PC12 cells (American Type Culture Collection, Manassas, VA, USA) were maintained at 37°C in an atmosphere containing 5% CO₂ in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 5% horse serum (Gibco; Thermo Fisher Scientific, Inc.), 100 µg/ml streptomycin and 100 U/ml penicillin. Nerve growth factor (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), at a final concentration of 100 ng/ml, was added to the medium 3 days prior to drug treatment to induce neuronal differentiation. Cells were treated with MPTP (Sigma-Aldrich; Merck KGaA) and/or PCs (cat. no. T2849; Target Molecule Corp., Boston, MA, USA).

The viability of cells was measured using the MTT assay. PC12 cells were cultured in 96-well plates at a density of 1×10⁴ cells/well. Cells were exposed to 150 µmol/l MPTP following treatment with 0.5, 1 or 5 µg/ml PCs for 24, 48, 72 or 96 h at 37°C. The cells were then incubated with MTT (0.25 mg/ml) at 37°C for 4 h, after which, MTT formazan products were dissolved in dimethyl sulfoxide and the absorbance was measured at 570 nm using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Cell apoptosis was detected using an Annexin V/Propidium Iodide (PI) Apoptosis Detection kit (Sigma-Aldrich; Merck KGaA), according to the manufacturer's protocol. Cells were exposed to 150 µmol/l MPTP following treatment with 0.5, 1 or 5 µg/ml PCs for 48 h at 37°C. Following this, these cells were harvested and 1×10⁶ cells were fixed using 4% polyformaldehyde for 30 min at 4°C. Following this, the cells were resuspended in 300 ml PBS and were stained with Annexin V-fluorescein isothiocyanate and PI (5 µg/ml each) in the dark for 15 min at 37°C. Apoptotic cells were analyzed by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA). FlowJo software (version 10; FlowJo LLC, Ashland, OR, USA) was used to calculate the apoptosis rate.

MMP alterations were measured using a JC-1 MMP Assay kit (Beyotime Institute of Biotechnology, Shanghai, China), according to the manufacturer's protocol. Briefly, cells were exposed to 150 µmol/l MPTP following treatment with 0.5, 1 or 5 µg/ml PCs for 48 h at 37°C. The medium was then replaced with PBS, and 1×10⁶ cells were incubated for 24 h with the JC-1 probe (10 µg/ml) at room temperature. JC-1 fluorescence was subsequently detected using a microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA) with an excitation and emission wavelength of 536–620 nm.

All animal handling procedures were conducted in accordance with the Guidelines for Laboratory Animal Research of Nanjing Medical University (Nanjing, China). The present study was approved by the Institutional Animal Care and Use Committee of Nanjing Medical University. A total of 20 male C57BL/6 mice (age, 9 weeks; weight, 20–22 g) were purchased from the Laboratory Animal Center of Nanjing Medical University. The mice were housed at 23±2°C and a relative humidity of 60±10% under a 12-h light/dark cycle, with free access to water and food. Mice were assigned to five groups: (i) Control group (n=4); (ii) MPTP (30 mg/kg) group (n=4); (iii) MPTP (30 mg/kg) + PC (300 mg/kg/day) group (n=4); (iv) MPTP (30 mg/kg) + PC (400 mg/kg/day) group (n=4); and (v) MPTP (30 mg/kg) + PC (500 mg/kg/day) group (n=4). PCs were intragastrically administered at 300, 400 or 500 mg/kg/day for 14 days consecutively, whereas the control group received an equivalent volume of saline. Treatment began 7 days prior to the initial MPTP treatment, from which point MPTP (20 mg/kg) dissolved in saline was intraperitoneally injected four times daily at 2 h intervals for a total of 7 days. All mice were sacrificed for further investigation 24 h after the last MPTP injection had been administered.

The pole test was used to measure motor behavior in the mouse model of PD. The pole test was performed as previously described (15), and began following 7 days of MPTP administration. Briefly, the mice were held on top of the pole (diameter, 8 mm; height, 55 cm; rough surface), and the time taken for the mice to climb down and place four feet on the floor was recorded as the time for locomotion activity (T-LA). Each trial had a cut-off limit of 30 sec. All measurements were performed three times to ensure accuracy.

Brain tissue preparation was performed as previously described (15,16). Briefly, 24 h after the last injection of MPTP, brains were obtained from the four mice in each group. One side of the brain was fixed in 4% paraformaldehyde for 72 h at 4°C, followed by incubation in 0.1 M phosphate buffer (pH 7.4) containing 25% sucrose at 4°C for 2–3 days. Following this, the brain tissues were frozen, and then substantia nigra tissues were then cut into 25 µm sections and stored in cryoprotectant at 4°C until further use in the immunohistochemistry (IHC) and terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL) experiments. For ROS and MMP assays, as well as western blotting, the other side of the substantia nigra was isolated and stored at −80°C until use.

