The acute retinal pigment epithelial 19 (ARPE-19) human RPE cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in high glucose Dulbecco's modified Eagle's medium (DMEM; HyClone; GE Healthcare Life Sciences, Logan, UT, USA) with 10% fetal bovine serum (Thermo Fisher Scientific, Inc., Waltham, MA, USA), penicillin (100 U/ml) and streptomycin (50 U/ml) in a 5% CO₂-humidified environment at 37°C.

The cells were seeded at a density of 4×10³ per well in 96-well plates and 8×10⁵ per dish in 60 mm dishes, and then cultured with lutein (Aladdin Chemical Co., Ltd., Shanghai, China) at concentrations of 0, 1, 5, 10 and 15 µM for 12 h at 37°C. Lutein was dissolved in dimethyl sulfoxide (DMSO; MP Biomedicals, Illkirch, France) with a stock concentration of 1 mM and maintained in the dark. Following washing once with PBS, the RPE cells were incubated in culture media containing 0, 200, 400, 600, 800, 1,000, 1,200, 1,600 and 2,000 µM H₂O₂ (Guangzhou Chemical Reagent Factory, Guangzhou, China) for 12 or 24 h at 37°C prior to the specific assays.

Following treatment with lutein or H₂O₂, the RPE cells were washed in PBS and incubated at 37°C in DMEM containing 0.25 mg/ml MTT. After 4 h, the MTT solution was removed and 150 µl DMSO was added to each well. The optical densities at 490 nm were read on a microplate spectrophotometer (Biotek Instruments, Inc., Winooski, VT, USA).

The RPE cells pretreated with lutein for 24 h were incubated with 800 µM H₂O₂ for another 24 h. The cell apoptosis, ROS levels and cell cycle were detected using a multicaspase kit, oxidative stress kit and cell cycle kit, respectively (Muse™; Merck Millipore, Darmstadt, Germany). All procedures were performed according to the manufacturer's protocols. The Muse™ Cell Analyzer software (version 1.3) was used for accurate statistical analysis.

Total RNA was extracted with TRIzol reagent (Takara Bio, Inc., Shiga, Japan). A 1 µg sample of RNA was reverse transcribed using a PrimeScript™ RT reagent kit (Takara Bio, Inc.), and the mixtures were incubated at 37°C for 15 min and 85°C for 5 sec. Subsequently, 1 µl (10 ng/µl) DNA was added to 5 µl SYBR Green I, 0.5 µl (10 µM) forward primer, 0.5 µl (10 µM) reverse primer and 3 µl ddH₂O, using the Universal qPCR kit (Kapa Biosystems, Wilmington, MA, USA). The qPCR was performed on a LightCycler® 96 sequence detection system (Roche Diagnostics, Basel, Switzerland) according to the manufacturer's protocol. The LightCycler® 96 application software version 1.1 was used for data collection and analysis. Relative quantitative analysis of interleukin (IL)-6, IL-8 and tumor necrosis factor-α (TNF-α) mRNAs were performed using the 2^−ΔΔCq method with normalization to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA (16). The system settings were as follows: Preincubation at 95°C for 60 sec and amplification at 72°C for 10 sec for 45 cycles, and melting at 97°C for 1 sec. The primer sets were designed as shown in Table I.

