The human lens epithelial cell line (SRA01/04) was obtained from Guangzhou JiNiu Biotechnology Co., Ltd. (Guangzhou, China). SRA01/04 cells were cultured in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum (both from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C and in a humidified atmosphere containing 5% CO₂. When the cells reached ~80% confluency, they were treated with 0.25% trypsin solution, centrifuged (106 × g, 5 min, room temperature) and plated for subculture or frozen for storage.

Cells were treated with a concentration gradient of freshly-prepared H₂O₂ (0–150 µM) to mimic aging in vitro. Prolonged exposure to H₂O₂ to stimulate senescence was conducted as previously described (28). Cells cultured in the same medium without H₂O₂ served as the control group. The medium was changed with fresh H₂O₂-containing medium every 3 days for a total of 2 weeks. All experiments were performed with cells of passages 4–6 to avoid the influence of natural senescence.

Cell morphology observation was used as a method to evaluate cell senescence. Treated cells were observed by light microscopy (Olympus Corporation, Tokyo, Japan) at magnification, ×20.

A growth curve was performed to examine cell viability over time in the experimental groups. SRA01/04 cells were seeded at 1×10³ cells/well in 96-well plates and incubated overnight with normal medium. The following day, cells were treated with a concentration gradient H₂O₂ for different periods of time (0, 1, 3, 5, 7, 9, 12 and 15 days), with 5 replicates for each experimental group and for each time point. When reaching the stated time point, cells were incubated for 4 h at 37°C with 20 µl MTT (5 mg/ml; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), then the medium was removed and 150 µl DMSO was added to dissolve the formazan products in each well. Absorbance was measured at 570 nm using a microplate reader (Thermo Fisher Scientific, Inc.).

A SA-β-gal staining kit (Beyotime Institute of Biotechnology, Haimen, China) was used, according to the manufacturer's instructions. SRA01/04 were seeded on 6-well plates and treated with low doses of H₂O₂ as aforementioned. At stated time points, and after washing with PBS, cells were incubated with 1 ml fixing solution at room temperature for 15 min, washed three times with PBS, and then incubated with 1 ml freshly prepared cell staining solution at 37°C overnight (without CO₂). Stained cells were analyzed by light microscopy by observing at least 10 random fields for each sample and manually quantifying the % of SA-β-gal positive cells.

Following the 2-week treatment period, cells were washed once in PBS and a cell suspension of 1×10⁶ cells/ml was fixed in 70% cold ethanol at 4°C for 12 h. The cells were then washed with PBS and incubated with 100 µl RNase A for 30 min at 37°C. Finally, cells were stained with 400 µl propidium iodide in the dark for 30 min at 4°C. Cell cycle analysis was performed using a FACScan flow cytometer equipped with CFlow Plus version 1.0.264.15 (both from BD Biosciences, San Jose, CA, USA).

Total RNA was isolated from cultured cells using TRIzol reagent (Thermo Fisher Scientific, Inc.) and converted into cDNA using the PrimeScript RT reagent kit with gDNA Eraser (cat no. RR047A; Takara Bio, Inc., Otsu, Japan), according to the manufacturer's instructions. The concentration of total RNA was measured using a NanoDrop 2000 (Thermo Fisher Scientific, Inc.). qPCR was performed on a LightCycler 480 real-time PCR system (Roche Diagnostics, Indianapolis, IN, USA) with the SYBR Premix Ex Taq II kit (cat no. RR820A. Takara Bio, Inc.). The primers used for qPCR were: SMP30, sense 5′-CCGTGGATGCCTTTGACTATGAC-3′ and antisense 5′-GTAACAGGCCACCCAGAGCTTC-3′; GAPDH, sense 5′-GCACCGTCAAGGCTGAGAAC-3′ and antisense 5′-TGGTGAAGACGCCAGTGGA-3′. The thermocycling conditions were set up according to the manufacturer's instructions: 95°C for 30 sec, followed by 40 cycles at 95°C for 5 sec and 60°C for 30 sec, followed by a melting curve analysis of 1 cycle at 95°C for 5 sec, 60°C for 1 min and 50°C for 30 sec. The melting curve analysis confirmed that the amplification contained no primer dimers or non-specific PCR products. The experiment was repeated six independent times in triplicates and the results were calculated using 2^−ΔΔCq method (29).

Cells were extracted using lysis buffer (Beyotime Institute of Biotechnology) at 4°C for 30 min. The concentration of total protein was measured by bicinchoninic acid assay (Beyotime Institute of Biotechnology). Protein samples (50 µg/well) were boiled, separated on 10% sodium dodecyl sulfate polyacrylamide gel, and then electrophoretically transferred onto polyvinylidene fluoride membranes. Blots were subsequently incubated for 1 h at room temperature with blocking buffer, which contained 5% (w/v) skimmed milk powder in TBS + 0.1% Tween-20 (TBST). The blots were subsequently incubated with antibodies targeting SMP30 (cat no. ab67336; dilution 1:1,000; Abcam, Cambridge, MA, USA) and β-actin (cat. no. 12262; dilution 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA) overnight at 4°C. Blots were then washed with TBST for 30 min. All blots were incubated with a IRDye 680RD goat anti-mouse secondary antibody (cat. no. 926-68070; dilution 1:15,000; Li-Cor Biosciences, Lincoln, NE, USA) for 2 h at room temperature. Following washing with TBST, protein signals were measured using the Odyssey Infrared Imaging System v3.0 (Li-Cor Biosciences). The experiment was repeated six independent times in triplicates.

