In the present study, SK-N-SH human neuroblastoma cells were purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China). The cells were cultured in high glucose Dulbecco's modified Eagle's medium (DMEM; Biological Industries, Kibbutz Beit-Haemek, Israel) containing 10% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin (all purchased from Biological Industries). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂.

The present study determined that ethanol volatilized over time under normal culture conditions, thereby reducing the ethanol concentration in the culture medium. Therefore, ethanol volatilization was determined per 24 h and appropriate quantities of ethanol were added to maintain relatively stable concentrations of ethanol in the medium. 5 ml culture medium containing ethanol (50, 100, 200 and 400 mM) was added into a 25-cm² cell culture flask. After 24 h at 37°C, the remaining ethanol concentration was detected using headspace gas chromatography (cat. no. GC-14A; Shimadzu Corporation, Kyoto, Japan), as described in a previous study (27). The quantity of daily ethanol volatilization was calculated using the following equation: Quantity of daily ethanol volatilization = initial quality - remaining quality.

SK-N-SH cells were cultured in a 25-cm² cell culture flask at a density of 1×10⁶ cells/ml. Ethanol was added into DMEM culture medium at 37°C. When cells reached an 80–90% confluence, according to the duration of ethanol treatment, SK-N-SH cells were divided into 24, 48 and 72 h groups, and according to the ethanol concentration, cells were divided into 0 (control group), 50, 100, 200 and 400 mM groups. Ethanol at 0 and 100 mM was also used to treat SK-N-SH cells for 2 days at 37°C and cells were categorized into memantine (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and non-memantine groups. The concentration of memantine used was 4 µM. According to whether protein expression of NR1 was downregulated by RNA interference (RNAi), cells were divided into NR1 short hairpin RNA (shRNA) and control shRNA groups.

SK-N-SH cells were seeded in 6-well plates at a low density (<10% confluence) in normal growth medium containing various concentrations of puromycin (cat. no. sc108071; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) (0, 1, 2, 3, 4, 6, 8 and 10 µg/ml) to determine the minimum concentration necessary to kill all untransfected cells. An NR1 shRNA plasmid (cat. no. sc-91941-SH; Santa Cruz Biotechnology, Inc.) was used in the RNAi. It is a pool of 3 target-specific lentiviral vector plasmids, each encoding 19–25 nucleotide (plus hairpin) shRNAs designed to knock down gene expression. The control shRNA plasmid (cat. no. sc-108060; Santa Cruz Biotechnology, Inc.) encodes a scrambled shRNA sequence that does not degrade any known cellular mRNA. The NR1 shRNA plasmid and the control shRNA plasmid were of the same vector type. Each plasmid contained a puromycin resistance gene for the selection of stable cells that express the desired shRNA. At 60–70% confluency, shRNA Plasmid Transfection Reagent (10/200 µl, cat. no. sc-108061; Santa Cruz Biotechnology, Inc.) was used to transfect the NR1 shRNA plasmid (5/200 µl) and control shRNA plasmid (5/200 µl) into cells, according to the manufacturers' protocol. Following 48 h transfection at 37°C, the medium was replaced with fresh DMEM medium containing puromycin (1 µg/ml) to select the transfected cells over 5 days at 37°C. At 90% confluency, total protein was extracted and the transfection efficiency was determined by western blotting. The sequences of NR1 shRNA and control shRNA (28) are presented in Table I.

Cell viability was determined using an MTS kit (Promega Corporation, Madison, WI, USA). SK-N-SH cells were seeded at a density of 4,000 cells/well and treated in 96-well plates for various durations. Following 24 h culture, the cells were washed with PBS three times and fresh DMEM medium (100 µl) and MTS (20 µl) reagent were added to the wells for 1 h at 37°C in the dark (25,26). The absorbance was measured at a wavelength of 490 nm on an ELx808 absorbance reader (BioTek Instruments, Inc., Winooski, VT, USA). To eliminate possible interference by alcohol, cells treated with the same concentrations of alcohol (0, 50, 100, 200 and 400 mM) and memantine (4 µM) but without addition of assay reagents were used as blank controls. All experiments were repeated at least five times.

