2.1. Reagents
2-Chloroethanol (2-CE) was obtained from Sinopharm Chemical Reagent Co., Ltd. (Ningbo, China), while reagents for the cell culture were purchased from Biological Industries (Beit-Haemek, Israel). The quantitative polymerase chain reaction (qPCR) assay kits were acquired from Takara (Japan). SB202190, pyrrolidine dithiocarbamate (PDTC), SR11302, and N-acetyl-l-cysteine (NAC) were purchased from Selleck (Houston, USA), APExBIO (Houston, USA), or Beyotime Biotechnology (Shanghai, China). Fluorocitrate (FC) and GIBH-130 (GI) were bought from Sigma (New Jersey, USA) and MedChemExpress (New Jersey, USA), respectively. Short hairpin RNA (shRNA) targeting the interleukin-1β (IL-1β) gene was designed and synthesized by Hanbio (Shanghai, China), while small interfering RNAs (siRNAs) targeting the tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), or CYP2E1 genes were designed and synthesized by RiboBio (Guangzhou, China).
2.2. Primary cultures of astrocytes and microglia
Astrocytes and microglia were purified from the cerebral cortices of 1–2-day-old postnatal Wistar rats that were obtained from the Animal Experiment Center of China Medical University, which were cultured as previously described [3, 14, 15]. Briefly, the cerebral cortices of rat brains were taken immediately after decapitation, and carefully cleaned of meninges and blood vessels. They were enzymatically and mechanically dissociated to generate a single cell suspension. The cells were then seeded and incubated at 37℃ in a 5% CO2-humidified atmosphere in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 20% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The medium was completely replaced every 3 d until the cells reached a confluence, which usually occurred after 12–14 d.
The microglia were isolated from confluent mixed glial cultures via the mild trypsinization [14, 15]. After 0.08% trypsinization, the microglia were firmly attached to the bottom of the culture plates, and the supernatant containing a detached intact astrocyte layer was collected and reseeded on culture plates pre-coated with Poly-L-Lysine. The astrocytes were purified via four passages to remove oligodendrocytes and neurons. By contrast, the microglia were incubated for 24 h with 200 ng/mL macrophage colony-stimulating factor (MCSF, Novoprotein Scientific, Shanghai, China) to stimulate their proliferation [16, 17]. Astrocyte and microglia purities were determined using glial fibrillary acidic protein (GFAP) and ionized calcium binding adapter molecule 1 (Iba1), respectively.
The highly aggressively proliferating immortalized (HAPI) microglia cell line purchased from Otwo Biotech (Shenzhen, China) was also used in this study. This cell line was obtained initially from mixed glial cultures prepared from 3–day-old rat brain and possessed the characteristics of microglia.
2.3. Treatment of rat astrocytes with 2-CE and preparation of astrocyte-conditioned medium (ACM)
First, astrocytes were treated with 2-CE at 7.5, 15, or 30 mM for 8 h or at 30 mM from 0 to 48 h to analyze changes in target proteins. Next, the cells were treated with NAC (5, 50, or 500 μM), FC (0.5, 1, or 2 μM), GI (1, 5, or 10 μM), SB202190 (1, 10, or 30 μM), PDTC (25 μM), or SR11302 (10 μM) for 1 h prior to the addition of 30 mM 2-CE. These inhibitors were dissolved in dimethyl sulfoxide (DMSO) and then diluted with DMEM containing 5% FBS. The final DMSO concentration was 0.1% at most and did not affect the behavior of the cells. Moreover, the cells were transfected with siRNAs of CYP2E1, TNF-α or iNOS using Lipofectamine 3000 Transfection Reagent (Invitrogen, Carlsbad, USA) or infected with IL-1β shRNA according to the manufacturer's instructions. Astrocytes were incubated with transfection media for 6 h, thereafter the complete media were replaced and incubated for 24 h before 2-CE exposure. The cells were harvested after 8 h treatment with 30 mM 2-CE for subsequent experiments.
For preparation of ACM, the treated astrocytes were washed with D-Hank’s solution, and then incubated with fresh medium for 6 h. Subsequently, the media were collected and filtered using 0.22 μm pore filters before addition into the cultures of primary microglia or HAPI cells. In this experiment, ACM from untreated astrocytes was used as the control. After 12 h incubation with ACM, the primary microglia and HAPI cells were collected for subsequent experiments.
2.4. Immunofluorescence
The cells were fixed with 4% paraformaldehyde for 20 min, permeabilized in 0.2% Triton for 10 min, and then blocked with 10% goat serum for 40 min at room temperature. Subsequently, astrocytes were incubated with rabbit anti-complement component 3 (C3) (1:100, ABclonal, Wuhan, China) and mouse anti-GFAP (1:250, Millipore, Billerica, USA), and microglia were incubated with rabbit anti-Iba1 (1:100, Bioss, Beijing, China), mouse anti-cluster of differentiation (CD) 86 (1:100, ABclonal), and mouse anti-arginase-1 (Arg-1) (1:100, Proteintech, Wuhan, China) at 4℃ overnight. Thereafter, the secondary antibody of goat anti-Rabbit-Alexa Fluor 488 (1:100, Proteintech) and goat anti-Mouse-Alexa Fluor 597 (1:100, Proteintech) were added for a 30 min incubation at 37°C. Finally, the nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). Images were obtained using a RVL-100 fluorescence microscope camera system (Echo Laboratories, USA).
