Reagents
DSS (MW 36–50 kDa) was purchased from MP Biomedicals LLC (Santa Ana, CA, USA). DMEM, fetal bovine serum (FBS), penicillin (100 unit/mL) and streptomycin (100 μg/mL) were obtained from Welgene, Inc. (Daegu, Korea). The following antibodies were used in these studies: anti-ZO-1, anti-Occludin, anti-Claudin-1, IL-6 (Thermo Scientific, Grand Island, NY, USA), and GAPDH (Santa Cruz, CA, USA). Fermented barley and soybean (BS) were obtained from Sempio Fermentation Research Center (Osong, Korea). Barley (Hordeum vulgare) was cultivated by the agricultural Technology Center of Yeonggwang-gun, Jeollanam-do, and Republic of Korea. Soybean (Glycine max (L.) MERR) was supplied by Sempio Foods Company (Seoul, Korea). Voucher specimens were deposited in the herbarium at the R&D Center of Sempio Foods Company. Briefly, fermentation was performed using enzymatically hydrolyzed barley (40 g/L) and soybean (40 g/L) mediums. The medium was autoclaved and pH was adjusted to 7.0 with 2 M ammonia solution. The pre-cultured yeast, P. jadinii (KFCC 11487P), was inoculated into the enzymatically hydrolyzed barley and soybean. Fermentation was respectively conducted at 30 °C with shaking at 20 × g for 48 h in a 5 L bioreactor, and samples were dried and stored at −18 °C [7]. BS powder was dissolved in phosphate buffered saline (PBS) and then was stored at −20 °C.
In vitro DSS treatment
Human colon carcinoma cell line (Caco-2) was obtained from the Korea Cell Line Bank (Seoul, Korea). Cells were grown at 37 °C in DMEM supplemented with 10% FBS, penicillin and streptomycin in a humidified atmosphere of 5% CO2. To test the effect of BS on DSS-treated Caco-2 cells, cells were seeded onto 12-well plates (SPL Life Science, Pocheon, Korea). After reaching 90–100% confluency, the Caco-2 cell monolayers were allowed to differentiate for an additional 14 days. Fully differentiated cell monolayers were incubated with or without 2% DSS in the absence or presence of 100, 200, and 400 μg/mL BS for 48 h. DSS was dissolved in culture media and filter-sterilized using a 0.45-μm filter [8–10].
Immunofluorescence assay
Cells grown on glass coverslips and frozen tissues were fixed and permeabilized in methanol or acetone at −20 °C. Cells or tissues were incubated with primary antibodies overnight at 4 °C, followed by incubation with FITC-labeled secondary antibody for 1 h at room temperature. Sections were then mounted with mounting medium containing 4, 6-diamidino-2-phenylindole (DAPI) for nuclear counterstaining. Images were observed by fluorescence microscopy. FITC and DAPI images were taken from the same area.
Western blot analysis
Whole cell and detergent-insoluble fractions were prepared as described previously [11]. Briefly, whole cell protein lysates and detergent-insoluble fractions were prepared in a modified RIPA buffer containing proteinase inhibitors and phosphatase inhibitors as described elsewhere [12]. Homogenates were spin down at 12,000 rpm at 4 °C for 20 min. Supernatants were collected for whole cell lysates and the pellets used as detergent-insoluble fractions. The pellets were dissolved in 0.1% SDS. Protein concentration of each samples was determined using BCA reagents (Thermo Scientific). Equivalent amounts of protein (20–80 μg) were loaded in 10% or 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gels and transferred by blotting to polyvinylidene fluoride membranes. The blot was incubated with primary antibodies against human ZO-1, Occludin, Claudin-1, or GAPDH. After washing, the blot was incubated with HRP-conjugated secondary antibodies. The protein–antibody complexes were detected by Absignal (Abclone, Seoul, Korea) according to the manufacturer’s recommended protocol.
Animal study
Six-week-old female C57BL/6 mice (weighing 20 ± 2 g) were received from the Orient Co. (a branch of Charles River Laboratories, Seoul, Korea). The mice were housed in a specific pathogen free (SPF) animal facility and acclimated under the conditions of 22 ± 2 °C, 40–60% relative humidity, and 12 h light/dark cycle for 7 days. Mice were divided into 4 groups of 5 mice each. The first group was vehicle-treated control and the second group was given drinking water with DSS only. The third and fourth groups of mice were treated with BS (100 and 200 mg/kg/day) through oral gavage for 3 days, then exposed to 5% DSS in their drinking water for 7 days to induce colitis. After DSS treatment, BS treatment groups were additionally administered BS for 4 days according to the experimental design. The study used the animal model to study the effects of fermented barley and soybean mixture during inflammation.
In vivo permeability assay
In vivo permeability assay was performed to assess barrier function using fluorescein isothiocyanate dextran (FITC-D). For each experiment, mice were divided into 4 groups of 5 mice each. The first group was vehicle-treated control and the second group was given drinking water with DSS only. The third and fourth groups of mice were treated with BS (100 and 200 mg/kg/day) through oral gavage for 3 days, then exposed to 5% DSS in their drinking water for 7 days to induce colitis. Briefly, food and water were withdrawn for 4 h, and mice were inoculated with FITC-D by oral gavage (20 mg/kg). After 4 h, mice serum was collected and fluorescence intensity was measured (excitation, 492 nm; emission, 525 nm). Detection of viable bacteria in mesenteric lymph nodes (MLNs) represented bacterial translocation from the lumen to the MLNs. The MLNs of left colonic regions were removed aseptically and were put into eppendorf tubes with 0.1-mL sterilized PBS and tissues were homogenized by micro grinder (RPI, Mount Prospect, IL, USA). The homogenates were plated on blood agar (Thermo Fisher Scientific, Lenexa, KS, USA) and incubated for 48 h at 37 °C. The number of colonie was counted and the ratio of bacterial translocation was presented for percentages.
Semi-quantitative RT-PCR and quantitative PCR (qPCR) with 16S rRNA for specific species
For semi-quantitative RT-PCR, 1 μg of RNA was used as a template for reverse-transcription using the Prime Script 1’st strand cDNA Synthesis kit (Takara; Kyoto, Japan). PCR was carried out with 20 ng of cDNA using a PCR pre-mixture (Takara). RT-PCR was performed to amplify genes using a cDNA template corresponding to gene-specific primer sets. The primer sequences used are as follows. Primer sequences are listed in the Additional file 1: Table S1. Fecal samples were collected before necropsy and immediately frozen in liquid nitrogen. Bacterial genomic DNA was extracted from fecal samples using a Wizard genomic DNA purification kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The abundance of specific intestinal bacterial groups was measured by qPCR. Genus- or species-specific 16S rRNA gene primers were used as described previously [13]. Primer sequences are listed in the Additional file 1: Table S2. 16S rRNA of Eubacteria was used as a housekeeping gene.
Statistical analysis
The results are analyzed by one-way analysis of variance (ANOVA) and differences were considered statistically significant at level of p-values < 0.05.
