Modeling method
MPTP was used to establish the PD mice (C57BL6) model. Each mouse in MPTP group received intraperitoneal injection of MPTP 20 mg/kg for 4 times in 7 days, while the same amount of normal saline was injected to each mouse in CON group. UC2288 group each mouse was injected with 10 mg/kg UC2288 and MPTP + UC2288 group treated MPTP (20 mg/kg) with UC2288 (10 mg/kg) for 4 times in 7 days by intraperitoneal injection under the guideline of National Institute of Toxicological Research Korea and Drug Administration for the human care and use of laboratory animals and approved by IACUC of Chungbuk National University CBNUR-1117-18. 1–2 h later after the last injection, the behavioral tests and biochemical experiments were conducted in succession.
Behavior Tests
Behavior changes were tested by Rota rod, Pole and gait tests as described elsewhere 32. Motor performance and coordination were examined using the Rota rod treadmill (MED Associates Inc., St. Albans, VT.), consisting of a 3.6-cm diameter cylindrical treadmill connected to a computer controlled stepper motor as described previously 33. The pole test trials were performed using the rough-surfaced wooden pole three times per animal and average values from three examinations were used for each animal. A gait test trial was performed on a bright runway (4.5 cm wide) and a dark goal box (20 × 17 × 10 cm) and stride length was measured as the distance between successive paw prints. Data was presented as the average of five strides for each animal.
Cresyl Violet Staining
The brains were post fixed in 4% paraformaldehyde for 24 h at 4 ℃, and then transferred to 30% sucrose solutions. 25-µm brain sections were thoroughly washed with phosphate-buffered saline (PBS), and then remove the excess fixative agent and then transferred to gelatin-coated slide glasses and stained with 0.1% cresyl violet (2–5 min) for the purpose of identifying cortical layers and cytoarchitectural features of the isocortical region. After this, the sections were washed in distilled water then dehydrated through ascending grades of ethanol, 50, 70, 90, and 100% ethanol for 2 min in each grade followed by 10 min immersion in a 1:1 mixture of absolute alcohol and xylene. They were cleared in xylene for 5–10 min and mounted in mounting medium (Cytoseal™ XYL; Thermo Scientific, Pittsburgh, CA).
Oxidative Stress Assay
The MDA concentration in brain tissue and neuronal cells were determined based on thiobarbituric acid (TBA) reactivity (Cell Biolabs, Inc. San Diego, CA) by the manufacture’s guidance. Briefly, trichloroacetic acid with HaCaT keratinocytes mixture were centrifuged, and then supernatant was related with TBA. The reactingr color was measured at 532 nm with a spectrophotometer. H2O2 binds to molybdenic acid complex was assessed using a commercially available kit (DCFH-DA, Sigma Aldrich) specifically as a H2O2 content at 405 nm, and the H2O2 content was then calculated. GSH-Px activity was determined by the velocity method using a GSH-Px kit (DCFH-DA, Sigma Aldrich). The change in absorbance during the conversion of GSH to GSSG was recorded spectrophotometrically at 412 nm as GSH-Px activity. SOD production of ARBEC was assessed with a superoxide anion assay kit (Catalog Number CS1000, Sigma, USA). The cells were grown in none serum medium, and they were treated with 100 µM MPP + for 24 h or with 10, 20, 50 nM UC2288. Cell suspensions (90 µl each) were added to the 96-well plate containing 5 µl of luminol solution (Catalog Number L5043) and 5 µl of enhancer solution (Catalog Number E4281), and the samples were mixed with a micropipette. After a 15 min incubation period, the luminescence intensity was measured with an Orion II Microplate Luminometer (Berthold, Germany).
Western Blot Analysis
The brain tissues were homogenized with lysis buffer (PROPREP; iNtRON, Sungnam, Korea; n = 8 mice per group) and centrifuged at 2,500 × g for 15 min at 4 ℃. Equal amounts of total protein (40 µg) isolated from brain tissues were resolved on 8 or 10% sodium dodecyl sulfate polyacrylamide gels and then transferred to nitrocellulose membranes (Hybond ECL; Amersham Pharmacia Biotech, Piscataway, NJ). Membranes were incubated at roomtemperature for 2 h with the following specific antibodies: anti-parkin, anti-ubiquitin, anti-ERK, anti-p-ERK, anti-p-P21, anti-p-P38, anti-P38, anti-p-JNK, anti-JNK (Cell Signaling Mol Neurobiol Technology, Inc., Beverly, MA), anti-BAX, anti-cleaved caspase-3 (1:1,000; Santa Cruz Biotechnologies, Inc., Santa Cruz, CA) and anti-β-actin (1:2,500; Sigma, St Louis, MO). Blots were then incubated at room temperature for 2 h with, corresponding peroxidase-conjugated anti-mouse or anti-rabbit antibodies (1:2,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Immunoreactive proteins were detected using an enhanced chemiluminescence (ECL) Western blotting detection system. The relative density of the protein bands was scanned densitometrically using My Image (SLB, Seoul, Korea) and quantified by Lab Works 4.0 (UVP, Upland, CA).