Cell apoptosis was measured using an Annexin V-fluorescein isothiocyanate (FITC)/PI apoptosis detection kit (BD Biosciences, San Jose, CA, USA). SK-N-SH cells were seeded at a density of 1×10⁶ cells/ml and treated in 25-cm² tissue culture flasks until an 80–90% confluence was observed. Following washing with PBS twice, cells were double-stained with FITC-conjugated Annexin V and PI for 15 min at 20°C in a Ca²⁺-enriched binding buffer in the kit. Cells were immediately analyzed on a flow cytometer in their staining solution. Annexin V and PI emissions were detected in the FL-1 (band pass, 530 nm; band width, 30 nm) and FL-2 (band pass, 585 nm; band width, 42 nm) channels. A total of 10,000–20,000 events were recorded per sample. BD FACSDiva V8.0.1 software (BD Biosciences) was used to analyze this data.

Intracellular Ca²⁺ was measured with the Ca²⁺-sensitive dye fura-2-acetoxymethyl ester (fura-2-AM; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) as previously described (29). SK-N-SH cells were seeded to 80–90% confluence and treated in 25-cm² tissue culture flasks. To prepare cell suspensions, the cells were washed twice with Hank's balanced salt solution (HBSS; Biological Industries), trypsinized (Biological Industries), centrifuged at a speed of 2,000 × g, for 5 min at room temperature) and resuspended in HBSS containing 20 g/l bovine serum albumin (Biological Industries), and the cell concentration was adjusted to 1×10⁶-1×10⁷ cells/ml. The survival rate of the cells was determined using a trypan blue staining cell viability assay kit (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufactures' protocol. A total of 0.1 ml cell suspension, containing 10⁵ cells was mixed with 0.4% trypan blue solution (0.1 ml). The mixture was then incubated for 3 min at room temperature. The quantity of living cells (which were not stained by trypan blue) were counted and determined to be >95%. SK-N-SH cells (1×10⁶-3×10⁶ cells/ml) were loaded with 3 mM fura-2-AM in HBSS at 37°C for 20 min. The cells were then washed once with HBSS and incubated for 1 h at 37°C in HBSS. The fluorescence of the cell suspension was monitored continuously using a F-4500 fluorescence spectrometer (Hitachi, Ltd., Tokyo, Japan) with excitation at 340 and 380 nm and emission at 500 nm. Triton X-100 (0.1%) and 10 mM EDTA were added to obtain the maximum and minimum concentrations of calcium, respectively.

SK-N-SH cells were seeded until an 80–90% confluence was reached and treated with ethanol (0, 50, 100, 200 and 400 mM) and memantine (4 µM) in 25-cm² tissue culture flasks. Cells were subsequently washed with Dulbecco's PBS (Biological Industries) and lysed on ice using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology) containing 1 mM phenylmethanesulfonyl fluoride (Beyotime Institute of Biotechnology). Lysates were harvested using cell scrapers, placed on ice for 30 min and then fragmented using an ultrasonicator. The lysates were centrifuged at 21,000 × g for 15 min at 4°C and quantified for total proteins using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology).

Equal quantities of protein (~30 µg) were separated using 10% SDS-PAGE and the separated protein was transferred onto polyvinylidene fluoride membranes. The membranes were blocked for non-specific binding with 5% non-fat dry milk in Tris-buffered saline containing 0.05% Tween-20 (TBS-T) for 2 h at room temperature and then probed with primary antibodies overnight at 4°C. The primary antibodies used were as follows: Rabbit anti-NR1 (1:500; cat. no. 13771-1-AP; ProteinTech Group, Inc., Chicago, IL, USA), rabbit anti-caspase-3 (1:200; cat. no. sc-98785; Santa Cruz Biotechnology, Inc.) and mouse anti-β-actin (1:1,000; cat. no. TA-09; OriGene Technologies, Inc., Rockville, MD, USA). Subsequently, the blots were washed with TBS-T three times (5 mins/wash) and incubated with the corresponding peroxidase-conjugated goat anti-mouse or anti-rabbit secondary antibodies (1:10,000; cat. nos. E030110-02 and E030120-02; EarthOx Life Sciences, Millbrae, CA, USA) for 2 h at room temperature. Protein bands were detected with enhanced chemiluminescence reagent (EMD Millipore, Billerica, MA, Germany). Chemiluminescent signals were detected and analyzed by a Tanon 5500 Chemiluminescent Imaging system (Tanon Science and Technology Co., Ltd., Shanghai, China). Band densities were analyzed semi-quantitatively using Image J 1.6.0 software (National Institute of Health, Bethesda, MD, USA).

Total RNA was extracted from cells using TRIzol^® (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and reverse transcribed into cDNA using a PrimeScript™RT Reagent kit (Perfect Real Time; Takara Biotechnology Co., Ltd., Dalian, China). qPCR was performed using a SYBR^® Premix Ex Taq™ II (TliRnaseH Plus; Takara Biotechnology Co., Ltd.) in a reaction volume of 20 µl on an ABI 7500 Real-Time PCR system (Thermo Fisher Scientific, Inc.) using the following thermocycling conditions: 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 34 sec, 95°C for 15 sec, 60°C for 60 sec and 95°C for 15 sec. The primer sequences used were as follows: β-actin forward, 5′-CTAACTTGCGCAGAAAACAAGAT-3′ and reverse, 5′-TTCCTGTAACAACGCATCTCATA-3′; and NMDAR1 forward, 5′-CGCCAACTACAGCATCAT-3′ and reverse, 5′-ATCGTCACAATCTTCAGTCT-3′. β-actin was used as the reference gene. The relative gene expression levels were represented as ΔΔ quantification cycle (ΔΔCq) and the fold change of gene expression was calculated via the 2^−ΔΔCq method (30). Experiments were repeated in triplicate.

GraphPad Prism version 6 (GraphPad Software, Inc., La Jolla, CA, USA) was used for statistical analysis. Measurement data are expressed as the mean ± standard error. One-way analysis of variance and Turkey's multiple comparisons test was used to compare differences between groups. The Wilcoxon rank sum test was used to compare differences among other types of data, including the percentage of apoptotic cells. P<0.05 was considered to indicate a statistically significant difference.

The ethanol volatilization per 24 h from 25-cm² flasks containing 5 ml culture medium with ethanol are presented in the Table II.

The relative expression levels of NR1 protein in the untransfected group and control shRNA group were similar (Fig. 1A and B). Compared with the control group, the relative NR1 protein expression level in the NR1 shRNA group was significantly lower (Fig. 1A and B). These results suggested that RNAi was successful and cells transfected with NR1 shRNA and control shRNA were used for subsequent experiments.

SK-N-SH cells were treated with increasing concentrations of ethanol for 24–72 h and then cell viability was measured. Compared with the 24 h/0 mM ethanol group, cell viability decreased with increasing ethanol concentration and time (Fig. 2A). Compared with the control group, the cell viability of the ethanol groups was significantly lower. Compared with the ethanol group, the cell viability of the ethanol + memantine and ethanol + NR1 shRNA groups was significantly higher (Fig. 2B).

SK-N-SH cells were treated with increasing concentrations of ethanol for 24–72 h. Annexin V-FITC/PI double staining was used to detect the apoptotic rate of cells. Apoptotic cells included early apoptotic and late apoptotic cells (Fig. 3A and B; Table III). Compared with the 0 mM ethanol groups at 24, 48 and 72 h, apoptosis was increased gradually with increasing ethanol concentration and time (Fig. 3A and B). Compared with the control group, apoptosis of the ethanol group was significantly higher. Compared with the ethanol group, apoptosis of the ethanol + memantine and ethanol + NR1 shRNA groups were significantly lower (Fig. 3C and D).

Western blotting revealed that the expression of cleaved caspase-3 was higher in the ethanol group compared with the control group, whereas expression of cleaved caspase-3 was significantly lower in the ethanol + memantine and ethanol + NR1 shRNA groups compared with the ethanol group (Fig. 4A and B).

Compared with the 0 mM ethanol groups at 24–72 h, the mean intracellular Ca²⁺ concentration increased with increasing ethanol exposure concentration and time (Fig. 5A). Compared with the control group, the mean intracellular calcium concentration in the ethanol group was significantly higher. Compared with the ethanol group, the mean intracellular calcium concentration in the ethanol + memantine and ethanol + NR1 shRNA groups was significantly lower (Fig. 5B).

SK-N-SH cells were treated with increasing concentrations of ethanol for 24–72 h. Whole proteins were extracted for western blotting and the relative expression levels of the NR1 protein was detected. β-actin was used as the internal reference. Compared with the 0 mM ethanol groups at 24–72 h, relative expression levels of the NR1 protein increased with increasing ethanol concentration and time (Fig. 6A-F). SK-N-SH cells were treated with increasing concentrations of ethanol for 24–72 h, and then the relative expression levels of NR1 mRNA were measured via RT-qPCR. Fig. 6G-I demonstrate that, compared with the 0 mM ethanol groups at 24, 48 and 72 h, the relative mRNA expression of NR1 increased gradually (excluding the 50 mM group at 24 h) with increasing ethanol concentration and time (P<0.05; Fig. 6G-I).