PPD (chemical structure shown in Fig. 1) was HPLC grade and purchased from the Ambo Institute (Seoul, Korea). All chemicals and solvents used were of the highest analytical grade available. Cell culture supplies and media, fetal bovine serum (FBS), phosphate-buffered saline (PBS) and penicillin-streptomycin were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Anti-cleaved capase-3 antibody (#9664) was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Hoechst 33258 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Annexin V-FITC was purchased from BD Biosciences (Flanklin Lakes, NJ, USA). Dihydroethidium (DHE) was purchased from Calbiochem (EMD Millipore; Billerica, MA, USA). Tetramethylrhodamine ethyl ester (TMRE), MitoSOX™ Red and MitoTracker Green FM were purchased from Invitrogen (Waltham, MA, USA).

The PC12 cell line was purchased from the American Tissue Culture Collection (ATCC; Rockville, MD, USA). The cells were cultured under sterile conditions at 37°C in a humid environment with 5% of CO₂ in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (both from Thermo Fisher Scientific), 4 mM glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin. The cultures were regularly checked and trypsinized when the cell confluence reached 85%.

To determine the half maximal inhibitory concentration (IC₅₀ value) of glutamate on the PC12 cells, 5×10³ cells in single cell suspensions were seeded in individual wells of 96-well plates and incubated for 24 h at 37°C prior to exposure to glutamate at the indicated concentrations (1, 5 and 10 mM) for 24 h. After determining that the glutamate IC₅₀ value was 5 mM, the cells were exposed to 5 mM glutamate for 24 h, 10 μM PPD, or a combination of both (5 mM glutamate plus 10 μM PPD). MTT solution was added to each well followed by incubation for 4 h at 37°C prior to removing the culture medium. DMSO was then added and mixed for 30 min at room temperature. Cell viability was determined by measuring the absorbance at 562 nm. The cell viability for each group was calculated as a percentage of that of the control group.

The cells were seeded at a density of 1×10⁵ cells/well into 24-well plates and were left to grow exponentially at 37°C in a 5% CO₂ atmosphere. The PC12 cells were then treated with the different drugs [untreated control (U), glutamine (Glut), PPD or Glut + PPD], and incubated for 12 h. Subsequently, the cells were fixed with 4% paraformaldehyde, washed with PBS 3 times and observed under an inverted microscope (Olympus CKX41; Olympus Corp., Tokyo, Japan).

To evaluate cell apoptosis, 1×10⁶ cells/well were plated on a coverslip in a 12-well plate in triplicate. The cells were treated with the different drugs [U, Glut, PPD or Glut + PPD] and kept in a CO₂ incubator at 37°C for 24 h. Following incubation and the addition of fresh medium, the cells were incubated with 5 μl/well of Hoechst 33258 (Sigma-Aldrich) solution at 37°C for 10 min, followed by observation under a laser confocal microscope (LSM-700; Carl Zeiss, Oberkochen, Germany). Strong fluorescence with shrunken nuclei was observed in the apoptotic cells, whereas weak fluorescence with normal-sized nuclei was observed in the non-apoptotic cells. The quantification of apoptotic cells was performed by capturing images in random fields and counting at least 200 cells in 4 random fields in each well.

Briefly, 30–50 μg of protein were resolved by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then electro-blotted onto polyvinylidene difluoride membranes for western blot analysis. The blots were probed with 1:1,000-diluted primary antibodies [anti-cleaved caspase-3 (#9664; Cell Signaling Technology, Inc.), actin (sc-7210; Santa Cruz Biotechnology, Dallas, TX, USA)] overnight at 4°C, followed by horseradish peroxidase-conjugated secondary antibodies (anti-rabbit IgG, HRP-linked antibody, #7074; Cell Signaling Technology, Inc.). The proteins were then visualized using enhanced chemiluminescent reagent (ECL; Millipore Corp., Billerica, MA, USA) and exposure to X-ray film.

The cells were analyzed by flow cytometry using a BD FACSCanto II flow cytometer as indicated by the manufacturer (BD Biosciences). Following 2 washes with PBS, the cells were fixed in 4% paraformaldehyde for 10 min at 37°C and permeabilized with 0.25% Triton X-100 in PBS for 10 min. The cells were stained with individual dye for 1 to 2 h at 4°C and the secondary antibodies (1:100) for 1 h on ice. Following 2 washes with PBS, the cells were fixed in 4% para-formaldehyde and assayed immediately. Flow cytometry data were collected using 10,000–30,000 cells and were analyzed using FlowJo software (Tree Star, Inc., Ashland, OR, USA).

