Primary human RPE cells and ARPE-19 cell line culture
Human RPE cells were obtained from eye bank donor eyes, which were cut across the posterior pole, and the vitreous and neural retina were removed. Eyes were obtained from three male human donors between 30 and 50 years of age. None of the donors had a known history of eye disease. The eyes at time of receipt were all integral. RPE cells were isolated within 4 to 16 h after death.The remaining eyecup was washed with phosphate buffered saline (PBS, 8.00 g NaCl, 0.20 g KCl, 0.24 g KH2PO4 and 1.44 g Na2HPO4 in 1 l distilled water, pH 7.4) and incubated with 0.025% trypsin-ethylene diamine tetraacetic acid (EDTA; Invitrogen-Gibco, Carlsbad, CA) in a humidified chamber at 37 °C. The cells then were scraped gently and seeded in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen-Gibco) containing 15% fetal bovine serum (FBS; Invitrogen-Gibco).
A human retinal pigment epithelial cell line (ARPE-19) was purchased from American Type Culture Collection (ATCC; Manassas, VA). The cells were maintained in DMEM/F12 medium (Invitrogen-Gibco) supplemented with 10% FBS (Invitrogen-Gibco) and 1% penicillin and streptomycin, and were cultured at 37 °C in a humidified atmosphere of 95% air and 5% CO2. The medium was changed every two days. The ARPE-19 cells were used within 10 generations. Since cells of lower generations were quite sensitive to oxidative stress.
The ARPE-19 cells were seeded in flat-bottomed microculture 96 well plate (15,000 cells/well) and allowed to adhere for 24 h. Dose–response assays were performed on the ARPE-19 cells to determine the 50% inhibiting concentration (IC50) of hydrogen peroxide (H2O2). The cells were treated with 0 to 500 μM H2O2 in serum-free and phenol-free DMEM-F12 medium for 24 h. The 30% H2O2 stock solution was used within its three-month expiration limit. Working solutions of H2O2 were made fresh and added to the medium.
CP55,940 (Sigma, St. Louis, MO) is a nonselective CB1 and CB2 cannabinoid receptor agonist. ACEA (Tocris Bioscience, Ellisville, MO) is a selective CB1 agonist. JWH015 (Cayman, Ann Arbor, MI) is a selective CB2 receptor agonist. ARPE-19 cells were preincubated with various concentrations of CP55,940, ACEA, and JWH015 for 15 min before exposure to 200 μM H2O2 for 24 h in serum-free, phenol-red-free DMEM and F12 media at 37 °C.
For each concentration of H2O2 and the compounds, five wells were analyzed. Each experiment was performed at least three times.
Total RNA was isolated from primary human RPE cells, ARPE-19 cells and, H2
(200 μM)-treated ARPE-19 cells using the RNeasy Total RNA System (RNeasy Mini Kit, Qiagen, Valencia, CA) following the manufacturer's recommendation, and then treated with RNase-free DNase I to remove any contaminating genomic DNA. The isolated RNA had optic density (OD) 260/280 ratios greater than or equal to 2.0 [30
]. To synthesize a cDNA template for PCR, we reverse transcribed 1 μg of total RNA with oligo-(dT) primer and reverse transcriptase (ReverTra Ace, Toyobo Co., Ltd., Osaka, Japan). The quality of first-strand cDNA was confirmed by PCR with β-actin
Real-time reverse transcription-polymerase chain reaction (Real-time RT–PCR)
Real-time RT–PCR was performed for quantitative analysis according to the standard protocol using the SYBR Green PCR Master Mix (Toyobo Co., Ltd., Osaka, Japan). PCR conditions for CB1 and CB2 were as follows: after initial denaturation at 95 °C for 5 min, 40 cycles were performed at 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min, followed by a 10 min extension at 72 °C. For FAAH, the conditions were as follows: after initial denaturation at 95 °C for 5 min, 40 cycles were performed at 94 °C for 30 s, 62 °C for 30 s, and 72 °C for 1 min, followed by a 10 min extension at 72 °C.
Primers used were shown in . Quantification analysis of CB1, CB2, and FAAH mRNA was normalized with β-actin as reference. The specificity of PCR amplification products was checked by performing a dissociation melting curve analysis. Relative multiples of changes in mRNA expression were determined with the relative comparative threshold (CT) method.
Primer sequences used for real time RT–PCR.
