SDA was obtained from Southern Medical University Integrated Hospital of Traditional Chinese Medicine (Guangzhou, China); 1-Methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP), G418, Doxycycline (Dox), Rapamycin (Rap), Chloroquine (CQ), MG132, Hoechst 33342, DAPI and rabbit anti-α-syn antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA.); Unless otherwise specified, all other chemicals and reagents used were of analytical grade.
2.2 Cell culture
PC12 cells were purchased from the American Type Culture Collection (ATCC, USA), were cultured in Dulbecco’s modiﬁed Eagle’s medium (DMEM) supplemented with 10% heat-inactivated horse serum (HS), 5% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin (Carlsbad, CA, USA) at 37oC in a 5% CO2 atmosphere incubator. PC12 cells transfected with A53T α-syn genes (PC12/α-syn cells) were stimulated with Dox to overexpress A53T α-syn and the PC12 stable overexpressing α-syn were cultured in DMEM containing 10% HS, 5% FBS and 200 μg/mL G418 (Sigma-Aldrich, MO, USA) at 37 oC in a 5% CO2 atmosphere incubator.
The male C57BL/6J mice (weighing 21-25 g, aged 10 weeks) and male SD rats (weighing 220-250 g), provided by Guangdong Medical Laboratory Animal Center, were used for the preparation of PD model. They were raised on a 12 h light/dark cycle with ad libitum access to food and water and were housed in clean cages under conditions of 24±1oC room temperature and 55±5% relative humidity. All experimental protocols were conducted according to guidelines of the experimental animal care and use committee of Southern Medical University (Guangzhou, China). The experimental protocols were approved by the Ethics Committee for Animal Experiments of Integrated Hospital of Traditional Chinese Medicine, Southern Medical University.
2.4 Serum samples
Briefly, 10 male SD rats were randomly divided into two groups. Rats in SDA group were intragastric administrated 900 mg/kg SDA twice a day for 5 days. Solvent saline was used in control group. Then on the 6th day, serum samples were collected and analyzed for PC12 cells protection.
2.5 Cell viability
CCK-8 assay was performed in a 96-well plate. Briefly, PC12 cells (1×104 cells/well) were incubated with different concentrations of SDA serum samples for 24 h and then exposure to 60 μM 6-OHDA (Sigma-Aldrich, MO, USA) for further 24 h. The absorbance was measured at 570 nm on a microplate reader. Cell viability was expressed as a percentage of control group.
2.6 Flow cytometer analysis
Annexin V-FITC/PI double staining analysis for apoptosis. For experiment group, PC12 cells were treated with 20% SDA serum for 24 h and then exposure to 60 μM 6-OHDA for further 24 h. After treatment, the cells were collected and stained with Annexin V-FITC/PI and subjected to flow cytometry analysis (BD Accuri C6). The positive control group was only treated with 6-OHDA and the negative control cells were cultured in complete medium.
2.7 Hoechst staining assay
PC12 cells were subcultured in a 35 mm confocal dish at a density of 5×104 cells/dish and allowed to adhere for 12 h. After washing with PBS for 3 times, the cells were fixed with 4% polyformaldehyde, incubated with Hoechst 33342 (5 mg/mL) for 5 min at 4oC, and then observed by fluorescence microscope.
2.8 siRNA transfection
PC12 cells transfected with A53T α-syn genes were stimulated with Dox resulting in overexpressing A53T α-syn (inducible PC12/α-syn cells), which were seeded in 6-well plates at a density of 2×105 cells/well and allowed to adhere for 12 h. Cells were transfected with Nrf2 siRNA or scrambled siRNA (Santa Cruz, CA, USA) for 48 h using SureFECT transfection reagent according to the manufacturer’s instructions. After induction by Dox (1 μg/mL) for 24 h to induce the expression of α-syn, cells were treated with SDA for another 24 h and then collected for western blot assay.
