Animals and model grouping
Wild-type zebrafish (AB strain, 4 months old) were chosen in this study (Nanjing Qi Wu Biotechnology Co., Ltd.). They were maintained under 14/10 h light/dark cycle at 28°C, following the previous method[13]. Natural fertilized and normal developed embryos were generated and cultured to 3 days post-fertilization (dpf) in an illumination incubator. All zebrafish experiments were carried out under the supervision of the Animal Ethics Committee of China Pharmaceutical University.
Zebrafish larvae were divided into six groups and treated from 3 dpf to 7 dpf: control group, model group, model + donepezil hydrochloride (DPZ) group, model + ACT groups. The control group was maintained in the medium with 0.2% DMSO and the model group was treated with 150 μM AlCl3 (pH 5.8). The model + DPZ group was co-treated with AlCl3 and 8 μM DPZ. The model + ACT groups were co-treated with AlCl3 and different concentrations of ACT (200, 100, 50 μM). ACT (HPLC purity ≥ 98%) was obtained from Baoji Herbest Bio-Tech Co., Ltd. (Baoji, China). AlCl3·6H2O and DPZ were purchased from Shanghai Aladdin Bio-Chem Technology Co., LTD. (Shanghai, China).
Behavioral analysis
Zebrafish larvae movements were recorded with a ViewPoint behavioral analyzer (Zebralab 2018, ViewPoint Life Sciences Co., Ltd.) at 28°C. Briefly, the behavioral parameters and result processing were consistent with the method we established earlier[13]. Here, average speed (AS), speed change (ΔS), dyskinesia recovery rate (DRR), and response efficiency (RE, %) were selected to evaluate dyskinesia recovery in zebrafish.
Determination of acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) activity
After treated from 3 dpf to 7 dpf, zebrafish larvae were collected to measure AChE and ChAT activity. Based on the manufacturer's protocol, the activity was detected by the enzyme-linked immunosorbent assay (ELISA) kits (MLBIO biotechnology Co. Ltd., Shanghai, China). And the protein concentrations of different samples were determined by BCA method.
Cell cultures and treatments
BV-2 cell line (immortalized murine microglial cell line) was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). They were cultured in DMEM (KeyGen Biotech Co., Ltd, Nanjing, China). The medium was supplemented with 10% FBS (Gibco, Grand Island, NY, USA), 100 U/mL penicillin, as well as 100 mg/mL streptomycin, with 95% air/5% CO2 at 37℃.
BV-2 cells were incubated with ACT (50, 25, 12.5 μM) or stimulating with Lipopolysaccharide (LPS, 1 μg/mL; Sigma-Aldrich, St Louis, MO, USA) for 24 h. Finally, all the cells or supernatant were collected for the various analyses.
Cell viability assay
Cell Counting Kit-8 (CCK-8) assay (JianCheng Bioengineering Institute, Nanjing, China) was used to evaluate the viability of BV-2 cells. The cells were seeded in a 96-well plate (1×104 cells/well, Wuxi NEST Biotechnology Co., Ltd.). Briefly, the medium was removed at the end of the treatment, and 100 μL of serum-free medium containing CCK-8 solution was added to each well for 2 h at 37℃. The absorbance was measured at 450 nm with a microplate reader (Bio-Tek Instrument, Winooski, VT, USA). Cell viability is expressed as a percentage of the control group. The experiment was repeated three times.
Nitric oxide (NO) production assay
NO was determined by measuring nitrite levels in the BV-2 culture supernatant using Griess reagent. Briefly, at the end of the treatment, the medium (100 μL) was transferred to a new 96-well plate. The same volume of Griess reagent was added to each well and reacted for 15 min in the dark. The absorption at 540 nm was determined by a Microplate Reader.
Inflammatory cytokines levels in supernatant
The concentrations of TNF-α, IL-1β and IL-10 in BV-2 cell supernatant were determined by ELISA kits according to the manufacturer's instructions (MLBIO biotechnology Co. Ltd., Shanghai, China).
Observation of cellular morphology
To determine the effect of ACT on BV-2 cells M1/M2 polarization, the cells were plated in 6-well dish and observed under the inverted microscope (Nikon ECLIPSE Ti2, Japan).
Cellular metabolism determination by HPLC-Q-TOF-MS analysis
BV-2 cells were seeded in 6-well dish separately (n=6/group). After treatment, the medium was removed, and the cells were washed three times with cold PBS. Then immediately exposed to liquid nitrogen to suppress cells metabolism. The cells were harvested with cold 80% methanol (1 mL/well) and the suspension were transferred to a 2 mL Eppendorf tube. To facilitate protein precipitation, vigorously vortexed for 1 min and centrifuged at 13,000 rpm for 15 min at 4°C. The cell suspension was transferred to a new 2 mL Eppendorf tube and dried under a stream of nitrogen and stored at -80°C until analysis. The dried residue was reconstituted in 150 μL of pre-cooled 25% acetonitrile. In order to ensure the stability and accuracy of the sequence analysis, equal volumes (10 μL) of each cell sample were combined as quality control (QC) samples. During metabolite detection, these samples were injected after every six cell samples to confirm their stability. A 1 μL aliquot was injected for HPLC-Q-TOF-MS.
HPLC-Q-TOF-MS analysis was performed on Agilent 1290 HPLC system connected with the Aglient 6530 Quadrupole Time-of-Flight (Q-TOF) mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). The separation was carried out on an ACQUITY UPLC BEH C18 column (2.1×100 mm, 1.7 μm). The mobile phase was composed of 0.1% formic acid-water (v/v; A) and acetonitrile (B).
