RPML-1640 medium (DMEM), phosphate-buffered saline (PBS), fetal bovine serum (FBS), and other cell culture reagents were purchased from Hyclone Laboratories (Logan, UT, USA). Potato dextrose agar medium (PDA), nutrient agar (NA) and MacConkey agar medium were the products from Difco Laboratory (Detroit, MI, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, MTT and other analytical grade reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA).
Preparation of bioprocessed rice bran extract (BPRBE)
The genetic identity of Lentinus edodes was confirmed by the Korean Center of Microorganisms (Seoul, Korea). Cultivation of the Lentinus edodes mushrooms and the bioprocessing of rice bran with added fungal mycelia were conducted according to our previously reported method [21, 22]. Briefly, the main liquid medium (3 L) containing rice bran (20 g/L) and dried soybean powder (2 g/L) was inoculated with the inoculum (10%) culture mycelia. The main liquid culture was then bioprocessed using 5 L fermenter at 28 °C and 150 rpm. After 7 days, the culture mass was ground in a colloid mill and the powder was then treated with 0.1 M lactic acid for 60 min, followed by treatment with an enzyme mixture for cell wall lysis. The acid- and enzyme-treated culture mass was then extracted with hot water at 90 °C and freeze-dried to a solid material. Non-bioprocessed rice bran extract (NPRBE), not subjected to the fungal mycelia fermentation but subjected to the acid and enzyme treatments, was used as a control formulation.
Component analysis by GC-MS
The dried extracts from above were derivatized using previously published methods [22, 23]. Briefly, samples were reacted at 60 °C for 1 h with 100 μL of methoxyamine hydrochloride in pyridine (20 μL/mL). The sample was then silylated with 100 μL of N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) (70 °C/1 h). GC/MS conditions were as follows: the gas chromatograph, model 6890GC (Agilent Technologies, Santa Clara, CA, USA) was equipped with a mass spectrometer detector 5975 and an apolar stationary-phase DB-1 column (100% dimethylpolysiloxane); (30 m × 0.25 mm; i.d. 0.25 μm film thickness). The starting oven temperature of the column at the time of injection, 70 °C, was maintained for 4 min and then ramped up at a rate of 10 °C/min until it reached a final temperature of 300 °C. The carrier gas (helium) had a flow rate of 1 mL/min. The injector and the detector were maintained at 250 °C. Injection volume was 1 μL, after being split 25 to 1. The mass spectrometer was set to electron ionization mode (70 eV). Identification of components was by both retention time, and comparison of the mass spectra to a commercial library .
Bacterial strain and culture conditions
Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) strain SL1344 was obtained from the National Collection of Type Cultures (Salisbury, Wiltshire, UK) and kept as frozen glycerol stock. The bacterial cell cultures were used as previously described .
In vitro bactericidal assay
The bactericidal effect against S. Typhimurium SL1344 in liquid culture was examined as follows: aliquots of BPRBE and NPRBE were added to 3 mL Salmonella culture to form three concentrations of BPRBE (1, 10, and 100 μg/mL, respectively). The assay was performed using an inoculum size of Salmonella at 1 × 107 colony-forming units (CFU). These were then allowed to grow for 12 h at 37 °C in nutrient broth. Cell growth profiles were determined spectrophotometrically by monitoring the turbidity of the culture at 600 nm at 2 h intervals for up to 8 h using a UV/VIS spectrophotometer (iMark™, Bio-Rad, Hercules, CA, USA).
Mammalian cell culture
Murine RAW 264.7 macrophage cells were obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FBS (v/v), 100 U/mL penicillin, and 100 μg/mL streptomycin. The cells were maintained at 37 °C with 5% CO2 in a humidified atmosphere.
