Toxicology and Applied Pharmacology 329 (2017) 40–47

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Toxicology and Applied Pharmacology

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Rebamipide ameliorates radiation-induced intestinal injury in amouse model

Sehwan Shim a, Hyo-Sun Jang a, Hyun-Wook Myung a, Jae Kyung Myung a,b, Jin-Kyu Kang a, Min-Jung Kim a,Seung Bum Lee a, Won-Suk Jang a, Sun-Joo Lee a, Young-Woo Jin a, Seung-Sook Lee a,b, Sunhoo Park a,b,⁎a Laboratory of Radiation Exposure & Therapeutics, National Radiation Emergency Medical Center, Korea Institute of Radiological and Medical Sciences, Seoul, Republic of Koreab Department of Pathology, Korea Cancer Center Hospital, Korea Institute of Radiological and Medical Sciences, Seoul, Republic of Korea

⁎ Corresponding author at: Korea Institute of RadioNowon-ro, Nowon-gu, Seoul 01812, Republic of Korea.

E-mail address: sunhoo@kirams.re.kr (S. Park).

http://dx.doi.org/10.1016/j.taap.2017.05.0120041-008X/© 2017 Elsevier Inc. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 10 March 2017Revised 10 May 2017Accepted 15 May 2017Available online 16 May 2017

Radiation-induced enteritis is amajor side effect in cancer patients undergoing abdominopelvic radiotherapy. Ra-diation exposure produces an uncontrolled inflammatory cascade and epithelial cell loss leading to impaired ep-ithelial barrier function. The goal of this study was to determine the effect of rebamipide on regeneration of theintestinal epithelia after radiation injury. The abdomens of C57BL/6 mice were exposed to 13 Gy of irradiation(IR) and then the mice were treated with rebamipide. Upon IR, intestinal epithelia were destroyed structurallyat the microscopic level and bacterial translocation was increased. The intestinal damage reached a maximumlevel on day 6 post-IR and intestinal regeneration occurred thereafter. We found that rebamipide significantlyameliorated radiation-induced intestinal injury. In mice treated with rebamipide after IR, intestinal barrier func-tion recovered and expression of the tight junction components of the intestinal barrier were upregulated.Rebamipide administration reduced radiation-induced intestinal mucosal injury. The levels of proinflammatorycytokines and matrix metallopeptidase 9 (MMP9) were significantly reduced upon rebamipide administration.Intestinal cell proliferation and β-catenin expression also increased upon rebamipide administration. Thesedata demonstrate that rebamipide reverses impairment of the intestinal barrier by increasing intestinal cell pro-liferation and attenuating the inflammatory response by inhibitingMMP9and proinflammatory cytokine expres-sion in a murine model of radiation-induced enteritis.

© 2017 Elsevier Inc. All rights reserved.

Keywords:Barrier functionIntestineRadiotherapyRebamipide

1. Introduction

The treatment of malignant tumors with radiation therapy also af-fects surrounding healthy tissues (Haydont et al., 2007). The gastroin-testinal tract, especially the small intestine, is particularly sensitive toradiation, which renders it vulnerable to collateral radiation fromradiotherapeutic treatments for abdominal and pelvic cancers(Bachmann et al., 2015). Radiation-induced enteritis reduces patients'qualities of life and increases treatment and social health care costs(Fyles et al., 1992; Abayomi et al., 2009). Although many studies haveexamined the radioprotective effects of various agents, there are no ef-fective clinical treatments for radiation-induced intestinal injury. There-fore, the development of effective therapeutic treatments to improvethe outcomes of radiation-induced enteritis is urgently needed.

Radiation-induced gastrointestinal injury is described as destructionof crypt cells, a decrease in villous height and number, and impaired ep-ithelial barrier function (Atasoy et al., 2010). Impaired intestinal barrier

logical & Medical Sciences, 75

function has been observed during the early stages of radiation-inducedgastrointestinal injury (Touchefeu et al., 2014). The intestinal epithelialbarrier regulates the penetration of substances, such as macromole-cules, bacteria, and other intralumen toxins (Deitch, 1990). Bacterialpenetration potentiates the development of septicemia, which is oneof several causes of death following radiation exposure. Thus, the epi-thelial barrier of the small intestine has promise as a therapeutic target.

