Samples of human Tenon's capsule were obtained from patients from the Zhongshan Ophthalmic Center (Sun Yat-sen University, Guangzhou, China), with the approval of the Ethical Committee of the Zhongshan Ophthalmic Center and in accordance with the Declaration of Helsinki. Informed consent was gained. Fresh tissues were cut into 1–2 mm pieces under a stereo microscope (×1.5), then placed in culture dishes with Dulbecco's modified Eagle's medium (DMEM)/F12 supplemented with 10% fetal bovine serum (both from Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C and 5% CO₂. After 8 days, primary cultures (~60% confluence) were passaged with 0.25% trypsin-EDTA (Invitrogen; Thermo Fisher Scientific, Inc.). Cells at passages 3–6 were used in subsequent experiments. Cell identification was performed by immunostaining with the fibroblast marker vimentin and cytokeratin (Abcam, Cambridge, UK). Briefly, the cells were seeded on coverslip with 80–90%, and were fixed with 4% paraformaldehyde at room temperature for 30 min. After washing with PBS, the cells were permeated with 0.1% TritonX-100 for 15 min at room temperature. After blocking with 5% BSA for 30 min at room temperature, cells were incubated with antibody against vimentin (cat. no. 5741; Cell Signaling Technology, Inc., Danvers, MA, USA; 1:200) at 4°C overnight. Cells were then incubated with the secondary antibody Alexa Fluor 488 of goat anti-rabbit IgG antibody (cat. no. ab150077; Abcam; 1:500) at room temperature for 1 h, The nuclei were stained with DAPI (5 mg/ml) at room temperature for 1 min. Immunofluorescence was visualized using a fluorescence microscope (LSM710; Zeiss AG, Oberkochen, Germany) at 512×512 pixel resolution. Images were collected using a ×20 apochromatic objective.

HTFs proliferation was determined by Cell Counting kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) assay. Briefly, a 96-well plate was seeded with HTFs (1×10⁴ cells/well) and incubated overnight at 37°C and 5% CO₂. Following treatment with TGF-β1 (5 ng/ml) alone or in the presence of 5 or 10 µM of U0126, 10 µl CCK-8 solution was added to each well and incubated at 37°C for 4 h. A microplate reader was used to measure the absorbance at 450 nm (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

HTFs (5×10⁵) were seeded into 6-well plates and incubated to 100% confluence. The cell monolayer was scratched with a 200-µl pipette tip and washed with PBS, following which fresh DMEM/F12 containing TGF-β1 (5 ng/ml) alone or combined with 5 µM of U0126 was added and the plates incubated for 24 h. The distance of cell migration was measured via phase-contrast microscopy (magnification, ×20).

A collagen contraction assay was performed using a Cell Biolabs Collagen Contraction Assay kit (Cell Biolabs, Inc., San Diego, CA, USA), according to the manufacturer's protocol. Briefly, HTFs (5×10⁵ cells/well) and 500 µl collagen preparation were added to a 24-well plate for polymerization. Following treatment with TGF-β1 (5 ng/ml) alone or combined with 5 µM of U0126 for 24 h, gels were detached from the wells and images were captured under a light stereo microscope (magnification, ×1.5). Analyses were performed using ImageJ software 2.1.4.7 (National Institutes of Health, Bethesda, MD, USA).

Extracts were prepared by lysing cells with radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) on ice for 10 min. Protein concentrations were quantified with a Micro bicinchoninic acid Protein Assay kit (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Protein lysates (30 µg) were separated via SDS-PAGE with 10% of separation gel and 3% stacking gel and then transferred to a polyvinylidene fluoride membrane (Thermo Fisher Scientific, Inc.). Proteins of interest were detected using primary antibodies against human α-SMA (cat. no. ab5694; 1:1,000) and GAPDH (cat. no. ab9485; 1:1,000; both Abcam), TGF-β1 (cat. no. sc-130348; 1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) Smad2 (cat. no. 5339; 1:1,000), Smad3 (cat. no. 9523; 1:1,000), phosphorylated (p)-Smad2/3 (cat. no. 8828; 1:1,000), p38MAPK (cat. no. 8690; 1:1,000), p-p38MAPK (cat. no. 4511; 1:1,000), ERK1/2 (cat. no. 4695; 1:1,000), p-ERK1/2 (cat. no. 4370; 1:1,000; all Cell Signaling Technology, Inc.) and zinc finger protein SNAI1 (cat. no. 3879; Cell Signaling Technology, Inc.; 1:1000), followed by incubation with mouse peroxidase-conjugated secondary antibodies (cat. no. sc-2357; Santa Cruz Biotechnology, Inc.; 1:2,000) for 1 h at room temperature. Specific proteins were visualized by enhanced chemiluminescence (cat. no. WBKLS0500; Merck KGaA, Darmstadt, Germany).

