Rebamipide ameliorates indomethacin-induced small intestinal damage and proton pump inhibitor-induced exacerbation of this damage by modulation of small intestinal microbiota

Tetsuya Tanigawa; Toshio Watanabe; Akira Higashimori; Sunao Shimada; Hiroyuki Kitamura; Takuya Kuzumoto; Yuji Nadatani; Koji Otani; Shusei Fukunaga; Shuhei Hosomi; Fumio Tanaka; Noriko Kamata; Yasuaki Nagami; Koichi Taira; Masatsugu Shiba; Wataru Suda; Masahira Hattori; Yasuhiro Fujiwara

doi:10.1371/journal.pone.0245995

. 2021 Jan 28;16(1):e0245995. doi: 10.1371/journal.pone.0245995

Rebamipide ameliorates indomethacin-induced small intestinal damage and proton pump inhibitor-induced exacerbation of this damage by modulation of small intestinal microbiota

Tetsuya Tanigawa ^1,^2,^*, Toshio Watanabe ¹, Akira Higashimori ¹, Sunao Shimada ^1,², Hiroyuki Kitamura ¹, Takuya Kuzumoto ¹, Yuji Nadatani ¹, Koji Otani ¹, Shusei Fukunaga ¹, Shuhei Hosomi ¹, Fumio Tanaka ¹, Noriko Kamata ¹, Yasuaki Nagami ¹, Koichi Taira ¹, Masatsugu Shiba ¹, Wataru Suda ³, Masahira Hattori ³, Yasuhiro Fujiwara ¹

Editor: Jan S Suchodolski⁴

PMCID: PMC7842908 PMID: 33507971

Abstract

Non-steroidal anti-inflammatory drugs (NSAIDs) induce small intestinal damage. It has been reported that rebamipide, a mucoprotective drug, exerts a protective effect against NSAID-induced small intestinal damage; however, the underlying mechanism remains unknown. In this study, we investigated the significance of the small intestinal microbiota in the protective effect of rebamipide against indomethacin-induced small intestinal damage in mice. A comprehensive analysis of the 16S rRNA gene sequencing revealed an alteration in the composition of the small intestinal microbiota at the species level, modulated by the administration of rebamipide and omeprazole. The transplantation of the small intestinal microbiota of the mice treated with rebamipide suppressed the indomethacin-induced small intestinal damage. Omeprazole, a proton pump inhibitor, exacerbated the indomethacin-induced small intestinal damage, which was accompanied by the alteration of the small intestinal microbiota. We found that the transplantation of the small intestinal microbiota of the rebamipide-treated mice ameliorated indomethacin-induced small intestinal damage and the omeprazole-induced exacerbation of the damage. These results suggest that rebamipide exerts a protective effect against NSAID-induced small intestinal damage via the modulation of the small intestinal microbiota, and that its ameliorating effect extends also to the exacerbation of NSAID-induced small intestinal damage by proton pump inhibitors.

Introduction

The emergence of video capsule endoscopy and balloon-assisted enteroscopy have enabled the precise diagnosis of diseases involved in small intestinal damage, and the clinical features of nonsteroidal anti-inflammatory drug (NSAID)-induced small intestinal damage have been clarified [1–6]. NSAIDs frequently induce small intestinal damage, which cause overt and occult bleeding, perforation, and stenosis of the small intestine [2, 7]. Our previous clinical study revealed that 25% of the chronic NSAID users from among the patients with rheumatoid arthritis had mild damage; more importantly, 27.8% had severe damage and significantly decreased hemoglobin levels [8].

