溴芬酸抑制转化生长因子-β1诱导的人翼状胬肉和结膜成纤维细胞的纤维化作用。

Bromfenac Inhibits TGF-β1-Induced Fibrotic Effects in Human Pterygium and Conjunctival Fibroblasts.

机构信息

Eye Center, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, People's Republic of China.

Key Laboratory of Ophthalmology of Zhejiang Province, Hangzhou, People's Republic of China.

出版信息

Invest Ophthalmol Vis Sci. 2019 Mar 1;60(4):1156-1164. doi: 10.1167/iovs.18-24743.

DOI:10.1167/iovs.18-24743

PMID:30908581

Abstract

PURPOSE

Nonsteroidal anti-inflammatory drugs (NSAIDs) have shown antifibrotic effects on several diseases. The aims of the present in vitro study were to investigate the antifibrotic effects of bromfenac (a kind of NSAID) on primary human pterygium fibroblasts (HPFs) and primary human conjunctival fibroblasts (HConFs), as well as to explore the possible mechanisms of these effects.

METHODS

The cells used in this study were primary HPFs and HConFs, and profibrotic activation was induced by transforming growth factor-beta1 (TGF-β1). Western blot, quantitative real-time PCR, and immunofluorescence (IF) assays were used to detect the effects of TGF-β1 and bromfenac on the synthesis of fibronectin (FN), type III collagen (COL3), and alpha-smooth muscle actin (α-SMA) in HPFs and HConFs; the changes of signaling pathways were detected by Western blot; cell migration ability was detected by wound healing assay; cell proliferation ability was detected by CCK-8 assay; and pharmaceutical inhibitions of the downstream signaling pathways of TGF-β1 were used to assess their possible associations with the effects of bromfenac.

RESULTS

Bromfenac suppressed the TGF-β1-induced protein expression of FN (0.59 ± 0.07 folds, P = 0.008), COL3 (0.48 ± 0.08 folds, P = 0.001), and α-SMA (0.61 ± 0.03 folds, P = 0.008) in HPFs. Bromfenac also attenuated TGF-β1-induced cell migration (0.30 ± 0.07 folds, P < 0.001), cell proliferation (0.64 ± 0.03 folds, P = 0.002) and the expression levels of p-AKT (0.66 ± 0.08 folds, P = 0.032), p-ERK1/2 (0.69 ± 0.11 folds, P = 0.003), and p-GSK-3β-S9 (0.65 ± 0.10 folds, P = 0.002) in HPFs. PI3K/AKT inhibitor (wortmannin) and MEK/ERK inhibitor (U0126) reduced the TGF-β1-induced synthesis of FN, COL3, and α-SMA in HPFs. All the results were similar in HConFs.

CONCLUSIONS

Bromfenac protects against TGF-β1-induced synthesis of FN, α-SMA, and COL3 in HPFs and HConFs at least in part by inactivating the AKT and ERK pathways.

摘要

目的

非甾体类抗炎药（NSAIDs）已显示出对多种疾病的抗纤维化作用。本体外研究的目的是探讨溴芬酸（一种 NSAID）对原代人翼状胬肉成纤维细胞（HPFs）和原代人结膜成纤维细胞（HConFs）的抗纤维化作用，并探讨这些作用的可能机制。

方法

本研究使用的细胞为原代 HPFs 和 HConFs，转化生长因子-β1（TGF-β1）诱导成纤维细胞增殖。Western blot、实时定量 PCR 和免疫荧光（IF）检测 TGF-β1 和溴芬酸对 HPFs 和 HConFs 中纤维连接蛋白（FN）、III 型胶原（COL3）和α-平滑肌肌动蛋白（α-SMA）合成的影响；Western blot 检测信号通路变化；划痕愈合试验检测细胞迁移能力；CCK-8 检测细胞增殖能力；TGF-β1 下游信号通路的药物抑制作用评估其与溴芬酸作用的可能相关性。

结果

溴芬酸抑制 TGF-β1 诱导的 HPFs 中 FN（0.59±0.07 倍，P=0.008）、COL3（0.48±0.08 倍，P=0.001）和α-SMA（0.61±0.03 倍，P=0.008）的蛋白表达。溴芬酸还减弱了 TGF-β1 诱导的细胞迁移（0.30±0.07 倍，P<0.001）、细胞增殖（0.64±0.03 倍，P=0.002）以及 p-AKT（0.66±0.08 倍，P=0.032）、p-ERK1/2（0.69±0.11 倍，P=0.003）和 p-GSK-3β-S9（0.65±0.10 倍，P=0.002）在 HPFs 中的表达水平。PI3K/AKT 抑制剂（wortmannin）和 MEK/ERK 抑制剂（U0126）降低了 TGF-β1 诱导的 HPFs 中 FN、COL3 和α-SMA 的合成。在 HConFs 中也得到了类似的结果。