All experiments were repeated at least three times. Statistical analysis was performed with SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). Results were expressed as mean ± standard deviation. Differences between groups were analyzed for significance by one-way analysis of variance followed by Fisher's least significant difference test. P<0.05 was considered to indicate a statistically significant difference.

Following observation by microscopy, no significant changes in cell morphology were observed between the untreated control group and the 25 µM H₂O₂ group (Fig. 1). Exposure to 50 and 75 µM H₂O₂ resulted in somewhat enlarged cell morphology and a slightly decreased cell density in 5–7 days (Fig. 1). Continuous culture for two weeks at 75 µM H₂O₂ resulted in a markedly decreased cell growth and a flattened cell morphology with accumulation of granular cytoplasmic inclusions compared with the control group (Fig. 1). At the dose of 100 and 125 µM H₂O₂, cells appeared shrank, elongated and a larger number of floating cells were observed, while after 10 days cells gradually became detached and disrupted (Fig. 1). At 150 µM H₂O₂ exposure, cells were all dead in the first 3 days (Fig. 1).

SA-β-Gal staining is routinely used to detect senescent cells, and positive staining appears as cytoplasmic blue granular coloring under microscopic observation. As illustrated in Fig. 2A, positively stained cells appeared following exposure of the cells to certain doses of H₂O₂ for two weeks. There were nearly no positive cells in the control untreated group (2.15±0.68%) and the % of positive cells observed increased in a dose-dependent manner compared with the control group (Fig. 2B). Only a few positive cells appeared in the 25 µM group (13.37±3.50%; Fig. 2B) and 50 µM group (40.23±6.58%; Fig. 2B). The % of positive cells increased dramatically in the 75 µM group (90.48±6.80%; Fig. 2B) and 100 µM group (92.10±3.84%; Fig. 2B). In the 125 and 150 µM groups, although positive staining was observed, most cells appeared non-viable following the two-week exposure (Fig. 2A).

Cell growth was evaluated by MTT assay over the period of 15 days. Untreated control SRA01/04 cells grew slowly on days 1–3 of culture, while at day 5, cell growth turned to a logarithmic phase (Fig. 2C). In the H₂O₂ exposure groups, the 25 µM group exhibited no effect on cell survival compared with the control group (Fig. 2C). In the 50 µM group, the growth curve declined compared with control (Fig. 2C). The growth curve of the 75 µM group exhibited a small increase in the first 5 days of exposure, but then cell growth was arrested and maintained in a low level for the rest of the treatment (Fig. 2C). Finally, in the 100, 125 and 150 µM groups, growth declined substantially compared with control and was almost at zero for the whole duration of the experiment (Fig. 2C).

Cell cycle analysis (Table I and Fig. 3A) was performed to examine the proportion of cells in each phase of the cell cycle. In the 25, 50, 75 and 100 µM groups, the % of cells in the G0/G1 phase were decreased significantly compared with the control group (Fig. 3B). Meanwhile, the 50, 75 and 100 µM groups exhibited significantly higher % of cells in the S phase compared with the control group (Fig. 3B), as well as a significant increase in the G2/M cell populations compared with the control group (Fig. 3B). The 125 and 150 µM groups were all cell debris (Fig. 3A). The cell proliferation index (PI), which was calculated as the sum of the % of cells in the S plus G2/M phases, was significantly increased in the 25, 50, 75 and 100 µM groups compare with the control group (Fig. 3C). The results demonstrated that prolonged exposure to 50, 75 and 100 µM H₂O₂ disrupted the normal cell cycle progress, arrested SRA01/04 cells in the S and G2/M phases and increased the PI value compared with control.

These above experiments were employed to evaluate cell senescence in response to prolonged exposure to H₂O_2. Based on the results of the present study, treatment with 75 µM H₂O₂ for two weeks resulted in chronic oxidative stress and simulated aging of SRA01/04 cells in vitro, without causing cell death. Therefore, the dose of 75 µM H₂O₂ was selected to induce in vitro aging for the remainder of the experiments. The doses of 25, 50 and 100 µM were used to investigate the different grades of chronic oxidative stress.

In order to investigate the expression of SMP30 in senescent HLECs, SRA01/04 were exposed to H₂O₂ for two weeks to simulate senescence induced by chronic oxidative stress, then mRNA and protein expression levels were examined by RT-qPCR and western blotting. RT-qPCR analysis demonstrated that expression of SMP30 mRNA levels were significantly decreased following H₂O₂ exposure compared with the control group, to 0.7, 0.74, 0.5 and 0.4-fold of the control in the 25, 50, 75, 100 µM H₂O₂ groups, respectively (Fig. 4A). However, there was no difference between the 25 and 50 µM H₂O₂ groups (P=0.921), and no difference between the 75 and 100 µM H₂O₂ groups (P=0.053). As presented in Fig. 4B, at the protein level, SMP30 expression was no different in the 25 µM group (P=0.695) and the 50 µM group (P=0.126) compared with control. However, SMP30 expression was significantly decreased by >6-fold in the 75 and 100 µM groups compared with the control (Fig. 4B). No significant difference was observed in the SMP30 protein expression between the 75 and 100 µM groups (Fig. 4B).