Animals
In this study, 96 C57BL/6 male mice (6–8 weeks old, weight 18–22 g) were obtained from SPF (Beijing) Biotechnology Co., Ltd (Beijing, China). Animals were housed in an animal house with a temperature of 20–24 °C, a humidity of 65-70%, and a light/dark cycle of 12 h, 5 animals were housed in each cage with free access to food and water. All experiments were conducted and approved by the Animal Ethics Committee of Guangdong Hospital of Traditional Chinese Medicine (No. 2020062).
Animal model and experimental groups
The mice were randomly divided into eight groups (n = 12), normal control group, model group, piperine low-dose group (10 mg/kg, ig, CAS:94–62-2, Sichuan Weikeqi Biological Technology Co., Ltd.), piperine medium-dose group (20 mg/kg, ig) and piperine high-dose group (40 mg/kg, ig), autophagy inhibitor group (3-methyladenine, 3-MA, 30 mg/kg, ip, CAS: S2767, Selleck, USA), autophagy activator group (rapamycin, 1 mg/kg, ip, CAS: S1039, Selleck, USA), and madopar group (112.5 mg/kg, ig, CAS: SH2443, Shanghai Roche Pharmaceutical Co., Ltd.) for 40 days, once a day. Except for the normal group, reserpine was subcutaneously injected with 0.1 mg/kg once 48 h for 40 days [11]. In addition, on the 20th day of drug-administrated, except for the normal group, bilateral common carotid artery ligation operation was performed under 10% chloral hydrate [350 mg/kg, ip, CAS: A600288-0250, Sangon Biotech (Shanghai) Co., Ltd.] anesthesia and lasted 2 h in all the other groups. When the mice were anesthetized, bilateral common carotid arteries were simultaneously clamped with artery clips for 10 min, then loosened and repeated for 3 times [12]. Finally, the mice were sutured and the PDD mice were established.
Open field test
After the last administration, mice were put into a self-made opaque open box (height 30 cm, bottom side length 72 cm, bottom side was divided into 64 small squares on average, side length of small squares was 9 cm, center 16 grids were central grids, and other grids were edge grids). The experiment lasted for 5 min, and only one mouse was tested at a time. Place the mouse gently in the center square of the self-made open box, and press the stopwatch at the same time to observe and record the behavior of the mouse, including: (1) the residence time of the center grid (the time when the mouse was placed in the center grid until all its four paws left); (2) cross the grid number; (3) the number of grooming (licking feet, scratching and washing face); (4) The number of hind legs standing (the two front paws were vacated at the same time and the hind paws landed on the ground for 1 time). Remove the excrement from the box before the next round of experiments. Calculate the distance of the central motion and the distance of the edge motion according to the number of the crossing lattice.
Pole test
After the last administration, a ball with the diameter of 2.5 cm was fixed at the top of the wooden pole which was at the length of 50 cm and at the thickness of 1 cm. The ball was wrapped with gauze to increase friction, and the wooden pole was placed vertically in a square plastic bowl. At the beginning of the experiment, the mice were placed on the ball, and the time spending on getting down from the ball and the time to climb the wooden pole was recorded. Each mouse was tested for 3 times and the average value was taken.
Sample collection
After the behavioral test, the mice were anesthetized and fixed with 10% chloral hydrate (350 mg/kg, intraperitoneal injection), and then perfused and fixed with 0.9% normal saline successively through the heart. The mice were killed with their heads severed, and the whole brain was removed within 1 min. The cortical, striatum and midbrain tissues were rapidly separated in ice water, weighed, and stored at – 80 °C for index detection.
ELISA analysis
The striatum from mice were homogenized according to the ratio of striatum to ice-normal saline 1:9, centrifuged at 12,000 r/min at 4 °C for 10 min, and the supernatant was extracted. The striatal of DA, MAOB, DDC, β-secretase, acetylcholinesterase (AChE), Aβ42, TNF-α and interleukin-6 (IL-6) levels of mice were determined by mouse ELISA kit (LOT: 07/2021, Shanghai Enzyme-linked Biotechnology Co., Ltd.), respectively. All kits were conducted according to the manufacturer's protocol.
Immunohistochemistry
The mesencephalon was fixed in 4% paraformaldehyde overnight. The mesencephalon was dehydrated by 80% (1 min × 2 times), 95% (3 min × 2 times) and 100% (5 min × 2 times) ethanol, followed by paraffin impregnation, paraffin embedding, and conventional paraffin section with thickness of 4 μm. Slices were immersed in 0.3% H2O2 for 30 min, then hot repaired in citrate buffer (0.01 mol/L, pH 6.0), washed with PBS buffer (5 min × 3 times). Slices were blocked in 5% BSA and incubated overnight at 4℃ with tyrosine hydroxylase (TH) primary antibody (CAS: ab137869, 1:100, Abcam, USA). This was followed by incubation with HRP-labeled secondary antibody (cas: ab205718, 1:1000, Abcam, USA) for 30 min at 37 °C. Then nuclear redyeing was done with hematoxylin, washed and dried with distilled water, sealed with neutral resin, and stored at 37 °C. The positive expression of TH was observed under light microscope, and the images were taken.
