2.Materials and methods
2.1 Chemicals and reagents
Antibodies against HO-1 (ab68477), GCLC (ab190685), GCLM (ab126704), p62 (ab109012), Pink1 (ab216144), Bax (ab32503), Bcl-2 (ab194583), TH (ab137869), PCNA (ab92552), β -actin (ab8226), Tuj1 (ab18207), anti-rabbit (ab6721) and anti-mouse (ab6789) were purchased from abcam (USA). Antibodies against Nrf2 (16396-1-AP) and LC3 (81004-1-RR) were purchased from proteintech (Wuhan, China). GSH/GSSG ratio detection assay kit (ab205811) and reactive oxygen species assay kit (ab113851) were bought from abcam (USA). RIPA buffer (89900), protease and phosphatase inhibitors (78442), nuclear and total protein extraction (78833), Bicinchoninic acid (BCA) protein assay kit (23225), the enhanced chemiluminescence reagents (34580), the MitoSOX™ red mitochondrial superoxide indicators (M36008), bovine serum albumin (BSA) (37520), Alexa Fluor 488(A11001) and Alexa Fluor 546(A10040) were purchased from Thermo Scientific (USA). 1-methyl-4-phenylpyridine iodide (MPP+) (D048), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (M0896), and poly-D-lysine hydrobromide (P6407) were bought from Sigma Aldrich (Shanghai, China). The cell counting kit-8 (CCK-8) assay kit (4483230) was bought from Adamas Life (Shanghai, China). Total SOD activity detection kit (S0101S) was purchased from Beyotime Biotechnology (Shanghai, China). JC-1 staining reagent (D22200-1) was purchased Bridgen (Beijing, China). 4, 6-2-2-phenyl indole (DAPI) (S2110) was bought from Solarbio (Beijing, China). Trypsin (15050065) and neurobasal medium (12348017) were purchased from Gibco. (USA). Dulbecco’s Modified Eagle Medium/Nutrient Mixture F12 Ham (DMEM/F12) (C3130-0500), fetal bovine serum (FBS) (C04001), and penicillin and streptomycin (P/S) mixture (C3420-0100) were purchased from VivaCell (Shanghai, China). For information on DACA, refer to supplementary material. All the other chemicals used were of analytical grade.
2.2 Animals and drug treatment
Pregnant C57BL/6 mice at 16–18 days gestation and adult male C57BL/6 mice were purchased from Department of Laboratory Animal Science, Kunming Medical University. All the animal-related operations were strictly in compliance with the relevant laws and regulations on the use and care of laboratory animals in China. The animal experiment part of this project was approved by the animal ethics committee of Kunming Medical University of Technology, and experiment was conducted as per the Guide for the Care and Use of Laboratory Animals at the Kunming Medical University, Kunming, China. Animals were raised in an environment temperature of 22 ± 2℃, 60% humidity and 12-hour light/dark cycle, with available food and water. PD models were prepared using intraperitoneal injection of MPTP. After 1 week of acclimatization under standard conditions, animals were divided into 5 groups (n=13). Group I, normal control, saline (i. p.); Group II MPTP (30 mg/kg, i. p.); Group III, MPTP treatment (30 mg/kg, i. p.) + DACA (20 mg/kg, i. g.); Group IV, MPTP treatment (30 mg/kg, i. p.) + DACA (40 mg/kg, i. g.); Group V, MPTP treatment (30 mg/kg, i. p.) + DACA (80 mg/kg, i. g.). The trial lasted 7 days.
2.3 Behavioral experiments
The effects of MPTP and DACA drug treatment on motor symptoms in mice were assessed using the pole test and the open field test, both conducted by the same operator. The baseline behavioral measurements of mice were recorded one day prior to the experiment. Behavioral assessments were performed one day after completion of the entire treatment regimen to evaluate the changes in behavior.
(1) Pole test: The experimental apparatus for the mice climbing pole consisted of a sturdy base, an elongated vertical pole measuring approximately 80 cm in length and 1 cm in diameter, and a spherical object. The mice were positioned with their heads facing up on the spherical object at the pinnacle of the pole. Timing commenced upon release of the animal by the experimenter and concluded when one hind-limb made contact with the floor. Three trials were conducted, and subsequently, an average score was calculated.
(2) Open field test(OFT) :The mice open field reaction box was 50cm × 50 cm × 50 cm with black inner wall, and the bottom surface was divided into 16 small squares of 12.5 cm × 12.5 cm. Animals were tested within the first 2–4 h of the dark cycle after being habituated to the testing room for 15 min. Videos were analyzed for the following parameters: time spent moving, distance traveled, number of times the central box is encroached (center entries), time spent in central box (center time), and time spent in corner box (corner time).
2.4 Primary neuron culture
The skull, blood, and meninges were carefully removed from the fetal mice brain. After cortical tissue was digested with 0.125% trypsin for 12min at 37°C, the cell suspension was passed through a filter with 40μm mesh size after termination of digestion and then centrifuged at 1000 rpm for 5min. The pellet was then resuspended in neurobasal medium. The cells were distributed on poly-D-lysine hydrobromide coated plates and half of the neural basal matrix was renewed every 2 days. After 7 to 8 days of culture, neurons were subjected to in vitro experiments.
2.5 Cell culture and drug treatment
The human neuroblastoma SH-SY5Y cell line was purchased from Procell Life Science and Technology and were maintained with DMEM/F12 medium with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin in a humidified incubator with 5% CO2 at 37°C. We changed the culture medium every two days and subcultured the cells when the density reached 80%. To investigate the protective effect of DACA on MPP+-treated SH-SY5Y cells and primary neuronal cells, we treated cells with PBS, MPP+, or DACA + MPP+ for 24 h. The concentrations of DACA were 5, 10, and 20 μM and the concentrations of MPP+ is 1mM.