The flow rate was set at 0.4 mL/min with the following optimal gradient elution condition: 0 to 2 min, 5% B; 2 to 20 min, 5% to 95% B (positive ion mode); 0 to 2 min, 5% B; 2 to 20 min, 5% to 95% B (negative ion mode). The operation parameters of the mass spectrometer were set as follows: gas temperature, 320℃; drying gas, 10 L/min; nebulizer, 35 psi; VCap, 4000 V; fragmentor, 120 V.
The raw data were operated under MassHunter Workstation Software version B.07.00 (Agilent Technologies, Santa Clara, CA, USA). The raw data were pre-processed by XCMS platform. Principal components analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) of the normalized data were conducted with MetaboAnalyst (https://www.metaboanalyst.ca). Combined with literature, the differential metabolites (VIP > 1, T-test P < 0.05) were identified on HMDB (https://hmdb.ca). Finally, pathway analysis was conducted with MetaboAnalyst.
Measurement of mitochondrial membrane potential (MMP)
MMP was detected using fluorescent probe JC-1 (Beyotime, China) in accordance with the manufacturer’s instructions. Briefly, cells from different groups were rinsed with PBS and incubated with JC-1 staining solution for 20 min at 37°C. After staining, cells were washed twice using staining buffer. Then, fluorescent signals were detected by flow cytometry (BD Accuri C6).
Measurement of mitochondrial adenosine 5'-triphosphate (ATP)
ATP concentration in mitochondria was detected by an ATP Assay Kit (Beyotime, China) in accordance with manufacturer’s instructions. Briefly, the culture medium of BV-2 cells from different groups were discarded, and cells were homogenized with lysis buffer on ice. The supernatant obtained after centrifugation (12,000 g, 5 min) was used to determine the ATP concentration. The luminescence (luciferase-catalyzed fluorescein reaction) was detected by EnVision Multimode Microplate Reader (PerkinElmer).
Measurement of intracellular reactive oxygen species (ROS) level
ROS Assay Kit (Beyotime, China) was used to measure ROS level. The cells from different groups were incubated with DCFH-DA (10 μM) for 20 min at 37°C. After probe loading, cells were washed three times with DMEM. Then, fluorescent signals were detected by flow cytometry (BD Accuri C6).
Transmission electron microscopy (TEM)
BV-2 cells were seeded in 6-well dish. The medium was removed and 1 mL of 2.5% glutaraldehyde was rapidly added to each well. Then the cells were transferred to a 1.5 mL Eppendorf tube and centrifuged at 1000 rpm for 3 min. The cells were fixed overnight with new 2.5% glutaraldehyde at 4°C. After fixation, dehydration and embedding, the cells were observed with an HT7800 transmission electron microscope (Hitachi, Tokyo, Japan).
RNA-seq and bioinformatic data analysis
Total RNA from BV-2 cells (n=3/group) were extracted using Trizol reagent (Vazyme Biotech, China) according to the reagent manufacturer's instructions. All analytical samples were sent to Majorbio (Shanghai Majorbio Bio-pharm Technology Co., Ltd.) for performing the RNA sequence assay. The data were analyzed on the free online platform of Majorbio Cloud Platform (www.majorbio.com). The parameters for the differential expression analysis were P-adjust < 0.05 and |log2FC| ≥ 1. The original sequence data have been submitted to the database of the NCBI Sequence Read Archive (SRA).
Quantitative real time polymerase chain reaction (qRT-PCR)
The total RNA of BV-2 cells in each group was harvested using 500 μL RNA-easyTM Isolation Reagent (Vazyme Biotech, China), and reverse transcription reaction was conducted with FastKing-RT SuperMix (TIANGEN Biotech, China). Reactions were performed according to the manufacturer's protocol. cDNA was subjected to qRT-PCR assays with specific primers and TransStart TOP Green qPCR SuperMix (TransGen Biotech, China). The primers are listed in Table S1 (see Additional file 1) and β-actin was used as the internal control. The 2-ΔΔCT method was used for quantitative analysis.
Western blot analysis
BV-2 cells were lysed by RIPA lysis buffer (KeyGen Biotech Co., Ltd, Nanjing, China) containing 1% Protease Inhibitor Cocktail (Thermo Fisher) to obtain total protein. A 10% SDS-PAGE was performed to separate the proteins, which were transferred to NC membranes. After blocking with 5% skimmed milk/BSA for 2 h, the membranes were incubated with AMPKα (Proteintech), p-AMPKα (Affinity Biosciences), PGC-1α (Proteintech), NF-κB (Proteintech), p-NF-κB (ABclonal) or GAPDH (ABclonal) antibodies in 5% TBST at 4℃ overnight. The membranes were incubated with a secondary horseradish peroxidase-conjugated antibody (ABclonal) for 1 h at room temperature. The high-sig ECL western blotting substrate (Tanon, China), Gel imaging system (Tanon, China) and ImageJ software were used to visualization and quantitation.
Molecular docking
Molecular docking analysis were performed using Autodock software (Version 4.2). The affinity between ACT and proteins were observed by AutodockTools software. The three-dimensional (3D) protein structures of AMPKα (PDB ID: 5g5j), and NF-κB (PDB ID: 4q3j) were retrieved from the Protein Data Bank (https://www.rcsb.org).
Statistical analysis
All data are expressed as the mean ± standard deviation (SD). The differences between the different groups were analyzed by one-way analysis of variance (ANOVA), followed by Tukey's multiple comparison test. P <0.05 was considered statistically significant.