Sodium carbonate (Na₂CO₃), sodium chloride (NaCl), potassium chloride (KCl), naphthylethylenediamine dihydrochloride (NED), ethanol, and methanol were purchased from Merck (Merck KGaA, Darmstadt, Germany). Visfatin, ethylenediaminetetraacetic acid (EDTA), sodium phosphate monobasic (NaH₂PO₄), sodium phosphate dibasic (Na₂HPO₄), dimethyl sulfoxide (DMSO), 4,5-diaminofluorescein (DAF-2), 3-morpholinosydnonimine hydrochloride (SIN-1), diethylenetriamine-pentaacetic acid (DTPA), Tris base, and trichloroacetic acid (TCA) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Agarose and Coomassie brilliant blue R-250 were purchased from Promega (Madison, WI, USA). The CellTiter 96 colorimetric assay kit was from Promega. The NucBuster and PROPREP protein extraction kits were from Novagen (Darmstadt, Germany). Protein extraction solution was supplied by Intron Biotechnology (Seongnam, Korea). The polyvinylidene fluoride membrane and ECL kit were from Amersham Pharmacia Biotech (Buckinghamshire, UK). Mouse PGE₂, TNF-α, IL-1β, IL-6, and human major capsid protein (MCP)-1 enzyme-linked immunosorbent assay (ELISA) kits were purchased from Endogen (Thermo Fisher Scientific Inc., Rockford, IL, USA). Anti-ICAM-1, goat anti-rabbit IgG-HRP, goat anti-mouse IgG-HRP, and donkey anti-goat IgG antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-NAMPT, anti-SIRT, anti-iNOS, anti-COX, anti-ERK1/2, anti-phospho-ERK1/2, anti-JNK, anti-phospho-JNK, anti-p38, anti-phospho-p38, anti-Bax, anti-c-Myc, anti-Bcl-2, anti-Bcl-xL, anti-cytochrome C, anti-caspase-3, anti-caspase-7, and anti-β-actin antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). All other chemicals used were of analytical grade.

Thirty-six, 7-week-old male ICR mice (30 g; Daehan Bio Link, Eumseong, Korea) were kept in groups of three per cage in a temperature-controlled (24°C) vivarium under a 12-hour light/dark cycle (lights on from 7 am to 9 pm) and allowed formula M07 (Daehan Bio Link) and tap water ad libitum. Animals were housed in the Laboratory Animal Center of Kosin University College of Medicine. Food was removed 45 hours before sham or true irradiation, but water was still available. All animals were kept in a specific pathogen-free facility at the Animal Center in accordance with the rules and regulations of the Institutional Animal Care and Use Committee of our institute.

Thirty-six mice were randomly divided into six groups (n=6 per group): a control group vehicle-treated before sham irradiation (C) that received oral administration of 1 mL of vehicle only, a control group vehicle-treated before irradiation (RC) that received an oral administration of 1 mL of vehicle, a group rebamipide-treated before irradiation (RM) that received orally administered rebamipide at a dose of 320 mg/kg of body weight, a group sucralfate-treated before irradiation (RU) that received orally administered sucralfate at a dose of 500 mg/kg of body weight, a group rifaximin-treated before irradiation (RN) that received orally administered rifaximin at a dose of 100 mg/kg of body weight, and a control group rebamipide-treated before sham irradiation (M) that received orally administered rebamipide at a dose of 320 mg/kg of body weight.

All the prescribed doses of reagents were dissolved in normal saline (vehicle). Mice were gavaged with a 1 mL suspension per dose five times every 10 hours before the sham or true irradiation. Rebamipide was kindly provided by Otsuka Pharmaceutical Co., Ltd. (Seoul, Korea), rifaximin was provided by Alfa Wassermann S.p.A. (Alanno, Italy), and sucralfate was provided by Fuji Chemical Industry Co., Ltd. (Toyama, Japan).

This study protocol was approved by the Ethical Review Committee of our institute, and experimental procedures in this study adhered to the Declaration of Helsinki for the care and use of laboratory animals.

After removal of the intestines, the tissue samples were flushed briefly with ice-cold saline and placed on filter paper. The large and small intestines were prepared, weighed, and snap-frozen in liquid nitrogen. Samples were stored at –80°C until analysis.

Irradiation was performed between 12:00 pm and 1:00 pm. Thirty-six mice were anesthetized with 7.5 mg ketamine hydrochloride (Yuhan Corporation, Seoul, Korea) and 2.5 mg xylazine hydrochloride (Bayer Korea Ltd., Seoul, Korea) per 100 g body weight by intraperitoneal injection. Sham-irradiated mice were also anesthetized under the same conditions. Three mice at a time were fixed in a supine position in the center of a specially designed, box-shaped, acrylic phantom containing two inner jelly-filled-bags to ensure density homogeneity (Fig. 1). A 6 MV photon beam from a Linac system (ClinaciX; Varian Medical Systems, Inc., Palo Alto, CA, USA) was delivered in the anterior to the posterior and the posterior to the anterior directions of the entire phantom after treatment planning to deliver a 4-Gy dose of radiation to the abdomen and pelvic region containing the large and small intestines in a uniform manner (Eclipse 8.1; Varian Medical Systems, Inc.).