Accumulating clinical evidence suggests that proton pump inhibitors (PPIs) exacerbate NSAID-induced small intestinal damage. PPIs are frequently prescribed with NSAIDs for prophylactic therapy against NSAID-induced upper gastroduodenal ulcers and bleeding [9]. However, our previous study has suggested that the use of PPIs was an independent risk factor for severe NSAID-induced small intestinal damage in patients with rheumatoid arthritis who were chronic NSAID users [8]. Endo and his colleagues also showed that PPIs were risk factors for low-dose aspirin-induced small intestinal damage [10]. A recent randomized, placebo-controlled trial in healthy volunteers revealed that rabeprazole increased the incidence and number of small intestinal damage induced by celecoxib [11]. Basic research has also demonstrated the deleterious effect of PPIs on NSAID-induced small intestinal damage [12–14].

The induction of dysbiosis in the small intestine by PPI is proposed as a possible mechanism by which PPI exacerbates NSAID-induced small intestinal damage. Dysbiosis is an abnormality in the composition of the microbial community in which the population of bacteria beneficial for the host health decreases and the population of pathogenic bacteria, typically present in small numbers, increases [15]. Wallace and his colleagues demonstrated, in basic experimental research, that PPI exacerbate NSAID-induced small intestinal damage in rats, and that this is accompanied by the induction of dysbiosis by PPIs [13, 16]. Conversely, the beneficial modulation of the small intestinal microbiota so that it is resistant to NSAID-induced small intestinal damage could be a good strategic option for the prevention and therapy of NSAID-induced small intestinal damage.

Rebamipide [(2 RS)-2-(4-chlorobenzoylamino)-3-(2oxo-1,2-dihydroquinolin-4-yl) propanoic acid] is a mucoprotective drug that has been used clinically for the treatment of gastritis and peptic ulcers [17]. Recent clinical evidence indicates that rebamipide exerts an ameliorating effect on NSAID-induced small intestinal damage [18–21]. Previous studies, including one by our research group, revealed that rebamipide suppressed indomethacin-induced small intestinal damage, which was accompanied by the alteration of the intestinal microbiota [22–24]. However, in the previous studies, it was not determined if the rebamipide-modulated small intestinal microbiota has the potential to ameliorate NSAID-induced small intestinal damage. It also remained unclear whether rebamipide suppressed the exacerbation of NSAID-induced small intestinal damage by PPIs.

In the present study, we aimed to establish the following in NSAID-induced small intestinal damage: i) whether rebamipide suppress the NSAID-induced small intestinal damage directly via the modulation of the small intestinal microbiota; and ii) the effect of rebamipide on the exacerbation of NSAID-induced small intestinal damage by PPIs via the modulation of the small intestinal microbiota. We found that rebamipide ameliorates indomethacin-induced small intestinal damage and omeprazole-induced exacerbation of this damage via the modulation of the small intestinal microbiota.

Materials and methods

Induction of small intestinal damage by indomethacin in mice

Seven-week-old specific-pathogen-free male C57BL/6 mice were purchased from Charles River Japan, Inc. (Yokohama, Japan). All animals were housed in polycarbonate cages with paper chip bedding in the filtered-air ventilated cage rack of an air-conditioned biohazard room with a 12-h light-dark cycle. All animals had free access to food and water. The animals were fed with standard rodent diet (CE-2; CLEA Japan Inc.,Tokyo, Japan). The animals of treatment groups were separately kept in individual polycarbonate cages after shipping through the study.

When performing invasive procedures including intravenous injection and euthanasia, anesthesia was always conducted using isoflurane anesthetizer (MK-A110D, Muromachi Kikai, Tokyo, Japan). Anesthesia was induced at 4% isoflurane with 20% oxygen using a polycarbonate chamber and maintained at 1.5–2.75% isoflurane with 20% oxygen using anesthetic mask. Euthanasia was performed by instant cervical dislocation under deep sedation. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All experiments were carried out with confirmation of The Regulations on Animal Experiments and with approval of The Institutional Animal Care and Use Committee of Osaka City University Graduate School of Medicine (protocol ID: 09037). All invasive procedures were performed under isoflurane anesthesia, and all efforts were made to minimize suffering.

