HCECs from cultures of <30 passages were provided by the Department of Plastic, Shandong Provincial Qianfoshan Hospital affiliated Shandong University (Shandong, China); the cells were purchased from the American Type Culture Collection (Manassas, VA, USA). The HCECs were cultured in a growth medium consisting of 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 IU/ml penicillin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), 100 IU/ml streptomycin (Sigma-Aldrich; Merck KGaA) and Dulbecco's modified Eagle's medium/F12 (Invitrogen; Thermo Fisher Scientific, Inc.). The cells were maintained in a humidified atmosphere with 5% CO₂ at 37°C. When the cells had formed a sheath on the bottom of the culture vessel, they were re-suspended by incubation with 0.125% trypsin, collected and seeded at a ratio of 1:2 in a fresh 25 cm² culture flask. They were then sub-cultured in 5 ml complete medium.

When the HCECs had grown to ~80% confluency, the residual growth medium was discarded and replaced with 1 ml pre-heated PBS. The cells were then gently agitated and washed with PBS. Next, the culture dish lids were removed and the cells were placed 12 cm below an UVB lamp (wavelength range, 250–350 nm; peak wavelength, 297 nm) for direct irradiation. The intensity of UV radiation directly measured at a 12-cm vertical distance below the UVB lamp was 20 µw/cm², and was calculated using the following formula: H=TxE; where H was the radiant exposure (J/cm²), t was the exposure duration (seconds) and E was the measured irradiance (W/cm²). Radiation dosages corresponding to UVB irradiation times of 1, 2 and 3 h were 0.072, 0.144 and 0.216 J/cm², respectively. Following irradiation, the cells were once again cultured in fresh medium.

Hst1 was obtained from the Chinese Peptide Company (Hangzhou, China). The sequence was DSH EKR HHG YRR KFH EKH HSH REF PFY GDY GSN YLY DN. Four doses of Hst1 (0, 10, 50 and 100 µg/ml) were added to the cells for 12 h prior to UV radiation treatment or other assays.

The viability of HCECs was measured by examining their general health and proliferation using the alamarBlue^® assay. HCECs (~10⁵) were seeded into the wells of 24-well plates and then incubated at 37°C with 5% CO₂ for 24 h. The cells were exposed to UV light when the cultures were ~75% confluent. Following UV exposure, the cultures were again incubated for 24 h. Subsequently, the medium was aspirated from each well and the cells were rinsed with 1 ml culture medium containing serum. After aspirating the residual medium, 1 ml 10% alamarBlue^® (Thermo Fisher Scientific, Inc.) plus growth medium without serum was added to each well, and the cells were incubated at 37°C for 3 h. Following incubation, the fluorescence in each well was measured at 530/590 nm using a Thermo Plate microplate reader (Rayto Life and Analytical Science Co. Ltd., Shenzhen, China).

The proliferation of cultured HCECs was evaluated using the MTT colorimetric assay, which is based on the chemical reduction of MTT by living cells to form formazan crystals. In brief, HCECs were seeded into 96-well tissue culture plates (2×10⁴ cells/well) and incubated at 37°C under 5% CO₂ for 0, 12, 24 or 48 h. Next, the cells were incubated in 100 µl MTT solution (5 mg/ml; cat. no. M6494; Invitrogen; Thermo Fisher Scientific, Inc.) for 3 h, and then washed with PBS. The purple formazan crystals formed in living cells were then solubilized with dimethylsulfoxide (Invitrogen; Thermo Fisher Scientific, Inc.). The absorbance of the formazan solution was measured at 450 nm using a Thermo Plate microplate reader (Rayto Life and Analytical Science Co. Ltd.).

Apoptosis assays were performed using the Annexin-V-FITC Apoptosis Detection Kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) according to the manufacturer's instructions. Early apoptotic cells were defined as annexin-V-positive or propidium iodide-negative cells. Analyses were performed using a Beckman Gallios Flow Cytometer (Beckman Coulter, Brea, CA, USA).

Total cellular RNA was isolated using TRIzol reagent (Takara Biotechnology Co., Ltd., Dalian, China). The first strand cDNA was synthesized using the PrimeScript™ 1st strand cDNA Synthesis kit (cat. no. 6110A; Takara Biotechnology Co., Ltd.). Next, RNA samples were subjected to qPCR analysis to measure their levels of IGF-1, B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X (Bax) mRNA. The primers used for IGF-1, Bcl-2, Bax and GAPDH are listed in Table I. qPCR reactions were conducted using the SYBR^® Fast qPCR mix (cat. no. RR430A; Takara Biotechnology Co., Ltd.). The PCR amplification conditions were as followings, pre-denaturation at 95°C for 30 sec, denaturation at 95°C for 5 sec and annealing at 60°C for 10 sec (40 cycles). The PCR products in 4 µl of the product mixture were separated by electrophoresis on a 1.25% agarose gel, the gel was stained with ethidium bromide solution for 15 min at <50°C. The densities of the various complementary DNA bands were analyzed by scanning their absorbance areas with an AlphaImager gel imaging and analysis system (ProteinSimple, San Jose, CA, USA). Alphalmager^® Ger Documentation (version 1.0; ProteinSimple) was used to assess the density the bands. Band intensity values for IGF-1, Bcl-2 and Bax were normalized to those of GAPDH and the relative expression was calculated using the 2^−ΔΔCq method (12).