CB1 and CB2 expression in the ARPE-19 cells was determined by immunofluorescent staining. In brief, ARPE-19 cells were grown to confluence in chamber slides (Nalgene-Nunc, Lab-Tek, Naperville, IL). The growth medium was aspirated and the cells were washed three times with PBS, and then fixed with 4.0% paraformaldehye for 20 min at 4 °C. After the cells had been washed with PBS, they were permeabilized with 0.2% Triton X-100 in PBS for 15 min at room temperature. Subsequently, CB1 and CB2 expression in the ARPE-19 cells was determined by immunofluorescent staining using a 1:100 dilution of anti-CB1 (rabbit polyclonal, Abcam, Cambridge, UK) or anti-CB2 (rabbit polyclonal, Abcam), respectively, for 6 h at 4 °C. After the cells had been rinsed with PBS, they were probed with 1:250 goat anti-rabbit FITC (Pierce, Rockford, IL) for 1 h at room temperature. Their nucleus was counterstained with 4’,6-diamidino-2-phenylindole (Molecular Probes, Invitrogen-Gibco, Carlsbad, CA). Images were obtained with a fluorescent microscope (Olympus IX 81) at 20X objective with 1.5X optical zoom (Olympus IX 81, Olympus Optical, Tokyo, Japan).
MTT assay for cell viability
An 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay is a qualitative index of cell viability. Mitochondrial and cytosolic dehydrogenases of living cells reduce the yellow tetrazolium salt (MTT) to produce a purple formazan dye that can be detected spectrophotometrically [31
]. After ARPE-19 cells were preincubated with various concentrations of CP55,940, ACEA, and JWH015 for 15 min followed by exposure to 200 μM H2
for 24 h. Then, MTT (Sigma, St. Louis, MO) was added to a final concentration of 0.5 mg/ml and incubated for 4 h at 37 °C. The culture medium was then removed and the remaining blue precipitate was solubilized in dimethyl sulfoxide, followed by reading absorbance at 570 nm in a plate reader using 630 nm as a reference (Spectra Max 340; Molecular Devices, Sunnyvale, CA). This reading was divided by the adjusted absorbance reading of untreated cells in control wells to obtain the percentage of cellular survival.
Reactive oxygen species determination
The intracellular reactive oxygen species (ROS) level is an important biomarker for oxidative stress. An increased ROS level generally indicates increased oxidative stress. Relative ROS production was determined by the formation of a fluorescent Dichlorofluorescein (DCF) compound on oxidation of the nonfluorescent, reduced DCF-DA [32
]. Cells were incubated with 10 μM DCF-DA at 37 °C for 30 min, and then washed twice with PBS. Relative fluorescence was measured using a fluorescence plate reader at 485 nm excitation and 535 nm emission wavelength (Wallace, Perkin-Elmer, Warrington, UK).
Western blot analysis
We plated the ARPE-19 cells into six-well plates (150,000 cells/ml). To evaluate the expression of CB1 and CB2 receptors and FAAH, we treated the cells with 200 μM H2O2 in serum-free and phenol-free DMEM and F12 medium for 24 h. As for the expression of PI3K/Akt and ERK1/2 protein, the cells were pretreated with or without 1 μM CP55,940 for 15 min and then exposed to 200 μM H2O2 for 24 h. After the treatment, the cells were rinsed twice with ice-cold PBS, then scraped into cell lysis buffer and centrifuged at 13400x g for 10 min at 4 °C. Protein levels were determined using the bicinchoninic acid (BCA) protein assay (Pierce). Next, 15 μg of total protein were solubilized in 2% sodium dodecyl sulfate (SDS) sample buffer, separated on 10% SDS-polyacrylamide gel by electrophoresis (SDS–PAGE) and transferred to nitrocellulose membranes by electroblotting. The blots were washed in Tris-buffered saline containing 0.1% Tween-20 and 5% nonfat dairy milk, and incubated in antibodies to 1:1000 dilution of rabbit polyclonal CB1 and CB2 receptors antibodies (Abcam), 1:1,000 dilution of mouse polyclonal FAAH antibody (Abcam), 1:3,000 dilution of rabbit polyclonal PI3K/Akt and ERK1/2 antibodies (Cell Signaling Technology, Beverly, MA), and 1:10,000 dilution of mouse monoclonal GAPDH antibody (Cell Signaling Technology) at 4 °C overnight. Blots were washed three times, incubated with 1:3,000 horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Pierce) or 1:20,000 HRP-conjugated goat anti-mouse IgG (Pierce) and developed using chemiluminescence (SuperSignal West Pico Luminescent; Pierce) according to the manufacturer’s instructions.
Data were presented as the mean±standard error of the mean (SEM) of the results of two or three separate experiments, as specified in the figure legends. Data were analyzed using ANOVA or a Student’s t-test with the SPSS software. A p-value <0.05 was considered significant.