2.1 Cell culture
A mouse BV2 microglial cell line was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All reagents and materials in the process were obtained from Life Technologies (CA, USA). The cells were cultured in minimum Eagle’s medium (MEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37℃ in a humidified environment with 5% carbon dioxide (CO2). The medium was renewed every 48 hours. In addition, the cells were regularly treated with 0.25% trypsin to transfer and place them in more culture bottles until enough cells were obtained. Finally, monolayers of the BV2 microglial cells were cultured on 24-well plates (1×105 cells/well) for further experiments.
2.2 Reagents and preliminary experiments
Palmitic acid (PA, one type of saturated fatty acids) was obtained from Sigma-Aldrich (USA), and was dissolved using 0.1 mmol/l sodium hydroxide at 70℃ for 5 minutes. Then, the solution is mixed using 10% bovine serum albumin at 55℃ for another 10 minutes, and was adjusted to a series of concentrations (0, 400, 800, 1200, 1600 µmol/l) using specific neuronal medium (NM, Cat. #1521).
TAK-242 (one type of TLR4 blocker) was purchased from MedChemExpress (USA), and was dissolved using 0.1% dimethylsulfoxide (DMSO) to produce several concentrations of the reagent (0, 50, 100, 200, 400, 800 nmol/l).
After that, as shown in Fig. 1, the prepared cells were separately treated with different concentrations of PA and TAK-242 for 12 hours. Cell activity was determined using CCK8 assay to assess the safety of the reagents. Expression of miR-124 and TLR4 was measured separately using qPCR and western blotting to evaluate the effectiveness of the reagents. As a result, 800 µmol/l of PA and 200 nmol/l of TAK-242 were determined to be the optimal concentrations for further experiments.
2.3 Grouping and cell transfection
The BV2 cells were divided into control group, PA group, PA + mir_mimic group, PA + mir_inhibitor group, PA + mir_ctl group, PA + TAK group, PA + nlrp_siR group and PA + nlrp_ctl group. Each group contained ten wells.
MiR-124 mimic, miR-124 inhibitor and negative control were mixed with Lipofectamine 2000 transfection reagent (RiboBio, Guangzhou, China), and were adopted for cell transfection separately in the PA + mir_mimic, PA + mir_inhibitor and PA + mir_ctl groups [20]. Specific experimental steps referred to the specification of the commercial kit. Cell transfection was performed in in 37℃, 5% CO2 incubator for 48 hours.
The cells in the PA + nlrp_siR and PA + nlrp_ctl groups were separately transfected with NLRP3 siRNA and control siRNA using Lipofectamine LTX transfection reagent (RiboBio, Guangzhou, China) according to the manufacturer’s protocols. [21]. Sense and anti-sense of NLRP3 siRNA were 5’-GCUUCAGCCACAUGACUUUTT-3’ and 5’-AAAGUCAUGUGGCUGAAGCTT-3’. Sense and anti-sense of control siRNA were 5’-UUCUCCGAACGUGUCACGUTT-3’ and 5’-ACGUGACACGUUCGGAG -AATT-3’. The cells were also incubated for 48 hours before further measurement.
2.4 Modeling and intervention
After cell transfection, all the BV2 cells except those in the control group were treated with 800 µmol/l PA for 12 hours to construct an in vitro model of high-fat diet [22]. Meanwhile, the BV2 cells in the PA + TAK group were treated with 200 nmol/l TAK-242 for 12 hours to inhibit the expression of TLR4.
2.5 Cell viability assay
After the intervention, cell viability was measured using a commercial CCK8 assay kit (#ab228554, Abcam, UK). Briefly, all of the wells to be tested were incubated with CCK-8 solution (10 µl) at 37℃ for 2 hours. Then, the absorbance of the specimens at 450 nm was determined using a Thermo Scientific microplate reader (Waltham, MA, US).
2.6 qPCR assay
After the intervention, expression of miR-124 was measured using qPCR assay. First, total RNA extraction was adopted using a commercial RNAeasy™ animal RNA isolation kit (centrifugal column type) (Beyotime, Shanghai, China). Second, the obtained RNA was reverse-transcribed into complementary DNA using a commercial PrimeScript™ RT Master Mix (Perfect Real Time) (Takara Bio, JPN). Third, the qPCR process was performed using TB Green™ Premix Ex Taq™ II (Tli RNaseH Plus) (Takara Bio, JPN) and Applied Biosystems 7500 Real-Time PCR System (Thermo Scientific, Waltham, MA, US). The PCR cycling stages (parameters) were listed as followed: denaturation (95˚C, 30 seconds), annealing (60˚C, 30 seconds) and elongation (72˚C, 30 seconds). Expression of mRNA was normalized by U6, and the latter was adopted as an endogenous control. Primer sequences of miR-124 were 5′-GCTAAGGCACGCGGTG-3′ and 5′-GTGCAGGGTCCGAGGT-3′. Primer sequences of U6 were: 5′-ATTGGAACGATACAGAGAAGATT-3′ and 5′-GGAAC -GCTTCACGAATTTG-3′.
2.7 Western blotting
Expression of several proteins was analyzed using western blotting. First, the cells were treated with RIPA Lysis buffer containing protease and phosphatase inhibitors. Second, the lysates were incubated at 0℃ for 12 minutes and centrifuged at 10,000 rpm for 5 minutes. Third, protein amounts of the obtained supernatants were measured using a commercial BCA assay kit (Abcam, Cambridge, UK). Fourth, 50 µg of proteins were separated using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and then moved to polyvinylidene fluoride (PVDF) membranes for 60 minutes. Fifth, the membranes were blocked using 5% non-fat milk for 2 hours, and were incubated at 4℃ for 16 hours with TLR4 (#14358, 1:1000), MyD88 (#4283, 1:1000), NF-κB p65 (#8242, 1:1000), NLRP3 (#15101, 1:1000), IL-1β (#31202, 1:1000), CD206 (#24595, 1:1000), Arg-1 (#93668, 1:1000), CD86 (#19589, 1:1000), iNOS (#13120, 1:1000) primary antibodies (Cell Signaling, USA). Sixth, the membrane were washed three times using PBS, and were incubated with anti-rabbit HRP-conjugated IgG secondary antibodies for 60 minutes (Cell Signaling, USA). Seventh, protein bands were visualized using enhanced chemiluminescence (Immolilon Western, USA), and the intensities of them were measured using image J software.
2.8 Flow cytometry
Pyroptosis rate was determined by a FAM-FLICA in vitro caspase-1 detection kit (ImmunoChemistry, USA) [23]. First, the cells were mixed with trypsin. Second, the cells were washed several times using phosphate buffer saline. Third, the cells were stained with 10 µl FAM-FLICA and 5 µl PI for 20 minutes in a dark environment at room temperature. Fourth, fluorescence intensity was measured using a Coulter Epics XL flow cytometer. Pyroptosis rate was calculated using a formula: number of double-positive cells / number of total cells × 100%.
2.9 Statistical analysis
Continuous variables in the study were expressed in the form of mean ± standard deviation. Difference of two continuous variables was measured using independent sample t test, and difference of more than two continuous variables was measured using ANOVA with LSD test. If P value was less than 0.05, the difference was statistically significant.