PC12 cells were purchased from the Cell Resource Center of the Shanghai Institute of Biological Sciences of the Chinese Academy of Sciences (Shanghai, China). The cells were plated at an initial density of 5×10⁴ cells/well onto 24-well culture plates to test cell viability, and at a density of 1×10⁶ cells/well onto 6-well plates in order to prepare samples for western blot analysis. The culture media used was Dulbecco’s modified Eagle’s medium containing 4,500 mg/l D-glucose, 584 mg/l L-glutamine and 110 mg/l sodium pyruvate (Thermo Scientific, Tewksbury, MA, USA), and also containing 1% penicillin and streptomycin (Invitrogen Life Technologies, Carlsbad, CA, USA) and 10% fetal bovine serum (PAA Laboratories GmbH, Linz, Austria). The cells were given 0.5 μM rotenone (Sigma, St. Louis, MO, USA) with or without co-treatment with different concentrations of NMN (N3501, Sigma Aldrich, USA). The cells were kept for 24 h in an incubator with 5% CO₂ at 37°C. In total, the following four concentrations were tested: 0.1mM, 1.0 mM, 5mM and 10mM, however, the effects of last three appeared to be similar. Thus, the minimum (0.1 mM) and maximum (1.0 mM) concentrations were selected in order to indicate the effect of improving the energy activity and survival rate of rotenone-treated PC12 cells/

Cell survival was measured by a quantitative colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma). Following drug treatment, PC12 cells were incubated for 4 h with 5 mg/ml MTT. Subsequently the cell cultures were lysed in dimethyl sulfoxide (DMSO; Sigma) for 15 min. The optical absorption at 570 nm was determined using a plate reader (Synergy2, Biotek, Winooski, VT, USA).

Using a previously described method (15), cell survival was quantified by the measurement of LDH activity in cell lysates. Cells were lysed for 20 min in lysing buffer containing 0.04% Triton X-100, 2 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 0.2 mM dithiothreitol, 0.01% bovine serum albumin and 0.1% phenol red. A total of 50 μl cell lysates at pH 7.5 were mixed with 150 μl 500 mM potassium phosphate buffer (pH 7.5) containing 1.5 mM NADH (Sigma) and 7.5 mM sodium pyruvate (Sigma). The change in the absorbance at 340 nm (A340 nm) was monitored over 90 sec. Percentage cell survival was calculated by standardizing the LDH activity of the sample cell lysates to the LDH activity of the lysates of the control (wash only) cell cultures.

LDH is a cytosolic enzyme that is released into the cell media upon cell lysis. Extracellular LDH activity is highly correlated with a major index of cell necrosis: the level of propidium iodide-positive cells (16). Therefore, extracellular LDH activity may be used for assessing the number of cells undergoing cell death (17). In the present study, the extracellular LDH activity of PC12 cells was assessed using a previously described method (16). Briefly, 100 μl extracellular media from the samples was mixed with 150 μl potassium phosphate buffer (500 mM, pH 7.5) containing 1.5 mM NADH and 7.5 mM sodium pyruvate. Changes in the A340 nm of the samples was monitored over 90 sec using a plate reader.

Following washing twice with phosphate-buffered saline (PBS), the cells were suspended in cold 1X binding buffer at a concentration of 1×10⁶ cells/ml. A total of 100 μl cell suspension was mixed with 10 μl Annexin V R-phycoerythrin (PE) conjugate (SouthernBiotech, Birmingham, AL, USA). Following incubation on ice for 15 min, 200 μl cold 1X binding buffer was added to each tube, followed by the addition of 10 μl 7-AAD (SouthernBiotech). The number of the cells in early- and late-stage apoptosis, and necrosis, was assessed by a flow cytometer (BD FACSAria II; BD Biosciences, Franklin Lanes, NJ, USA).

The nuclear size of cells was assessed by Hoechst staining (18). Following treatment, cells were fixed in 4% formaldehyde for 15 min at room temperature. After washing with PBS, the cells were stained with 20 μg/ml Hoechst 33258 (Sigma) in PBS for 20 min. Images of the stained nuclei were captured under a fluorescence microscope (Leica DMI3000B, Leica, Mannheim, Germany).

Using a previously described method (19), PC12 cells were lysed in radioimmunoprecipitation assay (RIPA) cell lysis buffer [50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate and 0.1% sodium dodecyl sulfate (SDS); Sigma] supplemented with 100 μM protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Membrane fractions were separated by centrifugation at 16,000 × g for 20 min at 4°C. Protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, Rockford, IL, USA). The samples were normalized to 30-μg of total protein extract and were fractionated by 10% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes using a standard technique. The membranes were blocked with 5% skimmed milk at 37°C for 1 h and incubated with rabbit PARP-1 (p116/p25) antibodies (1:1,000; Epitomics, Burlingame, CA, USA) at 4°C overnight. Horseradish peroxidase (HRP)-linked anti-rabbit immunoglobulin G (IgG; Epitomics) and enhanced chemiluminescence (ECL) substrate (Thermo Scientific) were added and the bands were visualized. The equal loading of samples was confirmed by stripping the membranes and reprobing them with goat polyclonal anti-actin IgG (1:400, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and HRP-conjugated rabbit anti-goat IgG (Epitomics). Levels of immunoreactive proteins were determined by densitometric scanning using a ChemiDoc XRS system (Bio-Rad, Hercules, CA, USA).

