ABSTRACT

Purpose

The overexpression of transforming growth factor-beta1 (TGF-β1) after surgical excision often leads to excessive fibrosis, indicating the recurrence of pterygium. The aims of the present in vitro study were to investigate the role of RhoA/ROCK signaling in regulating fibrotic effects of primary human pterygium fibroblasts (HPFs), as well as to explore the possible mechanisms of these effects.

Methods

Pterygium samples were obtained from surgery, and profibrotic activation was induced by TGF-β1. Cell proliferation was detected by CCK-8 assay; cell migration was detected by wound healing assay; quantitative real-time PCR and Western blot were used to detect the effects of TGF-β1 and the role of RhoA/ROCK signaling in the synthesis of alpha-smooth muscle actin (a-SMA), type I and III collagen (COL1 and COL3), and matrix metalloproteinase-9 (MMP9) in HPFs. The changes of signaling pathways were detected by Western blot; and pharmaceutical inhibition of RhoA/ROCK signaling and its downstream MRFT-A/SRF transcription pathway were used to assess their possible mechanism in HPFs fibrosis.

Results

ROCK inhibitor Y-27632 decreased TGF-β1-induced cell proliferation and migration, reduced the TGF-β1-induced expression of profibrotic markers in HPFs, and suppressed TGF-β1-induced nuclear accumulation of Myocardin-related transcription factor A (MRTF-A) as well as accompanied elevation of F/G-actin ratio in HPFs. MRTF-A/Serum response factor (SRF) inhibitor CCG-100602 attenuated the TGF-β1-induced α-SMA expression and reduced myofibroblast activation in HPFs.

Conclusions

RhoA/ROCK signaling played a pivotal role in TGF-β1-induced fibrosis and myofibroblast activation in HPFs at least in part by inactivating the downstream MRTF-A/SRF transcriptional pathway.

中文翻译：

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RhoA/ROCK 信号通过 MRTF-A 调节人翼状胬肉成纤维细胞中 TGF-β1 诱导的纤维化作用

摘要

目的

手术切除后转化生长因子-β1（TGF-β1）的过度表达常导致过度纤维化，提示翼状胬肉复发。本体外研究的目的是研究 RhoA/ROCK 信号在调节原代人翼状胬肉成纤维细胞 (HPF) 的纤维化作用中的作用，以及探索这些作用的可能机制。

方法

翼状胬肉样本取自手术，TGF-β1诱导促纤维化活化。CCK-8法检测细胞增殖情况；伤口愈合试验检测细胞迁移；采用实时定量 PCR 和蛋白质印迹法检测 TGF-β1 的作用以及 RhoA/ROCK 信号在 α-平滑肌肌动蛋白 (a-SMA)、I 型和 III 型胶原蛋白 (COL1 和 COL3) 合成中的作用) 和 HPF 中的基质金属蛋白酶 9 (MMP9)。Western blot检测信号通路的变化；和药物抑制 RhoA/ROCK 信号及其下游 MRFT-A/SRF 转录途径被用来评估它们在 HPFs 纤维化中的可能机制。

结果

ROCK 抑制剂 Y-27632 降低 TGF-β1 诱导的细胞增殖和迁移，降低 TGF-β1 诱导的 HPF 中促纤维化标志物的表达，并抑制 TGF-β1 诱导的心肌素相关转录因子 A (MRTF-A) 的核积累) 以及 HPF 中 F/G-肌动蛋白比率的伴随升高。MRTF-A/血清反应因子 (SRF) 抑制剂 CCG-100602 减弱了 TGF-β1 诱导的 α-SMA 表达并降低了 HPF 中的肌成纤维细胞活化。

结论

RhoA/ROCK 信号传导在 TGF-β1 诱导的 HPF 纤维化和肌成纤维细胞活化中起关键作用，至少部分通过灭活下游 MRTF-A/SRF 转录途径。