Animal experiments followed the standards established by the animal experiment committee of the Fourth Affiliated Hospital of China Medical University and approved by the ethics committee of the Fourth Affiliated Hospital of China Medical University. All animal experiments were conducted ifollowed the “Guidelines for the Care and Use of Laboratory Animals” .
Intracerebral hemorrhage (ICH) model establishment
ICH model was established in 48 female Sprague-Dawley (SD) rats (8 weeks old) purchased from Hunan SJA laboratory animal Co., Ltd (Hunan, China).
The ICH model was processed according to the literature , and the specific procedure was as follows: stereotactic intranasal injection of type VII collagenase (Sigma-Aldrich, St. Louis, Missouri, USA). After anesthesia, a burr hole was drilled at the injection site (3.0 mm left of the midline, 0.2 mm posterior to the bregma, and 6 mm below the skull) and type VII collagenase (dissolved in 0.23 µL brine) was slowly injected at a rate of 0.5 µL/min into the central striatum. Then the needle was kept at the injection site for another 10 minutes to prevent reflux. The skull was sealed using bone wax after craniotomy. Rats were assigned into sham group, ICH group, ICH + dimethyl sulfoxide (DMSO) group (treated with DMSO after the rat ICH modeling), ICH + Baicalein group (treated with 50mg/kg baicalein after the rat ICH modeling), ICH + H2O group (treated with H2O after the rat ICH modeling), ICH + N-acetylcysteine (NAC) group (treated with 5 mM NAC after the rat ICH modeling) , ICH + sh-negative control lentivirus (sh-NC) group (treated with sh-NC after the rat ICH modeling), and ICH + sh-NLRP3 group (treated with sh-NLRP3 after the rat ICH modeling),with 6 rats per group. The treatments of sh-NC, oe-NC, sh-NLRP3, and oe-NLRP3 were as follows : rats were given stereotactic microinjection of lentivirus particles at a dose of 2.0 µL (10–10 TU/mL) into the CAI region of the right hippocampus, 72 hours before ICH treatment. The treatments of H2O and baicalein were as follows: the same volume (1 mL) of H2O or baicalein was injected intraperitoneally at an interval of 12 hours for 3 days. After 1 and 3 days of baicalein treatment, the rats were subjected to neurology score evaluation. After 3 days of baicalein treatment, the rats were euthanized with excessive sodium pentobarbital. Half of the tissues were used for brain edema assessment, and the other half were used for histological staining, quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot (WB).
Modified neurological severity score (mNSS)
To assess neurological abnormalities in animals, a mNSS  was performed by two independent investigators who were unaware of the experimental treatment. The mNSS test is composed of motor, sensory, reflex, and balance tests. Neurological function scores ranged from 0–18 according to supplementary table 1 (normal = 0; maximum defect score = 18).
Assessment of cerebral edema
After euthanizing animals with an overdose of pentobarbital sodium (160 mg/kg body weight) , the brains were removed and divided into the contralateral and ipsilateral hemispheres and the cerebellum. Each tissue was weighed immediately to obtain a wet weight and then dried at 160°C for 24 hours to obtain a dry weight. The formula for calculating water content was as follows: [(wet weight - dry weight)/(wet weight)] × 100%.
Fluoro Jade - c (FJC) staining
The number of degenerated neurons was assessed by FJC staining. ICH sections were detected using FJC’s standby dilution staining kit (Biosensis Pty Ltd, Thebarton, South Australia). After rinsing with phosphate buffer saline (PBS), the sections were incubated in FJC working solution for 20 minutes according to the instructions, and then observed under a fluorescence microscope. The number of FJC-positive neurons was calculated as follows: three brain regions of the microscope field around hematoma were randomly selected from each rat and then the number of FJC-positive neurons was calculated using the ImageJ software (NIH, Bethesda, MD, USA).
Apoptosis was detected by TUNEL staining. In brief, sections were paraffined, dewaxed, hydrated and cleared in brain tissues in different treatment groups. The TUNEL staining reaction solution (Roche, Shanghai, China) was added and the apoptotic cells were calculated under the fluorescence microscope (Eclipse Ti-U, Nikon Co, Japan) and photographed.
