Reagents
Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), and penicillin-streptomycin were purchased from Gibco (Carlsbad, CA, USA). LF was from Sigma-Aldrich (St. Louis, MO, USA). Lipopolysaccharide (LPS) (Escherichia coli 0127, B8) was purchased from Sigma.
Cell culture
Macrophage RAW 264.7 cells were purchased (Pasteur Institute, Tehran) and cultured in DMEM medium, supplemented with 10% FBS, 1% of penicillin/streptomycin at 37 °C in 5% CO2 incubator. Cells at 80% confluence were subcultured and the passages 3–5 were subjected to the subsequent experiment.
Cell viability and morphology
Cell viability was measured by MTT assay. Briefly, RAW264.7 cells were grown into 96-well plates (104 cells/well) for an overnight. Then, cell treatment was done with LF (0, 10, 100, 500 and 1000 µg/mL) and incubated for 1 h, and then were treated with LPS (1 µg/mL). After 24 h, MTT solution (0.5 mg/mL) was added and incubated for further 4 h. Then, after dissolving of formazan crystals by DMSO, the optical density was measured at 570 nm (Bio-Tek, ELX 800, USA). The qualitative morphology of the cell cultures was also analyzed by a light microscope.
Detection of miRNA and mRNA expression
Total RNA with miRNA contents were isolated from the RAW264.7 cells by using TRIzol reagent (Sigma-Aldrich, Germany). Extracted RNAs quality was assessed using Nanodrop (Thermo Scientific). The cDNA synthesis was conducted with the cDNA synthesis kit (Takara, Shiga, Japan) by using Total RNA (1 µg). The stem-loop-based quantitative real-time RT-PCR (qRT-PCR) method was used for detecting miRNAs expression. The qRT-PCR analyses were performed using Takara SYBR® Premix Ex Taq (Takara, Japan) on a LightCycler® 96 System (Roche Diagnostics, Germany). miRNAs primers were designed according to the corresponding sequence from miRBase and NCBI database and were listed in Table 1. The 2−ΔΔCT method was applied for calculation of the relative expressions of the genes.
Table 1
Sequences of stem-loop RT primers, forward primers and reverse primers of target genes
miRNAs/Genes | Primer | Sequence |
miR-155 | RT | GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACCCCT |
Forward | GTATACTTAATGCTAATTGTG |
miR-146a | RT | GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAACCCA |
Forward | GTATACTGAGAACTGAATTCC |
U6 | Forward | CAACGGATATCTCGGCTCT |
Reverse | CAACTTGCGTTCAAAGACTC |
Universal primer | Reverse | GTGCAGGGTCCGAGGT |
IL-6 | Forward | ACAAAGCCAGAGTCCTTCAGA |
Reverse | TCCTTAGCCACTCCTTCTGT |
TNF-α | Forward | ACTGAACTTCGGGGTGATCG |
Reverse | TCTTTGAGATCCATGCCGTTG |
β-actin | Forward | AGAGGGAAATCGTGCGTGAC |
Reverse | CAATAGTGATGACCTGGCCGT |
HMGB1 | Forward | GCAAAGGCTGACAAGGCTCG |
Reverse | GATTTTGGGGCGGTACTCAGA |
Myd88 | Forward | GGCCTTGTTAGACCGTGAGG |
Reverse | GTGGGACACTGCTTTCCACT |
TLR4 | Forward | TCTGGGGAGGCACATCTTCT |
Reverse | CAGGTCCAAGTTGCCGTTTC |
Assessment of TNF-α and IL-6 expression
Cells were seeded in 24-well plates (106 cells/mL) for 24 h, and then were treated with LF and stimulated with LPS for indicated periods. The concentration of TNF-α and IL-6 in cell culture of LF with or without LPS groups was carried out the commercially available ELISA kit (ExCell Biotech, China).
Western blot
For western blotting, cells were collected and washed using ice-cold PBS. Thereafter, cells were homogenized by lysis buffer (Roche Diagnostics, Germany). Protein concentration was quantified using a NanoDrop (ND-1000 Spectrophotometer, USA). Extracted protein was separated by 12% SDS-PAGE and blotted to a PVDF membrane (Millipore, USA), then, blocked and hybridized with the specific antibodies (Santa Cruz, CA, USA). After incubation with secondary antibodies, the results were visualized by enhanced chemiluminescence (Thermo Fisher Scientific, USA) in the dark-room. The results were normalized to the GAPDH signal value in the same lane.
Data analysis
Results are expressed as the mean ± SD (n = 3). The one-way analysis of variance (ANOVA) with multiple comparisons was used to compare the differences between the groups using GraphPad Prism software (Version 6; GraphPad, CA). A p < 0.05 was considered to as significant