The intestinal epithelium is a single layer of columnar epithelial cellsthat separates the intestinal lumen from the underlying lamina propria.These epithelial cells are tightly bound together by intercellular junc-tional complexes that regulate paracellular permeability and that arecrucial for the integrity of the epithelial barrier (Ulluwishewa et al.,2011). Proinflammatory cytokines are major inducers of matrixmetallopeptidase 9 (MMP9) production in intestinal inflammationmodels (Gan et al., 2001; Sternlicht and Werb, 2001; Castaneda et al.,2005). MMP9 release during inflammation may lead to tight junctiondegradation and loss of mucosal integrity (Naito and Yoshikawa,2005; Kofla-Dlubacz and Iwanczak, 2010). Tight junctions, the majorcomponents of junctional complexes, seal paracellular spaces betweenepithelial cells and prevent paracellular diffusion of microorganisms andother antigens across the epithelium (Assimakopoulos et al., 2004; Garg

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et al., 2014; Shim et al., 2015). Therefore, during intestinal epithelial cellloss and inflammation, intestinal junctional complexes are destroyed(Dublineau et al., 2004; Prasad et al., 2005; Ulluwishewa et al., 2011;Wells et al., 2011; Miner-Williams and Moughan, 2016).

Rebamipide is a gastroprotective agent that is already in clinical usefor the treatment of gastric ulcers and gastritis (Han et al., 2015;Kamada et al., 2015). Rebamipide has been shown to suppress immuneresponses (Byun et al., 2014; Yamane et al., 2015) and regulate inflam-matory cell activation (Aihara et al., 1998; Nagano et al., 2001) in vari-ous inflammatory disease models. Numerous studies using anonsteroidal anti-inflammatory drug (NSAID)-induced intestinal in-flammationmodel have shown that rebamipide affects the release of in-flammatory cytokines and growth factors (Lai et al., 2015; Watanabe etal., 2015). Because abnormal inflammatory responses lead to impairedepithelial barrier function in radiation-induced enteritis, these findingssuggest that rebamipide may be a promising therapeutic agent for radi-ation-induced enteritis. The aim of this research was to investigate theeffect of rebamipide on intestinal barrier function in a mouse model ofradiation-induced enteritis.

2. Materials and methods

2.1. Animals

Male C57BL/6 mice aged 7 weeks were obtained from Harlan Labo-ratories (IN, USA). The mice were kept under controlled conditions, in-cluding constant temperature, and were allowed free access to regularchow and 3-stage filtered water. The Animal Investigation Committeeof the Korea Institute of Radiological and Medical Sciences approvedall of the animal experiments.

2.2. Irradiation and administration of rebamipide

Animals were anesthetized by intraperitoneal injection of 85 mg/kgof alfaxalone (Alfaxan®; Careside, Republic of Korea) and 10 mg/kg ofxylazine (Rompun®, Bayer Korea, Republic of Korea) and underwentwhole abdominal irradiation with 13 Gy of radiation at a dose rate of2 Gy/min using an X-RAD 320 X-ray irradiator (Softex, Republic ofKorea). The irradiated dose and dose rate were measured with anUNIDOS® E dosemeter (PTW-FREIBURG, Germany). After radiation ex-posure, animals were treated orally with 200 mg/kg/day (IR + Rb200)or 400 mg/kg/day (IR + Rb400) of rebamipide (Mucosta®, Otsuka, Re-public of Korea) for the duration of the experiment.

2.3. Bacterial translocation assay

The presence of viable bacteria in mesenteric lymph nodes (MLNs),which were harvested under sterile conditions, represented bacterialtranslocation from the lumen of the intestine. Equal aliquots of homog-enates were plated onto MacConkey agar (Becton Dickson, FranklinLakes, NJ), were incubated at 37 °C for 24 h, and the numbers of colonieson the plates were counted.