The brain tissues were paraffined and rehydrated, and stained with Nissl staining solution (Beyotime, C0117) at 50–60℃ for 40 minutes. After the tissues were washed with distilled water, recrystallized with gradient ethanol, and then cleared in 100% dimethylbenzene for 5 minutes. Then the tissues were sealed with neutral gum or other sealant. The tissues were observed under the light microscope.
Hematoxylin-Eosin (HE) staining
After embedding the tissue, the wax block was fixed on a conventional continuous section with a thickness of 4 um. The wax block was spread and pasted in water at 46℃, and then baked in a toaster at 72℃ for 2 hours. The sections were cooled for 10 minutes, then dewaxed with xylene I for 10 minutes, dewaxed with xylene II for 10 minutes, fixed with anhydrous ethanol I and II for 5 minutes, with 90% ethanol for 2 minutes, with 80% ethanol for 2 minutes, and fixed with 70% ethanol for 2 minutes, and flushed with water for 5 minutes, and stained with hematoxylin for 5–10 minutes. Next, the sections were flushed with water for 5 minutes, differentiated with hydrochloric acid alcohol for 2–3 s, blued with lithium carbonate for 10 minutes, stained with eosin for 2 minutes, dehydrated with 80% ethanol for 2 minutes, with 90% ethanol for 2 minutes, with anhydrous ethanol for 2 minutes, and cleared with xylene for 2 minutes. The sections were finally wiped, sealed with neutral resin and observed under the microscope.
The specimen was fixed with 10% formaldehyde, and paraffined embedded were sliced at 4 µm. The tissue sections were baked at 60T for 1 hour, dewaxed with conventional xylene, then dehydrated with gradient alcohol, incubated at 37℃ in 3% H2O2 (Sigma) for 30 minutes, washed with PBS, and boiled in 0.01 M citrate buffer at 95℃ for 20 minutes, then cooled to room temperature, and washed with PBS. The tissue sections were be sealed with normal sheep serum working solution for 37℃ for 10 minutes. Sections were incubated with NOD-like receptor protein 3 (NLRP3) (ab214185; 1:200, Abcam, Cambridge, MA, USA), caspase-1 (ab62698, 1:500, Abcam), and IL-1β (ab216995, 1:200, Abcam) antibodies at 4t for 12 hours. After washing with PBS, the corresponding biotin-labeled goat anti-rabbit secondary antibody was added, and the reaction was carried out for 10 minutes. After washing thoroughly, horseradish peroxidase labeled streptomycidin working solution (S-A/HRP) was added to react at room temperature for 10 minutes. The sections were visualized using Diaminobenzidine (DAB) and stored in a dark room for 8 minutes. The sections were be rinsed with tap water, stained with hematoxylin, dehydrated, cleared, sealed, and observed under light microscope. Nikon Imaging Software was used to count the positive cells. Three non-overlapping fields of equal area (200 x) were selected from each section to count the number of positive cells.
The slides were fixed with 4% paraformaldehyde for 15 minutes, soaked in PBS 3 times, and dried with absorbent paper. Normal goat serum was added to the slides and sealed at room temperature for 30 minutes. The blocking liquid on the slides was absorbed with absorbent paper and the slides without washing. The primary antibodies NLRP3, Caspase-1 and apoptosis-associated speck-like (ASC) were added to each slide for overnight incubation at 4℃. The slides were dipped and washed three times with phosphate buffered saline with 0.05% tween 20 (PBST), 3 minutes each, and incubated with Alexa Fluor 488 labeled goat anti-rabbit IgG (ab150077, Abcam, UK) and Alexa Fluor 647 labeled goat anti-rabbit IgG (ab150083, Abcam) at 37℃ for 1 hour in the dark. After rinsing the slides with PBS 3 times in the dark, stained with 5ug/mL DAPI for 5 minutes and then washed with PBS for 5 minutes ×3 times. Slides were sealed and stored at 4℃ in the dark. The results were observed with the software Nis-Elements Viewer using a confocal laser microscope (Zeiss LSM 510, Zeiss, Oberko, Germany).
