Animals
For all experiments, 110 male Sprague Dawley rats weighing 180–220 g were obtained from Guangzhou University of Chinese Medicine, and experiments were performed according to the guidelines for the ethical treatment of experimental animals. All rats were housed at room temperature (22–27 ℃) under a 12 h light/12 h dark cycle (8:00 am–20:00 pm). The animals were raised in the Specific Pathogen Free Animal Laboratory of the First Affiliated Hospital of the Guangzhou University of Chinese Medicine. All procedures were performed in accordance with the guidelines of the Experimental Animal Ethics Committee of the First Affiliated Hospital of the Guangzhou University of Chinese Medicine. The Sprague Dawley rats were randomly divided into nine groups: sham-operated, DPD model, pramipexole, β-asarone low-dose, β-asarone medium-dose, β-asarone high-dose, 3-MA, rapamycin, and PI3K inhibitor. The 3-MA, rapamycin, and PI3K inhibitor groups were administered 3-methyladenine (3.0 mg/kg), rapamycin (1.0 mg/kg), and LY294002 (0.75 mg/kg), respectively, dissolved in 1% DMSO by intraperitoneal injections for 4 weeks. The pramipexole group received pramipexole (0.15 mg/kg) dissolved in saline via oral administration for 4 weeks. The low, medium, and high β-asarone groups received β-asarone (7.5, 15, or 30 mg/kg, respectively) dissolved in Tween 80 by oral administration for 4 weeks.
Reagents
The main antibodies the following protein were used in this study: Anti-Beclin 1 (1:1000, Abcam, ab207612, USA), rabbit Anti-p62 (1:1000, Abcam, ab109012, USA), rabbit Anti-PI3 Kinase p85 alpha (1:1000, Abcam, ab191606, USA), rabbit Anti- PI3 Kinase p85 alpha (phospho Y607) (1:1000, Abcam, ab182651, USA), rabbit Anti- pan-Akt (1:1000, Abcam, ab8805, USA), rabbit Anti-pan-Akt (phospho T308) (1:1000, Abcam, ab8933, USA), rabbit Anti-mTOR (1:1000, Abcam, ab134903, USA), rabbit Anti-mTOR (phospho S2481) (1:1000, Abcam, ab137133, USA), Anti-GAPDH (1:3000, Kangwei Century Biotechnology Co., Ltd. China) and goat anti rabbit secondary antibodies (rabbit, 1:3000, Kangwei Century Biotechnology Co., Ltd.). Other reagents included 6-OHDA (MCE, 23926), pramipexole (Boehringer Ingelheim, 303983-12), 3-Methyladenine (MCE, #40214), rapamycin (MCE, S1039) and LY294002 (MCE, #42715). DA (China National Institute of Food and Drug Control, 100070-201507), L-DOPA (China National Institute of Food and Drug Control, 100170-201805), 5-HT (China National Institute of Food and Drug Control, 1111656-202002), 5-HIAA (Shanghai yuanye Bio-Technology Co., Ltd, K20M11B109867), HVA (Shanghai yuanye Bio-Technology Co., Ltd, A19J11L116289). Rat ELISA kits for ɑ-syn, Trp, MAOA, TPH, TH, DDC, 5-HTP and 5-HTPDC (Shanghai Zylian Biotechnology Co. , Ltd. 11/2011).
β-asarone was extracted from Acorus tatarinowii Schott according to a previously established procedure. The purity of β-asarone reached 99.55%, as confirmed by gas chromatography mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance detection [29].
6-Hydroxydopamine lesion
Rats were anesthetized with an intraperitoneal injection of 20% urethane (1.5 g/kg) and were placed in a flat skull position on a cotton bed on a stereotaxic frame (Stoelting, Wood Dale, USA) with the incisor bar fixed at 4.5 mm below the interaural line. Severe unilateral lesion of the nigrostriatal pathway was obtained by microinjection (SGE Analytical Science, Ringwood, Australia) of 6 µL of 6-OHDA (4 µg/µL, free base dissolved in a solution of 0.2 mg/mL L-ascorbic acid in 0.9% normal saline) at a rate of 0.2 µL/min into the medial forebrain bundle at the following coordinates according to Paxinos and Watson [30] (flat skull, as above): tooth bar: -2.3 mm, anteroposterior: -4.4 mm, mediolateral: 1.2 mm, and dorsoventral: -7.8 mm. The rats in the sham-operated group received the same volume of normal saline according to the same procedure, excluding the 6-OHDA lesion. The injection speed was 0.5 µL/min, and the needle was left in for 15 min. The rats were injected intraperitoneally with 0.5 mg/kg apomorphine 28 days after modeling. The successful PD model rats rotated to the healthy side by more than seven cycles/min.
