Glutamate, 6-OHDA, glucose oxidase and dithiothreitol were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from GE Healthcare Life Sciences (Logan, UT, USA). BD™ MitoScreen (JC-1) and an Annexin V/propidium iodide (PI) kit were purchased from BD Biosciences (San Jose, CA, USA). A calcium fluorescent probe (Fluo-3 AM) was purchased from Beyotime Institute of Biotechnology (Wuhan, China). Mouse anti-human anti-RIP kinase 1 (ab56815) and rabbit anti-human anti-RIP kinase 3 (ab180535) primary antibodies were obtained from Abcam (Cambridge, UK). Alexa Fluor 680-conjugated goat anti-mouse immunoglobulin G (IgG) (A28183) and Alexa Fluor 790-conjugated goat anti-mouse IgG (A28182) secondary antibodies were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA), and tetramethylethylenediamine was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Chemical reagents used for western blot analysis, including glycine, bisacrylamide, and acrylamide were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), and ammonium persulfate was obtained from Sangon Biotech Co., Ltd.

Human SH-SY5Y neuroblastoma cells were obtained from the China Center for Type Cultural Collection (Wuhan University, Wuhan, China). The cells were cultured in DMEM culture medium supplemented with 10% FBS and maintained at 37°C in a 5% CO₂ humidified atmosphere. Upon reaching 80% confluence, the cells were digested with 0.25% trypsin for 3 min until the majority of the cells detached. The cells were subsequently resuspended with DMEM culture medium containing 10% FBS. Following centrifuging at 97 × g for 10 min, the supernatant was removed and cells were resus-pended with fresh culture medium. The cell density was adjusted to 5×10⁵ cells per well for each 6-well plate or 1×10⁴ cells per well for each 96-well plate. Subculturing was performed every three or four days, upon the cells reaching 80% confluence.

One day after initial seeding, cells were exposed to different concentrations of glutamate (10, 15, 20, 25 or 50 mM) diluted in serum-free DMEM culture medium. Non-treated cells served as a negative control. The culture medium was removed following an exposure time of 2, 4, 6 or 8 h. The cells were washed twice with phosphate-buffered saline (PBS) or distilled water and collected for subsequent experiments.

One day after initial seeding, cells were exposed to varying concentrations of 6-OHDA (0.05, 0.25, 0.5, 1 or 2 mM) dissolved in serum-free DMEM culture medium. Non-treated cells served as a negative control. The culture medium was removed following an exposure time of 4, 8 or 12 h. The cells were washed twice with PBS or distilled water and collected for subsequent experiments.

One day after initial seeding, cells were exposed to different concentrations of glucose oxidase (0.005, 0.05, 0.5, 5 or 20 mg/ml) dissolved in serum-free DMEM culture medium. Non-treated cells served as a negative control. The culture medium was removed following an exposure time of 10, 20, 40 or 60 min. The cells were washed twice with PBS or distilled water and collected for subsequent experiments.

Cells were seeded into 96-well plates (1×10⁴ cells per well). Subsequent to the appropriate incubation time with the indicated compound concentrations, 100 µl MTT (0.5 mg/ml; Sangon Biotech Co., Ltd.) was added to each well. The cells were incubated for 4 h at 37°C, the medium was removed and 150 µl dimethyl sulfoxide (Sangon Biotech Co., Ltd.) was added to each well to resuspend the MTT metabolic product. The absorbance of the dissolved formazan was measured at 490 nm with a microplate reader (Model 550; Bio-Rad Laboratories, Inc., Hercules, CA, USA). Data were calculated from three independent experiments.

Cells were seeded into 6-well plates (5×10⁵ cells per well). Following the appropriate incubation period, the culture medium was removed and cells were washed with PBS. Cells were collected by centrifuging at 67 × g for 5 min. Following PBS washing, 3×10⁴ cells were resuspended with the staining solution containing 5 µl PI and 5 µl Annexin V. The samples were examined by flow cytometry (BD FACSCalibur™; BD Biosciences) after a 30-min incubation in the dark. Annexin V-positive/PI-negative cells were identified as early apoptotic cells, and Annexin V-positive/PI-positive cells were identified as late apoptotic and necrotic cells.

Cells were seeded into 6-well plates (5×10⁵ cells per well). Subsequent to the appropriate incubation period, the culture medium was removed and cells were washed twice with distilled water. Cells were probed with 500 µl Fluo-3a solution for 1 h at 37°C. Following incubation, the Fluo-3a solution was removed and 2 ml distilled water was added to resuspend the cells. Cells were collected by centrifuging at 67 × g for 5 min and resuspended with distilled water. The samples were examined by flow cytometry, using the BD FACSCalibur™.

Cells were seeded into 6-well plates (5×10⁵ cells per well). Following the appropriate incubation period, the culture medium was removed and cells were washed twice with PBS. Cells were probed with 500 µl 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolo-carbocyanine iodide (JC-1) staining solution for 30 min at 37°C. Following incubation, the JC-1 solution was removed and 200 µl PBS was added to resuspend the cells. The samples were then examined by flow cytometry, using the BD FACSCalibur™.