2.6 Cell viability
The cell viability was assessed using the CCK-8 assay, following the manufacturer’s instructions. 1× 104 SH-SY5Y cells per well or primary neuronal cells were plated in 96-well plates and incubated for 24 hours. The cells were then treated with different methods and conditions. Then, 100μl of culture medium containing 10mM CCK-8 was added to each well and incubated at 37°C for 2h. Absorbance was measured at 450nm using a microplate reader (HBS-1096B, DeTie, Nanjing, China).
2.7 Intracellular reactive oxygen species (ROS) detection
The procedures of ROS measurement were according to manufacturer’s instructions. After drug treatment, cells were incubated in serum-free medium containing 10 μM DCFH-DA for 20 min at 37°C. ​After three washes with serum-free media, the cells were photographed under an inverted fluorescence microscope and the mean fluorescence intensity was analyzed using the software ImageJ. The fluorescence intensity was also measured with Ex/Em=485/535nm using a multifunctional microplate reader.
2.8 Superoxide dismutase (SOD) activity assay
The intracellular SOD activity was measured with a total SOD activity detection kit, according to the manufacturer’s instructions. After drug treatment, the cells were washed once with PBS and collected by centrifugation. The cells were fully lysed in SOD sample preparation solution, and the supernatants were collected at 12,000 × g at 4°C for 5 min. The absorbance was detected at 450 nm and 600 nm (reference wavelength) with a multimode microplate reader, and the SOD activity was calculated.
2.9 GSH/GSSG Ratio Detection Assay
According to manufacturer’s instructions, the GSH/GSSG ratio was determined using a GSH/GSSG Ratio Detection Assay Kit. The GSH/GSSG assay use a proprietary non-fluorescent water-soluble dye that becomes strongly fluorescent upon reacting with GSH. Cell samples were prepared after drug treatment,and Its signal was read by a fluorescence microplate reader at Ex/Em = 490/520 nm.
2.10 Mitochondrial Superoxide assay
Mitochondrial superoxide level was determined using the MitoSOX™ Red Mitochondrial Superoxide Indicators according to the protocol provided. Cells were incubated in serum-free medium containing 10 μM red reagent for 20 min at 37°C. After three washes with serum-free media, the cells were photographed under an inverted fluorescence microscope and the mean fluorescence intensity was analyzed using the software ImageJ.
2.11 Mitochondrial membrane potential analysis
Mitochondrial membrane potential was detected by JC-1 staining. Cells were incubated with 10 μg/mL of JC-1 working solution for 20 min at 37 °C in the dark. The maximum excitation wavelength of JC-1 monomer was 514nm, and the maximum emission wavelength was 529nm. The maximum excitation wavelength and emission wavelength of JC-1 polymer were 585nm and 590nm. The images were captured by a fluorescence microscope (ECLIPSE Ts2-FL, Nikon, Janpan).
2.12 Western blotting
Brain tissue and cells were placed in RIPA buffer containing protease and phosphatase inhibitors (1%), and residual tissue was removed by sonication and centrifugation (13200rpm, 30 min, 4C). The procedure of nuclear and total protein extraction was according to the manufacturer’s instructions. Cell/tissue lysates were prepared, and protein concentrations were quantified using the BCA Protein Assay kit. Samples with equal amounts of protein were separated by polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes blocked in 5% skim milk for 2 h at indoor temperature. Then, the membranes were incubated with primary antibodies against Nrf2 (1:2000), HO-1 (1:5000), GCLC (1:5000), GCLM (1:5000), p62 (1:5000), Pink1 (1:1000), LC3 (1:1000), Bax (1:5000), Bcl-2 (1:1000), TH (1:5000), PCNA (1:5000) or β-actin (1:5000) overnight at 4 °C. The next day, membranes were washed three times with TBST and incubated with HRP-conjugated anti-rabbit (1:5000) or anti-mouse (1:5000) secondary antibodies for 2 hours at room temperature. Blots were visualized using the enhanced chemiluminescence reagents, and images were analyzed using ImageJ software.
2.13 Immunofluorescence (IF) staining
Immunofluorescence staining was performed on brain sections or cells. Mice perfusion using 0.9% NaCl solution and 4% paraformaldehyde (PFA), then remove the brain, with 4% paraformaldehyde fixed up for the night. After gradient dehydration in sucrose solution, brains were embedded and sectioned. The cells were fixed with 4% PFA at room temperature for 20 to 30 mintures. With 5% bovine serum albumin (BSA) (containing 0.25% Triton X - 100) blocking buffer in the closed for 1 hour at room temperature and then incubated with primary antibodies against Nrf2(1:500), TH (1:500) and Tuj1 (1:1000) overnight at 4 °C. The cells or brain tissue section were incubated with corresponding secondary antibodies Alexa Fluor 488(1:500) and/or Alexa Fluor 546(1:500). After the wash three times with PBS, use amino 4, 6-2-2 - phenyl indole (DAPI) redyeing for 5 minutes. Fluorescence was captured on Confocal laser scanning micros (AX | AX R with NSPARC, Nikon, Janpan). The ImageJ software was used to measure the inflorescence intensity.
2.14 Statistical analysis
GraphPad Prism 9 Software (San Diego, CA, USA) was used for the
statistical analysis of data. The results were performed by mean ± SEM. Compared multiple groups of data and used one-way ANOVA followed by Tukey’s post hoc test. A two-sided P value was used for all tests, andρ < 0.05 was considered statistically significant.