Cell viability assay
MTT staining as described by Mosmann , and previously used in this laboratory [22, 23], was used to evaluate the cytotoxicity of BPRBE and NPRBE. RAW 264.7 cells were seeded into 96-well tissue culture plate at a density of 1 × 104 cells/well and cultured at 37 °C in humidified air with 5% CO2 for 16 h. The cells were then treated with 3 concentrations of BPRBE and NPRBE (1, 10, and 100 μg/mL) for 48 h and stained with added MTT. Absorbance was read in a microplate reader (iMark, Bio-Rad, Hercules, CA, USA) at 570 nm, with a reference wavelength of 655 nm. Cell viability was expressed as the percentage of live cells relative to those in the control group treated with PBS.
Nitric oxide (NO) generation assay
Nitric oxide (NO) formation was measured by determining the concentration of its stable metabolite nitrite using a microplate assay as described by Murakami, et al. , a method previously used in this laboratory [23, 27]. RAW 264.7 cells (1 × 105 cells/well) in a 96-well tissue culture plate were treated simultaneously with lipopolysaccharide (LPS; 100 ng/mL) and either BPRBE or NPRBE at three concentrations (1, 10 and 100 g/mL each) for 48 h. After incubation, the culture medium was mixed with an equal volume of Griess reagent (1% sulfanilamide and 0.1% N-[1-naphthyl]ethylenediamine dihydrochloride in 5% phosphoric acid) at room temperature for 15 min. The absorbance was then measured at 570 nm using a microplate reader against a standard of sodium nitrite.
Phagocytotic uptake assay
To measure internalized bacteria in macrophages, RAW 264.7 cells were infected with the S. Typhimurium as described by Lu, et al. . To analyze the efficiency of bacterial uptake by macrophages, inoculum containing 1 × 104 CFU was added to macrophage cells (1 × 104 cells) pre-treated with three concentrations of BPRBE and NPRBE (1, 10, and 100 μg/mL) for 4 h, and then incubated for 60 min at 37 °C in humidified air with 5% CO2. Cells were washed once with RPMI-1640 medium after incubation and then treated to kill extracellular bacteria with the same medium containing 10% FBS and gentamycin (30 μg/mL) for 30 min. For viable cell counting, the infected macrophage cells were washed thrice and then lysed with sterile distilled water. Aliquots of lysates were then plated onto nutrient agar (NA) medium to measure CFUs. Phagocytosis efficiency of BPRBE and NPRBE was expressed as the fold increase of internalized bacteria relative to those without sample treatment.
Measurement of interferon (IFN)-β
After RAW 264.7 cells (1 × 105 cells) pre-treated with BPRBE and NPRBE (100 μg/mL) for 24 h were subsequently infected with S. Typhimurium (1 × 105 CFU) for 5 h, the cell-free culture medium was recovered and stored at − 20 °C until use. IFN-β release into the culture medium was measured using an ELISA kit (PBL Assay Science, Piscataway, NJ, USA). The absorbance of the reaction mixture at 420 nm was measured in a microplate reader.
Gastroenteritis animal model
Gastroenteritis was stimulated in test animals as described in a previous study . Pathogen-free female Balb/c mice, aged 6 to 8 weeks, were purchased from Orient Bio Inc. (Seoul, Republic of Korea) and were hosted in a stainless-steel cage. Conditions were as follows: the light/dark cycle repeated every 12 h; the temperature was maintained at 20–22 °C; and the relative humidity was maintained at 50 ± 10%. The mice were given free access to a pelleted commercial chow diet (catalog no. 5 L79; Orient Bio Inc.). Tap water was available ad libitum. After a one-week acclimation, mice were divided into control and experimental groups (n = 10), avoiding any intergroup difference in body weight. Test groups were administered BPRBE or NPRBE (10 mg/kg) via oral dietary intake for 2 wk. Gastroenteritis was stimulated in mice as described by Barthel, et al. . Briefly, 4-h fasting mice were administered streptomycin (20 mg in 75 μL water) by oral gavage. After 24 h, diet and water were again withdrawn for 4 h. Mice were then infected with S. Typhimurium strain SL1344 in 100 μL PBS (1 × 108 CFUs) by oral gavage. After an additional 2 h of fasting, food and water was offered. The mice were monitored for 48 h and then sacrificed by asphyxia with CO2 to remove organs. The protocol for the mouse studies was approved by the Ethics Committee for Animal Care and Use, Ajou University, Suwon, Republic of Korea.