Immunofluorescence staining
The mesencephalon was fixed in 4% paraformaldehyde for 24 h, then dehydrated, paraffin embedded and then cut into 30-μm sectioned. Dried the sections until dewaxing, rehydrated and repaired with hot antigen. After cooling, washed with PBS for 3 times, 5 min each time. Dropped 5% BSA (cas: ST025, Shanghai Biyuntian Biotechnology Co., Ltd.) into the sections and sealed at 37 °C for 2 h and then with the Beclin-1, LC3B, p62 and GFAP antibodies (1:100; cas: ab62557, ab192890, ab109012 and ab7260, Abcam, USA) overnight at 4 °C. The next day was incubated in fluorescent secondary antibody (1: 1000, cas: ab150081 and ab150117, Abcam, USA) and incubation at 37 °C for 30 min. And added DAPI solution (cas: C1002, Shanghai Biyuntian Biotechnology Co., Ltd.) incubated at 37 °C for 5 min, sealed the sections by fluorescence anti-quenching agent (cas: P0123, Shanghai Biyuntian Biotechnology Co., Ltd.) and observed under fluorescence microscope. The expression rate of fluorescent proteins was calculated by Image-J analysis software.
HE staining
The sections were dewaxed to water after conventional dewatering and embedding treatment. Stain with hematoxylin aqueous solution for 15 min, rinse with water and then stain with alcohol eosin solution for 3 min. After dehydration and transparency, seal the slices with neutral gum, and observe the changes of the histopathological structure of the midbrain under light microscope.
Real-time polymerase chain reaction (RT-PCR) analysis
Total RNA was extracted from the right striatum in each group using Trizol reagent (cas: 15596018, Invitrogen, USA) and reverse transcribed into cDNA according to standard protocol (cas: AG11706 and AG11701, Hunan Aikerui Biological Engineering Co., Ltd.). RT-PCR was performed by the reaction conditions were as follows: pre-denaturation at 94 °C for 30 s, denaturation at 94 °C for 10 s, annealing at 64 °C for 30 s, extension at 72 °C for 30 s, maintained a total of 40 cycles. The quantitative analysis of each mRNA level calculated by using 2−ΔΔCt method after normalizing (cycle threshold) values with that of β-actin. The primers were used as (Table 1 and Table 2).
Western blot analysis
The right striatum slice in each group were homogenized with RIPA lysis buffer (containing protease inhibitor) (cas: P0013, Shanghai Biyuntian Biotechnology Co., Ltd.) was added at a rate of 1:5 (m:v). After homogenized in ice bath and centrifuged at 14,000g for 20 min at 4 °C. Protein concentration was determined by a BCA protein quantitation kit (cas: P0012, Shanghai Biyuntian Biotechnology Co., Ltd.). Then, lysates containing 50 μg proteins were subjected to polyacrylamide gel electrophoresis at a constant voltage of 80 V and separated gel electrophoresis at a constant voltage of 110 V. Subsequently, transferred onto PVDF membranes and blocked with 5% BSA (cas: ST025, Shanghai Biyuntian Biotechnology Co., Ltd.) for 60 min on shaker platform at 37 °C. The blots were probed with the corresponding primary antibodies: TH, α-syn, HSP90, Beclin-1, LC3B, p62 and GAPDH (1:1000, cas: ab137869, ab212184, ab13492, ab62557, ab192890, ab109012 and ab8245, Abcam, USA). The next day, the blots were incubated with HRP-labeled secondary antibody (1:1000 dilution, cas: ab205719 and ab205718, Abcam, USA) for 2 h at 37 °C. ECL chromogenic agent (cas: P0018AM, Shanghai Biyuntian Biotechnology Co., Ltd.) was added and detected by gel imaging analysis system, the gray value of each target strip was analyzed, the ratio of each group to GAPDH gray value was calculated, and the differences among groups were calculated.
Statistical analysis
SPSS17.0 software was used for statistical analyses. Measurement data were represented as mean ± standard deviation. All the data were normally distributed and had homogeneous variances. One-way analysis of variance was used for comparison between groups. The rank-sum test was used to compare between groups for non-normal distribution or variances. Significance is determined on a criterion of P < 0.05.