PC12 cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Hyclone; GE Healthcare Life Sciences, Logan, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1% Antibiotic Antimycotic solution consisting of 10,000 U penicillin and 10,000 U streptomycin. The cells were incubated at 37°C in humidified atmosphere of 5% CO₂ and always used at 70–80% confluence.

The viability of PC12 cells was determined using a Cell Counting Kit (CCK-8; Dojindo Molecular Technologies, Inc., Shanghai, China). Cells were seeded into 96-well plates at a density of 1×10⁵/well. After 24 h culture, cells were treated with NaN₃ (0–80 mM) and incubated for 12, 24, 48 and 72 h to establish the cell injury model. Subsequently, 10 µl CCK-8 solution (Dojindo Molecular Technologies, Inc.) was added to each well and incubated for an additional 2 h under the standard conditions (37°C and 5% CO₂). The absorbance at a wavelength of 450 nm was determined by ELx808 Absorbance Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA). The cell viability was calculated according to the mean optical density of 6 wells. The experiments were conducted in triplicate. The appropriate concentrations of NaN₃ for use in subsequent experiments were determined according the results of the cell viability assays.

PC12 cells were exposed to 0, 10, 20 and 40 mM NaN₃ for 24 h. In order to distinguish programmed cell death from non-apoptotic cell death, nuclei were stained with 10 µg/ml DAPI (C1005; Beyotime Institute of Biotechnology). Briefly, cells were washed twice with PBS and then fixed with 4% paraformaldehyde at room temperature for 30 min. Subsequent to three washes, fixed cells were stained with DAPI (1:5,000) for 5 min and then washed with PBS. Fluorescence images were acquired with a Leica DMI fluorescence microscope (magnification, ×400).

An Annexin V-FITC/PI Apoptosis Detection kit (Beyotime Institute of Biotechnology) was used to determine apoptosis of cells according to the manufacturer's instructions. Experiments were repeated in triplicate and were performed as follows: The apoptotic rates of control PC12 cells (0 mM NaN₃) and PC12 cells exposed to 10, 20 and 40 mM NaN₃ for 24 h was measured by flow cytometry (FC500; Beckman Coulter, Inc., Brea, CA, USA). Statistical analyses were conducted with SPSS statistical software v.13.0 (SPSS, Inc., Chicago, IL, USA).

ΔΨm is a significant parameter of mitochondrial function. It was assessed using staining with JC-1, a fluorescent probe. Experiments were repeated in triplicate, and were performed as follows: Control PC12 cells were treated with 0 mM NaN₃; and experimental PC12 cells were exposed to 10, 20 and 40 mM NaN₃ for 24 h. Subsequently, according to the manufacturer's protocol (C2006; Mitochondrial Membrane Potential Assay kit; Beyotime Institute of Biotechnology, Haimen, China), cells were incubated with the medium containing JC-1 (1X) at 37°C for 20 min, the cells were washed three times with wash buffer and collected with fresh medium without serum. Concomitantly, the positive control was treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP), an inhibitor, (10 µM) at 37°C for 20 min. Then, the red/green fluorescence was determined by FCM (FC500; Beckman Coulter, Inc.). The ratios of red fluorescence intensity over green fluorescence intensity represented the levels of ΔΨm.

ROS in PC12 cells was assessed using a Reactive Oxygen Species Assay kit (S0033; Beyotime Institute of Biotechnology, Haimen, China). Intracellular ROS generation was assessed by means of 2′,7′-dichlorofluorescein diacetate (DCFH-DA), a fluorescent probe. Intracellular ROS oxidizes DCFH-DA, yielding the fluorescent compound 2′,7′-dichlorofluorescein (DCF), and DCF fluorescence intensity is considered to be parallel to the amount of formed ROS, according to the instructions of the ROS assay kit. Experiments were repeated in triplicate and performed as follows: Positive control were treated with specific concentration of Rosup (50 mg/ml); control PC12 cells were treated with 0 mM NaN₃; and experimental PC12 cells were exposed to 10, 20 and 40 mM NaN₃ for 24 h. In addition, PC12 cells were treated with DCFH-DA (10 mM) dissolved in serum-free DMEM (1:1,000) for 20 min at 37°C and then washed three times with serum-free DMEM. The positive control was treated with Rosup, to induce ROS production. The ROS production was determined by FCM (FC500; Beckman Coulter, Inc.). The Mean fluorescence intensities (MFI) represented the levels of ROS.