The mitochondrial ROS levels were measured using the mitochondrion-specific fluorescent hydroethidine-derivative dye, MitoSOX Red (10 μM; Invitrogen), as previously described (24). The cells were exposed to glutamate with or without treatment with PPD and then incubated with MitoSOX Red for 30 min at 37°C in 5% CO₂. The cells were washed twice with PBS, fixed with 4% paraformaldehyde, and analyzed under a confocal microscope. To measure the intracellular ROS levels, the cells were incubated for 30 min in Krebs-HEPES buffer containing DHE (Calbiochem) washed twice, and analyzed using a flow cytometer.

The cells (1×10⁶ cells/well) were prepared on sterilized glass coverslips (BD Biosciences) in triplicate. Following 24 h of treatment with the drugs (U, Glut, PPD or Glut + PPD), the cells were fixed in 4% paraformaldehyde in PBS for 10 min, permeabilized with 0.25% Triton X-100 in PBS for 10 min, and incubated with the primary antibody (Tomm20, ab56783; Cambridge, MA, USA) for 2 h at room temperature. The cells were washed to remove the excess primary antibody, and incubated with the appropriate fluorescently-labeled secondary antibodies for 1 h at room temperature. The nuclei were stained with DAPI and incubated for 5 min. After mounting, fluorescence images were captured using a confocal microscope (LSM 700; Carl Zeiss). To quantify the immunoreacted cells, the fluorescence intensity was measured in 10 randomly selected images.

The determine cell apoptosis, monolayer cultures of PC12 cells treated with the different drugs [U, Glut, PPD and Glut + PPD] were supplemented with nutrient medium. The cultures were incubated for 24 h. The dead cells were analyzed prior to and subsequent to exposure to glutamate and treatment with PPD using the Muse™ Annexin V and Dead Cell Assay kit (Muse™Cell Analyzer; Millipore Corp.) according to the manufacturer's instructions.

For TEM analysis, the cells were collected and fixed with 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 2 h, post-fixed with 1% osmium tetroxide for 1 h, washed, and stained for 1 h in 3% aqueous uranyl acetate. The samples were then washed again, dehydrated with graded alcohol, and embedded in Epon-Araldite resin (Canemco, Inc., Lakefield, QC, Canada). Ultrathin sections were cut on an ultramicrotome (Reichert-Jung, Inc., Cambridge, UK), counterstained with 0.3% lead citrate, and examined under a transmission electron microscope (HT7700; Hitachi, Ltd., Tokyo, Japan).

The ΔΨm of intact cells was measured as previously described (25), with some modifications. Briefly, the cells were washed with PBS and trypsinized. The protein concentration of the cells was adjusted to 0.2 mg/ml in DMEM without phenol red (Invitrogen Life Technologies), FBS and antibiotics. TMRE (200 nM; T669; Invitrogen-Molecular Probes) was added to the cell suspension. The cells were incubated at 37°C for 30 min in the dark. ΔΨm was measured by flow cytometry, and the data were analyzed using FlowJo software. TMRE fluorescence was measured using the FL2 channel (582 nm).

Mitochondrial mass was measured using fluorescence levels following staining with MitoTracker Green FM (100 nM; M7514; Invitrogen) at 37°C for 30 min. Subsequently, the cells were washed once in PBS and promptly used for flow cytometry. The percentage and mean fluorescence intensity level of the mass were calculated for each sample.

To visualize the mitochondria, the mitochondrial marker, Tomm20, was used, and was added with DMEM at 37°C for 30 min. The cover glasses were then washed twice with PBS and images were immediately captured using a confocal laser scanning microscope (LSM 700; Carl Zeiss). The images were evaluated as best-focus intensity projections.

Data obtained from independent experiments (means ± SD) were analyzed using a two-tailed Student's t-test. Differences were considered significant if P<0.05. 95% confidence intervals were computed, 1.96 x standard error in each direction. Statistical significance was evaluated using a log-rank (Mantel-Cox) test.

To determine the appropriate concentration of glutamate, the PC12 cells were exposed to various concentrations of glutamate for 24 h. Glutamate (1–10 mM) gradually reduced cell viability in a dose-dependent manner, and the cell viability (as shown by MTT assay) decreased by approximately 70% in the cells incubated with 5 mM glutamate for 24 h (Fig. 2A). Hence, the concentration of 5 mM glutamate was selected for use in the subsequent assays. The cells exposed to 5 mM glutamate were pre-treated with 5, 10 or 20 μM PPD (Fig. 2B). We wished to examine the possible beneficial effects of PPD on the viability of glutamate-exposed PC12 cells. The exposure of the neuronal PC12 cells to glutamate for 24 h resulted in cell damage, as shown by the changes in cell morphology, such as cell shrinkage (Fig. 2C). As shown by MTT assay, pre-treatment with PPD resulted in a higher cell viability compared with the glutamate-exposed cells not treated with PPD (Fig. 2D). Thus, treatment with PPD significantly improved the viability of the glutamate-exposed cells.