2.4. Intestinal permeability assay

Intestinal permeability to 4 kDa FITC-dextran (Sigma-Aldrich, St.Louis, MO)wasmeasured 6 days post-IR (n=5animals per group). An-imals were anesthetized with alfaxalone and xylazine, a midline lapa-rotomy incision was made, and the small intestine was exposed. A5 cm segment of distal small intestine was isolated between bulldogclamps and was injected with 12.5 mg of FITC-dextran that had beendissolved in 100 μl of phosphate buffered saline (PBS). Animals werekept under anesthesia for 30 min, and then blood was obtained via car-diac puncture. Blood samples were placed into serum separating tubesand centrifuged at 1000g for 15 min to obtain sera. FITC-dextran con-centrations were measured using a fluorescence spectrophotometer

(BioTek microplate reader) at excitation and emission wavelengths of495 nm and 520 nm, respectively.

2.5. Histological examination of the intestine

Terminal ileum samples were fixed with a 10% formalin solution,embedded in paraffin wax, and sectioned at a thickness of 4 μm. Sec-tions were stained with hematoxylin and eosin (H&E). For immunohis-tochemical analysis, sectionswere treatedwith 0.3% hydrogen peroxidein methyl alcohol for 20 min to block endogenous peroxidase activity.After three washes with PBS, sections were blocked with 10% normalgoat serum (Vector ABC Elite Kit, Vector Laboratories, Burlingame, CA)and incubated with antibodies to claudin-3 (Invitrogen, Carlsbad, CA),occludin (Santa Cruz, CA), β-catenin (BD Bioscience, NJ), and Ki-67(Acris Antibodies, Germany). After three subsequent washes with PBS,sections were incubated with horseradish peroxidase (HRP)-conjugat-ed secondary antibody (Dako, Carpinteria, CA). Detection was per-formed using a diaminobenzidine substrate (Dako) according to themanufacturer's instructions. Quantitative assessment of immunoreac-tivity was performed with i-solution software.

2.6. RNA extraction, reverse transcription, and real-time PCR quantification

Total RNA was extracted from small intestines using TRIzol Reagent(Invitrogen). Total RNA samples (1 μg) were reverse transcribed intocDNA using oligo (dT) 18 primers and Accu-Power RT PreMix (Bioneer,Daejon, Korea). SYBR Green I Master Mix (Roche Diagnostics, Mann-heim, Germany) was used for real-time RT-PCR reactions. The primerssequences were as follows: IL-1β sense 5′-GCAACTGTTCCTGAACTCA-3′ and antisense 5′-CTCGGAGCCTGTAGTGCAG-3′, IL-6 sense 5′-CTTGGGACTGATGCTGGTGA-3′ and antisense 5′-TGCAAGTGCATCATCGTTGT-3′, IL-4 sense 5′-CGGCATTTTGAACGAGGTC-3′ and antisense 5′-GAAAAGCCCGAAAGAGTCTC-3′, TNF-α sense 5′-GCCTCTTCTCATTCCTGCTT-3′ and antisense 5′-CACTTGGTGGTTTGCTACGA-3′, TGF-β1 sense 5′-CCCTATATTTGGAGCCTGGA-3′ and antisense 5′-CTTGCGACCCACGTAGTAGA-3′, MMP9 sense 5′-GCCCTGGAACTCACACGACA-3′ and antisense 5′-TTGGAAACTCACACGCCAGAAG-3′, Wnt3A sense 5′-TTCTTACTTGAGGGCGGAGA-3′ and antisense 5′-ACCCGTATCCCAGACAGGA-3′, β-catenin sense 5′-ACTGCTGGGACTCTG-3′ and antisense 5′-TGATGGCGTAGAACAG-3′,LIG4 sense 5′-TTAGTTGTATCCGCACGCT-3′ and antisense 5′-TGTGAGGAAGCCATAGAAGC-3′, Claudin-3 sense 5′-AAGCCGAATGGACAAAGAA-3′ and antisense 5′-CTGGCAAGTAGCTGCAGTG-3′, Claudin-4 sense 5′-CGCTACTCTTGCCATTACG-3′ and antisense 5′-ACTCAGCACACCATGACTTG-3′, ZO-1 sense 5′-AGGACACCAAAGCATGTGAG-3′ and antisense 5′-GGCATTCCTGCTGGTTACA-3′, Occludin sense 5′-GCTGTGATGTGTGTGAGCTG-3′ and antisense 5′-GACGGTCTACCTGGAGGAAC-3′, c-myc sense 5′-ACACGGAGGAAAACGACAAG-3′ and antisense 5′-AGAGGTGAGCTTGTGCTCGT-3′, and β-actin sense 5′-TCCCTGGAGAAGAGCTATGA-3′ and antisense 5′-CGATAAAGGAAGGCTGGAA-3′. All of the reactions were performed induplicate with 2 μl of cDNA on a LightCycler 480 II Real-time PCR plat-form (Roche). Relative mRNA levels were determined using the 2 -ΔΔCt

