2.1 Materials and Microglia cells
HSYA (purity>98%) was purchased from Chengdu DeSiTe Biological Technology Co., Ltd. (Sichuan, China). The BV-2 mouse microglia cell line was purchased from Shanghai Fuheng Biological Co., Ltd.(Shanghai, China). Aβ1-42 was purchased from Sigma-Aldrich(USA). Aβ1-42,HiLyteTM Flour 647was purchased from ANASPEC PEPTIDE(USA). LV-TREM2 RNAi was purchased from Shanghai Genechem Co.,Ltd.(Shanghai, China). Enzyme-linked immunosorbent assay (ELISA) kits for TNF-α were acquired by Enzyme-Linked Biotechnology(China). Enzyme-linked immunosorbent assay (ELISA) kits for IL-1β, IL-4, IL-13, and antibodies to CD11b were purchased from Boster (China). PowerUp™ SYBR™ Green Master Mix and RevertAid First Strand cDNA Synthesis Kit were purchased from Thermo(USA). UNIQ-10 Column Trizol Total RNA Isolation Kit was acquired from Sangon Biotech(China). HRP-conjugated AffiniPure Goat Anti-Mouse IgG (H + L), HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H + L), antibodies for IκB-α, p-IκB-α, NF-κB p65, and p-NF-κB p65 were supplied by Affinity (USA). Antibodies for TLR4 and Arg-1 were acquired from Santa Cruz Biotechnology(USA). Antibodies for TREM2 were purchased from Abcam(USA). Antibodies against beta-actin and goat anti-mouse AlexFluor488® were obtained from Zsbio(China). Goat anti-rabbit AlexFluor488® was obtained from Cell Signaling Technology(USA).
2.2 Preparation of Aβ1-42 solutions
To generate soluble oligomers, Aβ1-42 peptide was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP; Sigma- Aldrich) at a concentration of 1 mM and then incubated for 24 hours under a fume hood(T. Jiang et al. 2014). The residual peptide film was dissolved to a concentration of 5 mM in dry dimethyl sulfoxide (DMSO). For oligomeric conditions, the peptide was dissolved in the peptide in a serum-free DMEM high glucose medium to a final concentration of 100 uM and kept at 4 °C for 24 hours.
2.3 Cell Culture and Treatment
Immortalized mouse BV-2 microglia were cultured in DMEM (Gibco, USA)high glucose medium with 0.1% penicillin-streptomycin (Gibco, USA) and 10% fetal bovine serum (Zhejiang Tianhang Biotechnology Co., Ltd., China) in a humidified atmosphere containing 5% CO2 and 95% air at 37℃. The cells were sub-cultured for further passages when they reached 80% confluence and the culture medium were changed every two days. The cells in the logarithmic growth phase can be used in the experiment. Cells were pretreated with or without Aβ1-42 for 24 hours and treated with various concentrations of HSYA (1, 2.5, 5, 10, and 20 μM) for 24 hours.
2.4 Cell Viability Assay and Morphological Analysis
BV-2 cell viability was assessed by the MTT assay using a 96-well culture plate. BV-2 cells were inoculated with Aβ1-42 or treated with different concentrations of HSYA for 24 hours, and the appropriate concentration was selected for the follow-up experiment. Briefly, BV2 cells were seeded and pretreated with Aβ1-42 for 24 hours and treated with different concentrations of HSYA.MTT (Soleibao, China) solution (20 μL) was added to each well. After incubation at 37 °C for four hours. The liquid was discarded and 200 μL DMSO were added to shake for 10 minutes. The absorbance at 570 nm was read on the enzyme meter (Thermo, USA). For morphological analysis, the cells were imaged with the Zeiss inverted microscope (Axio observer A1, Zeiss, Germany) at 100× magnification.
2.5 Phagocytosis Assay
BV-2 cells were spread in a 6-well culture plate at a density of 1 × 106 per well. BV-2 cells were treated with different concentrations of HSYA (1, 2.5, 5, 10, and 20 μM) for 24 hours. 500 nM Aβ1-42 HiLyteTM Flour 647 (AnaSpec, USA)was added to each well. After incubation at 37 °C for four hours, the cells were harvested and washed with phosphate-buffered saline (PBS). Light scattering characteristics of each sample (1 × 105 cells) were analyzed by FACSCanto analyzer (BD Biosciences, USA).
2.6 Lentivirus Transduction
The lentivirus encoded the TREM2 shRNA sequence 5′-AGCGGAATGGGAGCACAGTCA-3′. Lentivirus containing TREM2 shRNA (LV-shTREM2) at 1 × 108 TU/ml were purchased from Genechem (Shanghai, China). BV-2 cells were plated into a 6-well culture plate(5 × 104 cells/well) and incubated overnight. The TREM2 lentiviral particles were used to infect the cells at an MOI of 10. After 12 hours of lentiviral adsorption and infection, the transfected cells were screened by the complete culture medium of 2.5 μg/mL puromycin (Soleibao, China). The lentivirus transduction efficiency was observed by a fluorescence microscope and the expression levels of TREM2 were validated using Western blot.
