The female C57BL/6 mice (6–8 weeks old) used in these experiments were purchased from Shanghai Model Organisms Center, Inc (Shanghai, China) and housed under pathogen-free conditions in Laboratory Animal Center of Xinxiang Medical University. All animal experimental procedures were performed with the approval of the Xinxiang Medical University Animal Care and Use Committee.
ALI mice model and treatment
To establish an in vivo SEB-induced ALI mouse model, SEB (BT202, Toxin Technologies, Sarasota, FL) dissolved in sterile PBS was injected intraperitoneally into age- and weight-matched female C57BL/6 mouse in a volume of 100 μl for a dose of 40 µg (Hegde et al. 2011). These mice were randomly divided into three groups (n = 10 per group): vehicle group, SEB + vehicle group, and SEB + BBR group. For the treatment group, these mice received an intragastric administration with BBR (Sigma-Aldrich, St. Louis, MO, USA) dissolved in PBS through oral gavage at 100 mg/kg body weight in a volume of 100 μl every other day for 5 days prior to SEB injection. The mice in vehicle group and SEB + vehicle group received an intragastric administration with 100 μl PBS every other day for 5 days prior to SEB injection. Mice were monitored daily and euthanized 24 h after SEB injection. Blood and serum were separated by centrifugation (4 °C, 3200×g for 20 min) and stored at − 20 °C before use. The liver tissue samples were collected for histological analyses.
Biochemical and histological analyses
The serum levels of liver marker enzymes including alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum were measured using commercially available diagnostic kits (Alanine aminotransferase assay kit; cat no. C009-2; Aspartate aminotransferase assay kit; cat no. C010-2; Nanjing Jiancheng Bio Co., Ltd., Nanjing, China). The collected liver tissues were fixed in 10% formalin, embedded in paraffin, and cut into 5 µm sections. Subsequently, the sections were deparaffinized in xylene, rehydrated in alcohol (100, 95, and 90%) and stained with hematoxylin–eosin (H&E). The stained sections were examined under a Nikon E600 light microscope (Nikon, Tokyo, Japan) at 40× magnification.
Splenocytes isolation and detection
Splenocytes were prepared by aseptically removing the spleens from naive C57BL/6 mice. Spleens were homogenized into single-cell suspensions using a Stomacher 80 Biomaster blender (Seward, Davie, FL). The resulting suspension was centrifuged at 1600×g for 30 min and then subjected to red blood cell lysis (Sigma-Aldrich) according to the manufacturer’s instructions. The single splenocytes were collected and plated in a 96-well plate in RPMI 1640 media supplemented with heat inactivated 10% fetal bovine serum, 10 mM l-glutamine, 10 mM HEPES, 50 µM β-mercaptoethanol, and 100 µg/ml penicillin/streptomycin at a density of 1 × 106 cells/well at 37 °C. Splenocytes were stimulated with PBS or 1 µg/ml SEB, followed by treatment with BBR (1, 2, 4, or 8 μM) or 100 nM tichostatin A (TSA) for 24 h. To assess activation, splenocytes collected from in vitro culture were stained with anti-mouse CD69 antibody (Biolegend, San Diego, CA, USA). Flow cytometry analysis was conducted in splenocytes using a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA) and the number of splenocytes was analyzed by Cell Quest Pro (BD Biosciences).
Cell preparation and flow cytometry
Liver infiltrating mononuclear cells were separated and counted 24 h after SEB administration by Percoll density separation, as described (Hegde et al. 2011). To determine the phenotypical characteristics of the liver infiltrating cells and splenocytes isolated as above, cells were stained with the following fluorescent-conjugated antibodies: fluorescein isothiocyanate (FITC)-conjugated anti-CD8 (clone: 53-6.7), anti-CD3 (clone: 145.2 C11), phycoerythrin (PE)-conjugated anti-CD4 (clone: GK 1.5), anti NK1.1 (clone: PK136), from Biolegend, and PE-conjugated anti-Vβ8 from Ebioscience (San Diego, CA, USA). Stained cells were analyzed using Beckman Coulter 500 Flow Cytometer (Indianapolis, IN).
Cytokines analysis in culture supernatant and serum
Cell supernatants were collected from treated splenocytes after 24 h. The cytokine levels of IFN-γ, TNF-α, IL-6 and IL-2 in the isolated serum samples from mice or cell supernatants of the treated splenocytes were analyzed and quantified using individual enzyme-linked immunosorbent assay (ELISA) kits (Biolegend).
MTT assay was performed to evaluate BBR-induced cytotoxicity against splenocytes. Briefly, the treated splenocytes were plated into a 96-well plate at approximately 1 × 106 cells per well at 37 °C and cultured with fresh RPMI 1640 media containing different doses of BBR (1, 2, 4, or 8 μM). Following incubation for 24 h, 20 μl of MTT solution (5 mg/mL, Sigma-Aldrich) was added into each well and incubated at 37 °C for another 4 h. Subsequently, the culture medium was removed and 150 μl of dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. The absorbance at 490 nm was detected using a microplate reader (Bio-Rad, Hercules, CA, USA).
The HDAC activity was estimated using a colorimetric HDAC activity assay kit (BioVision Research Products, Mountain View, CA, USA). Briefly, proteins were extracted from treated splenocytes using RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) and quantified by BCA Protein Assay Kit (Pierce, Rockford, IL, USA). 100 μg of total extracts dissolved in a final volume of 85 μl ddH2O were added into the 96-well plates. Then, 10 μl of 10× HDAC assay buffer were added to each well, followed by 5 μl of the colorimetric substrate. After incubation at 37 °C for 1 h, 10 μl of lysine developer was added and incubated for 30 min at 37 °C to arrest the reaction. The absorbance was obtained by an ELISA plate reader at 405 nm.
Quantitative RT-PCR (qRT-PCR) analysis
Total RNA from the treated splenocytes was extracted using TRIzol reagent (Qiagen, Hilden, Germany) and quantified using the Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). For the detection of mRNA, 2 μg of total RNA was reversely transcribed into cDNA using PrimeScript RT reagent Kit (Bio-Rad, Hercules, CA, USA). Subsequently, RT-PCR was performed with SYBR Prime Script RT-PCR Kits (TaKaRa, Otsu, Shiga, Japan) on an ABI PRISM 7900 Real-Time system (Applied Biosystems, Foster City, CA, USA), with GAPDH as an endogenous control. The reaction protocol was as follows: denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation 95 °C for 30 s, annealing at 60 °C for 1 min and extending at 72 °C for 30 s. The relative gene expression was calculated using the 2−∆∆Ct method.
Total protein was extracted from the treated splenocytes using RIPA lysis buffer and protein concentration was detected by BCA Protein Assay Kit. Cellular extracts (20 µg per lane) were loaded onto 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline for 1 h at room temperature and then incubated with the primary antibody against acetylated histone H3 lysine 9 (H3K9Ac) (1:500; Abcam, Cambridge, MA, USA) and histone H3 (1:1000; Cell Signaling Technology, Beverly, MA, USA) at 4 °C overnight. After washing with TBST for 3 times, the membranes were further incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (1:1000; Abcam). Subsequently, signals were detected using an enhanced chemiluminescence detection kit (Pierce).
All results were expressed as the mean ± standard deviation (SD). All statistical analyses were performed using SPSS version 17.0 (SPSS, Inc., Chicago, IL, USA) software with unpaired Student’s t-test or one-way analysis of variance (ANOVA). P values < 0.05 were considered to indicate a statistically significant difference.