Cellular levels of malondialdehyde (MDA; cat. no. A003-4) and superoxide dismutase (SOD; cat. no. A001-1) were detected using ELISA kits (both Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions. In brief, 100 µl of a prepared standard solution or sample was added to each well of a sample plate and incubated overnight at 4°C. The plate was then washed four times with buffer, and subsequently, 100 µl biotinylated primary antibody was added to each well, followed by incubation at room temperature (RT) for 1 h. The plates were then washed four times with buffer and incubated with peroxidase-conjugated secondary antibody for 45 min. Next, the plates were washed three times with buffer, and 100 µl tetramethylbenzidine was added to each well to induce a color reaction at RT, which was stopped after 30 min. The optical density of each well at 450 nm was determined using an automated microplate reader.

Cells were harvested in radioimmunoprecipitation assay lysis buffer and centrifuged at 13,200 × g for 15 min at 4°C. The supernatant fractions were collected and the protein concentration was determined by the Bio-Rad Protein Assay kit II (cat. no. 5000002; Bio-Rad Laboratories, Inc., Hercules, CA, USA). Protein samples (40 µg/lane of total protein) were separated by 12% SDS-PAGE and electro-transferred to polyvinylidene difluoride membranes (cat. no. IPVH00010; EMD Millipore, Billerica, MA, USA). The membranes were then incubated with 5% non-fat skimmed milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h. Next, the membranes were incubated overnight with anti-IGF-1 (1:1,000; cat. no. ab9572; Abcam, Cambridge, UK), anti-Bcl-2 (1:1,000; cat. no. 2872), anti-Bax (1:1,000; cat. no. 5023) and anti-GAPDH (1:2,000; cat. no. 97166) (all Cell Signaling Technology, Inc., Danvers, MA, USA) primary antibodies at 4°C, followed by a subsequent incubation with horseradish peroxidase-conjugated goat anti-mouse (cat. no. A4416) and anti-rabbit (cat. no. A6154) secondary antibodies (both 1:10,000; Sigma-Aldrich; Merck KGaA) for 1 h at room temperature. The membranes were then rinsed with TBST, blots were visualized with enhanced chemiluminescence western blot detection reagents (cat. no. 321096; Thermo Fisher Scientific, Inc.) and then exposed to an X-ray film (Kodak, Rochester, NY, USA). The resultant protein bands were scanned using a Gel-doc2000 imaging system (Bio-Rad Laboratories, Inc.) and analyzed using Quantity One 1-D Analysis software (version 4.6.9; Bio-Rad Laboratories, Inc.).

All data were analyzed using Predictive Analytics Software for Windows (version 18.0; IBM Corp., Armonk, NY, USA). Statistical significance was assessed by Student's t-test and one-way analysis of variance followed by Tukey's post-hoc test. Statistical values are expressed as the mean ± standard error of the mean. P<0.05 was considered to indicate a statistically significant difference between groups. All experiments were performed at least three times.

The percentage of HCECs undergoing apoptosis increased with increasing exposure to UV light (Fig. 1A). While 29.1% of cells exposed to UV radiation for 2 h had entered apoptosis (early and late apoptosis), this percentage increased to 48.4% after 3 h of exposure, indicating that UV radiation can greatly damage HCECs (Fig. 1B). Furthermore, cells exposed to UV radiation for 3 h demonstrated a significant loss of viability as measured by alamarBlue^® assay, while a 1 h exposure had little or no effect (Fig. 1C). Simultaneously, a UV-free group was set as a control, and cells in this group were treated under the above conditions apart from being irradiated with UV light. Cell apoptosis slightly increased in the UV-free group with time; however, compared with this group at 0 h, no significant differences were demonstrated. Based on these results, a 2 h UV radiation exposure period (total radiation dosage, 0.144 J/cm²) was selected for use in the subsequent UV-induced cell damage model.

Three concentrations of Hst1 (10, 50 and 100 µg/ml) were used to treat the HCECs prior to UV radiation. Cell proliferation, which was assessed by the MTT assay was increased in the 10, 50 and 100 µg/ml Hst1 treatment groups. Cell proliferation was significantly induced in the 50 µg/ml Hst1 treatment group compared with the control group (Fig. 2A). However, cell proliferation in the 100 µg/ml Hst1 treatment group was markedly lower compared with the 10 µg/ml Hst1 treatment group, but it remained higher than that in the control group. Furthermore, by performing an MTT assay the proliferation of irradiated HCECs pre-treated with Hst1 was demonstrated to be increased compared with that of untreated irradiated cells. The same cells were also subjected to analysis of cellular SOD and MDA levels. The results revealed that the Hst1 treatment groups had lower SOD levels (Fig. 2B), but higher MDA levels (Fig. 2C) than the control group (0 µg/ml and UV irradiated). When pre-treated with 50 µg/ml Hst1, the intracellular content of SOD was significantly decreased, while the MDA concentration was increased when compared with that in the control group. Therefore, 50 µg/ml was selected as the concentration of Hst1 to be used in the subsequent experiments.

It was then examined how Hst1 protected HCECs against UV-induced damage. qPCR analysis demonstrated that the mRNA expression levels of IGF-1 (Fig. 3A) and Bcl-2 (Fig. 3B) were upregulated after pre-treatment of HCECs with 50 µg/ml Hst1 prior to irradiation, while and Bax was downregulated (Fig. 3C). Furthermore, Hst1 inhibited irradiation-induced apoptosis in HCECs (Fig. 4A and B). In addition, Hst1 was found to affect IGF-1, Bcl-2 and Bax protein expression in a similar way to the corresponding mRNA levels: The expression of IGF-1 and Bcl-2 in HCECs treated with Hst1 was significantly upregulated when compared with that in non-treated control HCECs. By contrast, Bax expression was significantly downregulated in Hst1-treated cells after irradiation (Fig. 4C-E). These results suggested that Hst1 may be a potential prophylactic therapeutic agent to protect against UV damage. Further studies may demonstrate that Hst1 improves corneal epithelial wound healing in UV-damaged eyes.