Materials
Protease inhibitor mixture solution and ethylenediaminetetraacetic acid (EDTA) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Phenylmethylsulfonyl fluoride (PMSF) was purchased from Sigma Aldrich Co., Ltd. (St. Louis, MO, USA). 2′,7’-Dichlorofluorescein diacetate (DCFH-DA) was obtained from Molecular Probes (Eugene, OR, USA). ECL Western Blotting Detection Reagents and pure nitrocellulose membranes were supplied by GE Healthcare (Piscataway, NJ, USA). Rabbit polyclonal antibodies against Nrf2, HO-1, p-p38, p-ERK1/2, NF-κBp65, and mouse monoclonal antibodies against, and p-IκBα, COX-2, iNOS, TNF-α, IL-6, histone, and β-actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Rabbit anti-goat, goat anti-rabbit, and goat anti-mouse immunoglobulin G (IgG) horseradish peroxidase (HRP)-conjugated secondary antibodies were acquired from Santa Cruz Biotechnology, Inc. All other chemicals and reagents were purchased from Sigma Aldrich Co., Ltd. (St. Louis, MO, USA).
Plant materials
Rhei Rhizoma was purchased from Ominherb Co. (Youngcheon, Korea). A voucher herbarium specimen has been deposited at the Herbarium of DaeguHaany University and was identified by Prof. S.S. Roh, the herbarium leader of this university. Dried slices of Rhei Rhizoma (100 g) were extracted with distilled water (1,000 mL) at room temperature for 2 h, and the solvent was evaporated in vacuo to give an extract with a yield of 23.1 %, by weight, of the original Rhei Rhizoma.
Analysis of Rhei rhizoma by HPLC chromatogram
The water extract of Rhei rhizoma (1 mg) was dissolved in 1 mL of 50 % methanol with multi-vortexing. We injected 50 μL of the sample into a reverse-phase HPLC using a ZORBAX Eclipse XDB-C18, Analytical 4.6 X 150 mm, 5-μm, with a column temperature of 25 °C. Mobile phase component A = methanol and B = water (10 mM 1-hexanesulfonic acid sodium). The gradient conditions were as follows: 15 % A; 0 min, 50 % A; 15 min, 30 % A, 30 min. The flow rate was 2.0 mL/min. The UV absorbance from 254 nm was monitored using an Agilent 1200 series with an 2998 Photodiode Array Detector from Waters Co. (Manchester, UK). All peaks were assigned by carrying out co-injection tests with authentic samples and comparing them with the UV spectral data. Sennoside A was detected from Rhei rhizoma. The measurement was repeated three times. Representative HPLC result is illustrated in Fig. 1.
Experimental animals and treatment
Six-week-old male Sprague–Dawley rats (B.W. 180 g - 200 g) were purchased from Samtako (Osan, Korea). Rats were maintained under a 12-h light/dark cycle, housed at a controlled temperature (24 ± 2 °C) and humidity (about 60 %). After adaptation (1 week), the rats (n = 24) were divided into four groups, avoiding any inter-group differences in body weight. The normal and vehicle-treated reflux esophagitis groups were given water, while the other groups were orally administered Rhei Rhizoma extract at a dose 125 or 250 mg/kg body weight daily using a stomach tube for 7 consective days (n = 6 in each group). The oral doses were determined in a preliminary study, which demonstrated biological activity without toxicity [22]. At the end of the administration period, the rats were fasted for 18 h prior to surgical procedures and kept in raised mesh-bottom cages to prevent coprophagy. And then rats were anaesthetized with an injection of Zoletil at 0.75 mg/kg (Virbac S.A. France). A midline laparotomy was performed to expose the stomach, and then both the pylorus and transitional junction between the forestomach and corpus were first exposed, and later ligated with a 2–0 silk thread but without a plyoric ring, contrary to the procedure originally proposed by Omura et al. [23]. The vagus nerves were left intact. At 6 h after the surgery, all rats were sacrificed. The entire esophagus was removed immediately and examined for gross mucosal injury. The esophageal tissue was immediately frozen in liquid nitrogen and blood samples were collected by vena cava puncture from anesthetized rats. Subsequently, the esophagus and serum were kept at −80 °C until analysis.
Esophageal lesion ratio
The rat esophagus was cut with scissors in a longitudinal direction from the gastroesophageal junction to the pharynx after sacrifice. The inner mucous was washed away with 0.9 % NaCl and the remaining tissue was laid out on paper. Thereafter, the dissected esophagus was photographed with an optical digital camera (Sony, Tokyo, Japan) and analyzed using the i-solution lite software program. The gross mucosal damage ratio was calculated as follows: the gross mucosal damage ratio (%) = [width of area with esophageal mucosal damage (mm2)/width of total area of esophagus (mm2)] × 100.
