BHB was purchased from Ildong Pharmaceutical Co., Ltd. (Seoul, Korea). Protein expression was detected using the following primary antibodies from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA): Rabbit polyclonal IL-1β (dilution, 1:500; cat. no. sc-7884); goat polyclonal IL-6 immunoglobulin G (IgG) (dilution, 1:500; cat. no. sc-1265); goat polyclonal TNF-α IgG (dilution, 1:500; cat. no. sc-1351); rabbit polyclonal cyclooxygenase-1 (COX-1; dilution, 1:1,000; cat. no. sc-7950); and goat polyclonal cyclooxygenase-2 IgG (COX-2; dilution, 1:1,000; cat. no. sc-1745) (all purchased from Santa Cruz Biotechnology, Inc.). The following primary antibodies were also used: Rabbit polyclonal inducible NOS (iNOS; dilution, 1:1,000; cat. no. ab15323); rabbit monoclonal neuronal NOS (nNOS; dilution, 1:1,000; cat. no. ab76067) (both purchased from Abcam, Cambridge, UK); and rabbit monoclonal endothelial NOS (eNOS; dilution, 1:1,000; cat. no. 32027; Cell Signaling Technology, Inc., Beverly, MA, USA). Horseradish peroxidase (HRP-conjugated goat anti-mouse IgG (dilution, 1:2,000; cat. no. sc-2005), HRP-conjugated goat anti-rat IgG (dilution, 1:2,000; cat. no. sc-2006) and HRP-conjugated donkey anti-goat IgG (dilution, 1:2,000; cat. no. sc-2020) were used (all purchased from Santa Cruz Biotechnology, Inc.).

A total of 30 male Sprague-Dawley rats, weighing between 240–250 g, were purchased from Samtako Laboratory Animal Company (Osan, Korea) and housed for 1 week in the animal facility for acclimation. Constant environmental conditions were maintained with a temperature of 23±1°C, humidity of 55% and a 12-h light/dark cycle. Following acclimation, the rats underwent a fast for the 24 h prior to the experiments. The present study was approved by the Institutional Animal Care & Use Committee of Korea University (approval number, KUIACUC-2013-181).

Gastric ulcers were induced in all rats by direct injection of acetic acid. The rats were anesthetized using tiletamine/zolazepam (10 mg/kg; intramuscular injection; Zoetis, Inc., Florham Park, NJ, USA) and xylazine (5 mg/kg; intraperitoneal injection; Bayer AG, Leverkusen, Germany). A longitudinal incision of 2 cm was made in the upper abdomen. The stomach was then exposed and directly injected with 2 cm³ of 60% acetic acid solution, as described by Okabe et al (6). After 45 sec, the gastric contents were aspirated by syringe. The abdomen was sutured and oral intake of food and water was permitted following closure. Following gastric ulcer induction, the rats were randomly divided into 6 groups, with 5 rats per group. The groups were organized as follows: Control, no BHB treatment; BHB 100; 300; or 1,000 mg/kg treatment; L-NAME (Sigma-Aldrich, St. Louis, MO, USA) 70 mg/kg treatment; and L-NAME 70 mg/kg treatment with BHB at 1,000 mg/kg. The drugs were dissolved in 2 ml 5% dextrose water (DW; JW Pharmaceutical Corporation, Seoul, Korea) and administered orally once per day for 5 days. The rats in the control group were administered 2 ml 5% DW without BHB.

A total of 5 days after the induction of gastric ulcers, the rats were sacrificed using CO₂. The stomachs were dissected, gently incised along the longer curvature, opened and rinsed with phosphate-buffered saline (PBS) to remove the gastric contents. The gastric mucosa lesions were macroscopically examined with a magnifier using a metric measurement scale. The areas of the ulcerous lesions were measured in mm² using the lesion index (7).