We then wished to examine whether glutamate-induced cytotoxicity is mediated by apoptotic processes, using Hoechst 33342 staining, Annexin V staining and caspase-3 antibody. Hoechst 33342 staining revealed that treatment with PPD significantly decreased the amount of condensed chromatin compared with the glutamate-exposed cells not treated with PPD (Fig. 3A). Pre-treatment with PPD also significantly decreased the percentage of Annexin V-positive cells, compared the glutamate-exposed cells not treated with PPD (Fig. 3B). The results of western blot analysis also revealed that treatment with PPD prevented the glutamate-induced cleavage of caspase-3 (Fig. 3C), consistent with the results regarding the inhibition of apoptosis in the PPD pre-treated cells. Thus, PPD plays a crucial role in reducing the cytotoxic effects of glutamate by inhibiting apoptosis.

Given that ginsenosides are able to reduce ROS generation, and that the glutamate-induced apoptotic pathway is linked to ROS of cytosolic and mitochondrial origin (26), in this study, we investigated the effects of PPD on the accumulation of ROS in glutamate-exposed PC12 cells. We measured cytosolic and mitochondrial ROS formation by flow cytometry and fluorescence microscopy using DHE and MitoSOX Red, a fluoroprobe that selectively detects ROS formation in live cells and mitochondria. The addition of PPD suppressed the glutamate-induced production of ROS, including the DHE-reactive superoxide anion (O₂⁻) (Fig. 4A). Cytosolic ROS was measured by flow cytometric analysis of the DHE-stained cells. In additional experiments, mitochondrial ROS production was estimated using MitoSOX Red, a cell-permeable probe that is non-fluorescent when chemically reduced, but fluoresces following cellular oxidation and the removal of acetate groups by cellular esterases (Fig. 4B). The mitochondrial ROS levels were significantly decreased following treatment with PPD (Fig. 4C). PPD scavenged the mitochondrial ROS produced by glutamate-induced mitochondrial damage. These results suggest that PPD exerts major effects on cell survival through its antioxidant effects.

A recent study reported that glutamate is associated with mitochondrial damage (27). Thus, to determine whether glutamate induces mitochondrial damage, we first examined mitochondrial morphology. Mitochondrial morphology was examined using Tomm20 (mitochondrial outer membrane) staining following treatment with glutamate for 24 h. A significant change in mitochondrial morphology was observed at 24 h in the glutamate-exposed cells. Treatment of the glutamate-exposed cells to PPD led to a substantial increase in the proportion of mitochondria, compared to the cells exposed to glutamate and not treated with PPD (Fig. 5A). We also analyzed the changes in mitochondrial morphology using TEM (Fig. 5B). Ultrastructural imaging revealed that the rate of swollen mitochondria and broken mitochondrial matrix was increased in the glutamate-exposed cells, whereas treatment with PPD resulted in an apparent increase in the number normal mitochondria compared with the glutamate-exposed cells not treated with PPD. Thus, treatment with PPD may protect the mitochondria against glutamate-induced damage.

Treatment with ginsenosides has been shown to improve mitochondrial function in various cells (28,29). However, in neuronal cells, it is not known whether treatment with PPD is capable of controlling mitochondrial function, density and morphology, particularly following exposure to glutamate. Thus, in this study, to examine the protective role of PPD in maintaining mitochondrial function, we analyzed the MMP, as well as the changes in mitochondrial mass. The MMP was measured by flow cytometry using a potentiometric fluorescent probe (TRME) following exposure of the cells to glutamate (Fig. 6A). Significant depolarization of the mitochondria was observed at 24 h and sustained until 24 h. The MMP was significantly higher in the cells pre-treated with PPD than in the cells exposed to glutamate and not treated with PPD. PPD protected the cells against glutamate-induced mitochondrial damage by regulating the MMP.

To further elucidate the possible mechanisms underlying the effects of PPD, we evaluated the mitochondrial mass. MitoTracker green staining indicated that treatment with PPD controlled mitochondrial mass following exposure to glutamate. The mitochondrial mass decreased by approximately 70% in cells exposed to glutamate and not treated with PPD (Fig. 6B). Thus, PPD improved mitochondrial function, as indicated by changes in MMP and mitochondrial mass, suggesting that PPD plays a role in maintaining mitochondrial functional.