method with β-actin as the internal control. +1.5 fold cut off is consid-ered for upregulation in mRNA expression levels.

2.7. Enzyme-linked immunosorbent assay

After general anesthesia, blood samples were collected from the in-ferior vena cava, placed in heparin tubes, and centrifuged at 2000g for

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20 min at 4 °C. Plasma supernatants were collected and stored at −80°C. Plasma diamine oxidase (DAO) concentrations were quantified bysandwich enzyme immunoassay using the Quantikine ELISA kit(Cusabio, MD, USA) according to the manufacturer's instructions. Ab-sorbance at 450 nm was measured using a microplate reader (BioTekmicroplate reader).

2.8. Statistical analysis

All of the data were expressed asmean±SD, statistical significanceswere evaluated by Student's t-test, and p values b 0.05 were consideredstatistically significant.

3. Results

3.1. Enteritis was induced by exposing mice abdomens to radiation

Histopathological changes and bacterial translocation were exam-ined in MLNs on days 4, 6, and 10 after IR (Fig. 1). Epithelial loss, short-ening of villi, and fewer crypts were observed in the small intestine onday 4, and these changes were at their maximum on day 6 (Fig. 1A).The irradiated group (IR) presented elevated bacterial translocation onday 4 when compared with the control group, and the maximum levelof bacterial translocation was seen on day 6 (p b 0.05, Fig. 1B). Thecrypt-villi structure and the intestinal barrier were regenerated by day10post-IR. These data indicate that irradiation damaged the small intes-tinal mucosal barrier, which resulted in loss of intestinal epithelium in-tegrity. In further experiments, we analyzed the effects of rebamipideon radiation-induced intestinal injury on day 6 post-IR.

3.2. Rebamipide ameliorated radiation-induced intestinal injury

To examine the putative role of rebamipide in protection from radi-ation-induced intestinal injury, we compared histologically the smallintestines of irradiated mice with and without rebamipide treatment.

Fig. 1. Time course of radiation-induced enteritis. C57BL/6 mice were exposedabdominally to 13 Gy of IR. (A) H&E staining of the small intestine on day 4, 6, and 10post-IR. (B) MLNs collected 4, 6, and 10 days after IR were analyzed for bacterialtranslocation (5 mice per condition). The bars represent mean values and the error barsshow standard deviations. *p b 0.05 versus control group.

Fig. 2. Effects of rebamipide on radiation-induced structural damage in the intestine.Tissues from the small intestines of C57BL/6 mice exposed abdominally to 13 Gy of IRand treated with saline or rebamipide (Rb) were collected on day 6 post-IR (5 mice percondition) and H&E stained (A) for villus height (B) and crypt number (C). The barsrepresent mean values and the error bars show standard deviations. *p b 0.05 versuscontrol group; +p b 0.05 versus IR group.

On day 6 post-IR, mucosal structural damage in the small intestine of ir-radiated mice was reversed upon treatment with rebamipide (Fig. 2A).The heights of villi and numbers of crypts per mm were measured onday 6 post-IR. The heights of villi in the IR group were lower than theheights of villi in the control group. However, treatment withrebamipide after irradiation attenuated the reduction in the heights ofthe villi (control: 324.2 ± 27.6 μm; IR: 216.9 ± 50.4 μm; IR + Rb200:300.4± 59.3 μm; IR+Rb400: 324.8± 45.8 μm; p b 0.05; Fig. 2B). In ad-dition, the number of crypts in the rebamipide treatment groups (IR+ Rb200: 8.8 ± 2.0, IR + Rb400: 9.8 ± 3.7) were greater than in theIR group (3.9 ± 1.2; P b 0.05; Fig. 2C).

