Reagents and antibodies
LPS, HEPE, ranitidine hydrochloride, protease inhibitor cocktail, DCFH-DA and DMSO were purchased from Sigma (St. Louis, MO, USA). The cell viability, proliferation, and cytotoxicity assay kits were purchased from DoGenBio Co., Ltd (Guro-gu, Seoul, South Korea). DMEM, FBS, and penicillin and streptomycin were purchased from Welgene (Namcheon-ro, South Korea). Griess reagent was purchased from Promega (Fithchburg, WI, USA). DAPI (Sigma-Aldrich, St.Louis, MO, USA). DCTM Protein assay reagent, polyvinylidene fluoride membranes, and bovine serum albumin standard were purchased from Bio-Rad Laboratories (Hercules, CA, USA). The phenylmethylsulfonyl fluoride, and ethylenediaminetetraacetic acid were purchased from Corporation Cleveland (OH, USA). iNOS, COX-2, IL-1β, TNF-α, β-actin antibodies, m-IgGκ BP-HRP secondary antibody and Luminol reagent were purchased from Santa Cruz Biotechnology (Delaware Ave, CA, USA). The p-IκBα, IκBα, p-NF-κB p65, NF-κB p65, p-SAPK/JNK, SAPK/JNK, p-p38 MAPK, p38 MAPK, p-p44/42 MAPK (Erk1/2), p44/42 MAPK (Erk1/2), and Lamin B1 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).
Buds from C. japonica plants were collected in the mountain area of Jeju, South Korea, and authenticated by the Korean Institute of Oriental Medicine (KIOM). Dried buds were suspended in 10 volumes of 75% ethanol and extracted three times for two hours each in a circulation distillation unit at 50°C. The ethanol extracts were filtered through advance filter paper, freeze-dried, pulverized, and stored at -80°C until use.
DPPH and ABTS radical scavenging assay
The DPPH radical scavenging assay was conducted using the method of Liyana-Pathirana C and Shahidi F . For this, DPPH (0.2 mM) in methanol and different concentrations of CJE (25, 50, 100 μg/mL) were mixed in a 1:1 ratio. The mixture was incubated at 37℃ for 30 min, and then the absorbance at 517 nm was measured using a microplate reader (Multiscan spectrum, Thermo Scientific).
The ABTS radical scavenging assay was conducted using the method of Jing et al . Here, ABTS (0.7 mM) and 2.4 mM potassium persulfate (K2S2O8) solution were mixed in a 2:1 ratio and reacted at room temperature for 16 h in the dark. The absorbance of the mixture at 734 nm was measured using a microplate reader, and the mixture was used when the value was in the range of 0.70±0.02. ABTS and different concentrations of CJE (50, 100, 200 μg/mL) were mixed in a 1:9 ratio. The absorbance at 734 nm was measured using a microplate reader (Multiscan spectrum, Thermo Scientific).
RAW264.7 cells were obtained from the American Type Culture Collection and cultured in medium containing DMEM, 10% FBS, 100 units/ml penicillin, and 100 μg/mL streptomycin. Cells were incubated in a 5% CO2 and 95% air environment at 37°C. The cells were grown to logarithmic phase for the experiments.
Determination of intracellular reactive oxygen species (ROS)
To measure intracellular ROS content, RAW264.7 cells were plated at a concentration 3 × 105 cells/mL. CJE at concentrations of 12.5, 25, and 50 μg/mL was added and incubated for 1 h, and LPS (1 μg/mL) was added for co-treatment for another 8 h. Then DCFH-DA (10 uM) solution was added to cells. After 30 min, the fluorescence intensity was measured by multimode microplate reader (Perkin Elmer, Gyeonggi-do, South Korea) at excitation wavelength 485 nm and emission wavelength 535 nm.
Cell viability and cell morphology observation and quantitation assay
To determine cell viability, RAW264.7 cells were seeded at a concentration of 5 × 105 cells/mL in a 96-well plate. Cells were pre-treated with CJE at concentrations of 50, 100, and 200 μg/mL and incubated for 1 h. Then, LPS (1 μg/mL) was added to the plate, and the cells were further incubated for 18 h. Cell viability was measured with the proliferation and cytotoxicity assay kit. Cell morphology was observed by inverted microscope (Nikon, Melville, NY USA) and quantified by the Image J program. The calculation formula was as follows: Roundness factor (RNF) = (A: cell area, dmax: maximum diameter).
Nitric oxide assay
To determine the production of nitrite in culture media, RAW264.7 cells were seeded at a concentration of 5 × 105 cells/mL in a 96-well plate. Cells were pre-treated with CJE at concentrations of 50, 100, and 200 μg/mL and incubated for 1 h. Then, LPS (1 μg/mL) was added, and the plate was further incubated for 18 h. Production of nitrite was measured in the Griess reagent according to the manufacturer`s instructions. Nitrite concentration was determined at 540 nm using NaNO2 as a standard curve. Each treatment was carried out in three replicates.
IL-1β ELISA kit assay
Concentration of IL-1β in culture media was measured using human IL-1β/IL-1F2 Immunoassay kit (R&D Systems, MN, USA) according to the product datasheets.