The expression levels of COXs (COX-1 and COX-2), cytokines (IL-1β, IL-6 and TNF-α) and NOS (nNOS, eNOS and iNOS) were measured using western blot analysis. The gastric tissues were frozen using liquid nitrogen and stored at −80°C. Samples were pulverized by a mortar and pestle, then mixed with radioimmunoprecipitation assay buffer (a lysis buffer) and centrifuged at 14,200 × g for 15 min. The supernatants were collected and the protein content was determined using a Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, Inc., Hercules, CA, USA). A similar mass of total protein was loaded from each sample onto a 5–12% sodium dodecyl sulfate gel and transferred to polyvinylidene fluoride membranes using electrophoresis. The membranes were blocked with a blocking buffer (5% skimmed milk in PBS) for 1 h at room temperature, then incubated with the primary antibody. Following several washes with PBS-Tween 20 over 30 min, the membranes were incubated with the secondary antibody specific to the primary antibody for 1 h at room temperature. Following several additional washes with PBS-Tween 20 over 30 min, detection was performed using an enhanced chemiluminescence kit (Pierce ECL Western Blotting Substrate; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and the images were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The intensity of each band was compared with that of the internal control, β-actin.

Data were processed and analyzed using SPSS, version 20.0 (IBM SPSS, Armonk, NY, USA). Statistical comparisons were performed using a Student's t-test. P≤0.05 was considered to indicate a statistically significant difference.

The control group developed ulcerous lesions (Fig 1). Rats that received BHB at doses of 100, 300 and 1,000 mg/kg demonstrated reductions in mucosal injury of 7.8, 10.7 and 19.3%, respectively, compared with the control (Fig. 2); the area of ulcerous lesions significantly decreased in the group treated with 1,000 mg/kg BHB (Fig. 2). L-NAME aggravated the acetic acid-induced ulcerous lesions, observed macroscopically (Fig. 1E), but the effect of L-NAME was somewhat reversed when it was administered with 1,000 mg/kg BHB (Fig. 1F). The L-NAME + BHB group exhibited significantly reduced lesion area compared with the L-NAME group (P<0.05; Fig. 2).

The expression levels of COX-1 and −2 are reported in Fig. 3. COX-2 expression was decreased in the BHB groups compared with the control group; however, a significant difference was only observed at 1,000 mg/kg (P<0.05; Fig. 3C). No significant difference in COX-1 expression was observed between the BHB treatment and control groups.

The expression levels of pro-inflammatory cytokines are reported in Fig. 4; and the expression levels of IL-1β, IL-6 and TNF-α were not observed to be significantly different following BHB treatment.

The effect of BHB on eNOS, iNOS and nNOS protein expression was also assessed by western blot (Fig. 5A). All BHB treatments significantly increased eNOS expression compared with the control group (P<0.05; Fig. 5B), but no significant differences were identified in the expression levels of nNOS or iNOS between the groups (Fig. 5C and D).

As presented in Fig. 6, COX-1 protein expression levels increased in rats administered L-NAME compared with the control group (P<0.05), and the co-treatment with BHB significantly decreased the expression of COX-1 compared with the L-NAME group (P<0.05). COX-2 protein expression levels increased in rats in the control group, and the administration of BHB significantly decreased COX-2 protein expression in both groups, with or without L-NAME (P<0.05). As presented in Fig. 7, L-NAME administration significantly increased the expression of the inflammatory cytokine TNF-α (P<0.05 vs. control group), whereas combined treatment suppressed its expression (P<0.05 vs. L-NAME group). No significant difference was observed in the expression of IL-1β and IL-6 with either drug.

In the presence of L-NAME, eNOS levels did not differ between those groups that were or were not administered BHB (P<0.05; Fig. 8A and B). The expression levels of nNOS were decreased in the L-NAME group compared with levels in the control group; and compared with the L-NAME group, these levels were significantly increased in the L-NAME + BHB group (P<0.05; Fig. 8A and C). By contrast, the expression levels of iNOS were suppressed by BHB + L-NAME co-treatment compared with L-NAME treatment alone (P<0.05; Fig. 8A and D).