转化生长因子β1诱导结膜成纤维细胞中NADPH氧化酶4的表达及纤维化反应

Transforming Growth Factor β1-Induced NADPH Oxidase-4 Expression and Fibrotic Response in Conjunctival Fibroblasts.

作者信息

Brown Karl David, Shah Manisha H, Liu Guei-Sheung, Chan Elsa C, Crowston Jonathan G, Peshavariya Hitesh M

机构信息

Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, Victoria, Australia 2Ophthalmology, University of Melbourne, Department of Surgery, Melbourne, Australia.

出版信息

Invest Ophthalmol Vis Sci. 2017 Jun 1;58(7):3011-3017. doi: 10.1167/iovs.16-20633.

DOI:10.1167/iovs.16-20633

PMID:28605812

Abstract

PURPOSE

Fibrotic scarring after ocular surgeries and chemical burn injuries can impede clarity of the cornea and cause vision impairment. Transforming growth factor β (TGFβ) signaling pathway is known to mediate fibrotic scarring, and NADPH oxidase-derived reactive oxygen species has been shown to be an effector molecule that facilitates TGFβ1-mediated responses. The present study explores the expression profile and functional importance of NADPH oxidase (Nox) in conjunctival fibroblasts. In addition, the effect of curcumin on the TGFβ1-induced NADPH oxidase expression and collagen synthesis was also investigated.

METHODS

The mRNA expression of Nox isoforms in rabbit conjunctival fibroblasts was measured by real-time PCR. The production of hydrogen peroxide (H2O2) and total collagen by these cells was measured by Amplex Red assay and Picro-Sirius red assay, respectively. Nox4 was knocked down by adenovirus-mediated siRNA targeting Nox4 (Adv-Nox4i).

RESULTS

We describe for the first time that conjunctival fibroblasts express mRNA encoding for Nox2, Nox3, Nox4, and Nox5. TGFβ1 was found to induce Nox4 mRNA expression and total collagen release by these cells (P < 0.05; n = 4), and both responses are blocked by Smad3 inhibitor SIS3. Suppressing Nox4 gene transcription with Adv-Nox4i completely attenuated TGFβ1-stimulated H2O2 release and collagen production by conjunctival fibroblasts (P < 0.05; n = 4-6). Similarly, curcumin also inhibited TGFβ1-induced Smad3 phosphorylation, Nox4-derived H2O2 production, and total collagen synthesis by conjunctival fibroblasts (P < 0.05; n = 4-6).

CONCLUSIONS

The present study suggests that TGFβ1-mediated production of collagen by conjunctival fibroblasts involves Nox4-derived H2O2 pathway and this effect of Nox4 is abrogated by curcumin. This mechanism might be exploited to prevent fibrotic scarring after surgeries and chemical burn injuries in the eye.

摘要

目的

眼部手术和化学烧伤后的纤维化瘢痕会妨碍角膜清晰度并导致视力受损。已知转化生长因子β（TGFβ）信号通路介导纤维化瘢痕形成，并且已证明烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶衍生的活性氧是促进TGFβ1介导反应的效应分子。本研究探讨了NADPH氧化酶（Nox）在结膜成纤维细胞中的表达谱及其功能重要性。此外，还研究了姜黄素对TGFβ1诱导的NADPH氧化酶表达和胶原蛋白合成的影响。

方法

通过实时聚合酶链反应（PCR）检测兔结膜成纤维细胞中Nox亚型的信使核糖核酸（mRNA）表达。分别通过Amplex Red法和天狼星红苦味酸染色法检测这些细胞中过氧化氢（H2O2）的产生和总胶原蛋白的含量。通过腺病毒介导的靶向Nox4的小干扰RNA（Adv-Nox4i）敲低Nox4。

结果

我们首次描述结膜成纤维细胞表达编码Nox2、Nox3、Nox4和Nox5的mRNA。发现TGFβ1可诱导这些细胞中Nox4 mRNA表达和总胶原蛋白释放（P<0.05；n = 4），并且这两种反应均被Smad3抑制剂SIS3阻断。用Adv-Nox4i抑制Nox4基因转录可完全减弱TGFβ1刺激的结膜成纤维细胞H2O2释放和胶原蛋白产生（P<0.05；n = 4 - 6）。同样，姜黄素也抑制TGFβ1诱导的结膜成纤维细胞Smad3磷酸化、Nox4衍生的H2O2产生和总胶原蛋白合成（P<0.05；n = 4 - 6）。