2.1 Cell sources and ethics statement
Primary cultured RPE cells were isolated from three healthy men, aged 45, 49 and 35, within 24 h of their accidental deaths. the eyes were taken after keratoplasty at the Affiliated Hospital of Zunyi Medical Uni- versity. All experimentation adhered to the ethical standards established by the unit's Human Test Committee and approved by the Ethics Committee of Zunyi medical university, China (1-111).
2.2 Patient involvement
Patients were not directly involved in the design of this study.
2.3 Reagents and instruments
The following reagents and instruments were used: nifedipine (Sigma, Germany); a Reactive Oxygen Species Assay Kit (Beyotime, China); Dulbecco’s modified Eagle medium/F12 (HyClone, USA); fetal bovine serum (HyClone, USA); trypsin-EDTA solution, 0.25% (HyClone, USA); penicillin-streptomycin, liquid (Gibco, USA); LysoTracker Red DND-99 (Sigma, Germany); dimethyl sulfoxide (Solarbio, China); a DAB Substrate Kit (Zsbio Commerce Store, China); A2E (presented by the First Affiliated Hospital of Shanghai Jiaotong University); a medical blue light tube (Yingze, Tianjin); a high-speed tabletop centrifuge (Eppendorf, Germany); a Beckman Gallios flow cytometer (Beckman, USA); a Lysosome Isolation Kit (Invent, USA); and a Thermo U3000 Thermo Q Exactive Plus (Thermo, USA).
2.3.1 Primary culture of human RPE cells
Briefly, eye scissors were used to cut the eyeballs approximately 5~6 mm behind the corneal limb, and then the anterior segment was discarded. The vitreous body was removed, and the retinal neuroepithelium was carefully removed to isolate eye cups. The eye cups were digested with 0.25% trypsin for 30 min at 37 °C in a 5% CO2 atmosphere incubator. The cells were collected and resuspended in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Then, the cells were cultured in a 25 cm2 culture flask at a density of 5×104 cells/ml and kept at 37° in 5% CO2. The RPE cells were passaged at a ratio of 1:2, and cells at generation 4~6 were used for experiments. Each group comprised cells of the same generation from the same individual.
RPE cells were grown on glass coverslips coated with 0.01% polylysine. The cells were covered, fixed with precooled acetone for 10 min and incubated with 3% hydrogen peroxide for 15 min. After three washes with distilled water, the cells were permeated with 1% Triton-X for 30 min at room temperature and washed again with PBS. Then, the cells were incubated with goat serum for 10 min, followed by mouse anti-human RPE65 antibody overnight at 4°C and secondary goat anti-mouse IgG antibodies for 1h at 37°C. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI).
2.3.3 Exposure of RPE cells to blue light and A2E treatment
The human RPE cells from the fourth to sixth generation used for experiments were randomly divided into the following five groups: control group(CON), blue light group(BL), A2E-loaded group(A2E), A2E-loaded + blue light group(A2E+BL), and A2E-loaded + blue light + nifedipine group(A2E+BL+NIF). Before the cells were exposed to blue light, A2E was diluted to a final concentration of 25 µM in DMEM, and the cells were incubated with A2E for 2 h at 37 °C and then washed three times to remove extracellular A2E. Nifedipine at a final concentration of 10-5 mol/L was added 1 h before light exposure. Then, the RPE cells were exposed to blue light at an intensity of 2000 ± 500 lux for 6 hours, and the cells were cultured for 24 hours after exposure. Groups A and C were treated under the same conditions and wrapped in tin foil. Using a TES1330A illuminometer to measure the light intensity, the light intensity was adjusted by changing the distance between the culture flask or culture plate and the blue light tube.
2.3.4 Reactive oxygen species (ROS) levels in human RPE cells
After RPE cells were processed with blue light and A2E, they were washed twice with PBS, following which DCFH-DA diluted to a final concentration of 10 μM in DMEM was added and incubated at 37 °C for 20 minutes. Then, the cells were digested and centrifuged at 1000 rpm, the supernatant was discarded, and the pellet was resuspended in 300 µL of PBS containing 3% calf serum at a density of 106 cells/ml. The ROS concentration was analyzed by flow cytometry with the pass band filter set at 488 nm for excitation, and the emission of DCFH was detected at 525 nm. The data were analyzed with FlowJo software. In this experiment, the cells from 49 years old donor eyeball were used.
3.1 Isolation of cytoplasm and lysosomes
After completing the treatment described above, the cells were collected and washed once with cold PBS. The supernatant was completely removed, and the pellet was resuspended in buffer A. The cell suspension was incubated on ice for 10 min. The tube was vigorously vortexed for 20 seconds, following which the cell suspension was immediately transferred to the filter cartridge and centrifuged at 16000 × g for 30 seconds. Thee filter was discarded, and the pellet was resuspended by vigorous vortexing for 10 seconds. The sample was centrifuged at 2000 × g for 3 min (the pellet contained the nuclei, large cellular debris and some unruptured cells), and then all supernatant was transferred to a fresh 1.5 ml microfuge tube and centrifuged at 4 °C for 15 min at 11000 × g. The pellet contained mainly mitochondria and cellular debris. After centrifugation, the supernatant was carefully transferred to a fresh 1.5 ml tube and spun at 16000 × g at 4 °C for 30 min. The supernatant consisting of the cytoplasm was collected.
In addition, the pellet was resuspended in cold buffer A and centrifuged at 2000 × g for 4 min. The supernatant was carefully transferred to a fresh 1.5 ml tube. Buffer B was added to the tube, which was vortexed briefly to mix well (the supernatant to buffer B ratio was 2:1). The tube was incubated on ice for 30 min and centrifuged at 11000 × g for 10 min. The supernatant was removed completely, and the pellet was a highly enriched lysosome fraction. The experiment used cells from the eyeballs of 35 years old donors.
3.2 A2E levels in cytoplasm and lysosomes
Methanol was added to the cytoplasm obtained as described above, and the mixture was centrifuged twice at 4 °C for 15 min at 16000 × g. The supernatant was used for high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS). A reverse-phase C18 column (2.1×50 mm, 1.7 μm, Waters, UK) was used for the entire HPLC analysis. The mobile phase consisted of a 0.1% formic acid-water solution and acetonitrile. The injection volume for each run was 5 µL. The samples were eluted at a flow rate of 0.4 ml min-1.
Lysosomes obtained as described above were dissolved in ultrapure water, and methanol was then added to the suspension and centrifuged twice at 4 °C for 15 min at 16000 × g. The supernatant was used for HPLC-MS, and the detection method was as above.
3.3 A2E fluorescence in lysosomes
The coverslips were placed in 35 mm culture dishes, and 100 µL of cell suspension was added at a density of 105 cells/ml in 2 ml of culture medium. After processing with blue light and A2E, the cells were washed twice with PBS. Then, LysoTracker red DND-99 at a final concentration of 50 nM was added, and cells were stained in the dark for 20 min at 37°C. Next, the RPE cells were fixed with 4% paraformaldehyde for 20 min and washed with PBS three times, and the cover slips were fixed on slides. The fluorescence intensity of A2E in lysosomes was observed and imaged using a Leica laser scanning confocal microscope. Cells from 35-year-old eyeball donor.
4.1 Statistical analysis
These experiments were repeated at least 3 times. Data are expressed as the mean ± standard deviation, and SPSS 18.0 software was applied to assess differences among groups with one-way analysis of variance followed by the least significant difference (LSD) test. Differences with a P-value < 0.05 were considered statistically significant.