Experiments were repeated in triplicate and performed as follows: Control PC12 cells were treated with 0 mM NaN₃; and experimental PC12 cells were exposed to NaN₃ at different concentrations (10, 20 and 40 mM) for 24 h. Subsequently, the cellular ATP content was determined using a Firefly Luciferase ATP Assay kit (Beyotime Institute of Biotechnology) according to the protocol of the manufacturer.

PC12 cells were exposed to 0, 10, 20 and 40 mM NaN₃ for 24 h, lysed in radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology) containing a protease inhibitor and centrifuged at 13,362 × g for 10 min at 4°C in order to collect the supernatants. Subsequently, the protein concentrations were determined using BCA kit (Pierce; Thermo Fisher Scientific, Inc.). Equal amounts of proteins (90 µg) were subjected to 10 and 12% SDS-PAGE, and then transferred onto polyvinylidene fluoride membranes (0.45 µm) using a Semidry Electro-transfer Unit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Following blocking with 5% bovine serum albumin in TBS containing 0.1% Tween-20 (TBST) for 2 h at room temperature, the membranes were incubated with primary antibodies against Pgc-1α (Abcam, Cambridge, MA, USA; 1:500), Nrf-2 (Abcam; 1:500), Cox IV (Abcam; 1:1,000), Tfam (Abcam; 1:1,000), procaspase-3 (Abcam; 1:500), Nrf-1 (Cell Signaling Technology, Inc., Danvers, MA, USA, 1:500), pan-calcineurin A (CaN; Cell Signaling Technology Inc.; 1:1,000), phosphorylated (p)-CaMKII (Cell Signaling Technology; 1:1,000), p-p38 MAPK (Cell Signaling Technology, Inc.; 1:1,000), p-extracellular signal-regulated kinase (Erk)1/2 (Cell Signaling Technology, Inc.; 1:1,000), B-cell lymphoma-2 (Bcl-2)-associated X protein (Bax; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; 1:200), Bcl-2 (Santa Cruz Biotechnology, Inc.; 1:200) and cytochrome c (Santa Cruz Biotechnology, Inc.; 1:200) at 4°C overnight. The membranes were then washed with TBST and incubated with horse-radish peroxidase (HRP)-conjugated secondary antibodies (rabbit; cat. no. A0208; 1:1,000; or mouse; cat. no. A0216; 1;1,000; both Beyotime Institute of Biotechnology) for 1 h at room temperature. Immunoreactive bands were visualized by the enhanced chemiluminescence system (Clinx Science Instruments Co., Ltd., Shanghai, China) and quantitatively analyzed with Image J version l.32 J (National Institutes of Health, Bethesda, MD USA), and β-actin was selected as the loading control.

All statistical analyses were conducted with SPSS statistical software v.13.0 (SPSS, Inc., Chicago, IL, USA). Data are expressed as the mean ± standard deviation standard error of the mean. The statistical significance of differences between groups was determined by one-way analysis of variance followed by Bonferroni post-hoc tests. P<0.05 was considered to indicate a statistically significant difference. Each experiment was repeated least three times.

The effect of NaN₃ exposure on the proliferation of PC12 cells was assessed by CCK-8 assay. Cells were challenged with different concentrations (0–80 mM) of NaN₃ for 12–72 h. Table I and Fig. 1A-D indicate that the cell viability was decreased by NaN₃ in a concentration-dependent manner. Almost 100% of the cells died following exposure to 80 mM NaN₃ for 72 h, indicating that NaN₃ markedly induced cell death, and the cytotoxicity of NaN₃ was detected in a dose- and time-dependent manner. Exposure to NaN₃ at a concentration of 20 mM for 24 h caused marked cell death in the PC12 cells (50%). Therefore, the cells cultured for 24 h were used for subsequent experiments.