Determination of reactive oxygen species (ROS)
The slides were fixed with 4% paraformaldehyde for 15 minutes, and then soaked with PBS for 3 times. The PBS was dried with absorbent paper, and normal goat serum was added to the slides and then the slides were sealed at room temperature for 30 minutes. The blocking solution was absorbed by absorbent paper without washing. Each slide was dripped with DCF-DA diluent and incubated at 37℃ for 30 minutes, and observed under a light microscope (Olympus, Tokyo, Japan). Fluorescence intensity was evaluated using Image Pro Advanced 6.0 software (NIH, Bethesda, MD, USA).
Enzyme linked immunosorbent assay (ELISA)
The expression of inflammatory cytokine interleukin-1 (IL-1) (1210122, IL-1β ELISA kit 96T, Dakewe, Shenzhen, China), tumor necrosis factor-α (TNF-α) (1217202, TNF-α ELISA kit 96T, Dakewe), and IL-10 (1311002, IL-10 ELISA kit 96T, Dakewe) in serum of rats was detected. The levels of malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in serum of rats were also measured according to the instructions of ELISA kits (Bio-Swamp, Wuhan, China). Specific steps can refer to the operation manual. Briefly, 100 µL antibody dilution buffer were incubated with the biotinized antibody working solution (1:100, 100 µL/well) for 2 hours, the value of optical density (OD) was measured at 450 nm, and the results were obtained by comparing with the standard and blank control. The experiment was repeated 3 times.
Total RNA was extracted using TRIzol (Invitrogen, Car, USA). RNA was reverse transcribed into cDNA using PrimeScript RT kit (RR037A, Takara, Japan). The reaction system was 10 µL. Then, the reaction solution was taken for fluorescence quantitative PCR according to the instructions of the SYBR®Premix ExTaqTMⅡ kit (RR820A, TaKaRa) using a real-time quantitative fluorescence PCR system (ABI 7500, ABI, Foster City, CA, USA). Using GAPDH as an internal reference, the relative expression of each target gene was calculated by the 2−ΔΔCt method . The relevant primers were designed by Shanghai Sangon Bio (Shanghai, China) (Table 1).
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Western blot (WB)
Tissues were collected by trypsin digestion and lysed with the enhanced radio immunoprecipitation assay lysate (Boster, Wuhan, China) containing protease inhibitors, and then the protein concentration was determined with the bicinchoninic acid (BCA) protein quantitative kit (Boster, Wuhan, China). Proteins were isolated with 10% SDS-PAGE, and the isolated proteins were transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were sealed with 5% bovine serum albumin (BSA) for 2 hours to block non-specific binding. Diluted primary antibody NLRP3 (ab214185, 1:1000, Abcam), Caspase-1 (ab62698, 1:1000, Abcam), ASC(ab180799, 1:1000, Abcam) and GAPDH (ab9485, 1:2500, Abcam) were added, respectively, and incubated overnight at 4℃. After washing the membranes, HRP-labeled sheep anti-rabbit secondary antibody (ab6721, 1:2000, Abcam) was added into the membranes and incubated for 1 hour. Next, the membranes were added with enhanced chemi-luminescence (ECL) working solution (EMD Millipore, MA, USA) at room temperature for 1 minute. Then the excess ECL reagent was removed, the membranes were sealed with the plastic wrapped, and X-Ray film was put in the dark box for 5–10 minutes exposure for blotting development and fixation. Image J analysis software (NIH) was used to quantify the grayscale of each band in Western blot images, and GAPDH was used as an internal reference. Each experiment was repeated 3 times.
SPSS version 19.0 (IBM Corp. Armonk, NY, USA) was used for statistical analysis. All data were in compliance with normal distribution and homogeneity of variance. Data were presented as mean ± standard deviation. Unpaired t test was used to compare the data between two groups, and one-way analysis of variance (ANOVA) was used to compare the data among multiple groups. Tukey's was used for post hoc test. A value of P < 0.05 indicated the difference was statistically significant.