To induce small intestinal damage, 10 mg/kg of indomethacin (Sigma Chemical Company, St. Louis, MO) with vehicle (0.5% carboxymethylcellulose) was orally administered by gavage with intragastric feeding tube to non-fasted animals. For the evaluation of macroscopic damage, 1% Evans blue was injected intravenously 30 min before sacrifice in order to delineate the mucosal damage; after sacrifice, the small intestine was collected and opened along the antimesenteric attachment side of the lumen. The macroscopic mucosal damage was defined as ulcer or erosion clearly delineated by Evans blue dye. The macroscopic mucosal damage was measured in a masked fashion by the investigator (A. H.). The shape of macroscopic mucosal damage was round or elliptical shape, therefore we measured the major axis and minor axis with a digital precision caliper, multiplied major and minor axis, summed for each of the small intestine, and used the value as the lesion index. For histological evaluation, each of the small intestinal tissue samples that exhibited typical mucosal damage was fixed with 10% buffered formalin, and 4-μm thick tissue sections were mounted on glass slides, and subjected to hematoxylin and eosin (H&E) staining.

Histological evaluation of small intestinal damage

Tissue sections stained with H&E were viewed under high power using a white-light microscope. For each mouse, at least 10 random villi in the injured areas were scored independently in a masked fashion by two investigators (H.K. and T.K.). For evaluation, a modified histological scoring system was used [25]. The histological score ranged from 0 to 13 and was divided into the following six categories: epithelium (0 = normal, 1 = flattened, 2 = loss of epithelial continuity, 3 = severe denudation), villus shape (0 = normal, 1 = short and rounded, 2 = extremely short and thick), villus tip (0 = normal, 1 = damaged, 2 = severely damaged), stroma (0 = normal, 1 = slightly retracted, 2 = severely retracted), inflammation (0 = no infiltration, 1 = mild infiltration, 2 = severe infiltration), and crypt status (0 = normal, 1 = mild crypt loss, 2 = severe crypt loss).

Experimental groups and transplantation of small intestinal microbiota

Donor mice for the transplantation of the small intestinal microbiota received oral administration of vehicle (0.5% carboxymethylcellulose) or rebamipide (300 mg/kg body weight), which was supplied by Otsuka Pharmaceutical Co. (Tokyo, Japan) once daily for 1 week. Twenty-four hours after the final drug administration, the animals were sacrificed, and the contents of the ileum were collected.

In the first series of the experiments, the recipient mice were treated with antibiotics (penicillin/streptomycin/ampicillin) for 7 d. Penicillin and streptomycin (Sigma-Aldrich Co. LCC., Japan) were given in bottle water (penicillin: 6000 U/L of water, streptomycin 0.6 g/L of water) and ampicillin [800 mg/kg body weight (BW), Sigma-Aldrich) was administered once per day by gavage with intragastric feeding tube. Twenty-four hours after the last administration of ampicillin, the water bottle containing penicillin and streptomycin was switched for a bottle of pure water, and the mice were subjected to the transplantation of small intestinal microbiota by the oral administration of the ileal content obtained from vehicle- or rebamipide-treated mice. The content of the ileal lumen was suspended in normal saline at a concentration of 100 mg/mL; the suspension was centrifuged at 1,000 rpm for 5 min and the supernatant was collected. The mice were administered 0.4 mL of the supernatant by gavage with intragastric feeding tube. After 5 d, the mice were administered 10 mg/kg of indomethacin and the intestinal damage was evaluated 24 h after administration of indomethacin.

In the second series of the experiments, to assess the influence of omeprazole on indomethacin-induced small intestinal damage and the effect of rebamipide-modulated microbiota on the exacerbation of indomethacin-induced small intestinal damage by omeprazole, the mice were treated with the same antibiotics as the first series of experiments for 7 d. At 24 h after the final antibiotic treatment, the mice were divided into three groups: (1) the mice transplanted with small intestinal microbiota obtained from vehicle-treated mice and administered with vehicle for 5 d; (2) the mice transplanted with small intestinal microbiota obtained from vehicle-treated mice and administered with omeprazole (140 mg/kg BW, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) for 5 d; and (3) the mice transplanted with small intestinal microbiota obtained from rebamipide-treated mice and administered with omeprazole (140 mg/kg BW) for 5 d. After 5 d, the mice in all groups were administered indomethacin (10 mg/kg BW) and 24 h later the intestinal damage was evaluated. Omeprazole and indomethacin were administered by gavage with an intragastric feeding tube.