3.3. Rebamipide reversed radiation-induced damage to the intestinalbarrier

Next, to determinewhether rebamipide treatment after IR improvedepithelial barrier function, we assessed intestinal barrier function onday 6 post-IR using bacterial translocation and FITC-dextran absorptionassays. In the bacterial translocation assay, bacterial colony counts inMLNs from the IR group were higher than in the control group (p b

0.05, Fig. 3A). However, bacterial colony counts reduced significantly

Fig. 3. Effects of rebamipide on radiation-induced impairment of the intestinal barrier. Samples from C57BL/6 mice exposed abdominally to 13 Gy of IR and treated with saline orrebamipide (Rb) were collected on day 6 days post-IR (5 mice per condition). (A) MLNs were collected and evaluated for bacterial translocation. (B) Sera were collected and evaluatedfor FITC concentration. The bars represent mean values and the error bars show standard deviations. *p b 0.05 versus control group; +p b 0.05 versus IR group; ++p b 0.05 versus IR+ Rb200 group.

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and in a dose-dependent manner with rebamipide treatment. In addi-tion, the concentration of FITC-dextran in sera was lower in therebamipide treatment group than in the IR group (p b 0.05, Fig. 3B).

Because paracellular diffusion of microorganisms across the epithe-lium is prevented by tight junctions that seal the paracellular spaces be-tween epithelial cells, we analyzed the mRNA levels of tight junctionalmolecules in the small intestine on day 6 post-IR (Fig. 4A–D). mRNA

Fig. 4. Effects of rebamipide on intestinal junctional complexes. Tissues from the small intestines(Rb) were collected on day 6 post-IR (5mice per condition). Total RNA was isolated and quantioccludin (C), and zona occuldens-1 (ZO-1) (D). The bars representmean values and the error baF) Immunohistochemical staining for claudin-3 (E) and occludin (F) protein expression on day

levels of claudin-3, claudin-4, and occludin, which aremolecules withinparacellular junctional complexes in intestinal epithelial cells (Lee,2015), were lower in the IR group than in the control group (p b 0.05,Fig. 4A–C). However, the level of ZO-1, intracellular junction complexesof intestinal epithelial cells, was not significantly changed among theexperimental groups (Fig. 4D). The claudin-3 and occludinmRNA levelswere higher in the rebamipide treatment groups than in the IR group (p

of C57BL/6mice exposed abdominally to 13Gy of IR and treatedwith saline or rebamipidetative real-time PCR was performed to measure expression of claudin-3 (A), claudin-4 (B),rs show standard deviations. *p b 0.05 versus control group; +p b 0.05 versus IR group. (E,6 post-IR. Nuclei, purple; labeled cells, brown.

44 S. Shim et al. / Toxicology and Applied Pharmacology 329 (2017) 40–47

b 0.05, Fig. 4A, C). Furthermore, claudin-3 and occludin immunostainingwas performed to examine protein expression of these intestinal barriermolecules in the small intestine. Claudin-3 and occludin expressionwere clearly observed throughout the membranes of epithelial cellswith surface villi (Fig. 4E, F). Thus, IR reduced expression of junctionalproteins in epithelial cells with villi, and this reduced expressionwas at-tenuated with rebamipide treatment. These data suggest thatrebamipide treatment reverses intestinal barrier dysfunction by accel-erating synthesis of tight junctionalmolecules during radiation-inducedenteritis.

3.4. Rebamipide suppressed MMP-9 expression in the irradiated smallintestine

MMP-9, which is released from intestinal epithelial cells in responseto proinflammatory cytokines (Naito and Yoshikawa, 2005;Kofla-Dlubacz and Iwanczak, 2010), degrades junctional molecules,and leads to loss of mucosal integrity. Because rebamipide has beenshown to function as an anti-inflammatory agent in various intestinalinflammation models (Aihara et al., 1998; Byun et al., 2014; Han et al.,2015; Kamada et al., 2015; Lai et al., 2015), we analyzed the effect ofrebamipide on MMP9 expression in our abdominally irradiated mousemodel (Fig. 5).