Histological examination in the esophagus
For microscopic evaluation, the opened esophagus was cut to isolate the middle segment. This segment was fixed in 10 % neutral-buffered formalin and, after embedding in paraffin, cut into 2-μm sections and stained using hematoxylin and eosin (H/E). The stained slices were subsequently observed under an optical microscope and analyzed using the i-Solution Lite software program (Innerview Co. Korea).
Measurement of gastric pH
After sacrifice, the stomach of each rat was washed with 1 mL of 0.9 % NaCl (pH 7.4) using a 1,000-μL micropipette. The pH of the collected gastric juices were measured using a pH meter (EcoMet, iSTEK Co., Seoul, Korea).
Measurement of ROS and TBARS levels in the esophagus
Esophageal ROS levels were measured employing the method of Ali et al. [24]. Esophageal tissues were homogenized on ice with 1 mM EDTA-50 mM sodium phosphate buffer (pH 7.4), and then 25 mM DCFH-DA was added to homogenates. After incubation for 30 min, the changes in fluorescence values were determined at an excitation wavelength of 486 nm and emission wavelength of 530 nm. The TBARS level was estimated according to the method of Mihara and Uchiyama [25].
Preparation of nuclear and post-nuclear fractions
Nuclear protein extraction was performed according to the method of Komatsu [26]. In brief, esophageal tissues were homogenized with ice-cold lysis buffer containing 5 mM Tris–HCl (pH 7.5), 2 mM MgCl2, 15 mM CaCl2, and 1.5 M sucrose, and then 0.1 M dithiothreitol (DTT) and protease inhibitor mixture solution were added. After centrifugation (10,500 x g for 20 min at 4 °C), the pellet was suspended with extraction buffer containing 20 mM 2-[4-(2-hydroxyethyl)-1-piperazyl] ethanesulfonic acid (pH 7.9), 1.5 mM MgCl2, 0.42 M NaCl, 0.2 mM EDTA, and 25 % (v/v) glycerol, and then 0.1 M DTT and protease inhibitor mixture solution were added. The mixture was placed on ice for 30 min, and then the nuclear fraction was prepared by centrifugation at 20,500 × g for 5 min at 4 °C. The post-nuclear fraction was extracted from the esophagus of each rat, as described below. In brief, esophageal tissue was homogenized with ice-cold lysis buffer (pH 7.4) containing 137 mM NaCl, 20 mM Tris–HCl, 1 % Tween 20, 10 % glycerol, 1 mM PMSF, and protease inhibitor mixture solution. The homogenate was then centrifuged at 2,000 × g for 10 min at 4 °C. The protein concentration in each fraction was determined using a Bio-Rad protein kit (Bio-Rad Laboratories, Hercules, CA, USA).
Immunoblotting analyses
For the estimation of Nrf2, NF-κBp65, and histone, 10 μg of protein from each nuclear fraction was electrophoresed through 8–10 % sodium dodecylsulfate polyacrylamide gel (SDS-PAGE). Separated proteins were transferred to a nitrocellulose membrane, blocked with 5 % (w/v) skim milk solution for 1 h, and then incubated with primary antibodies to Nrf2, NF-κBp65, and histone, respectively, overnight at 4 °C. After the blots were washed, they were incubated with anti-rabbit or anti-mouse IgG HRP-conjugated secondary antibody for 1.5 h at room temperature. Also, 10–15 μg of protein of each post-nuclear fraction of HO-1, p-p38, p-ERK1/2, IκBα, COX-2, iNOS, TNF-α, IL-6, and β-actin was electrophoresed through 8–15 % SDS-PAGE. Each antigen-antibody complex was visualized using ECL Western Blotting Detection Reagents and detected by chemiluminescence with Sensi-Q 2000 Chemidoc (Lugen Sci Co., Ltd., Gyeonggi-do, Korea). Band densities were measured using ATTO Densitograph Software (ATTO Corporation, Tokyo, Japan) and quantified as the ratio to histone or β-actin. The protein levels of groups are expressed relative to those of normal rats (represented as 1).
Ethical approval
Ethical approval for the study was granted by the University of Daegu Haany Ethical Committee on the 06/02/2015 with certificate number DHU2015-009.
Statistical analysis
The data are expressed as the mean ± standard deviation. Significance was assessed by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test (SPSS 11.5.1 for Windows, 2002, SPSS Inc., Chicago, IL, USA). Values of P < 0.05 were considered significant.