RAW264.7 cells were plated on a square glass coverslip at a concentration of 3 × 105 cells/mL in a 6-well plate. Cells were pretreated with CJE (200 μg/mL) for 1 h, and LPS (l μg/mL) was added for another 30 min. Cells were harvested for fixing, permeating, and blocking as described in our previous study. Cells were incubated with the primary antibody of p65 (1:200) overnight at 4 ℃ and then incubated with the second antibody (m-IgGk BP-FITC 1:1000) for 2 h at room temperature. After staining the nuclei with DAPI, images were collected using super resolution confocal laser scanning microscope (magnification, 63 ×).
Preparation of RAW264.7 cells protein extracts
RAW264.7 cells were plated at a concentration of 1 × 106 cells/mL in a 6-well plate. The normal control group cells were treated with medium, and the LPS-treated group cells were stimulated by LPS (1 μg/mL) only. The co-treatment group cells were pre-treated with different concentrations of CJE (100 or 200 μg/mL) for 1 h, and then LPS (1 μg/mL) was added. All cells were incubated for 30 min or 18 h at 37°C in a 5% CO2 and 95% air incubator. The cells were washed with ice-cold phosphate-buffered saline (PBS) three times and then lysed with 100 μL of whole protein cell lysis buffer [5 M NaCl, 0.5 M EDTA, 1 M Tris (PH 8.0), 10% NP-40, 10% sodium deoxycholate, and 10% SDS]. The tubes were placed on ice for 15 min, vortexed every 5 min, and then centrifuged at 13000 rpm for 15 min. The supernatant containing the protein was collected and stored at -80°C until analysis.
Rat maintenance and surgery of acute reflux esophagitis
Seven-week-old male Sprague-Dawley rats (180 ± 20 g) were used according to the animal welfare recommendations of the Institutional Animal Care and Use Committee of Chonbuk National University Laboratory Animal Center in South Korea. Rats were kept in standard rat cages and supplied food and water ad libitum in a facility maintaining a 12 h light/dark cycle and a controlled 21°C to 25°C temperature and 35% to 60% humidity environment. Rats were acclimated for one week under these conditions and subsequently divided into four groups (n = 8) randomly: normal group, RE control group, medication group, and positive control group. Rats were fasted for 18 h before surgery. Two h before surgery, the normal group rats did not receive oral administration, and the RE control group rats were orally administered saline; the medication group rats were orally administered CJE at a dose of 200 mg/kg body weight, and the positive control group rats were orally administered ranitidine at 40 mg/kg body weight. The rats were anesthetized nasally. A 2 cm incision was made in the rats’ abdomens to expose the stomach, and both the junction of the stomach and forestomach and the pylorus were ligated using a 3-0 silk thread. Care was taken to ensure that the vagus nerve remained intact [14, 15]. All rats were sacrificed 4.5 h after the surgery. The stomach and esophagus were removed to determine the ratio of esophageal injury. The esophageal tissue was stored at -80°C until analysis.
Esophageal damage ratio
The esophageal and gastric tissue was placed on paper and digitally photographed after esophageal mucus was washed away with 0.9% saline. The esophageal injury was analyzed using Image J software. The esophageal lesion index was calculated as follows: lesion index (%) = [area of esophageal damage (mm2)/total area of esophagus (mm2)] × 100.
Esophageal histological study
A 4-5 mm section of esophageal tissue was removed, placed in 4% NBF fixative, and fixed with paraffin. Slices were 5 μm thick and stained with hematoxylin/eosin. Images were acquired with a Leica microscope at 10 X magnification.
Preparation of cytoplasmic and nuclear protein fractions from esophageal tissue
The nuclear and cytoplasmic proteins of esophageal tissue were extracted according to our previous method . In brief, esophageal tissues were homogenized in ice-cold lysis buffer containing 10 mM HEPES, 10 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, and protease inhibitor mixture solution. Samples were stained on ice for 30 min. After centrifugation at 13000 rpm for 2 min at 4°C, the supernatant with the cytoplasmic proteins was collected. The remaining pellet was re-suspended with extraction buffer containing 50 mM HEPES, 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 1% (v/v) glycerol, 1 M DTT, and protease inhibitor mixture solution. Following incubation on ice for 30 min, the sample was centrifuged at 13000 rpm for 10 min at 4°C. The supernatant with the nuclear protein fraction was collected and stored at -80°C until analysis.
Western blot analysis
Equal amounts of quantitative proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with different percentages and transferred to PVDF membranes. The membranes were blocked with 5% skim milk for 2 h at room temperature, washed with ice-cold PBS, and then incubated with primary antibodies (1:1000) overnight at 4 °C. Finally, the membranes were treated with HRP-conjugated secondary antibodies (1:10000) for 1.5 h with gentle shaking at room temperature. Bands were visualized using a Luminol reagent, a 1:1 ratio of solutions A and B, and Bio-Rad image software (NY, USA).
All data are expressed as mean ± standard deviation. One-way ANOVA followed by LSD multiple comparisons analyses were performed using SPSS 12.0K for Windows. A P-value < 0.05 indicated a significant difference.