GPS was purchased from Ankang Dongke Maidisen Nature Pharmaceutical Co. (purity > 99%, confirmed by HPLC analysis) (Xi’an, China) [14,29]. GP was obtained from the Wonkwang Food Manufacturing Co. (Geochang, Korea) and a voucher specimen of the herbal leaves of GP was deposited at the herbarium of the College of Pharmacy, Chungbuk National University (Cheongju, Korea). The air-dried leaves of GP (1 kg) were extracted with ethanol (80%, v/v) and the ethanol extracts were evaporated to dryness under reduced pressure and temperature (GP-EX, 97.2 g, yield, 9.7%, w/w).
L-DOPA, 6-OHDA, benserazide hydrochloride, and apomorphine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rabbit polyclonal primary antibodies and anti-rabbit IgG HRP-linked secondary antibodies against ERK1/2, phospho-ERK1/2, ∆FosB and β-actin were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). All other chemicals were of analytical grade.
Rats (Sprague–Dawley, male, 200–250 g) were purchased from Samtako (Osan, Korea) and housed under standard conditions of temperature (23 ± 2°C), humidity (60 ± 5%), and illumination (12-h light–dark cycle lighted on at 07:00) with ad libitum access to standard rat food and water. All experimental procedures were approved by the guidelines of Animal Ethics Committee of Chungbuk National University Laboratory Animal Research Center (Approval no. CBNUA-708-141-01) and were conducted according to the National Institutes of Health (NIH) guidelines.
Unilateral 6-OHDA lesion
Unilateral 6-OHDA lesions were conducted as described previously [11,17]. The rats were anesthetized intraperitoneally with Zoletil 50 (100 mg/kg, Virbac, Carros, France) and placed in a stereotaxic stand (David Kopf Instruments, Tujunga, CA, USA). The coordinates for the medial forebrain bundle were measured accurately (antero-posterior, AP: −2.5 mm; medio-lateral, ML: +2.0 mm; dorso-ventral, DV: −8.5 mm; relative to bregma). Next, 6-OHDA (8 μg/2 μl in saline solution containing 0.05% of L-ascorbic acid) was single injected into the left medial forebrain bundle at 1 μl/min using a Hamilton syringe. After the injection, the needle was left in place for 5 min before being retracted in order to allow for complete diffusion of the medium. The rats were left in the stand until they had recovered from the anesthesia. In order to assess the efficacy of the lesion, all rats were tested for apomorphine (0.5 mg/kg, s.c.)-induced rotation at 3 weeks after the 6-OHDA lesions. Rats showing more than 150 rotations/30 min were selected for this study . In these states, the striatal levels of dopamine in 6-OHDA-lesioned rats decreased to 40.9–47.1% as compared with control group (dopamine levels of control group, 7.12 ± 0.75 ng/mg tissue).
The experimental rats were randomly divided into five groups (n = 8–10 per group) 6 weeks after the 6-OHDA lesion . The 6-OHDA-lesioned groups were treated with saline (0.9%, i.p.) or both L-DOPA (25 mg/kg, i.p.) and benserazide (15 mg/kg, i.p.) at 10 am once a day for 22 days. In addition, the 6-OHDA-lesioned groups treated with L-DOPA (25 mg/kg, i.p.) were treated with either GPS (25 and 50 mg/kg) or GP-EX (50 mg/kg) orally (p.o.) 30 min prior to L-DOPA administration once a day for 22 days  (Figure 1). After the last day of GPS, GP-EX and L-DOPA treatment, rats were tested for last AIMs score and contralateral rotation. Finally, the rats were sacrificed for biochemical analysis including ∆FosB expression and ERK1/2 phosphorylation.
Rats were monitored for AIMs according to previously published procedures and methods [17,37]. After treatment with L-DOPA, rats were observed individually for 1 min every 20 min for 180 min period following dose of L-DOPA. Observation was performed by trained observers who were blinded to the animal groupings and experimental conditions. The AIMs were scored for exhibition of the following four categories: (1) axial AIMs, twisting movement of the neck, trunk and head toward the side contralateral to the 6-OHDA lesion; (2) limb AIMs, repetitive jerky movements or dystonic posturing of the forelimb contralateral to the 6-OHDA lesion; (3) orolingual AIMs, purposeless jaw movements and contralateral tongue protrusion without the presence of food or other objects; (4) locomotive AIMs, increased circular locomotion with contralateral side bias. During the 1 min observation period, the four subtypes were scored on a scale from 0 to 4 in each rat based on the following criteria: 0, not present; 1, present for less than half of the observation time; 2, present for more than half of the observation time; 3, present all the time but suppressible by threatening stimuli; 4, present all the time and not suppressible. The axial, limb, and orolingual AIMs were calculated by adding each of the individual dyskinesia scores as body AIMs (total AIMs). The body AIMs were also expressed by the area under the curve of the each AIMs parameter. Locomotive AIMs rating took into account the rat’s circular movements on a flat floor using its all four limbs, which were counted by only complete 360° turns. The contralateral rotation was counted for 1 h and started at 20 min after L-DOPA .
The rats were deeply anesthetized with Zoletil 50 (100 mg/kg, Virbac, Carros, France) and sacrificed by rapid decapitation 1 h after L-DOPA treatment. The brains were quickly removed and the striatum was isolated on ice and homogenized in a lysis buffer. Protein extracts from the striata of PD rats were prepared from the left striata, and 20 μg protein from each rat was used for western blotting . The primary antibodies used were rabbit polyclonal primary antibody (1:1000 dilution) against ∆FosB, phospho-ERK1/2, ERK1/2 and β-actin. Proteins in samples (20μg) were separated using 10–15% sodium dodecyl sulfate-poly acrylamide gel electrophoresis. Proteins were transferred to polyvinylidene difluoride membrane at 300 mA for 1 h. The blots were blocked for 1 h at room temperature in a fresh blocking buffer (TBS-T containing 5% bovine serum albumin [BSA]) and then incubated overnight at 4°C using primary antibodies diluted 1:1000 in TBS-T with 5% BSA, and for 1 h at room temperature using secondary antibodies (anti-rabbit IgG HRP-linked antibodies, 1:5000 dilution in TBS-T with 5% BSA), according to a standard procedure. The blots were then washed, and the transferred proteins were incubated with ECL substrate solution (Amersham Pharmacia Biotech, Inc., Piscataway, NJ) for 5 min, according to the manufacturer’s instructions, and visualized with a radiographic film.
Protein amounts were determined by a bicinchoninic acid protein assay kit using BSA (Pierce Protein Research Products, Rockford, IL). Behavioral data and group comparisons of dyskinesia intensity scores were analyzed by non-parametric Kruskal-Wallis one-way ANOVA test for multi-group comparisons at each day and Friedman repeated measures ANOVA test for two-group comparisons unless otherwise indicated. Biochemical data were also analyzed by one-way ANOVA followed by Tukey’s test. All data were expressed as mean ± S.E.M. with P values of < 0.05 being considered statistically significant.