CUMS procedure
The CUMS procedure was modified from previously published reports [31-32]. Except for the sham-operated group, all experimental animals were exposed to a series of mild stressors including (1) cage tilt (45°) for 24 h, (2) food deprivation for 24 h, (3) water deprivation for 24 h, (4) tail clamping for 3 min, (5) soiled cage (500 mL of water in 100 g maize-cob bedding) for 24 h, (6) overnight illumination, and (7) forced swimming at 4 °C for 5 min. The above stimulations were applied randomly to ensure unpredictability in the experiment. The entire stimulation process was performed over 4 continuous weeks.
Sample collection
The rats were anesthetized via an intraperitoneal injection of 20% urethane (1.5 g/kg) 1 h after the last drug administration. Subsequently, the rats were perfused with normal saline; perfusion ended when colorless liquid outflow was observed from the right atrial appendage. The rats were sacrificed after perfusion. The striatum, hippocampus, prefrontal cortex, and midbrain of the injured hemisphere (left cerebral hemisphere) were harvested for HPLC, enzyme-linked immunosorbent assay (ELISA), and western blot analyses. Lastly, remaining samples were harvested and fixed in 0.25% glutaraldehyde for transmission electron microscopy analysis.
HPLC
Midbrain tissue was weighed, homogenized in 5% perchloric acid, incubated on ice for 15 min, and centrifuged at 13000 g for 20 min at 4 ℃. Neurotransmitter levels were quantified using HPLC with fluorometric detection (HPLC-FD) (Water Alliance 2695 Separations Module; 2475 Fluorescence Detector, Waters, Milford, USA). The excitation and absorption wavelengths were 280 and 330 nm, respectively. The analysis was performed using a 5 µm Symmetry C18 column (150 mm × 4.6 mm; Waters) with a column temperature of 30 ℃ and a flow rate of 1 mL/min. The mobile phase consisted of 0.1 mol/L KH2PO4 and methanol at pH 3.5. For the gradient elution, the percentage of KH2PO4 was decreased from 100% to 82% between 0 and 18 min, and the percentage of methanol was increased from 0% to 18%. The column was equilibrated with the mobile phase for 30 min before analysis. Data are expressed as nanogram serotonin per milligram of tissue [16].
ELISA
The mesencephalon and hippocampus of rats in the sham-operated, DPD model, pramipexole, and low-, medium-, and high-dose β-asarone groups were homogenized at a ratio of 1:9 (wt:vol) in ice-cold phosphate-buffered saline (PBS) (pH 7.4, 4 ℃) and centrifuged at 3500 g at 4 ℃ for 15 min to obtain the cellular proteins in the supernatant. The ELISA was performed according to the manufacturer’s instructions. Data are expressed as pg/mg of tissue.
Western blot
Samples from the striatum and hippocampus were homogenized at a ratio of 1:10 (wt:vol) in ice-cold lysis buffer and centrifuged at 13,000 g at 4 ℃ for 15 min to obtain the cellular proteins in the supernatant. The concentration of total extracted protein was determined using a bicinchoninic acid (BCA) protein assay. Proteins (55 μg) were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a PVDF membrane. Membranes were blocked using QuickBlock™ blocking buffer (Biyuntian Biotechnology Co. , Ltd. China) for 15 min. Next, the membranes were incubated overnight at 4 °C with the following primary antibodies: rabbit anti-Beclin 1, rabbit anti-p62, rabbit anti-PI3K p85 alpha, rabbit anti-pan-Akt, rabbit anti-mTOR, and rabbit anti-GAPDH. The blots were then incubated with horseradish peroxidase-conjugated secondary antibodies at 23 ℃ for 2 h. The bound secondary antibodies were visualized using an enhanced chemiluminescence kit and ChemDoc XRS System with Quantity One software. The blots were repeated at least thrice for each condition. After development, the band intensities were quantified using the ImageJ 1.6.0. software.
Transmission electron microscopy
To further clarify the effects of β-asarone on autophagy, transmission electron microscopy was performed in the prefrontal cortex. Coronal sections (2 μm) were obtained using a vibratome (LEICA EM UC7; Leica Microsystems, Wetzlar, Germany). Sections were used to observe dopaminergic neuron morphology under a transmission electron microscope (H-7650, Hitachi, Tokyo, Japan), as previously described [33].
Statistical analyses
Data were fitted with a Gaussian curve and expressed as mean ± standard deviation (SD). Statistical differences among different groups were determined by one-way analysis of variance followed by a least significant difference post hoc test for multiple comparisons. Data are expressed as median quartile spacing, and statistical differences among different groups were determined by the Kruskal–Wallis test. Differences were considered statistically significant at P < 0.05. All statistical analyses were performed using SPSS 24.0.