Immunohistochemistry And Immunofluorescence
Immunohistochemistry and immunofluorescence analyze were done as describe elsewhere 32. Using paraffin-embedded tissue sections after incubated with p-p21 anti-mouse IgG antibody (1:1000 dilution; Vector Laboratories, Burlingame, CA, USA) After subsequently washed and incubated with avidin-conjugated peroxidase complex (ABC kit, 1:200 dilution; Vector Laboratories), the tissue section was stained with 3, 3′- diaminobenzidine tetrahydrochloride (DAB, 0.02%) as the chromogen. And evaluated via light microscopy (Olympus, Tokyo, Japan). For immunofluorescence examination, sections were incubated with anti-mouse secondary antibody labeled with Alexa-Fluor 568 (1:400 dilution; Invitrogen). The final images were acquired using a confocal laser scanning microscope (TCS SP2, Leica Microsystems AG, Werzlar, Germany).
Levels Of IL-6, IL-1β And TNF-α Of Protein
According to the instruction of manufacture, the level of IL-6, IL-1β and TNF was measured by a sandwich enzyme-linked immunosorbent assay (ELISA) as described elsewhere 34. The IL-6, IL-1β and TNF level in the blood were expressed as picogram of cytokines per milligram of protein.
Cell Culture And Treatment
Pregnant SD rats are delivered by DBL Company 48 h before sacrify in a calm room. Embryos were removed from pregnant (day 17) SD rats (DBL, South Korea) under intraperitoneal pentobarbitol (Sigma-Aldrich, UK) anesthesia. Cortices were dissected, and treated with papain (10 U/mL, Sigma-Aldrich, UK), then exposed for 5 min in Phosphate Buffer Saline (PBS) solution containing: DNAse I (Invitrogen, Thermo Fisher Scientific, Waltham, USA) and B27 (Gibco, Thermo Fisher Scientific, Waltham, USA), and D-glucose (33 mmol/L, Sigma-Aldrich, UK). Cells were then dissociated by trituration and filtered through a membrane (70 lm, BD Falcon). Cells were then purified with a BSA solution (8%, Sigma-Aldrich, UK) diluted in Neurobasal A-25 (Invitrogen, Thermo Fisher Scientific, Waltham, USA). (0.1 mg/mL, Sigma-Aldrich, UK). For each experiment, cortices from 8 to 12 embryos per rat are mixed. Experiments were reproduced three to eight times. Cells were grown in poly-D-lysine coated dishes Neurobasal (Eurobio) supplemented with B27 medium (Invitrogen, Thermo Fisher Scientific, Waltham, USA), containing 2 mmol/L glutamine, 0.1% penicillin and streptomycin (Gibco, Thermo Fisher Scientific, Waltham, USA), 250 U/mL amphotericin (Invitrogen, Thermo Fisher Scientific, Waltham, USA), and 1 mmol/L lactic acid (Sigma-Aldrich, UK) at a density of 6 × 105 cells per cm2. The percentage of neuronal cells in our culture system is more than 90% judged with cell morphology and immunostaining measurement. Cells were incubated at 37 °C and 95% humidity with 5% CO2. When the cells reached ∼80% confluence, the cells were then treated either with freshly prepared MPP + in DMSO (100 nM final concentration).
TUNEL Assay
Cell Death Detection Kit (Roche Diagnostics GmbH, Mannheim, Germany) was used for TUNEL assay according to the manufacturer's instructions. briefly, after fixation of 25-mm cryosections with 4% paraformaldehyde, 0.1% NaBH4 and 0.1 Triton X-100 was treated The slides were then incubated for at least 1 h with mixture reaction buffer containing deoxynucleotidyl transferase and FITC– dUDP (Roche, Reinach, Switzerland). For 40, 60-diamidino-2-phenylindole dihydrochloride (DAPI) staining, the slides were then incubated for 15 min at room temperature in the dark with a mounting medium for fluorescence containing DAPI (Vector Laboratories, Cambridgeshire, UK). The tissues were then examined through a fluorescence microscope (Leica Microsystems AG, Wetzlar, Germany), and the nuclei were visualized via DAPI staining.
Statistical Analysis
GraphPad Prism 4 software (GraphPad Software, La Jolla, CA) was used for statistical ananlysin. A one-way analysis of variance (ANOVA) was applied for assessment of difference among the graph. Once the significant was found, the differences were further analyzed by the Dunnett’s test. Data are presented as mean ± SD, a value of P < 0.05 was considered to be statistically significant.