Cells were seeded into 6-well plates (5×10⁵ cells per well). Following the appropriate incubation period, the culture medium was removed and cells were washed twice with PBS. Total protein was extracted from cells using radioimmunoprecipitation analysis lysis buffer containing 1 mM phenylmethylsulfonyl fluoride (both Sangon Biotech Co., Ltd.) and maintained for 30 min on ice. After centrifuging at 13,160 × g for 4 min at 4°C, the supernatant was carefully collected and stored at −20°C until use. Protein concentrations (1 µg/µl) were measured using the BCA Protein Assay kit (Beyotime Institute of Biotechnology). Equal quantities of protein extracts (50 ng) were then separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Sangon Biotech Co., Ltd.). The membranes were blocked with Tris-buffered saline plus Tween-20 (TBS-T; 0.1% Tween-20) containing 5% w/v non-fat dry milk (Sangon Biotech Co., Ltd.), then incubated with the primary antibodies (dilution, 1:1,000) at room temperature for 2 h. After washing with TBS-T, the membranes were incubated with secondary antibodies (dilution, 1:1,000) for 1 or 2 h at room temperature. To quantify the protein level, the expression bands of the target proteins were analyzed using the Odyssey Infrared Imaging System (LI-COR Biotechnology, Lincoln, NE, USA). Densitometric values obtained from western blotting were used to conduct statistical analysis and the housekeeping protein, GAPDH served as an internal control.

Data were analyzed by SPSS statistical software package, version 5 (SPSS, Inc., Chicago, IL, USA) and all data are presented as the mean ± standard deviation. Statistical significance was determined using the two-way analysis of variance comparison test and P<0.05 was considered to indicate a statistically significant difference.

The effect of glutamate, 6-OHDA and glucose oxidase on cell viability was examined. A significant reduction of cell viability was detected when cells were exposed to glutamate doses >20 mM (Fig. 1A; P<0.05). Cell viability was significantly decreased in cells exposed to concentrations of 6-OHDA >0.5 mM at all time points (Fig. 1B; P<0.05). Furthermore, cell viability was significantly reduced following exposure to >5 mg/ml glucose oxidase (Fig. 1C; P<0.05). Overall, a prolonged incubation duration or increased concentration decreased cell viability for the three stimuli that were assessed in the SH-SY5Y cells.

Apoptosis and necrosis induced by glutamate, 6-OHDA and glucose oxidase was investigated using Annexin V/PI staining followed by flow cytometry. Glutamate treatment induced early apoptosis and necrosis in the SH-SY5Y cells (Fig. 2A). Apoptosis was significantly induced after cells were exposed to 25 mM glutamate for 1 h (P<0.05). In addition, exposure to 20 and 50 mM glutamate significantly increased the percentage of necrotic cells (P<0.05). The percentage of necrotic cells was significantly increased when compared with untreated cells after 1 h of incubation with 1, 2 or 5 mM 6-OHDA (Fig. 2B; P<0.05). By contrast, early apoptosis was significantly induced following glucose oxidase incubation (Fig. 2C; P<0.05). These data indicate that glutamate induced apoptosis and necrosis, whereas 6-OHDA only induced necrosis and glucose oxidase only induced early apoptosis.

Cells were stained with Fluo-3a following exposure to glutamate, 6-OHDA or glucose oxidase to determine the resulting intracellular calcium ion level. The intracellular calcium concentration was observed to be dose-dependently elevated following exposure to glutamate (15–25 mM) for 1 h, (Fig. 3A; P<0.05 vs. the control). However, no significant difference was identified in the intracellular calcium concentration of cells treated with 10 or 50 mM glutamate when compared with the control cells (P>0.05). Furthermore, 6-OHDA (>0.05 mM) and glucose oxidase (>0.05 mg/ml) dose-dependently increased the intracellular calcium concentration (Fig. 3B and C; P<0.05). These results indicate that glutamate, 6-OHDA and glucose oxidase exposure increases intracellular calcium ion concentrations. Increased intracellular calcium ion concentrations may result in cell damage.

The changes in mitochondrial membrane potential of cells exposed to glutamate, 6-OHDA and glucose oxidase were determined by JC-1 staining. As shown in Fig. 4A, low doses of glutamate (10 or 15 mM) significantly decreased the mitochondrial membrane potential in SH-SY5Y cells (P<0.05), whereas high doses of glutamate (25 or 50 mM) significantly increased the mitochondrial membrane potential in the cells (P<0.05). Concentrations of 6-OHDA that were >0.25 mM and glucose oxidase concentrations that were >0.005 mg/ml significantly increased the mitochondrial membrane potential of the cells (Fig. 4B and C; P<0.05). These findings indicate that the mitochondrial membrane potential response varied depending on the concentration of glutamate (high or low), whereas 6-OHDA and glucose oxidase exposure of any concentration increased the mitochondrial membrane potential in the SH-SY5Y cells. Elevated mitochondrial membrane potential may contribute to neuronal cell death.

The expression levels of RIP kinase 1 and RIP kinase 3 (key signaling molecules in necrosis) following exposure to glutamate, 6-OHDA and glucose oxidase were determined by western blotting. The expression of RIP kinase 1 was significantly upregulated in a dose-dependent manner as a result of glutamate treatment (Fig. 5A; P<0.05). However, no significant difference was detected in the expression level of RIP kinase 3 in cells that were treated with glutamate. In addition, 6-OHDA and glucose oxidase treatment did not alter the levels of either RIP kinase 1 or 3 (data not shown). This data indicates that the RIP kinase 1 signaling pathway may be involved in glutamate-induced excitatory toxicity.