Following three 10 min washes in PBS with 0.05% Tween-20 (PBST), sections were incubated for 1 h at 37°C with PBST containing 2% bovine serum albumin (Sigma-Aldrich; Merck KGaA). Sections were subsequently incubated overnight at 4°C with anti-TH antibody (1:1,000; cat. no. 25859-1-AP; ProteinTech Group, Inc., Chicago, IL, USA), followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG) secondary antibody (1:5,000; cat. no. 10285-1-AP; ProteinTech Group, Inc.) for 1 h at 37°C and amplification with a DAB Vectastain ABC kit (Vector Laboratories, Inc., Burlingame, CA, USA), which was performed according to the manufacturer's instructions. Finally, sections were analyzed using a light Leica DM2700 P microscope (magnification, ×40; Leica Microsystems, Inc., Buffalo Grove, IL, USA). Quantification of TH activity was performed by counting the number of TH-immunoreactive (TH-IR) cells in 10 independent visual fields in the SNpc, and by measuring the optical density of TH-IR fibers in the ST using ImageJ software (version 1.48; National Institutes of Health, Bethesda, MD, USA).

Tissue sections were washed in PBS and subsequently fixed for 30 min with 4% paraformaldehyde at room temperature. Following one wash with PBS, PBS containing 0.1% Triton X-100 was added to the sections for 2 min in order to lyse the cells at room temperature. Sections were subsequently washed once with PBS and mounted onto slides, and 3% H₂O₂ was added to the slides for 5 min at room temperature. Slides were then rinsed and then incubated with 50 µl TUNEL detection solution (Roche Diagnostics, Basel, Switzerland) for 60 min at room temperature. The TUNEL reaction was visualized by chromogenic staining with DAB (0.75 mg/ml; Sigma-Aldrich; Merck KGaA) at room temperature for 20 min. Sections were imaged and ten visual fields were analyzed using a light Leica DM2700 P microscope (Leica Microsystems, Inc.). The percentage of cell death was determined by calculating the number of TUNEL-positive cells within a total of 100 cells in one visual field using ImageJ software (version 1.48; National Institutes of Health, Bethesda, MD, USA).

ROS was measured with the fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA; Sigma-Aldrich; Merck KGaA). Cells were exposed to 150 µmol/l MPTP following treatment with 0.5, 1 or 5 µg/ml PCs for 48 h at 37°C. The medium was then replaced with PBS, and 1×10⁶ cells were incubated with 10 µmol/l H₂DCFDA at 37°C for 30 min. Substantia nigra tissues were treated with collagenase (5 mg/ml) and then the cells were dislodged in the solution using a pipette. PC12 cells or single cell suspension of substantia nigra homogenate was incubated with 10 µmol/l H₂DCFDA at 37°C for 30 min. The cells were subsequently washed twice with PBS and dissolved in 1% Triton X-100. Fluorescence was measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm, using a fluorescence microplate reader.

PC12 cells were exposed to 150 µmol/l MPTP following treatment with 0.5, 1 or 5 µg/ml PCs for 48 h at 37°C. Proteins from PC12 cells or substantia nigra were prepared as described previously (17). Protein concentration was measured using bicinchoninic acid assays (Beyotime Institute of Biotechnology, Shanghai, China) and adjusted to the same final concentration. Protein samples (20 µg/lane) were separated by 12% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. Membranes were blocked with 5% skim milk in 50 mM Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h at room temperature, and membranes were incubated overnight at 4°C with the following primary antibodies in the same blocking solution: TH (cat. no. 2792), c-Jun N-terminal kinase (JNK; cat. no. 9252), phosphorylated (p)-JNK (cat. no. 9255), c-Jun (cat. no. 9165), p-c-Jun (cat. no. 3270), B-cell lymphoma 2-like protein 11 (Bim; cat. no. 2933), cleaved caspase-3 (cat. no. 9654), cleaved poly (ADP-ribose) polymerase (PARP; cat. no. 94885) and GAPDH (1:2,000; cat. no. 2118) (all 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA). Subsequently, membranes were washed with TBST and incubated with horseradish peroxidase-conjugated goat-anti-rabbit IgG (1:10,000; cat. no. 7074; Cell Signaling Technology, Inc.) or goat-anti-mouse IgG (1:10,000; cat. no. 7076; Cell Signaling Technology, Inc.) for 1 h at room temperature in TBST containing 5% skim milk. Cross-reactivity was visualized using enhanced chemiluminescence western blotting detection reagents (Sangon Biotech Co., Ltd., Shanghai, China) and was analyzed by densitometry using Tanon 5200 software (Tanon Science and Technology Co., Ltd., Shanghai, China).

