Protective effect of Rhei Rhizoma on reflux esophagitis in rats via Nrf2-mediated inhibition of NF-κB signaling pathway

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).

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.

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.

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.

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).

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).

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].

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).

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 for the study was granted by the University of Daegu Haany Ethical Committee on the 06/02/2015 with certificate number DHU2015-009.

Figure 2 shows the results on morphological examination of the esophagus. Morphological changes such as hyperemia and multiple erosions were observed in reflux esophagitis rats. Damage in normal rats was not apparent. The oral administration of Rhei Rhizoma led to a marked decrease of gross mucosal damage (p < 0.05). In addition, esophageal tissue stained with H&E revealed no microscopic mucosal changes in normal rat (Fig. 3a). The normal esophagus exhibited a thin epithelial layer with squamous cells and inflammatory cells were not observed in the submucosal layers. By contrast, 6 h subsequent to the induction of RE, RE control rats developed large coalesced longitudinal ulcers in the esophagus sections. Mucosal damage and hyperemia of the epithelial layers and edema and hemorrhage in the mucosa and submucosa were observed in the RE control rats (Fig. 3b). However, the administration of Rhei Rhizoma 125 or 250 mg/kg showed less severe pathological changes (Fig. 3c and d, respectively).

The reflux-induced esophagitis rats displayed a marked decrease in the gastric pH, as shown in Fig. 4. However, the gastric pH was not changed by Rhei Rhizoma administration.

As shown in Table 1, the levels of oxidative stress-related biomarkers, ROS and TBARS, in the esophagus of RE control rats were markedly higher than those of normal rats (p < 0.01), whereas, by the administration of Rhei Rhizoma at a dose of 250 mg/kg to esophagitis rats, the elevated levels were markedly decreased nearly to the levels of normal rats (p < 0.01). The 125 mg/kg Rhei Rhizoma-treated rats also showed a significant decrease (ROS; p < 0.05, TBARS; p < 0.01).

Figure 5 showed that esophageal expressions of Nrf2 and HO-1 in RE control rats were significantly decreased compared with those of normal rats (p < 0.001). However, Rhei Rhizoma administration adversely regulated the nuclear Nrf2 and cytosolic HO-1 expressions in the esophagus of reflux-induced esophagitis rats. Overall, the ameliorative effects of Rhei Rhizoma 250 mg/kg treatment were superior to those when 125 mg/kg treatment. Especially, our data showed that Rhei Rhizoma stimulated not SOD-1 and catalase but Nrf2 pathway. (Additional file 1: Figure S1)

MAPK-related protein expressions were augmented in the esophagus of RE control rats compared to the normal rats (p < 0.001), but the oral administration of Rhei Rhizoma significantly decreased the expressions of p-p38 and p-ERK1/2 in a dose-dependent manner, as shown in Fig. 6.

As shown in Fig. 7, protein levels such as p-IκBα (p < 0.001) and NF-κBp65 (p < 0.01) were enhanced in the esophagus of RE control rats, whereas these elevated levels were significantly reduced in Rhei Rhizoma-treated RE rats dose-dependently. Especially, the NF-κBp65 level was lowered nearly to that of normal rats by 250 mg/kg Rhei Rhizoma treatment (p < 0.01). Moreover, the expression levels of COX-2, iNOS, TNF-α were also enhanced in the esophagus of RE control rats in a dose-dependent manner by Rhei Rhizoma treatment (Fig. 8). Rhei Rhizoma 250 mg/kg treatment effectively suppressed more than 125 mg/kg treatment. However, IL-6 showed a tendency to decrease without significance.