The nitrite concentrations in the IR-treated mice were measured as an indicator of nitric oxide (NO) production using the Griess reagent system. A nitrite standard curve was made by making serial dilutions of nitrite solution with phosphate buffered saline (PBS) buffer. Fifty microliters of supernatant were added to wells and mixed with 50 μL of sulfanilamide solution, followed by a 5- to 10-minute incubation at room temperature in the dark. The same volume of NED (Merck KGaA) solution was added to all wells, followed by another 5 to 10 minutes incubation at room temperature in the dark. The absorbance of the mixture was measured at 540 nm with a microplate plate reader (Molecular Devices, Sunnyvale, CA, USA).

The amounts of TNF-α, IL-1β, IL-6, PGE₂, and MCP-1 were measured using the appropriate ELISA kits according to the manufacturer’s instructions for each kit.

Proteins were isolated from whole mice intestines using lysis buffer (PBS containing 0.05% Triton X-100, 0.15 M NaCl, 2 mM EDTA, 1 mM phenylmethylsulfonylfluoride, and 20 μg aprotinin/mL) for immunoblotting. Protein concentrations were determined using a commercial protein assay kit (Bio-Rad). Equal protein concentrations were subjected to 7.5%–12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes (Amersham Pharmacia Biotech). After blocking the membranes with PBS containing 5% non-fat dry milk for 1 hour at room temperature, each membrane was incubated with a specific primary antibody overnight at 4℃. After washing twice with PBS containing 0.1% Tween-20 (PBST), each membrane was immunoblotted with horseradish peroxidase-conjugated anti-mouse, anti-rabbit, or anti-goat IgG antibodies (Santa Cruz Biotechnology) for 1 hour at room temperature, followed by washing three times in PBST and visualization by enhanced ECL (Amersham Pharmacia Biotech). Vision Works Image Software (UVP, Cambridge, UK) was used to measure band intensities.

Nuclear proteins were extracted with slight modifications of the protocol described by Dignam et al. [11]. A portion of the large and small intestines was homogenized in buffer containing 0.6% IGEPAL, 0.15 M NaCl, 10 mM Tris-HCl (pH 7.9), 1 mM EDTA, and 0.1% protein inhibitor cocktail; vortexed; kept on ice for 5 minutes; and centrifuged at 500 ×g for 5 minutes at 4℃. Pelleted nuclei were resuspended in 60 mL of extraction buffer (10 mM Hepes [pH 7.9], 0.1 mM EDTA, 1.5 mM MgCl₂, 420 mM NaCl, 25% glycerol, 1 mM dithiothreitol, and 0.33% protein inhibitor cocktail). Following gentle mixing and incubation on ice for 20 minutes, samples were centrifuged at 500 ×g for 5 minutes at 4℃. The supernatant was transferred to new tubes and stored at –70℃. The protein concentrations of the samples were determined using a commercial protein assay kit (Bio-Rad). For the electrophoretic mobility shift assay (EMSA), we end-labeled NF-κB-specific oligonucleotide with [γ-³²P]-ATP using T₄ polynucleotide kinase (Promega) and purified using a microspinG-25 column (Amersham Inc., Piscataway, NJ, USA). An EMSA was performed according to the accompanying instruction manual provided by Promega. Ten mg of nuclear protein and 2 to 4 mL of binding buffer were mixed and incubated for 10 minutes once. Then 1 mL of ³²P-labeled NF-κB and 2 mL of loading buffer were added to the nuclear protein mixtures, followed by a 30-minute incubation at room temperature. DNA-protein complexes were separated from the unbound DNA probe by electrophoresis through 4% polyacrylamide gels using 0.5X Tris-borate-EDTA buffer (pH 8.0) as the running buffer. The gels were dried and exposed to an X-ray film for 2 hours at room temperature, and the bands were quantitated using Vision Works Image Software (UVP).

All experiments were performed at least three times by conducting each assay in triplicate. Data were analyzed using SPSS version 20.0 for Windows (IBM Corp., Armonk, NY, USA), and are expressed as the mean±standard deviation. The significance of differences between groups was evaluated using analysis of variance (Tukey test), and p<0.05 was considered to be statistically significant.

We investigated the patterns of expression of nicotinamide phosphoribosyltransferase (NAMPT) in the large and small intestines of mice in response to IR treatment and rebamipide, sucralfate, or rifaximin administration. As shown in Fig. 2, protein expression of NAMPT was decreased after IR treatment compared to that of control mice, whereas the administration of rebamipide, sucralfate, and rifaximin attenuated the IR-induced decrease in NAMPT expression (all p<0.05).