We measured mRNA levels of the proinflammatory cytokines TNF-α, IL-6, IL-1β, TGF-β1, and IL-4 in tissues isolated from the small intes-tines of our experimental groups (Fig. 5A-E). The mRNA levels of the

Fig. 5. Effects of rebamipide on proinflammatory cytokines. Tissues from the small intestines o(Rb) treatment were collected on day 6 post-IR (5mice per condition). Total RNAwas isolated aTNF-α (C), TGF-β1 (D), IL-4 (E), and MMP9 (F). The bars represent mean values, and the error

proinflammatory cytokines were higher in the IR group than in the con-trol group. This irradiation-induced increase in proinflammatory cyto-kine mRNA expression was reversed in mice treated with rebamipide.Moreover, MMP-9 expression, which has been shown to be involvedin the inflammatory response to irradiation (Hosgorler et al., 2016),was suppressed in the small intestines of the groups treated withrebamipide (Fig. 5F). These data suggest that rebamipide suppressesMMP9 expression during radiation-induced intestinal injury byinhibiting the production of proinflammatory cytokines.

3.5. Rebamipide improved proliferation of intestinal epithelial cells in the ir-radiated small intestine

Immunostaining of Ki-67, a cellularmarker of proliferation,was per-formed to assess whether rebamipide induces proliferation of intestinalcells in the irradiated intestine (Fig. 6A). We counted 40.88 ± 5.66 Ki-67-positive cells per field (1 × 104 μm2) in control mice, whereas wecounted only 21.04 ± 3.49 Ki-67-positive cells per field in irradiatedmice. We counted 30.49 ± 6.45 Ki-67-positive cells per field in the200 mg/kg/day rebamipide-treated group (IR + Rb200) and 36.99 ±7.56 Ki67-positive cells per field in the 400 mg/kg/day rebamipide-treated group (IR + Rb400; p b 0.05; Fig. 6B).

Because high concentrations of DAO have been found in epithelialcells of the small intestine (Bieganski et al., 1983) and because plasmaDAO concentrations have been shown to decrease upon IR-induced in-testinal injury (Ely et al., 1985; DeBell et al., 1987), DAO has been

f C57BL/6 mice exposed abdominally to 13 Gy of IR and treated with saline or rebamipidend quantitative real-time PCr was performed tomeasure expression of IL-1β (A), IL-6 (B),bars show standard deviations. *p b 0.05 versus control group; +p b 0.05 versus IR group.

Fig. 6. Effects of rebamipide on intestinal cell proliferation. Samples from C57BL/6 miceexposed abdominally to 13 Gy of IR and treated with saline or rebamipide (Rb) werecollected on day 6 post-IR (5 mice per condition). (A) Small intestines were Ki-67immunostained and evaluated for the number of immunoreactive cells per crypt. (B)Plasma DAO concentrations were measured. The bars represent mean values and theerror bars show standard deviations. *p b 0.05 versus control group; +p b 0.05 versus IRgroup.

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suggested as a candidate marker to measure the integrity of the intesti-nal epithelium (Fukudome et al., 2014). The normal concentration ofplasma DAO is 62.57 ± 9.27 unit/L, and the plasma DAO concentrationin the IR group was reduced to 15.05 ± 6.79 unit/L (p b 0.05). Uponrebamipide treatment, the plasma DAO concentrations increased to29.83 ± 5.06 unit/L in the IR + Rb200 group and 32.15 ± 5.90 unit/Lin the IR + Rb400 group on day 6 post-IR (p b 0.05, Fig. 6C). Thesedata indicate that rebamipide administration after radiation-inducedintestinal injury promotes proliferation of intestinal epithelial cells.