The cell proteins were extracted on ice using lysis buffer solution (20 mM Tris HCl, 150 mM NaCl, 10% glycerol, 1% NP-40, 2.1 g NaF, 1 mM PMSF, 1 mM Na₃VO₄, 10 µg/ml aptotin, 2 µg/ml leupeptin and 420 ml H₂O) containing phosphatase inhibitor (PhosSTOP; Merck Millipore). Protein concentrations were measured using a bicinchoninic acid assay kit (catalog no. C503021, Sangon Biotech Co., Ltd., Shanghai, China). A total of 20 µg protein from each sample was loaded onto 10% gels and subjected to SDS-PAGE, prior to transfer onto a nitrocellulose membrane for 75 min. The membrane was sealed with 5% defatted milk for 1 h, incubated with primary antibodies at 4°C overnight, and washed with 1X TBST for 10 min three times. The membrane was then incubated with secondary antibodies for 1 h at room temperature and washed with 1X TBST for 10 min three times. The ECL reagent Immobilon™ Western (EMD Millipore, Billerica, MA, USA) was added to the membranes for 1–3 min, and the immunofluorescence reaction was observed using a western blot luminescence imaging system (Tanon-5200; Tanon, Shanghai, China) and image analysis software (Gel Image System Ver. 4.2.5; Tonon). The antibodies used included tubulin α (cat. no. AF7010; Affinity Biotech, Kansas, MO, USA, GAPDH (cat. no. 60004–1-Ig; Proteintech Group, Inc., Wuhan, China), cyclin-dependent kinase 1 (CDK1; cat. no. BM1028; Boster Biotech, Wuhan, China), cyclin B1 (cat. no. BM0766; Boster Biotech) and cell division cycle 25C (CDC25C; cat. no. BM2728; Boster Biotech). The primary antibodies were diluted 1:500 and an anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (catalog. no. 7076S) or an anti-rabbit IgG Alexa Fluor® 555-conjugated secondary antibody (catalog no. 4413) from Cell Signaling Technology, Inc., Danvers, MA, USA, were diluted 1:2,000.

Each experiment was performed in triplicate. All analyses were performed using SPSS version 22.0 (IBM SPSS, Armonk, NY, USA). The data are expressed as the mean ± standard deviation and were statistically compared using Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

The cytotoxicity of lutein was assessed using an MTT assay for 3 days. Different concentrations of lutein were added to the cultural media of RPE cells. Compared with the control group of RPE cells cultured without treatment, the cell viability and proliferation of the RPE cells remained unchanged with concentrations of lutein up to 15 µM (Fig. 1A).

The RPE cells were treated with different concentrations of H₂O₂ (0–2,000 µM) for 24 h. Cell viability was also evaluated using an MTT assay. The results revealed that the cell viability reduced to ~50% of that in the control when the concentration of H₂O₂ reached 800 µM (Fig. 1B). Based on this result, 800 µM was selected as the concentration for inducing apoptosis and the production of ROS.

In the experiments, H₂O₂ reduced the cell viability of the RPE cells to 43.66% of the control. Lutein reversed the reduction in cell viability in a dose-dependent manner. When pretreated with lutein at concentrations of 5, 10 and 15 µM, the cell viability of the RPE cells was increased to 49.95, 65.39 and 74.32 of the control, respectively (Fig. 2A).

The expression levels of total caspases in the RPE cells increased to 66.3% when the cells were exposed to H₂O₂, compared with 11.1% in the control group. Lutein inhibited the increased expression of total caspases in a concentration-dependent manner. Following retreatment with lutein at concentrations of 5 and 10 µM, the expression of total caspases in RPE cells reduced to 49.3 and 26.9%, respectively. (Fig. 2B).

In the RPE cells treated with H₂O₂, the ROS levels increased to 65.21%, compared with 10.76% in the control group. Lutein reversed the elevation in ROS levels. The ROS levels reduced to 52.8 and 42.4% when the RPE cells were pretreated with 5 and 10 µM lutein, respectively (Fig. 2C).

In the present study, H₂O₂ markedly increased the expression levels of the IL-6, IL-8 and TNF-α inflammatory cytokines in the RPE cells (Fig. 3). When the RPE cells were pretreated with lutein at a concentration of 10 µM, the transcription levels of these inflammatory cytokines were also elevated, although pretreatment with lutein at a concentration of 5 µM did not alter the expression of these inflammatory cytokines.

When the concentration of H₂O₂ reached 400 µM, cell cycle arrest of the RPE cells was observed in the G₂/M phase. (Fig. 4A). It was found that, in the RPE cells treated with 600 µM H₂O₂, the proportion of cells in the G₂/M phase was 47.3%, compared with 35.9% in the control group. Lutein reversed the increased proportion of cells in the G₂/M phase in a concentration-dependent manner. When the cells were pretreated with 5 and 10 µM lutein, the proportions of RPE cells in the G₂/M phase were reduced to 40.8 and 33.4%, respectively (Figs. 4B and 5A).

When the RPE cells were treated with H₂O₂, the expression levels of CDK1 and CDC25C were inhibited, and the protein expression of of cyclin B1 was increased in the cells. However, the inactivation of CDK1 and CDC25C, and increase of cyclin B1 were attenuated when lutein was added to the cells (Fig. 5B and C).