Determination of bacterial load in organs and feces
Bacterial infection of the organs, and the presence in the feces, was determined as described in a previous study . The cecum, mesenteric lymph node (MLN), spleen, and liver of the sacrificed animals were removed aseptically. The cecum was used after removing its content. The organs were weighed and then homogenized in 1 mL of sterile PBS with the aid of a tissue homogenizer maintained at 4 °C. Diluents of the homogenates were processed to count MacConkey agar medium supplemented with streptomycin (50 g/mL).
Fecal samples were collected at days 1–2 after gavage of S. Typhimurium in the mice from the Salmonella gastroenteritis model. The number of bacteria per gram of feces was determined as follows: Aliquots (100 μL) of fecal suspensions were serially diluted in PBS and then plated on the streptomycin-supplemented MacConkey agar medium. Cells were counted following incubation overnight at 37 °C.
Reverse transcription polymerase chain reaction (RT-PCR)
Total cellular RNA was extracted using an Acid Phenol GTC-chloroform method as described by Chomczynski and Sacchi , and previously employed in this laboratory [22, 23, 27]. For RT-PCR, total RNA (1 μg) was incubated with AMV reverse transcriptase (6 U) and oligo (dT18) as primer (100 ng). DNA amplification from the iNOS gene was then primed in a reaction mixture containing dNTP mix (400 μM), Taq polymerase (2.5 U) and primer set (20 μM each): sense primer, 5’-ATGCCGAAGCAAACATCAC-3′; antisense primer, 5’-TAATGTCCAGGAAGTAGGTG-3′. Amplification was conducted in a thermocycler (PTC-200, MJ Research Inc., Reno, NV, USA) using the following program: one cycle for 5 min at 94 °C, 30 cycles for 30 s at 94 °C, 45 s at 58 °C, and 45 s at 72 °C, and finally one cycle for 5 min at 72 °C. The DNA was then fractionated with agarose gel electrophoresis and quantified using a gel documentation system (model LAS-100CH, Fuji Photo Film Co., Tokyo, Japan).
Western blot analyses were performed using the following antibodies: rabbit anti-mouse iNOS polyclonal antibody from Santa Cruz (Dallas, TX, USA) and mouse anti-Actin monoclonal antibody from Millipore Corp. (Billerica, MA, USA), and all rabbit monoclonal antibodies raised against Beclin-1, Atg5, Atg12, Atg16L, LC3A/B, and phospho-IRF-3 from Cell Signaling Tech. (Danvers, MA, USA).
Western blot analysis
The Western Blot technique was the same as described in previous studies [22, 23]. The BPRBE- and NPRBE-treated RAW 264.7 cells were lysed to extract total cellular proteins using RIPA buffer (50 mM Tris Cl, 150 mM NaCl, 15 NP-40, 0.5% sodium deoxycholate, 0.1% SDS and 1 mM EDTA, pH 7.4). Protein was quantified using a Bio-protein Kit (Bio-Rad) with bovine serum albumin (BSA) as a standard. Proteins from the cell extract (30 μg) were separated on 10% SDS-polyacrylamide gels and electrophoretically transferred onto the Millipore nitrocellulose membrane. After blocking with 5% skim milk, the membrane was incubated with primary antibodies to react for at least 3 h. The secondary antibody reaction with HRP-conjugated anti-IgG antibody was then performed under the same conditions. Blots were developed using the ELC detection kit (Pierce, Rockford, IL, USA). Quantification was achieved using a gel documentation system (model LAS-100CH, Fuji Photo Film Co., Tokyo, Japan).
Analyses were run in triplicate, and expressed as the mean ± SD. Significant differences between means were determined by the ANOVA followed by Tukey’s test using the Statistical Analysis Software package SAS (Cary, NC, USA). p < 0.05 is regarded as significant.