3.6. Rebamipide upregulated Wnt/β-catenin signaling in the irradiatedsmall intestine

To investigate whether rebamipide treatment affects the signalingpathway for intestinal regeneration, mRNA levels of Wnt3A and β-ca-tenin in small intestines were measured and found to be reduced in re-sponse to IR, whereas upon rebamipide treatment, Wnt3A and β-catenin mRNA levels were significantly upregulated when comparedwith the IR group on day 6 post-IR (Fig. 7A, B). In addition, β-cateninprotein expression in the small intestine was evaluated by

immunohistochemistry andwas clearly observed throughout themem-branes of intestinal epithelial cells (Fig. 7E). In the IR group, expressionof β-catenin in the villi and crypts of intestinal epithelial cells were re-duced, and this IR-induced reduction ofβ-catenin expressionwas atten-uated upon rebamipide administration.

It has recently been demonstrated that LIG4 and c-myc are upregu-lated by Wnt/β-catenin signaling in irradiated intestinal epithelial cells(Bettess et al., 2005; Jun et al., 2016), thus we also analyzed expressionof LIG4 and c-myc in the small intestines of our experimental groups(Fig. 7C, D). The mRNA levels of LIG4 and c-myc in the IR group werelower than in the control group (p b 0.05, Fig. 7A–C). In therebamipide-treated groups, the LIG4 and c-myc mRNA levels were sig-nificantly higher than in the IR group. These data suggest thatrebamipide activates the Wnt/β-catenin signaling pathway, whichfunctions in intestinal regeneration.

4. Discussion

Enteritis is the most common side effect of radiation therapy for thetreatment of abdominal cancer, and the susceptibility of the small intes-tine to radiation-induced damage is the limiting factorwhen determiningthe prescription dose of radiation therapy. However, to our knowledge,there are no widely used methods to reduce the severity of radiation-in-duced enteritis. Herein, we provide evidence that treatment withrebamipide after IR protects against radiation-induced intestinal injury.

Administration of rebamipide showed structural recovery of thesmall intestine in irradiated mice. In addition, treatment withrebamipide reduced bacterial translocation across the intestinal epithe-lium and restored expression of junctional complexes in the intestineindicating that rebamipide exerts beneficial effects on intestinal barrierfunction inmice. These beneficial effects can be explained by twomech-anisms: (1) suppression of inflammatory cytokine production andMMP9 expression in the small intestine and (2) an increase in intestinalepithelial cell proliferation.

In this study, themaximum impairment of the intestinal barrier wasreached onday 6 post-IR. In the rebamipide treatment groups, the struc-tural damage induced by irradiation, such as reduced heights of smallintestinal villi and the number of intestinal crypts, were reversed.These results show that rebamipide attenuates radiation-inducedbowel damage.

Impairment of the intestinal barrier leads to activation of the immuneresponse and a loss of solutes,which leads to leak-flux diarrhea (Sandle etal., 1990; Podolsky, 2002), thuswe investigated intestinal barrier functioninmice treatedwith rebamipide. Irradiation significantly increased bacte-rial translocation in MLNs and increased FITC-dextran concentrations insera. In contrast, rebamipide administration after irradiation significantlyreduced bacterial translocation and the serum concentration of FITC-dex-tran. Moreover, expression of intestinal junctional complex molecules,such as occludin and claudin-3, significantly increased after rebamipidetreatment, which demonstrates that rebamipide restores the damagedstructures in intestinal barriers thatwere inducedby irradiation. These re-sults indicate that rebamipide improves barrier function by upregulatingsynthesis of intracellular junctional molecules.

Because inflammation that occurs after radiation may result in vari-ous symptoms, including impairment of the intestinal barrier, reducinginflammation is generally beneficial. We observed that inflammation inthe small intestines of mice in the rebamipide treatment group wasmilder that in the small intestines of mice in the IR group. We alsoshowed that mRNA levels of the proinflammatory cytokines TNF-α, IL-1β, and IL-6, aswell asMMP-9,were higher in the irradiated small intes-tine. Further, MMP-9 is the most abundantly expressed protease in in-jured intestinal tissues (Tarlton et al., 2000; Castaneda et al., 2005).MMP-9 is released from intestinal epithelial cells in response to proin-flammatory cytokines and is responsible for the loss of mucosal integri-ty (Naito and Yoshikawa, 2005). Among proinflammatory cytokines,TNF-α and IL-1β are the primary inducers of MMP protein production