2.5. Western blot
The cells were scraped and lysed in radioimmunoprecipitation assay buffer supplemented with protease and phosphatase inhibitor cocktails. Total protein concentrations were analyzed by bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Waltham, USA). Protein extracts (40 μg) were separated using 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis before being transferred to polyvinylidene difluoride membranes (Millipore). The blots were probed overnight at 4℃ with the primary antibody of C3 (1:1000, ABclonal), S100A10 (1:1000, Immunoway Biotechnology, Plano, USA), IL-1β (1:1000, Abcam, Cambridge, USA), TNF-α (1:1000, Abcam), iNOS (1:500, Abcam), CYP2E1 (1:1000, Millipore), p-p38 MAPK (1:500, Cell Signaling Technology, Danvers, USA), p38 MAPK (1:1000, Cell Signaling Technology), p-p65 (1:500, Cell Signaling Technology), p-Inhibitor of κBα (p-IκBα) (1:1000, Cell Signaling Technology), p-c-Jun (1:500, Cell Signaling Technology), CD86 (1:1000, ABclonal), Arg-1 (1:1000, ABclonal), or β-actin (1:1000, ABclonal), followed by a horseradish-peroxidase-conjugated secondary antibody for 1 h. The membranes were visualized using an enhanced chemiluminescence kit. The intensity of each band was quantified via densitometry. Equal loading of protein was confirmed using β-actin.
2.6. qPCR
Total RNA in the cells was extracted using Trizol Reagent (Takara), and their concentrations were confirmed using a Nanophotometer N60 Touch (Implen, München, Germany). For the reverse transcriptase reactions, 500 ng RNA was used as a template by using the PrimeScript RT reagent kit (Takara). The expression levels of IL-1β, TNF-α, iNOS, CYP2E1, CD86, Arg-1, CD200R, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were quantified using SYBR Premix Ex Taq II (Takara) with the QuantStudio 6 Flex Real-time PCR System (Life Technologies, Carlsbad, USA). All the specific primers used for the qPCR are listed in Table 1. The running program was carried out for 30 s at 95℃ followed by 40 cycles of 5 s at 95℃ and 34 s at 60℃. Triplicate PCR reactions were prepared for each sample. Changes in gene expression (fold change) were calculated using the 2−ΔΔCt method with normalization to GAPDH.
2.7. Enzyme-linked immunosorbent assay (ELISA)
The levels of mature IL-1β and TNF-α in culture media were assessed via ELISA kits (Elabscience Biotechnology, Wuhan, China) according to the manufacturer’s protocol. During the co-culture of microglia with ACM, the ACM were sampled from astrocytes and detected via ELISA kits prior to being applied onto microglia.
2.8. Flow cytometry
For analysis of microglia polarization, the microglia were incubated at room temperature for 30 min with the fluorescently labeled antibodies CD86-APC (1:200, BioLegend, San Diego, USA) and CD200R-FITC (1:200, BioLegend). After incubation, the cells were washed, centrifuged and then resuspended in PBS to a final volume of 500 μL. Flow cytometry was performed on a FACSCantoⅡ apparatus (BD Biosciences, Bedford, USA). The obtained data were analyzed using FlowJo software (version 10.1, BD Biosciences).
2.9. Levels of reactive oxygen species (ROS) in the cells
The fluorescent probe of 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA, Beyotime) was used for quantifying ROS levels in the cells, and measured using a multi-functional microplate reader (BioTek Instruments, Winooski, USA) at excitation and emission wavelengths of 488 nm and 535 nm, respectively. To provide statistical data, the mean measured fluorescence intensity for the control was set to 100%. The flow cytometer was also used to fluorescently quantify the cells. The fluorescent images of ROS were visualized and acquired using a RVL-100 fluorescence microscope camera system.
2.10. Quantification of NO production
The cumulated NO production was assessed via the assay kit of Griess reaction (Elabscience Biotechnology), which detects the level nitrite (NO2-), a stable metabolite of NO in the cells.
2.11. Statistical analysis
Data are presented as the mean ± standard deviation (SD) and analyzed using SPSS 20.0 (IBM, Armonk, USA). Four to six independent experiments using primary cells of different origins were performed. Student’s t-test (two-tailed) was used for comparing the differences between two groups, while one-way ANOVA followed by Student-Newman-Keuls tests were applied for determining the significant differences among multiple groups. P < 0.05 was defined as statistically significant.