Contributed equally
The pathological alterations of Parkinson's disease (PD) predominantly manifest as a loss of dopaminergic neurons in the substantia nigra, which may be caused by oxidative stress damage. Proanthocyanidins (PCs) are a class of compounds found in various plants, which have significant antioxidant and free radical-scavenging activity. The present study investigated the protective effects and underlying mechanisms of PCs in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD model
Parkinson's disease (PD) is a common chronic degenerative disease of the nervous system, which is primarily characterized by a substantial loss of substantia nigra dopaminergic neurons, leading to a reduction of dopamine (DA) levels in the striata, accompanied by cognitive impairment and functional defects (
Oxidative stress is thought to be a main cause of dopaminergic neuron degeneration in PD (
Proanthocyanidins (PCs) are natural phenolic compounds that are present in various plants. PCs have gained increasing attention in the fields of nutrition and medicine, due to their antioxidative, anti-inflammatory (
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 (
Brain tissue preparation was performed as previously described (
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 (
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 (
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 (
JNK/c-Jun signaling is commonly activated by various stress stimuli, and is a known mediator of cell apoptosis under various pathophysiological conditions (
As presented in
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 (
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 (
In order to investigate the effects of PCs on dopaminergic neurons, an MPTP-induced experimental model of PD was established
PD is a movement disorder characterized by progressive loss of nigrostriatal dopaminergic neurons. Therapeutic strategies that slow or stop the neurodegenerative processes of PD are urgently required. The identification of polyphenolic compounds or polyphenols with potential neuroprotective properties has increased considerably during the last few years. Catechins, such as epigallocatechin-3-gallate, have been reported to exert several actions on the CNS, including anxiolytic, sedative and neuroprotective effects on animal models of Alzheimer's disease and PD. Notably, PCs are composed of catechin and epicatechin oligomers (
Overwhelming evidence has indicated that the apoptotic death of nigrostriatal dopaminergic neurons is initiated by oxidative stress (
Evidence indicates that activation of JNK regulates ROS-induced neuronal apoptosis (
In conclusion, PCs may represent a safe and affordable intervention for the clinical treatment of PD. PCs effectively prevented mitochondrial apoptosis, ROS production and JNK activation in neurons. The results of the present study provided experimental evidence to support the potential use of PCs as a therapeutic agent in PD.
Not applicable.
No funding was received.
The datasets used or analysed during the current study are available from the corresponding author on reasonable request.
XX, HC and JX conceived and designed the study. HC, JX, YL, PH and CL performed the experiments. JJ, XM and SL analyzed the data. XX and XM wrote the manuscript. All authors read and approved the manuscript.
The present study was approved by the Institutional Animal Care and Use Committee of Nanjing Medical University.
Not applicable.
The authors declare that they have no competing interests.
Protective effects of PCs on MPTP-induced cytotoxicity and apoptosis in PC12 cells. (A) Chemical structure of PCs. (B) Cell viability was determined using MTT assays. (C) Flow cytometric analysis of the effects of PCs on MPTP-induced apoptosis. Three independent experiments were performed. **P<0.01 vs. untreated control cells; ##P<0.01 vs. the MPTP-only treated group. FITC, fluorescein isothiocyanate; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; OD, optical density; PCs, proanthocyanidins; PI, propidium iodide; Q, quadrant.
Effects of PCs on MPTP-mediated ROS generation and mitochondrial dysfunction in PC12 cells. Cells were treated with MPTP in the absence or presence of 0.5, 1 or 5 µg/ml PCs for 24 h. (A) ROS levels were detected with the fluorescent probe, 2′,7′-dichlorodihydrofluorescein diacetate. (B) Mitochondrial membrane potential was measured using the fluorescent probe JC-1. (C) Increased MMP was observed in the control group and treatment with MPTP significantly suppressed the MMP in PC12 cells; however, pretreatment with PCs significantly attenuated suppressed levels of MMP. Three independent experiments were performed. **P<0.01 vs. untreated control cells; #P<0.05, ##P<0.01 vs. the MPTP group. FITC, fluorescein isothiocyanate; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PCs, proanthocyanidins; ROS, reactive oxygen species.
Western blot analysis of the JNK/c-Jun signaling pathway in PC12 cells. Cells were treated with MPTP in the absence or presence of 0.5, 1 or 5 µg/ml PCs for 24 h. Three independent experiments were performed. **P<0.01 vs. untreated control cells; #P<0.05, ##P<0.01 vs. the MPTP group. Bim, B-cell lymphoma 2-like protein 11; JNK, c-Jun N-terminal kinase 1; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; p, phosphorylated; PARP, poly (ADP-ribose) polymerase; PC, proanthocyanidins.
Effects of PCs on MPTP-induced movement impairment in mice. PCs (300, 400 or 500 mg/kg/day) were orally administered for 7 days. Subsequently, MPTP was intraperitoneally injected once every day for 7 days. Following the last MPTP injection, the pole test was conducted and mouse weight was recorded. (A) Alterations in mouse weight in each group were recorded. (B) Latency time on the climbing pole was recorded with a 30 sec cut-off limit. Three independent experiments were performed. *P<0.05 vs. the untreated control group; #P<0.05 and ##P<0.01 vs. the MPTP group. MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PCs, proanthocyanidins.
Effects of PCs against MPTP-induced neurotoxicity
Effects of PCs on MPTP-induced apoptosis. (A) A TUNEL assay was performed to detect apoptosis, and TUNEL-positive cells were detected (magnification, ×200). (B) ROS levels were measured using the fluorescent probe, 2′,7′-dichlorodihydrofluorescein diacetate. (C) JNK/c-Jun signaling pathway proteins were detected via western blot analysis. Three independent experiments were performed. **P<0.01 vs. the untreated control group; #P<0.05, ##P<0.01 vs. the MPTP group. Bim, B cell lymphoma 2-like protein 11; JNK, c-Jun N-terminal kinase 1; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; p, phosphorylated; PARP, poly (ADP-ribose) polymerase; PCs, proanthocyanidins; TUNEL, terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling.