2a. Materials and methods
In our previous study, we showed that TREM-1 inhibition improves neurological deficits and brain edema in subarachnoid hemorrhage [20].
2.1a. Cellular materials
Experimental cells: BV-2 cells (Solebo)
2.2a. Cellular experimental methods
2.2.1. Experimental cell groups
Normal group, siRNA group, monascin group, Xuezhikang group
2.2.2. Cell culture
BV-2 cells were cultured in a humidified incubator with 5% CO2 at 37 °C. After 2-3 days, Dulbecco’s Modified Eagle Medium (DMEM) was replaced, and non-adherent cells were removed. When the primary cultures reached 80% confluence, cells were harvested using 0.25% trypsin–EDTA solution and sub-cultured. Thereafter, the third passage of cells was selected for subsequent experiments.
2.2.3. Screening of Monascin/Xuezhikang concentration and the transfection sequence of Nrf2-siRNA by qPCR
RT-PCR was used to detect the Nrf2-mRNA levels, screen its best sequence, and the best drug concentrations of monascin or Xuezhikang. Exactly 1 ml aliquots of 5, 15, 30 μmol/L monascin or 100, 200, 500ug/ml Xuezhikang was added to a 12-well plate with three multiple holes in each group, and Nrf2 interference vector(siNrf2) were synthesized (GenePharma, Shanghai, China), then transfected by incubating in DMEM containing GP-transfect-Mate (GenePharma, Shanghai, China).
Total RNA was extracted using the Trizol reagent. First-strand cDNA was synthesized using the PrimeScript RT Master Mix Kit (ABI-Invitrogen, Thermo Fisher Scientific, Grand Island, NY, USA), while β-Actin was used as the internal control. The quantification of endogenous control mRNA levels was performed using TaqMan assays. The data were analyzed using the 2-△△Ct method.
The Nrf2-siRNA (100 nM) primer sequences screened by RT-PCR exhibited sense chain GCAGGACAUGGAUUUGAUUTT and antisense chain AAUCAAAUCCAUGUCCUGCTG. Then, 15μmol/L monascin or 200ug/ml Xuezhikang were selected for subsequent experiments.
2.2.4. Cell immunofluorescence
The immunofluorescence was performed as previo usly described[21]. The BV2 microglial cells were treated using Nrf2-siRNA, monascin, and Xuezhikang, respectively. The cells were collected and fixed with 4% paraformaldehyde, permeabilized using 0.5% Triton X-100, then blocked with goat serum for 60 min. The samples were overnight incubated with primary antibodies (anti-Nrf2, anti-Trem1, anti-Trem2, anti-CD80, anti-CD206, Abcam) at 4 ℃, incubated with a fluorescent dye-conjugated secondary antibody in the dark for 1 h, then stained using 4′,6-diamidino-2-phenylindole (DAPI). Three different visual fields were randomly selected for each sample and photographed under a fluorescence microscope (LeicaDmil, Germany).
2.2.5. Phenotype of BV-2 cells detected by flow cytometry
The BV-2 cells were inoculated and 100 µL cell suspension with a final concentration of 10 × 106 cells/mL was pipetted into a round-bottom Eppendorf tube. After incubation with the primary antibody CD80/CD206 (anti-CD80 1:50, anti-CD206 1:50 Abcam) for 2h, the microglial cells were washed then incubated using a fluorescent-labeled secondary antibody for 1h. The total percentage of CD206 and the relative ratio of CD206 to CD80 were determined through flow cytometry. Notably, CD206 was shown as APC in the Q1 quadrant; CD80 was displayed as fluorescein isothiocyanate (FITC) in the Q4 quadrant. The common mark was in the Q2 quadrant. The percentage of CD206 and CD80 (%) = 100 × (Q1+Q2)/(Q2+Q4) was the phenotype of the microglia. The phenotype of the cells was evaluated by quantitative percentage.
2.2.6. Detection of Nrf2, Trem1, Trem2, CD80, CD206, TNF-α, and BDNF protein expressions via Western Blot (WB)
The BV2 cells were collected and treated with radioimmunoprecipitation (RIPA) lysis buffer and measured using a bicinchoninic acid (BCA) kit as previously described[8]. Equal amounts of protein were loaded on an SDS polyacrylamide gel, electrophoresed, then transferred to polyvinylidene fluoride (PVDF) membranes, which were then overnight incubated with primary antibodies (anti-Nrf2, anti-Trem1, anti-Trem2, anti-CD80, anti-CD206, anti-TNF-α, and anti-BDNF, Abcam) at 4 °C. The membranes were incubated with a horseradish peroxidase-conjugated secondary antibody and visualized through chemiluminescence. Densitometry was performed to quantify the signal intensity using ImageJ software (https://imagej.net).
2.2.7.1. Observation of phagocytosis of fluorescent bioparticles by immunofluorescence
The BV2 cells were treated with 10 mg/mL zymosan fluorescent bioparticles (Alexa Fluor594 conjugate; zymosan: BV-2 = 40:1) for 1 h,then fixed with 4% paraformaldehyde, before being permeabilized with Triton X-100. Then, the samples were placed in 10% goat serum for 2 h, overnight incubated with anti-Iba1 antibody (1:100; Abcam) at 4 °C, then incubated with a FITC-conjugated secondary antibody. The phagocytosed bioparticles were observed using a fluorescence microscope.
2.2.7.2. Observation of phagocytosis of erythrocytes
After separation and purification of erythrocytes, they were counted and co-cultured with microglia at a ratio of 1:40 (microglia: erythrocytes). Microglia swallowing erythrocytes were observed using a microscope (Olympus CK40–32PH, Japan).
2b. Materials and methods
In our previous study, we found that Nrf2 agonist -monascin facilitates hematoma clearance, alleviates cerebral edema, and exerts neuroprotection after ICH[18,19].
2.1b. Animal materials
Experimental male Nrf2-/-C57BL/6 mice (4–5 weeks old) were purchased from Cyagen Model Biological Research Center (Taicang) Co.,Ltd. (Suzhou, China), the other male C57BL/6 mice (4–5 weeks old) were purchased from the Animal Experimental Center of Shanxi Medical University. All animal experiments were approved and conducted as per the guidelines of the Ethics Committee of Shanxi Medical University, Shanxi, China. All surgical procedures were performed under anesthesia, and every effort was expended to minimize suffering.
2.2b. Animal experimental methods
2.2.1. Experimental design
A total of 42 mice were randomized to the following groups: sham (n = 8), ICH+ vehicle (n = 9), Nrf2-/- +ICH(n = 8), ICH+ monascin(10mg/kg/day, twice, n = 8), ICH+ Xuezhikang (0.2g/kg/day, twice, n = 9). Dead animals were replaced before final assessment. All gavages were administered by gastric perfusion 6 h after ICH for 72 hours.
2.2.2. ICH Model
The experimental ICH model was induced by injecting collagenase type IV (0.5 units in 2 μl saline) into the basal ganglia region using stereotaxic instruments. Mice were fixed on a stereotaxic apparatus under chloral hydrate anesthesia (10ml/kg, intraperitoneally); the skull was exposed to reveal bregma. A1-mm cranial bur hole was drilled in the skull (coordinates: 0.9 mm posterior to the bregma, 1.5 mm lateral to the midline), then, collagenase was infused into the right basal ganglia (4 mm deep from the dura mater) using a microinjector. The needle remained in place for an additional 15 min to prevent “back-leakage”. The Sham-operated mice were syringed with equivalent dosages of physiological saline. After surgery, the skull hole was sealed using bone wax and the incision was sutured. Animals were allowed to recover after successful ICH induction that was confirmed by Rosenberg’s neurological score [22].
2.2.3. Neurobehavior tests
As previously described[6,18-19], before sacrificing the animals for tissue collection, they were subjected to neurofunctional assessments using the modified Garcia tests. Notably, the modified Garcia scale involves an 18-point sensorimotor assessment that includes six individual tests. Each test has a score ranging from 0 to 3, with a maximum score of 18. The individual tests evaluate spontaneous activity, response to side stroking, vibrissae touch, climbing, lateral turning, and forelimb walking.
2.2.4. Immunofluorescence detection
After intraperitoneal anesthesia, heart perfusion was performed using ice Phosphate-buffered saline (PBS) and 4% paraformaldehyde. The brain tissues of mice were removed on ice then placed in a 4% paraformaldehyde overnight at 4℃.Sucrose PBS buffer (20% and 30%) was used for dehydration at 4 °C until tissues were fully penetrated. After fixed embedding of OCT (optimal cutting temperature compound), frozen sections were coronally cut into 4-μm slices. The immunofluorescence methods were based on previously described methods.
2.2.5. Western blot
Total protein from brain tissue was collected, and their concentrations were determined using a BCA kit with a procedure similar to the foregoing cellular experiment methods.
2.3. Statistical analysis
All statistical and graphical analyses and were performed using SPSS 22.0 (IBM, Armonk, NY, USA) and GraphPad Prism 7.0 (https://www.graphpad.com/scientific-software/prism) software. All data were presented as the mean ± SEM (standard error of the mean). One-way analysis of variance (ANOVA) was performed for comparisons among multiple groups, whereas the SNK-q test was used for pairwise comparison between groups. A P-value of less than 0.05 P< 0.05 was considered statistically significant.