In DAPI staining, the morphological changes of PC12 cells were observed by fluorescence microscopy following exposure to different concentrations of NaN₃. The fragmentation of cell nuclei exposed to NaN₃ for 24 h was observed, and cell nuclei shrinkage and chromatin condensation were increased slightly with the concentration of NaN₃ in PC12 cells as compared to the control. In these cells, apoptotic cells were smaller and brighter compared with normal cells. Chromatin condensation and nuclear fragmentation were also observed, whereas blue nuclei of viable cells were identified in the control group. In addition, the number of apoptotic cells and the intensity of green fluorescence were increased in cells exposed to NaN₃ (Fig. 2).

Dead cells or late apoptotic cells that have lost cell membrane integrity may be stained by propidium iodide. Due to the loss of this cell membrane integrity, Annexin V-FITC may enter into the cytoplasm and combine with the phosphatidylserine inside of the cell membrane, exhibiting green fluorescence in dead cells. Fig. 3 demonstrates that the numbers of apoptotic cells were 5.03, 8.02, 43.77 and 78.67% (P<0.05) in the PC12 cells following exposure to NaN₃ at different concentrations (0, 10, 20 and 40 mM) for 24 h, respectively. These results additionally confirmed that NaN₃ induced cell apoptosis in a dose-dependent manner.

Mitochondria are generally considered key regulatory organelles involved in cell viability. Therefore, ΔΨm was used as the indicator of mitochondrial function. PC12 cells were stained with JC-1, a cationic dye that exhibits potential-dependent accumulation in mitochondria. Red fluorescence (J-aggregates), representing active mitochondria with stable membrane potential, was observed in a small number of cells exposed to increasing concentrations of NaN₃. By contrast, green fluorescence (J-monomer), representing apoptotic mitochondria with damaged ΔΨm, was observed in a number of cells. The ratio of red and green fluorescence gradually decreased in the control group (Fig. 4).

It is well known that mitochondria are the primary source of cellular ROS, and ROS serve an important role in activation of apoptotic signaling. Therefore, the role of ROS in NaN₃-induced PC12 cell death was additionally investigated. Fig. 5 indicates that the fluorescence intensities in control cells and cells exposed to NaN₃ at various concentrations (0, 10, 20 and 40 mM) for 24 h were 3.65, 6.99, 18.63 and 39.35, respectively, indicating that NaN₃ exposure significantly increased the production of mitochondrial ROS compared with the control group (P<0.05). These results suggested that NaN₃-induced apoptosis improved the production of intracellular ROS.

To evaluate the ATP content in PC12 cells exposed to NaN₃, the relative luminescence unit (RLU) was quantitatively determined by a luminometer. Fig. 6 indicates that NaN₃ inhibited mitochondrial ATP production and gradually decreased the cellular ATP level (P<0.05).

The Pgc-1α expression at the protein level was significantly decreased following NaN₃ exposure compared with the control (P<0.05). In addition, Nrf-1, Tfam, p-Erk1/2, Nrf-2 and Cox IV are well-known downstream targets of Pgc-1α dynamics in various cell types (8). The present study identified that NaN₃ exposure significantly inhibited Pgc-1α dynamics in a dose-dependent manner (P<0.01; Fig. 7A).

In addition, NaN₃ exposure upregulated the expression levels of Bax and cytochrome c, while it downregulated the expression levels of Bcl-2 and procaspase-3 (P<0.05). The ratio of Bax/Bcl-2 was also significantly increased compared with the control group (P<0.05; Fig. 7B).

The protein expression levels of other important members, including CaN, CaMKII, p-CaMKII, p38 MAPK and p-p38 MAPK, were also assessed. The results indicated that NaN₃ increased the expression of CaN and the phosphorylation of CaMKII and p38 MAPK compared with the control group, while the expression levels of total CaMKII and p38 MAPK were not changed. In addition, the ratios of p-CaMKII/CaMKII and p-p38/p38 MAPK in the NaN₃ group were significantly increased compared with those in the control group (P<0.01; Fig. 7C).