Next, we evaluated how down- and upregulation of NAMPT by IR treatment and administration of rebamipide, sucralfate, and rifaximin, affected silent information regulator factor 2-related enzyme (Sirt) 1 (Fig. 3), poly-ADP-ribose polymerase (PARP)-1 (Fig. 4), and peroxisome proliferator-activated receptor (PPAR)γ (Fig. 5) expression in the large and small intestines of mice. Downregulation of NAMPT by IR treatment caused a significant decrease in Sirt1 expression and an increase in PARP-1 expression; however, the relative increase in NAMPT levels induced by the administration of rebamipide, sucralfate, and rifaximin significantly increased Sirt1 expression and decreased PARP-1 expression compared to IR treatment only (p<0.05). These results indicate that NAMPT plays an important role in the upregulation of Sirt1 and the downregulation of PARP-1.

Because downregulation of Sirt1 and upregulation of PARP-1 by IR induced a proinflammatory response, we examined how IR treatment and administration of rebamipide, sucralfate, or rifaximin affected the activation of NF-κB and phosphorylation of mitogen-activated protein kinases (MAPKs), which are key signaling mediators of inflammation in the large and small intestines. Compared to the controls, activation of NF-κB (Fig. 6) and phosphorylation of MAPKs (Fig. 7) were significantly induced by IR treatment; in contrast, levels of these signaling molecules were effectively attenuated following the administration of rebamipide, sucralfate, or rifaximin (all p<0.05). Taken together, these results suggest that upregulation of Sirt1 and downregulation of PARP-1 following the administration of rebamipide, sucralfate, and rifaximin reduced the expression of key mediators of the inflammatory signaling pathway, potentially by suppressing the expression of inflammatory cytokines via inhibition of NF-κB and MAPK signaling in the large and small intestines.

To examine the effect of IR treatment and rebamipide, sucralfate, and rifaximin administration on NF-κB and MAPK signaling-mediated inflammation, we next examined the expression of the proinflammatory cytokines TNF-α, IL-1β, and IL-6; the chemokine MCP-1; the metabolic proteins iNOS, COX-2, and PGE₂; and the intercellular adhesion molecule 1 (ICAM-1) in the large and small intestines (Figs. 8-11). The production of TNF-α, IL-1β, and IL-6 was increased by IR treatment, while the administration of rebamipide, sucralfate, or rifaximin significantly attenuated their expression to levels similar to those of control mice (p<0.05) (Fig. 8). The pattern of expression of the chemokine MCP-1 was similar to that observed for the proinflammatory cytokines (Fig. 9). The expression and production of metabolic proteins iNOS, COX-2, and PGE₂ were significantly induced by IR treatment, while administration of rebamipide, sucralfate, or rifaximin markedly attenuated the IR-induced increase in expression of these proteins (p<0.05) (Figs. 10, 11). The expression of the adhesion molecule ICAM-1 followed a similar pattern (Fig. 12). Taken together, our results indicate that IR-stimulated activation of the NF-κB and MAPK signaling pathways plays a crucial role in inflammation, and downregulation of NF-κB and MAPK signaling by administration of rebamipide, sucralfate, and rifaximin may ameliorate IR-induced proinflammatory responses in the large and small intestines.

Because downregulation of the inflammatory response inhibits cell apoptosis, we hypothesized that the rebamipide, sucralfate, or rifaximin-induced downregulation of the inflammatory response would protect the large and small intestines from IR-induced apoptosis. Ca²⁺ is known to coordinate endoplasmic reticulum-mitochondrial interactions that regulate apoptosis. We examined the expression of proapoptotic and antiapoptotic genes in response to [Ca²⁺] oscillations in IR-treated large and small intestines. As shown in Fig. 13, [Ca²⁺] oscillations were significantly increased by IR treatment but attenuated by the administration of rebamipide, sucralfate, or rifaximin. Furthermore, expression of the proapoptotic genes Bax and c-Myc and the antiapoptotic genes Bcl-2 and Bcl-xL was potently suppressed and induced, respectively, by the administration of rebamipide, sucralfate, or rifaximin (Figs. 14, 15). These results suggest that downregulation of the inflammatory response by NAMPT positively regulates apoptosis in IR-treated large and small intestines.

Downregulation of NAMPT induces apoptosis, which is an energy-consuming process. To investigate the effect of NAMPT on IR-induced apoptosis, we evaluated the release of cytochrome C and the expression of caspase 3 and caspase 7. The release of cytochrome C was significantly increased by IR treatment, while it was markedly attenuated by the administration of rebamipide, sucralfate, or rifaximin to levels similar to those of control mice (p<0.05) (Fig. 16). In addition, expression of caspase 3 and caspase 7 was significantly elevated by IR treatment compared to that of the control group; however, administration of rebamipide, sucralfate, or rifaximin decreased the IR-induced increase in expression of these caspases (p<0.05) (Fig. 17). These results further demonstrate that NAMPT is an effective protectant against IR-stimulated large and small intestine damage and that the underlying mechanism of NAMPT-mediated protection involves downregulation of cytochrome C and the caspase cascade at the protein level.