ATP levels were quantified using an ATP Bioluminescence assay kit HS II (Roche Diagnostics) according to the manufacturers’ instructions. Cells were lysed with cell lysis reagent (Roche Diagnostics) and 50 μl lysates were mixed with 150 μl luciferase assay reagent (Roche Diagnostics). The luminescence was detected using a plate reader (Biotek Synergy 2; BioTek Instrument, Inc., Winooski, VT, USA). The protein concentrations of the samples were determined by a BCA assay. The ATP concentrations of the samples were calculated using an ATP standard and normalized against the amount of protein in the samples.

Following drug treatment, the cells were carefully washed with PBS. The levels of NAD⁺ were measured using a plate reader according to a previously described method (4). Cells were extracted in 0.25 ml 0.5 N HClO₄, scraped, neutralized with 3 M KOH and 100 mM sodium phosphate buffer (pH 7.0). The levels of NAD⁺ were assessed based on the reduction of MTT to formazan by NADH, which was generated by enzymatic cycling with alcohol dehydrogenase. The rate of optical density (OD) increase at 560 nm was determined by examining the samples immediately and 20 min after the addition of the sample extracts.

All data are presented as mean + standard error. Data were assessed by one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls post hoc test. P<0.05 was considered to indicate a statistically significant difference.

The present study determined the effect of NMN treatment on rotenone-induced changes in the survival rate of PC12 cells using MTT, intracellular LDH and extracellular LDH assays. Treatment with rotenone for 24 h decreased the survival rate of PC12 cells in a concentration-dependent manner, as assessed by MTT assay (Fig. 1A). Since 0.5 μM rotenone induced an ~60% decrease in MTT reduction, this concentration of rotenone was used in subsequent experiments. Co-treatment of the cells with NMN (0.1 or 1 mM) and rotenone led to a significantly higher survival rate of the PC12 cells when compared with the survival rate of the PC12 cells treated with rotenone alone (Fig. 1B). The effect of treatment with NMN alone is shown in Fig. 1C.

An intracellular LDH assay was also conducted to determine the effects of NMN and rotenone on cell survival. Treatment of the cells with rotenone caused a concentration-dependent reduction in intracellular LDH activity, which is an index of cell survival (Fig. 2A). This effect was significantly attenuated by NMN co-treatment (Fig. 2B). Cell death was assessed by extracellular LDH assay. It was observed that rotenone concentration-dependently induced the death of the PC12 cells (Fig. 2C), which was significantly attenuated by NMN co-treatment (Fig. 2D).

Flow cytometry-based Annexin V/7-AAD staining was conducted to determine the early and late apoptosis and necrosis of PC12 cells treated with rotenone and NMN. Phosphatidylserine (PS) is expressed on the outer leaflet of the plasma membranes of early-stage apoptotic cells, which may be stained by labeled Annexin V. Late-stage apoptotic and necrotic cells lose the integrity of their plasma membranes, which become permeable to the fluorescent dye 7-AAD (20). Thus, Annexin V⁻/7-AAD⁻, Annexin V⁺/7-AAD⁻, Annexin V⁺/7-AAD⁺ and Annexin V⁻/7-AAD⁺ cells are defined as normal, early-stage apoptotic, late-stage apoptotic and necrotic cells, respectively. Treatment of the cells with 1 mM NMN (Fig. 3A) did not significantly affect the level of cell death compared with that of the control cells (Fig. 3B). Treatment of the cells with 0.5 μM rotenone led to a marked increase in the number of Annexin V⁺/7-AAD⁻ cells, as well as increases in the numbers of Annexin V⁺/7-AAD⁺ and Annexin V⁻/7-AAD⁺ cells, compared with the control group (Fig. 3C). Co-treatment of the cells with rotenone and NMN led to significant reductions in the numbers of Annexin V⁺/7-AAD⁻ and Annexin V⁺/7-AAD⁺ cells when compared with the rotenone treatment alone (Fig. 3D). Quantification of these results indicates that NMN significantly decreases the numbers of early- and late-stage apoptotic cells (Fig. 3E).

Nuclear condensation is an indication of cellular apoptosis and may be observed through Hoechst 33258 staining. Furthermore, PARP-1 is cleaved by active caspase-3 during caspase-3-dependent apoptotic processes. To further determine whether NMN is able to decrease rotenone-induced apoptosis, the levels of nuclear condensation and PARP-1 cleavage in rotenone-treated PC12 cells were assessed. It was observed that rotenone concentration-dependently induced nuclear condensation, and this effect was significantly attenuated by NMN co-treatment (Fig. 4). Western blot analysis also revealed that rotenone concentration-dependently reduced the levels of PARP-1 (Fig. 5A and B). The rotenone-induced reductions in the levels of PARP-1 were significantly attenuated by NMN co-treatment (Fig. 5C and D).

Since NMN is a precursor of NAD⁺, the present study aimed to determine whether NMN treatment enhances the intracellular levels of NAD⁺ in PC12 cells. It was observed that treatment of PC12 cells with 0.5 mM rotenone led to a significant reduction in the intracellular levels of NAD⁺, which was prevented by co-treatment with 1 mM NMN for 24 h (Fig. 6).

To investigate the mechanisms underlying the protective effect of NMN on rotenone-induced cell death, the effect of NMN treatment on the intracellular levels of ATP in rotenone-treated cells was determined. Rotenone concentration-dependently reduced the intracellular levels of ATP in the cells (Fig. 7A); this effect was significantly attenuated when the cells were co-treated with NMN (Fig. 7B).