Bromfenac Inhibits TGF-β1-Induced Fibrotic Effects in Human Pterygium and Conjunctival Fibroblasts
Bromfenac Inhibits TGF-β1-Induced Fibrotic Effects in Human Pterygium and Conjunctival Fibroblasts
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DOI：
10.1167/iovs.18-24743
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发表时间：
2019-03-01
影响因子：
4.4
通讯作者：
Yao, Ke
中科院分区：
文献类型：
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作者：
Chen, Kailin;Lai, Kairan;Yao, Ke
目的.非甾体抗炎药（NSAID）已显示出对几种疾病的抗纤维化作用。本研究旨在观察溴芬酸（一种非甾体抗炎药）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，并探讨其可能的作用机制。本研究中使用的细胞是原代HPF和HConFs，促纤维化活化由转化生长因子-β 1（TGF-β 1）诱导。采用Western blot、实时荧光定量PCR和免疫荧光（IF）检测TGF-β 1和溴芬酸对纤维连接蛋白（FN）、III型胶原（COL 3）和α-平滑肌肌动蛋白合成的影响Western blot检测HPFs和HConFs中α-SMA的表达，创伤愈合实验检测细胞迁移能力，Western blot检测HPFs和HConFs中α-SMA的表达，创伤愈合实验检测HCFs和HConFs中α-SMA的表达。CCK-8法检测细胞增殖能力;药物对TGF-β 1下游信号通路的抑制作用用于评估其与溴芬酸作用的可能关联。溴芬酸抑制TGF-β 1诱导的HGF中FN（0.59 ± 0.07倍，P = 0.008）、COL 3（0.48 ± 0.08倍，P = 0.001）和α-SMA（0.61 ± 0.03倍，P = 0.008）的蛋白表达。溴芬酸也减弱TGF-β 1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）和p-AKT的表达水平（0.66 +/- 0.08倍，P = 0.032），p-ERK 1/2（0.69 +/- 0.11倍，P = 0.003），和p-GSK-3 β-S9（0.65 +/- 0.10倍，P = 0.002）。PI 3 K/AKT抑制剂（wortmannin）和MEK/ERK抑制剂（U 0126）减少TGF-β 1诱导的HPF中FN、COL 3和α-SMA的合成。结论：HConFs细胞的上述结果与HConFs细胞相似。溴芬酸至少部分地通过灭活AKT和ERK途径来防止TGF-β 1诱导的HPF和HConF中FN、α-SMA和COL 3的合成。
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-beta 1). Western blot, quantitative real-time PCR, and immunofluorescence (IF) assays were used to detect the effects of TGF-beta 1 and bromfenac on the synthesis of fibronectin (FN), type III collagen (COL3), and alpha-smooth muscle actin (alpha-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-beta 1 were used to assess their possible associations with the effects of bromfenac.RESULTS. Bromfenac suppressed the TGF-beta 1-induced protein expression of FN (0.59 +/- 0.07 folds, P = 0.008), COL3 (0.48 +/- 0.08 folds, P = 0.001), and a-SMA (0.61 +/- 0.03 folds, P = 0.008) in HPFs. Bromfenac also attenuated TGF-beta 1-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-3 beta-S9 (0.65 +/- 0.10 folds, P = 0.002) in HPFs. PI3K/AKT inhibitor (wortmannin) and MEK/ERK inhibitor (U0126) reduced the TGF-beta 1-induced synthesis of FN, COL3, and a-SMA in HPFs. All the results were similar in HConFs.CONCLUSIONS. Bromfenac protects against TGF-beta 1-induced synthesis of FN, a-SMA, and COL3 in HPFs and HConFs at least in part by inactivating the AKT and ERK pathways.