Fig. 7. Effects of rebamipide onWnt/β-catenin signaling. Tissues from the small intestines of C57BL/6mice exposed abdominally to 13 Gy of IR and treatedwith saline or rebamipide (Rb)were collected on day 6 post-IR (5 mice per condition). Total RNA was isolated and quantitative real-time PCR was performed to measure expression of Wnt-3A (A), β-catenin (B), LIG4(C), and c-myc (D). The bars represent mean values and the error bars show standard deviations. *p b 0.05 versus control group; +p b 0.05 versus IR group. (E) Immunohistochemicalstaining for β-catenin protein expression. Nuclei, purple; labeled cells, brown.

46 S. Shim et al. / Toxicology and Applied Pharmacology 329 (2017) 40–47

(Gan et al., 2001; Sternlicht and Werb, 2001; Nee et al., 2004). ThemRNA levels of TNF-α, IL-1β, and MMP9 were significantly reduced inthe rebamipide treatment group. These results suggest that rebamipideadministration after radiation-induced enteritis alleviates inflammationand improves mucosal integrity in the small intestine.

Because recovery frommucosal injury relies on cellular proliferation,we evaluated proliferation of intestinal epithelial cells in mice treatedwith rebamipide. Cellular proliferation wasmeasured by Ki-67 labeling.Ki-67 labeling analysis showed a reduction in the proliferation of smallintestinal cells after irradiation, and proliferation was upregulated afterrebamipide administration. Although the amount of proliferation didnot vary significantly between the two groups treated with differentrebamipide dosages, both groups showed significantly increased cellproliferation compared with the IR group. The plasma DAO level is acandidate marker for measuring radiation-induced epithelial cell dam-age (Ely et al., 1985; DeBell et al., 1987). The DAO plasma level in themouse intestine decreased after IR and recovered to a normal levelafter rebamipide administration indicating that intestinal injury was re-duced and the integrity of the intestinal epithelium improved in micetreated with rebamipide.

Wnt/β-catenin signaling regulates proliferation of small intestinalstem cells. Loss of β-catenin leads to a rapid loss of intestinal epithelialcells beginning with crypt loss, inhibition of cellular proliferation, andan increase in enterocytic differentiation. In this study,Wnt3Aandβ-ca-tenin mRNA levels and intestinal cell proliferation decreased after IR,and rebamipide administration reversed these reductions in gene

expression and cell proliferation. We also investigated expression ofLIG4 and c-myc, which are signaling targets of Wnt/β-catenin. Recentstudies have demonstrated that Wnt/β-catenin signaling upregulatesLIG4, a DNA repair gene, in irradiated intestinal epithelial cells (Jun etal., 2016) and that c-myc is associated with proliferation and formationof intestinal crypts (Pinto et al., 2003; Bettess et al., 2005). Expression ofLIG4 and c-myc were reduced after IR, and this reduction was reversedupon treatmentwith rebamipide. These results suggest that rebamipideactivatesβ-catenin signaling to promote intestinal cell proliferation andregeneration.

In conclusion, rebamipidemay ameliorate inflammation in the smallintestine and may improve the structure of junctional complexes be-tween small intestinal epithelial cells during radiation-induced intesti-nal injury. Rebamipide administration may also increase cellproliferation and regeneration of the intestinal epithelium by activatingWnt/β-catenin signaling. Although a higher dose of rebamipide did notshow an equivalent increase in benefits, rebamipide has been shown tobe safer than conventional drugs, including NSAIDs, immunosuppres-sants, and TNF-α blockers (Zhang et al., 2013). Furthermore,rebamipide has recently been shown to suppress invasion of cancercells (Kang et al., 2013). Therefore, rebamipide is a promising therapeu-tic for patients with radiation-induced enteritis.

Disclosure statement

The authors have no competing interests.

47S. Shim et al. / Toxicology and Applied Pharmacology 329 (2017) 40–47

Financial support

This study was supported by a grant of the Korea Institute of Radio-logical and Medical Sciences (KIRAMS), funded by Ministry of Science,ICT and Future Planning, Republic of Korea (1711045573/50535-2017).

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