Materials
BAC (purity ≥ 98%) and RAW264.7 cells were provided by Yousi Scientific Co., Ltd., and the Chinese Academy of Sciences (Shanghai, China). LPS from Escherichia coli and TPCK (NF-κB inhibitor) were obtained from Sigma (St. Louis, MO, USA). SB202190 (p38 MAPK inhibitor), U0126 (ERK inhibitor), SP600125 (JNK inhibitor), and Cell-Counting Kit-8 (CCK-8) were obtained from Medchem Express (St. Louis, MO, USA). Trizol and phenylmethanesulfonyl fluoride (PMSF) were acquired from Ambion (CA, USA) and Aladdin (Shanghai, China). HiScript Reverse Transcriptase and SYBR Green Master Mix were provided by VAZYME (Nanjing, China). ROS Assay Kit, BCA Protein Assay Kit, radioimmunoprecipitation assay (RIPA), and phosphatase inhibitor cocktail were acquired from Beyotime (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco (Grand Island, NY). Anti-NF-κB, anti-JNK, anti-p38, anti-ERK1/2, anti-TAK1, anti-p-NF-κB, anti-p-JNK, anti-p-p38, anti-p-ERK1/2, and anti-p-TAK1 antibodies were purchased from Abcam (Cambridge, UK). Anti-IκBα and anti-p-IκBα antibodies were obtained from Affinity Bio Reagents (Golden, CO, USA).
RAW264.7 Murine Macrophage Culture
Culture medium was used to dilute the stock solution of LPS to 1 μg/mL before use. Cells cultured in DMEM containing 10% FBS at 37°C in a 5% CO2 moist atmosphere were pretreated with the indicated concentrations of BAC for 1 h before co-treatment with 1 μg/mL LPS at different time points.
CCK-8 Assay
RAW264.7 cells were plated at 1.5×104 cells/well in 96-well plates overnight and treated with BAC (0.1, 1, 10, 100 and 500 μM) for 1 h before 1 μg/mL LPS stimulation in triplicate. Then, 10 mL of CCK-8 buffer was added to each well, and cells were incubated at 37°C for 24 h. FlexStation 3 microplate reader (Molecular Devices, CA, USA) was used to detect the absorbance at the wavelength of 450 nm.
NO Assay
NO levels in activated macrophages were determined using Griess reagent, which could measure the nitrates and nitrites concentrations. U0126, SB202190, TPCK, and SP600125 was administered to cells separately with or without BAC (10 μM) for 1 h before LPS (1 μg/mL) stimulation for an additional 24 h period. Then, 50 μL of Griess reagent was mixed with 50 μL of culture medium in 96-well plates and the reaction was left to proceed for 5 min. After collecting the supernatant, an NO Assay Kit (Jiancheng Bioengineering Institute, Nanjing, China) was used to determine the NO production in activated macrophages, and a microplate reader was used to detect the absorbance at 540 nm.
ROS Assay
Cells were pretreated with BAC (1, 10, and 100 μM) for 1 h before LPS (1 μg/mL) stimulation for 24 h to detect the intracellular ROS levels. The media were then removed before cells were incubated with DCFH-DA (10 μM) for 20 min. Olympus BX53 fluorescence microscope (Olympus, Japan) was used to measure ROS’s level as the mean fluorescence intensity after sufficient washing. Excitation and emission were detected at the wavelengths of 488 nm and 525 nm, respectively.
ELISA
Cells were pretreated with BAC (1, 10, and 100 μM) for 1 h before LPS (1 μg/mL) stimulation for 24 h in the medium to detect pro-inflammatory cytokine levels. ELISA kits (Elabscience, Wuhan, China) were used to determine the production of IL-6, IL-1β, TNF-α, and PGE2 following the manufacturer's instructions.
Quantitative Real-Time PCR Detecting
After treatment, total RNA was extracted with Trizol and converted to cDNA using the HiScript Reverse Transcriptase, according to the manufacturer’s recommendations. The primer sequences used in our study were as follows: β-actin (sense: CACGATGGAGGGGCCGGACTCATC, antisense: TAAAGACCTCTATGCCAACACAGT); IL-1β (sense: TCAGGCAGGCAGTATCACTC, antisense: AGCTCATATGGGTCCGACAG); TNF-α (sense: GCCTATGTCTCAGCCTCTTCT, antisense: TTGTGAGTGTGAGGGTCTGG); and IL-6 (sense: GGAGTTCCGTTTCTACCTGG, antisense: GCCGAGTAGACCTCATAGTG). SYBR Green Master Mix and ABI QuantStudio 6 Real-Time PCR System were used to quantitate the relative mRNA concentrations. β-actin was used as a reference gene, and data from the relative gene expression were analyzed using the 2-ΔΔCt method.
Western Blot Assay
After BAC or/and LPS treatment, macrophages were washed three times with ice-cold phosphate-buffered saline (PBS), and the whole-cell proteins were extracted using RIPA buffer supplemented with PMSF and phosphatase inhibitor cocktail. Subsequently, the total protein concentration was measured using the BCA protein assay. After separation by electrophoresis, 40 μg protein was transferred onto the PVDF membranes (Millipore, Billerica, MA, USA), which were then blocked for 2 h with 5% nonfat milk Tris-buffered saline Tween 20 (TBST). Subsequently, membranes were incubated with specific primary rabbit antibodies and washed five times with TBST, followed by incubation for 2 h with goat anti-rabbit IgG HRP at room temperature. Finally, each membrane was washed with TBST and visualized using the ECL substrate (Beyotime, Shanghai, China).
Immunofluorescent Assay
After BAC (100 μM) pretreatment for 1 h, LPS stimulation for 20 min, macrophages were stimulated by LPS (1 μg/mL) for 30 min to detect the location of NF-κB p65 in activated macrophages. After washing twice with PBS for 20 min, cells fixed with 4% paraformaldehyde for 15 min were permeabilized with 0.5% Triton X-100 for 30 min. Then cells were blocked with normal goat serum before incubation with rabbit anti-NF-κB p65 antibody (dilution 1:100) at 4°C overnight. After incubation with Cy3-labeled goat anti-rabbit IgG (dilution 1:100) as secondary antibodies in the dark for 30 min and sufficient washing, cells were stained with DAPI (0.5 μg/mL) for 10 min. Stained cells were visualized under a BX53 fluorescence microscope (Olympus, Japan).
Statistical Analysis
All data were presented as mean ± SD. Statistical analyses were performed using SPSS v22 software (IBM, Armonk, NY, USA). P-values of < 0.05 were considered statistically significant.