2.9 MPTP-induced mice PD model
The mice PD model was created by acute MPTP protocol . Briefly, MPTP, a selective DA neurons neurotoxin, was injected (30 mg/kg/day i.p.) for 5 days to selectively destruct DA neurons and induce experimental Parkinsonism. After 3 days for a resting period, SDA treatment groups (100, 300 and 900 mg/kg), Selegiline (10 mg/kg) or equal volume of saline were administered orally once daily for 5 days for 2 consecutive weeks to PD mice.
2.10 Behavior test
At the end of treatment, mice were tested behaviorally (pole, rotarod and open field tests) to evaluate the different aspects of parkinsonism.
The pole test was used to evaluate the coordination of mice limbs . The pole (length 50 cm, diameter 1 cm) was fixed one 1.5 cm-diameter ball in its upper end and wrapped in gauze to prevent slipping. The trial of mice descending to the bottom platform was performed three successive times with 1 h interval. The time was recorded and average value of three trials was calculated for statistical analysis.
Rotarod test was also used to measure the movement and coordination of PD model mice . Mice were positioned on the rotarod (Anhui Zheng Hua Instrument Co., Ltd., China), and then tested on the revolving rod at the speed of 5 rpm for up to 120 s. Automatically recorded time, mice first fell off the rod, was designated as latency. Mice were tested three successive trials with 1 h interval. The trial was performed three successive times with 1 h interval and average time was calculated for statistical analysis.
The open field test was performed in an open field apparatus (length 50 cm, width 50 cm, height 40 cm), which consists of clear plexiglass walls and floor. Briefly, mice were placed individually in an acrylic apparatus with a floor divided into equal squares (length 25 cm, width 25 cm), then left to freely explore the arena for 5 min and movement distance was recorded for statistical analysis .
2.11 TH immunofluorescence
Briefly, the brains were collected, fixed with 4% PFA (v/w = 10:1) overnight at 4oC, dehydrated with graded sucrose solution (10 to 30%), OTC-embedded and sliced to make 25 μm coronal sections encompassing the entire SNpc for TH staining. Sections were permeabilized with 0.3% Triton X-100 for 15 min, and then blocked with 5% BSA in PBS for 1 h at room temperature. After they were incubated with anti-TH antibody (Millipore, Billerica, MA, USA) overnight at 4oC, the sections were incubated with anti-rabbit secondary antibody conjugated with FITC (Beverly, MA, USA) for 60 minutes at 37°C. Photographs were taken under a fluorescence microscope (BX51, Olympus Corp, Japan).
2.12 HPLC analysis
Endogenous level of DA and its metabolites (DOPAC, 3,4-dihydroxyphenylacetic acid and HVA, homovanilic acid) were measured using reverse-phase HPLC. Briefly, striatal tissues were dissected rapidly on ice, immediately frozen in liquid nitrogen and stored at -80oC for further biochemical analysis. The weighed striatum was sonicated in 0.1 N perchloric acid, the homogenate was centrifuged at 12000 g for 10 min at 4oC, and then 20 μL of supernatant collected was injected into an HPLC-electrochemical detection system equipped with Agilent Eclipse Plus C18 reverse phase column (4.6×150 mm). The endogenous level of DA and its main metabolites was expressed as ng/mg tissue weight.
2.13 Western blotting
Samples were homogenized with RIPA buffer containing 1 mM PMSF and 1% halt phosphatase inhibitor cocktail on ice. After centrifugation, the supernatants were retrieved and protein concentrations were measured with a BCA kit. 30 μg protein per sample was separated by 10-15% SDS-PAGE and transferred to PVDF membranes. After blocking with 5% skim milk for 1 h, the membranes were incubated with various primary antibodies overnight at 4oC and then incubated with secondary antibodies at room temperature for 2 h. The protein bands were detected using an enhanced chemiluminescence plus kit (Amersham Bioscience, Aylesbury, UK). β-actin was used as the loading control and the immunoreactivity of each protein was performed using Care stream Molecular Imaging Software.
2.14 Statistical analysis
All experimental were analyzed by the GraphPad Prism software 5 (GraphPad, San Diego, CA, USA). One-way ANOVA and Tukey’s multiple comparison test methods were used in the analysis method. The data were expressed as mean ± SEM. P < 0.05 was considered to be significant statistically. All experiments were repeated at least three independent times.