Duloxetine (duloxetine hydrochloride, Cat#HT-B0161A), trolox (Cat#HY-101445), folic acid (Cat#HY-16637), rifampicin (Cat#HY-B0272), retinoic acid (RA) (Cat#HY-14649), rapamycin (Cat#HY-10219), chloroquine (Cat#HY-17589) and necrostatin-1 (Cat#HY-15760) were purchased from MCE (MedChemExpress, Shanghai, China). Z-VAD (Cat#S7023) and ferrostatin-1 (Cat#S7243) were purchased from Selleck Chemicals (Houston, TX, USA).
Mouse neuroblastoma cells (N2a cells) were purchased from ATCC (American Type Culture Collection), and mouse neural progenitor cells (C17.2 cells) were purchased from ECACC (European Collection of Cell Culture). N2a cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Cat#10099141, Gibco, Thermo Fisher Scientific, California, USA), 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in a humidified atmosphere of 5% CO2. C17.2 cells were cultured as described for the N2a cell protocol except that 5% horse serum was added to the medium (Cat#26050070, Gibco). The two cell lines were grown to 80% confluence, and then passaged by trypsin at a ratio of 1:4. The cell morphology of duloxetine-induced cytotoxicity and neurite outgrowth was recorded using a living-cell imaging system: a Leica MC170HD microsystem (Leica Microsystems, Wetzlar, Germany).
Cell viability assay
Cells were seeded in 96-well plates at a density of 2×105 cells per well, grown for 24 h, and then treated with the drugs according to time-dependence or dose-dependence protocols. Each treatment was conducted in triplicate. After the drug treatments, the cell viability was assayed using a Cell Counting Kit-8 (CCK-8) (Cat#CK04-3000T, Dojindo Laboratories, Japan) according to the manufacturer’s instructions. CCK-8 uses the sensitive colorimetric WST-8 assay to determine the number of viable cells. WST-8 is a highly water-soluble tetrazolium salt, with the chemical designation of 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt.
N2a cells were treated with the various indicated concentrations of duloxetine for 24 h. Triplicate wells of 6-well plates containing 1×103 cells were treated with various concentrations of duloxetine and maintained for another 21 days. The colonies were fixed with methanol, stained with a 0.1% crystal violet solution (Cat#G1064, Solarbio, Beijing, China) in 1 h at room temperature, and counted. The colony formation assay was repeated three times.
N2a cell differentiation
N2a cells were differentiated by a protocol that involved RA addition and serum withdrawal. The differentiation medium was DMEM supplemented with 20 μM RA, 100 U/ml penicillin and 100 μg/ml streptomycin. Neurites were identified as cell processes greater than two cell body diameters in length. Differentiated cells were defined as those bearing neurites. The percentage was statistically analyzed by counting 180 cells in six randomly chosen fields per well. The neurite length was defined as the distance from the cell body to the tips of the neurites. The length of the longest neurite was measured in at least 50 cells in five randomly chosen fields using ImageJ software. To evaluate the cell toxicity, N2a cells that were committed to differentiation with RA were treated with the addition of 12.5 μM duloxetine or 12.5 μM duloxetine plus 10 μM rifampicin for 24 hours. Statistical comparisons of the cell morphology were conducted between control, RA, RA+duloxetine and RA+duloxetine+rifampicin groups after a 24-hour treatment (n=3). The cell viability and cell morphology were recorded each day during the full differentiation period (n=3). Furthermore, the events associated with cell cycle and cell death were analyzed at various time points in the control and RA groups to permit interpretation of the key changes during the N2a cell differentiation. Statistical analyses of cell death (n=4), cell cycle (n=4) and biochemical changes (n=3) were conducted in the control, RA, RA+duloxetine groups after the 24-hour treatment.
Lactate dehydrogenase assay
Lactate dehydrogenase (LDH) is an intracellular enzyme that is released to the supernatant during cell death. The LDH release into the incubation medium after cell membrane damage was measured using an LDH diagnostic kit (Cat#C0016, Beyotime, Shanghai, China) according to manufacturer’s instructions. There were three repeats of each group for statistical analysis.
Lipid peroxidation assay
The lipid peroxidation level was determined by measuring the concentration of malondialdehyde (MDA), which is the end product of lipid peroxidation and reacts with TBA to form a fluorescence adduct. The total MDA quantities were determined using a Lipid Peroxidation MDA Assay Kit (Cat#S0131, Beyotime) according to manufacturer’s instructions. The total protein content was determined using the Pierce BCA Protein Assay Kit (Cat#23227, Thermo Fisher Scientific). The MDA level for each group was the total MDA divided by the total protein. There were three repeats of each group for statistical analysis.
Cell death assay
Cell death was determined by flow cytometric analysis of annexin V- and propidium iodide (PI)-positive cells and photography of TUNEL (TdT-mediated dUTP Nick-End Labeling) positive cells. For flow cytometry, the cell samples were trypsinized to single cells and stained using an annexin V-FITC apoptosis-detection kit (Cat#C1062M, Beyotime) according to manufacturer’s instructions. The populations of annexin V- and PI-positive cells were measured using flow cytometry (AttuneTM NxT Acoustic Focusing Cytometer, A24863, Thermo Fisher Scientific). For photography, the cells were fixed with 4% paraformaldehyde and stained using a TUNEL-FITC kit (Cat#11684817910, Roche, Basel, Switzerland) according to manufacturer’s instructions. DAPI staining was used to determine the total cell number. For statistical analysis, the positive ratios were determined in three visual fields using fluorescence microscope photography (TE2000, Nikon, Tokyo, Japan).
Cell cycle analysis
The populations of cells in the phases of the cell cycle were determined by DNA content as indicated by staining with PI. The cell samples were fixed with 70% cold ethanol at 4°C overnight. PI staining was conducted using a cell cycle and apoptosis analysis kit (Cat#C1052, Beyotime) according to manufacturer’s instructions. Flow cytometric analysis of the fluorescence intensity was used to evaluate the various phases of the cell cycle.
Enzyme-linked immunosorbent assay
Enzyme-linked immunosorbent assays (ELISA) were used to determine the serotonin (Cat#BAE-5900, Rocky Mountain Diagnostics, Colorado springs, CO, USA) norepinephrine (Cat#BAE-5200, Rocky mountain diagnostics), cytochrome P450 (CYP) 1A2 (Cat#xyD294Ra, IBL-America, Minneapolis, MN, USA) and CYP2D6 (Cat#xyD302Ra, IBL-America) levels. For the cell culture supernatant assays, samples of the medium were collected and immediately frozen at −80°C. For the assays of the intracellular levels, the cells were collected and lysed using liquid nitrogen, and the cell lysate dilutions were determined on the basis of the total protein assayed using the BCA kit. The concentrations were calculated according to standard curves prepared on the same ELISA plates according to the manufacturer’s instructions. There were three repeats of each group for statistical analysis.
The means ± SD were used to express quantitative results, and scatter plots were used to express proportional results. The comparisons of two groups were statistically analyzed using unpaired Student’s t-tests. ANOVA (Analysis of Variance) was used for comparisons among multiple groups, such as those for time- or dose-dependent changes. P values of < 0.05 were considered statistically significant for all.