Particle preparation and characterization
The PM2.5 sampling point was located in Tangshan (118°10′26′E longitude, 39°37′46′N latitude) of Hebei Province. The preparation of PM2.5 was followed the previous report[22]. PM2.5 was characterized with high-resolution transmission electron microscopy (HR-TEM, JEM-2100, Japan, accelerating voltage of 200 kV) and scanning electron microscopy (SEM, Japan, accelerating voltage of 30kV).
PM2.5 data sources
The Real-Time PM2.5 data in the location (118°10′26′E longitude, 39°37′46′N latitude) was downloaded from the TAP (http://tapdata.org.cn/) database. In TAP database, PM2.5 and its components data (Sulfate (SO42−), nitrate (NO3−), ammonium (NH4+), organic matter (OM), and black carbon (BC)) were estimated by combining information from other sources, including ground observations, satellite aerosol optical depth, chemical transport model simulations, meteorological factors, land use data, et al. and being calculated based on machine learning, with the out-of-bag cross-validation R2 of 0.83.
2.3 Hippocampal astrocytes culture
Mice born within 24 hours were used to collect hippocampus under a dissecting (stereo) microscope (Olympus, Japan). The hippocampal tissue was made into single cells, inoculated in culture flask encapsulated with 0.03% polylysine (PLL, Thermofisher, USA), and maintained with DMEM/F12 medium (Gibco, USA) containing 12% FBS (Gibco, USA) and 1% penicillin/streptomycin (Thermofisher, USA) at 37°C with 5% CO2. Purificated and cultured astrocytes growing into confluent which was termed as F0 generation. Astrocytes in F2 generation were used for further in vitro experiments. The flow chart of astrocytes primary culture is shown in Figure S4A.
2.4 PM2.5 and dimethyl fumarate (DMF) / phytochemicals treatment in vitro PM2.5 treatment
Astrocytes were exposed to different doses of PM2.5 (0, 5, 10, 20, 40, 80, 160 µg/mL) for 48 h and then the cell viability was measured with Cell Counting Kit 8 (Biosharp, China) following the manufacturer’s instructions.
DMF / phytochemicals treatment
Dimethyl fumarate (DMF) is a well-known Nrf2 agonist [41]. To determine the concentration of DMF, and three phytochemicals (procyanidin (PC, Xi'an xuhuang Bio-Tech, China), lyceum barb arum polysaccharide (LBP (Xi'an Xuhuang Bio-Tech, China), and phillyrin (Xi'an xuhuang Bio-Tech, China)) in further study, astrocytes were exposed to different concentrations of DMF, PC, LBP, or phillyrin for 2 h. After treatment, the cell viability was measured with Cell Counting Kit 8 (Biosharp, China) followed by the manufacturer’s instructions.
2.5 Animal and Treatments
All animals were treated in compliance with the Animal Use and Ethics Committee of Capital Medical University. All experimental treatments followed the 3R principle. All mice were purchased from the Gempharmatech Biotechnology (Nanjing, China). Mice were housed in a specific pathogen-free (SPF) animal facility under a 12-h dark/light cycle with free access to food and water. The ambient temperature is 22 ± 2℃. The relative humidity of the environment is maintained at 60 ± 10%. Male mice were used for all experiments in this study, and littermates were allocated to experimental groups randomly. All mice used in this study were maintained on the C57BL/6J background. After a 1-week adaptation period, mice were exposed to control (filter room air, FRA) and real-world PM2.5 for 15 weeks respectively. The real-ambient PM2.5 exposure system has been designed according to the previous study[28]. The concentration of ambient PM2.5 was measured in outdoor and inside IVC.
pAAV-GfaABC1D-GFP-miR shRNA (Nfe212)-WPRE (serotype AAV2/5, 8.83×1012 genome copies (GC)/mL) were purchased from OBIO Technology (Shanghai, China). The mice were anesthetized by isoflurane (flow velocity: 300–500 mL/min) and stereotaxic injected with the viruses (flow velocity: 50 nl/min, volume: 500 nl) into the bilateral hippocampus of adult mice (KOPF brain stereotactic locator, Germany). The coordinates for the stereotaxic injection were as follows: hippocampus (anterior-posterior: ± 2.1mm; mediolateral: ± 1.8 mm; dorsoventral: ± 1.5 mm from bregma and dura, flat skull). Mice were allowed to recover in cages which were placed partially on a low-voltage heating pad for 2 hours.
PC was dissolved in a phosphate-buffered solution (PBS). Control group mice were orally administered to PBS and PC respectively. The remaining two groups with PM2.5 exposure were exposed to PC (100 mg/kg/d) and PBS for 15 weeks respectively.
2.6 RNA extraction and quantitative real-time PCR (qRT-PCR) analysis
Total RNA of astrocytes and murine hippocampus was extracted with RNA isolation reagent (Invitrogen, USA) following the manufacturer’s instructions. RT-qPCR analysis was performed with the SYBR Premix Ex Taq kit (Takara, Japan) using the Quant Studio 6 Flex system (Applied Biosystems, USA). β-actin was used to quantify the relative expressions of target genes with the 2−ΔΔCt method. All the experiments were performed independently in triplicates.