Total RNA was extracted from cultured cells using RNeasy spin columns (Qiagen Ltd., Crawley, UK) according to the manufacturer's protocol. cDNA was synthesized by reverse transcriptase from total RNA with PrimeScript RT Master Mix (Takara Bio Inc., Otsu, Japan). The RT reaction was conducted at 25°C, 5 min, followed by 55°C, 10 min, and the reaction was terminated by heating at 85°C for 5 min. qPCR was subsequently performed using the SYBR^® Premix Ex Taq^™ kit (RR820A; Takara Biotechnology Co., Ltd). The thermocycling conditions for qPCR is as follows: Initial denaturation at 95°C for 30 sec; 40 cycles of denaturing the cDNA template at 95°C for 10 sec and annealing/extension at 60°C for 30 sec. The gene expression levels were normalized to GAPDH according to the 2^−∆∆Cq relative quantification method (15). The primers used in the experiment were as follows: GAPDH forward, 5′-GGAGCGAGATCCCTCCAAAAT-3′ and reverse, 5′-GGCTGTTGTCATACTTCTCATGG-3′; α-SMA forward, 5′-AAAAGACAGCTACGTGGGTG-3′ and reverse, 5′-GCCATGTTCTATCGGGTACTTC-3′; Snail forward, 5′-TCGGAAGCCTAACTACAGCGA-3′ and reverse, 5′-AGATGAGCATTGGCAGCGAG-3′; TGF-β1 forward, 5′-GGCCAGATCCTGTCCAAGC-3′ and reverse, 5′-GTGGGTTTCCACCATTAGCAC-3′.

Cells were seeded onto coverslips once they reached 40–60% confluence. Upon treatment with the indicated agents, cells were fixed in 4% paraformaldehyde for 15 min at room temperature and then permeated with 0.1% Triton X-100 for 10 min. Following blocking with 3% bovine serum albumin (Sigma-Aldrich; Merck KGaA) for 1 h at room temperature, the cells were incubated with a primary antibody against α-SMA (cat. no. ab5694; Abcam; 1:200) or cytokeratin (cat. no. 5741; 1:200; Cell Signaling Technology, Inc.) at 4°C overnight, and then exposed to the Alexa Fluor^® 488-conjugated green fluorescence secondary antibodies (cat. no. ab150077; 1:500) or Alexa Fluor^® 555-conjugated of red fluorescence secondary antibodies (cat. no. ab150158; 1:500; both Abcam) for 1 h at room temperature. The nuclei were stained with DAPI (5 mg/ml) for 1 min at room temperature and images were captured using a Leica fluorescence microscope (magnification, ×20).

All experiments were performed at least three times and the data are presented as the means ± standard deviation. Comparisons among multiple groups were performed by one-way analysis of variance with Tukey's post-hoc test using GraphPad Prism version 7.0 (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

First, the present study aimed to identify the primary cultured HTFs. The cells were grown in a monolayer and exhibited a typical spindly, generally flat and elongated shape (Fig. 1). To identify HTFs at a molecular level, immunostaining using vimentin and cytokeratin, cell surface markers for fibroblasts and the epithelium, respectively, was performed. As indicated in Fig. 1, the HTFs exhibited extensive staining for vimentin and not for cytokeratin, suggesting that the isolated HTFs were of high purity.

