑肌肌动蛋白（α-SMA）、F-肌动蛋白和肌球蛋白轻链（MLC）磷酸化水平的变化。使用RhoA-G-LISA和RhoA-ELISA试剂盒评估TGF-β1刺激和GA处理后RhoA/ROCK的激活和表达，同时分别通过蛋白质免疫印迹法检测MYPT1的磷酸化水平以及ROCK1和ROCK2的表达。
GA以剂量和时间依赖性方式显著抑制TGF-β1刺激的HSF收缩。此外，GA明显减弱了TGF-β1增强的α-SMA表达、F-肌动蛋白形成和MLC磷酸化。TGF-β1显著刺激RhoA激活，但不改变HSF中RhoA的表达。然而，GA预处理后再进行TGF-β1刺激，RhoA的激活和表达均明显降低。此外，GA抑制ROCK活性，但不影响TGF-β1刺激后其表达。
这些结果表明，GA通过下调RhoA/ROCK信号级联反应，进而抑制α-SMA表达、F-肌动蛋白形成和MLC磷酸化，展现出预防TGF-β1刺激后HSF收缩的潜力。