2.7 Western blotting assay
Total proteins were extracted by a total protein extraction kit (Thermo Fisher, USA) from primarily cultured astrocytes and murine hippocampus. A total of 15 µg proteins of each sample were loaded to 8% or 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were blocked with 5% bovine serum albumin (BSA) for 1 h and incubated with primary antibodies anti-C3 (1: 1000 dilution; Cell Signaling Technology, USA), anti-Nrf2 ((1: 800 dilution; Abcam, England), anti-GFAP (1: 1000 dilution; Cell Signaling Technology, USA), anti-HO-1 (1: 1000 dilution; Proteintech, China), anti-NQO-1 (1: 1000 dilution; Proteintech, China), and anti-β-actin (1: 5000 dilution; GenStar, China) 12h followed by incubation with HRP-conjugated secondary antibodies for 1 h. The images were developed by using the Super ECL Plus Kit (Yeasen, China).
2.8 Histopathological and Immunohistochemistry (IHC) staining analysis
Mouse brain tissues were collected and fixed with 4% PFA. After dehydration, the sample was embedded in paraffin and cut into 3 µm paraffin sections. The sections were gradient dewaxed and stained by hematoxylin-eosin (H&E). For IHC staining, microwave antigen retrieval was performed in citric acid buffer (PH = 6.0) and tissue sections were cooled down in TBST containing 5% BSA for 30 min at room temperature., Then the sections were incubated with the primary antibodies (anti-GFAP: 1:250, anti-C3: 1:500, anti-Nrf2: 1:200, anti-Iba1: 1:600, anti-CD68: 1:100) for 24 h at 4°C. Thereafter, the sections were incubated in the suitable biotinylated secondary antibodies in blocking solution (3%) for 1 h at room temperature and developed with DAB (Nakasugi Jinqiao, China). Images were scanned with Zeiss Laser Scanning Microscope 710 (Carl Zeiss, Germany). Non-overlapping high-power fields (HPF, 400×) were captured by Panoramic SCAN (3DHISTECH, Hungary). Immunostaining intensity or number of GFAP+ cells in each field was evaluated.
2.9 Immunofluorescence (IF) staining
After dewaxing, antigen repairing, and endogenous peroxidase blocking, slices of brain tissue were incubated with 5% BSA at room temperature for 30 min. Astrocytes were fixed with 4% PFA and permeabilized sealed with 5% BSA containing 0.3% Triton X-100 for 1h. After washed, samples were incubated with mouse anti-GFAP (1: 250 Signaling Technology, USA) was mixed incubated at 4 ℃ for 36 h counterstaining with rabbit anti-C3 (1: 200) or rabbit anti-Nrf2 (1: 200), followed by 1 µg/mL of goat anti-rabbit IgG secondary antibody conjugated with Alexa Flour 488 and goat anti-rabbit IgG secondary antibody conjugated with Alexa Flour 555 (1: 400, Signaling Technology, USA), and stained with DAPI. The images were taken with a fluorescent microscope (Zeiss, Germany).
2.10 Flowcytometry assay
After PM2.5 exposure, astrocytes were collected and loaded the probe with Reactive Oxygen Species (ROS) Assay Kit (Beyotime, China) according to the manufacturer’s instructions. The number of ROS+ astrocytes was detected by flow cytometry ( BD, U.S.A). All assays were performed independently for three times.
2.11 Measurement of antioxidant indexes
Cells were collected and lysed with RIPA lysis solution. The levels of superoxide dismutase (SOD) and catalase (CAT) in astrocytes were measured respectively with SOD Assay Kits (Beyotime, China) and CAT Assay Kits (Beyotime, China) following the manufacturer’s instructions.
2.12 Enzyme-linked immunosorbent assay (ELISA)
Peripheral blood serum was collected from mice, and the protein level of S100β was measured according to the manufacturer's instructions utilizing mouse S100β ELISA kits (Beyotime, China).
2.13 Open field test (OFT)
OFT was performed in a 42 × 42 × 42 cm polyvinyl chloride box, and the movement of mice were taken by a camera (Litong, China). Each mouse was tested and recorded for 3 min. The videos were analyzed by using EthpVision XT 17.0 (Noldus, Netherlands).
2.14 Sucrose preference test (SPT)
To determine the degree of depression, we measured the sucrose preference of mice with 1% sucrose solutions. The amount of pure water (Mwater) and sucrose solution (Msucrose solution) consumed by mice within 24 h was measured. The equation for sucrose preference is as follows: Sucrose preference (%) = M sucrose solution/ (M water + M sucrose solution) ×100%
2.15 RNA-seq analysis
Total RNA was extracted from the hippocampus of the mouse by trizol. Transcriptome sequencing experiments were delegated to Novogene Bioinformatics (Beijing, China). The basic principle of sequencing was depends on Sequencing by Synthesis. In the data analysis phase, differentially expressed genes with a cut-off as adjusted p-value < 0.05 and |log2FoldChange| > 1.5 were identified for further analysis. WikiPathways analyses were carried out by the Metascape database (https://metascape.org/gp/index.html).
2.16 Statistical analysis
The data analysis was performed using GraphPad Prism 8.0 and presented as mean ± SEM. Comparison between the two groups were performed by Student’s t-test, or Whitney U test for immunohistochemical data. Comparisons between multiple groups, one-way or two-way ANOVA were performed. P < 0.05 was set as the significance level.