Cell culture and treatments
FuHeng Biology (FuHeng Biology, Shanghai, China) provided the BV-2 microglial cell line in mice. At 37 °C and 5% CO2 in humidified air, BV-2 microglia were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% sodium pyruvate. At least 12 hours prior to treatment with or without the test reagents lipopolysaccharide (LPS), Dex, pyrrolidine dithiocarbamate (PDTC), and N-acetyl-L-cysteine (NAC), BV-2 cells were inoculated at a density of 4 × 106 cells/well in 6-well plates.
To determine cytotoxicity, BV-2 cells were seeded at a density of 1 × 104 cells per well in 96-well plates and treated with various concentrations of LPS (0, 10, 50, 100, 200, 400, 800 ng/L) (Sigma-Aldrich, St. Louis, MO, USA) for various times (6, 12, 24 h) or with various concentrations of Dex (0, 0.1, 1, 5, 10, 20 μM) for 24 h. To test if Dex has anti-inflammatory activity against BV-2 cells, the cells were treated with 10 μM Dex and then subjected to 100 ng/mL LPS. PBS-treated cells served as the control group to ensure experimental rigor.
Cell viability
Cell viability was determined using a CCK-8 test kit (Beyotime Institute of Biotechnology, Nanjing, China). For treatment, BV-2 cells were seeded into each well of a 96-well microtiter plate, followed by the addition of 10 μL of CCK-8 solution and incubation at 37 °C for 4 hours. The absorbance of each well was then determined at 490 nm (OD490) using a microplate reader (BioTek Instruments, Vermont, USA). Three separate tests were conducted three times each.
ELISA
The cytokines IL-1β and TNF-α were determined in cell culture supernatants using an ELISA kit (R&D Systems, Minneapolis, Minnesota, USA) in accordance with the manufacturer's instructions.
Total RNA extraction and real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR)
Trizol reagent was used to extract total RNA from BV-2 cells (Life, Massachusetts, USA). To synthesis cDNA, we utilized a reverse transcriptase kit and an SYBR Premix Ex Taq kit (Takara, Tokyo, Japan) in a CFX96 Touch real-time PCR system (Bio-Rad, California, USA). The expression of β-actin served as a control to standardize the quantity of cDNA in the various samples. The comparable Ct method was used to assess real-time qPCR products according to the manufacturer's recommendations. The primer sequences for qRT-PCR were listed in Table 1.
Table1. Sequences of the primers used in the experiments
qRT-PCR Primers
|
Sequences
|
Mouse Nlrp3
|
Forward 5’-GAAGAAGAGTGGATGGGTTTG-3’
|
Reverse 5’-CTGCGTGTAGCGACTGTTGAG-3’
|
Mouse Caspase-1
|
Forward 5’-GACAAGGCACGGGACCTATGT-3’
|
Reverse 5’-CAGTCAGTCCTGGAAATGTGC-3’
|
Mouse IL-1β
|
Forward: 5’-GTGTCTTTCCCGTGGACCTT-3’
|
Reverse: 5 ‘-CGTTGCTTGGTTCTCCTTG-3’
|
Mouse β-actin
|
Forward: 5’-GGGAATGGGTCAGAAGGACT-3’
|
Reverse: 5’-TTT GAT GTC ACG CAC GAT TT-3’
|
Western blotting
Total proteins were isolated from BV-2 cells according to the manufacturer's procedure using protein extraction kits (Sigma, St Louis, MO, USA). BCA protein assays (Beyotime Institute of Biotechnology, Nanjing, China) were used to measure the protein content in the lysate supernatant. For electrophoresis, equal quantities of protein (20 μg) each well were loaded onto 12 percent sodium dodecyl sulfate-polyacrylamide gels and then transferred to polyvinylidene difluoride membranes (PVDF; Millipore, Bedford, MA, USA). After blocking nonspecific binding sites on the membrane for 1 hour at room temperature with 5% nonfat milk in TBS containing 0.1% Tween-20 (TBS-T), the membranes were incubated overnight at 4 °C with the following primary antibodies: Nlrp3 (1:1000, ab214185, Abcam, Shanghai, China), Caspase-1 (1:800, ab138483, Abcam, USA), p65 (1:1000, #8242, CST, MA, USA) and GAPDH (1:1000, #5174, CST, MA, USA). Following three washes with TBS-T, the membranes were incubated for 1 hour at room temperature with horseradish peroxidase-labeled secondary antibodies (1:2000, CST, MA, USA). Luminescent liquid was subsequently added, and photographs were taken with a gel imager. Finally, we used a chemiluminescence (ECL) system to visualize the relative expression level of the proteins and used ImageJ software to analyze the intensities of the bands, with GAPDH as an internal control.
ROS analysis
We measured ROS fluorescence intensities using a ROS staining kit (Beyotime Institute of Biotechnology, Nanjing, China) according to the manufacturer's procedure. Briefly, cells were seeded in a 6-well plate and then washed three times with PBS after drug treatment. The cells were then incubated for 20 minutes at 37 °C in the dark with 2 mol/L DCFH-DA probe. After washing with PBS, the nuclei were counterstained with Hoechst 33342 (Sigma, St Louis, MO, USA). A fluorescent microscope was used to determine the fluorescence intensity of BV-2 cells.