Activated fibroblasts generally exhibit a high degree of proliferation and migration. To ascertain whether the inhibition of MEK signaling may suppress the HTF proliferation induced by TGF-β1, HTFs were incubated with TGF-β1 (5 ng/ml) alone or in the presence of 5 or 10 µM of the MEK1/2 inhibitor U0126. Cell proliferation was determined using a CCK-8 assay. It was identified that treatment with TGF-β1 for 24 h significantly increased the proliferation of HTFs (P<0.01; Fig. 2A), which was abrogated by pre-treatment with U0126. (P<0.05; Fig. 2A). However, no significant difference in the effects between 5 and 10 µM treatments was observed. Notably, treatment with U0126 alone had no significant effect on cell viability (Fig. 2B). Next, the effect of U0126 on HTF migration was evaluated using a scratch wound assays. As indicated in Fig. 2, cell migration was significantly increased upon treatment with TGF-β1 for 24 h (relative wound width=44.2±4.5% TGF-β1 vs. 57.6±2.1% vehicle control; P<0.001; Fig. 2C). This effect was also substantially attenuated by pre-treatment with 5 µM U0126 (relative wound width =73.6±6.1% TGF-1β+U0126 vs. 41.1±4.8% TGF-1β; P<0.01; Fig. 2C). Collectively, these results indicated that U0126 may markedly inhibit HTF proliferation and migration simulated by TGF-β1.

The transdifferentiation of fibroblasts into myofibroblasts is considered a pivotal step during fibrosis. At the molecular level, this process is characterized by the upregulation of α-SMA expression (16). To determine the effect of U0126 on the TGF-β1-induced conversion of fibroblasts into myofibroblasts, the changes in α-SMA mRNA and protein expression were evaluated via RT-qPCR and western blot analysis, respectively. As indicated in Fig. 3A and B, while TGF-β1 exposure increased α-SMA mRNA and protein expression, as expected, this effect was compromised by 5 µM U0126 (P<0.0001). Additional immunofluorescence was performed to monitor the expression of α-SMA following treatment with 5 µM U0126, anda similar result was obtained (Fig. 3C). In addition to α-SMA, previous studies have indicated that Snail, a transcription factor that serves a critical role in epithelial-mesenchymal transition (EMT), is also involved in the switching of fibroblasts into myofibroblasts. Therefore, the effect of U0126 on the expression of Snail induced by TGF-β1 was examined. The results demonstrated that, while untreated HTF cells exhibited low levels of Snail expression, incubation with 5 ng/ml TGF-β1 resulted in a marked increase in Snail expression (Fig. 4A and B). Similarly, 5 µM U0126 largely attenuated TGF-β1-induced Snail mRNA and protein expression (Fig. 4A and B). At the functional level, activated HTFs contribute to contraction of the extracellular matrix, a hallmark of scar formation (17). Therefore, whether U0126 may inhibit the collagen matrix contraction response induced by TGF-β1 was determined. As expected, TGF-β1 treatment lead to enhanced collagen matrix contraction compared with the vehicle control (P<0.0001; Fig. 4C). Treatment with 5 µM U0126 significantly decreased the collagen matrix contraction induced by TGF-β1 (P<0.0001; Fig. 4C). Altogether, these results support the hypothesis that U0126 may potently prevent HTF transdifferentiation into myofibroblasts.

TGF-β1 has been demonstrated to mediate the canonical Smad2/3 and the non-canonical MAPK pathways in various cell types. To reveal the mechanism underlying U0126-inhibition of HTF activation, the activation status of these signaling pathways in HTFs was evaluated. Smad2/3 and P38MAPK phosphorylation levels were detected by western blot analysis, and the results suggested that treatment with 5 ng/ml TGF-β1 induced activation of the Smad2/3 signaling pathway, as reflected by the increased levels of TGF-β1 mRNA and protein expression and Smad2/3 phosphorylation, which was in agreement with previous studies (18,19) (Fig. 5A). Furthermore, the phosphorylation of non-canonical p38MAPK and ERK1/2 proteins was also stimulated by TGF-β1 (Fig. 5B). Exposure to 5 µM U0126 markedly impaired TGF-β1-induced TGF-β1 mRNA and protein expression, and the phosphorylation of Smad2/3, p38MAPK and ERK1/2 (Fig. 5A and B). Therefore, the data suggested that U0126 prevents HTF activation through suppression of the Smad2/3, p38MAPK and ERK1/2 signaling pathways.