2.7 Determination of cytokine levels by ELISA
Measure inflammatory factors such as TNF-α (Enzyme-Linked Biotechnology, China), IL-1β (Boster, China), IL-4 (Boster, China), IL-13 (Boster, China) released into the culture medium. NC and LV-shTREM2 BV-2 cells were inoculated into a 6-well culture plate(1× 106 cells/well). The cells were incubated with Aβ1-42 (1 μM) for 24 hours and treated with HSYA (5 μM) for 24 hours. The supernatant was collected and the concentrations were measured by ELISA according to the manufacturer’s instructions. Optical density (OD) was measured at 450nm using a microplate reader (Thermo, USA)
2.8 Quantitative PCR (qPCR) Assay
Total RNA was extracted using the UNIQ-10 Column Trizol Total RNA Isolation kit (Sangon Biotech, China). According to the standard protocol, the isolated RNA was treated with PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa Bio INC, China) to eliminate genomic DNA and reverse-transcribed into single-stranded cDNA. Specific primers (Sangon Biotech, China) were used to amplify target genes by using QuantiNovaTM SYBR Green PCR kit (Qiagen, Germany). The Rotor-Gene Q system (QIAGEN, Malaysia) for qPCR analysis. Reaction conditions: pre-denaturation 95 °C, 10 min; 95 °C, 10s, 60°C, 45s, 40 cycles. Each sample was analyzed in triplicate, and calculate the relative expression of mRNA after normalizing IL-6 and IL-10. By comparing the CT value of the target gene with that of GAPDH, the relative change of gene expression level was 2−ΔΔCt. All primer sequences used are listed in Table 1.
Table 1
Primers sequences used for qPCR.
Gene
|
Forward primer
|
Reverse primer
|
IL-6
|
5′-TTCTTGGGACTGATGCTGGTG-3′
|
5′-CACAACTCTTTTCTCATTTCCACGA -3′
|
IL-10
|
5′-TTACCTGGTAGAAGTGATGCCC-3′
|
5′-GACACCTTGGTCTTGGAGCTTA -3′
|
GAPDH
|
5′-AAGAGGGATGCTGCCCTTAC-3′
|
5′-CCATTTTGTCTACGGGACGA -3
|
2.1 Western blot Analysis
NC and LV-shTREM2 BV-2 cells were plated into a 6-well culture plate at a density of 1 × 106 per well and treated as mentioned above. Protein of TREM2 was extracted by membrane protein extraction kit (Sangon Biotech, China). Other cultured cells were lysed with RIPA buffer(Solarbio, China) supplemented with protease and phosphatase inhibitors, scraped off; the flasks, and collected for protein extraction. The lysates were incubated on ice for 30 min, centrifuged at 4 °C at 12000 rpm for 20 minutes, and the supernatant was collected. The protein concentration was determined using the protein analyzer Q5000 (Thermo, USA) and quantitatively denatured. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane at 23 V. After blocking with 5% skim milk or BSA in TBS-T for one hour, the PVDF membranes were incubated overnight at 4°C with the following primary antibodies: rat monoclonal anti-TREM2 (1:1000, Abcam, USA), rabbit monoclonal anti-TLR4 (1:1000, Santa, USA), rabbit polyclonal anti-NF-κB (1:750, Affinity, USA), rabbit polyclonal anti-p-NF-κB p65 (1:750, Affinity, USA), rabbit polyclonal anti-IκB-α (1:750, Affinity, USA),rabbit polyclonal anti-p-IκB-α (1:750, Affinity, USA), mouse monoclonal anti-β-actin (1:1000, ZSGB-BIO, China). On the next day, the membrane was washed four times with TBS-T buffer for five minutes each time and incubated for 60 minutes with anti-rabbit or anti-mouse, horseradish peroxidase (HRP)-conjugated immunoglobulin G (IgG) secondary antibody diluted in TBS-T(1: 10000). Finally, the membranes were washed four times with TBS-T buffer for five minutes each time. The protein signals were detected with an enhanced chemiluminescence kit (Thermo Fisher Scientific, USA) and detected with EC3 Imaging System (Ultra-Violet Products Ltd., UK). Image-Pro Plus6.0 image processing software was used to quantitatively analyze the gray value of the signal.
2.9 Immunofluorescence Assay
NC and LV-shTREM2 BV-2 cells were plated onto glass coverslips in 24-well(1 × 105 cells/well) culture plate and treated as mentioned above. The cells were washed twice with PBS and fixed with 4% paraformaldehyde (Solarbio, China) for 10 minutes at room temperature(25~ 30 °C) followed by permeabilization with 0.3% Triton X-100 (Solarbio, China) for 20 minutes. Cells were then blocked with 5% goat serum (Solarbio, China) in PBS for 30 minutes followed by incubation with rabbit polyclonal anti-CD11b (1:50, Cell Signaling Technology, USA), mouse monoclonal anti-Arg-1 (1:50, Santa, USA), and rabbit polyclonal anti-iNOS (1:200, Cell Signaling Technology, USA) at 4°C overnight. The next day, the cells were washed three times with PBS-T and incubated with goat anti-rabbit AlexFluor488® (1:1000, Cell Signaling Technology, USA) or goat anti-mouse AlexFluor488® (1:50, Zsbio, China) at room temperature in the dark for one hour. The cellular nuclei was counterstained with 1 mg/ml PI (Solarbio, China) for 10 minutes in the dark and mounted with 50% glycerol (Solarbio, China). Fluorescence images were acquired using a confocal laser(LSM510, Zeiss, Germany). The quantification of the fluorescence intensity was performed by analyzing the fluorescence images using the ImageJ software.
2.10 Statistical analysis
Statistical analysis was performed using SPSS software 22.0(IBM, Inc., Armonk, NY, USA). One-way analysis of variance (ANOVA) followed by the Tukey test was used to assess the statistical significance of differences between groups. The results are expressed as the mean ± standard error (SEM). P<0.05 was considered a significant difference. P<0.05 is considered statistically significant.