All data were analyzed using Prism software 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). Data are expressed as the mean ± standard error of the mean. All experiments were performed in triplicate. Statistical evaluation of the results was performed by one-way analysis of variance followed by Bonferroni's correction. P≤0.05 was considered to indicate a statistically significant difference.

The simplest structure of PCs is a dimer formed by catechin, L-Epicatechin or catechin and L-Epicatechin, which is highly soluble in water and may be easily absorbed. Furthermore, PCs also have an important role in scavenging free radicals (18). The chemical structure of a PCs dimer formed from catechin is presented in Fig. 1A. To exclude the possibility that PCs induced PC12 cell toxicity, cell viability was determined in response to various concentrations of PCs at 24, 48, 72 and 96 h using the MTT assay, and the results revealed that PCs did not induce toxicity in PC12 cells (data not shown). The data demonstrated that MPTP markedly inhibited PC12 cell proliferation compared with the control, and this effect was gradually counteracted by increasing concentrations of PCs (Fig. 1B). Furthermore, the apoptotic rate for each group was assessed by flow cytometry. The typical quadrant analysis results obtained from PC12 cells, treated with or without PCs prior to MPTP treatment, are presented in Fig. 1C. Compared with the control group (3.0%), the percentage of apoptotic cells was significantly increased in the MPTP treatment group (22.5%). Conversely, PC pretreatment reduced the apoptotic percentage from 22.5 to 15.2% (0.5 µg/ml), 12.7% (1 µg/ml) and 7.5% (5 µg/ml). It was therefore concluded that PCs may reduce MPTP-induced apoptosis.

Mitochondria are the major source of ROS in various mammalian cells, and excessive production of ROS in the mitochondria disrupts normal redox signaling. In addition, MMP is a marker of mitochondrial function, which is also involved in apoptosis (19). The production of ROS in PC12 cells was analyzed using a H₂DCFDA fluorescence assay. As presented in Fig. 2A, exposure to MPTP increased ROS levels in PC12 cells. Pretreatment with PCs significantly inhibited the accumulation of ROS induced by MPTP. These results suggested that PCs protected mitochondrial function and suppressed ROS production in PC12 cells. Furthermore, enhanced MMP was observed in the control group and treatment with MPTP significantly reduced MMP in PC12 cells; however, pretreatment with PCs markedly restored reduced MMP (Fig. 2B and C).

JNK/c-Jun signaling is commonly activated by various stress stimuli, and is a known mediator of cell apoptosis under various pathophysiological conditions (20). Therefore, MPTP-induced cell apoptosis and the potential protective effects of PCs were examined by western blotting. Administration of MPTP significantly increased p-JNK/JNK and p-c-Jun/c-Jun expression ratios. Furthermore, proapoptotic proteins Bim, cleaved caspase-3 and cleaved PARP were detected; MPTP significantly increased the expression of these proteins, whereas PC pretreatment inhibited this increase (Fig. 3).

As presented in Fig. 4A, the PD mouse model group were of a lower weight compared with the control group; however, this effect was reduced following treatment with PCs. To determine the effects of PCs on MPTP-induced bradykinesia, a pole test was performed on day 7 after MPTP injection. In the MPTP group, T-LA was significantly prolonged to 7.2 sec on day 7, compared with the control group. However, on day 7, T-LA was significantly shortened in the 300, 400 and 500 mg/kg PC-treated groups to 5.9, 6.1 and 4.0 sec, respectively, compared with MPTP alone (Fig. 4B).

The neuroprotective action of PCs and the functional viability of dopaminergic neurons in the substantia nigra pars compacta were further assessed by determining the expression of the rate-limiting enzyme for DA biosynthesis, TH. As evidenced by IHC and western blot analysis (Fig. 5A and B), the expression of TH was reduced in MPTP mice compared with the control group. Conversely, TH expression in PC-pretreated mice was more pronounced compared with in MPTP-induced mice. TH is a specific marker protein for the identification of midbrain dopaminergic neurons (21). Therefore, these results demonstrated that PCs may protect against neuronal loss in a mouse model of PD, thus suggesting that PCs exert a neuroprotective effect in vivo.

Analysis of TUNEL staining in the substantia nigra further suggested that the control and PC-pretreated groups presented with fewer TUNEL-positive cells compared with in the MPTP group (Fig. 6A). ROS levels were subsequently detected. As presented in Fig. 6B, MPTP exposure led to a significant elevation in ROS levels in primary mice substantia nigra cells compared with in the control group. Pretreatment with PCs inhibited ROS generation in the MPTP group. Furthermore, western blot analysis demonstrated that MPTP increased JNK/c-Jun signaling pathway protein expression, whereas PCs reversed this increase (Fig. 6C).