The protocol is summarized in Fig 1.

Sample collection and DNA extraction

The content of the small intestinal lumen was collected immediately after removing the intestine from the mice. The samples were frozen with liquid nitrogen and stored at −80°C until use. In brief, prior to DNA extraction, each sample was suspended in 15 mL PBS buffer (Thermo Fisher Scientific K.K., Tokyo, Japan) and sample suspension was filtered through a 100-μm mesh nylon filter (Corning Inc., New York, NY, USA) to remove eukaryotic cells and other debris. The debris on the filter was washed twice using a plastic bar with PBS buffer. The bacteria-enriched pellet was obtained by centrifugation of the filtrate at 9,000 g for 10 min at 4°C. The pellet was washed once with 35 mL PBS, and once again with TE20 buffer (10 mM Tris-HCl (Sigma-Aldrich Co. LCC., Tokyo, Japan), 20 mM EDTA (Thermo Fisher Scientific K.K., Tokyo, Japan)); then, DNA was extracted.

High molecular weight DNA was extracted from the samples by enzymatic lysis [26, 27]. The bacterial pellets suspended in 35 mL TE20 were centrifuged at 9,000 g for 10 min at 4°C. The pellets were resuspended in 0.4 mL TE20 and the suspension was incubated with a final concentration of 15 mg/mL lysozyme (Sigma-Aldrich Co. LCC.) and 2,000 units/mL of purified achromopeptidase (FUJIFILM Wako Pure Chemical Co., Osaka, Japan) for 2 h at 37°C with gentle shaking. The mixture was further incubated in the solution to a final concentration of 1% SDS (Sigma-Aldrich Co. LCC.) and 1 mg/mL of proteinase K (Merck & Co. Inc., USA) for 1 h at 55°C. The lysate was gently mixed with an equal volume (500 μL) of phenol/chloroform/isoamyl alcohol (Nippon Gene Co. Ltd., Tokyo, Japan) for 10 min, and the mixture was centrifuged at 9000 g (12000 rpm) for 10 min at room temperature. The aqueous supernatant was transferred to a new clean tube, and DNA was precipitated by the addition of 3 M sodium acetate (Nippon Gene Co. Ltd.) to the final concentration of 0.3 M, followed by the addition of an equal volume of isopropanol (FUJIFILM Wako Pure Chemical Co.). The DNA was pelleted by centrifugation at 9000 g (12000 rpm) for 10 min at 4°C, rinsed once with 75% ethanol, and dissolved in 500 μL of TE buffer. After the DNA was treated with 10 mg/mL RNase A (Nippon Gene Co. Ltd.) for 30 min at 37°C, 0.6 volumes of PEG solution (20% polyethylene glycol 6000 and 2.5 M NaCl; Hampton Research Corp., Aliso Viejo, CA, U.S.) was added, mixed gently, and kept on ice for at least 10 min. The DNA was pelleted by centrifugation at 9000 g (12000 rpm) for 10 min at 4°C, rinsed twice with 75% ethanol twice, and dissolved in 50 μL of TE buffer. The DNA concentration was measured using a Qubit Broad Range and High Sensitivity (Thermo Fisher Scientific K.K.).

16S rRNA gene sequencing

The 16S rRNA analysis of DNA samples was performed as previously described [27]. Briefly, PCR was performed using 27Fmod 5′-AGRGTTTGATYMTGGCTCAG-3′ and 338R 5′-TGCTGCCTCCCGTAGGAGT-3′ to amplify the V1–V2 region of the 16S rRNA gene [26]. The amplified DNA samples (~330 bp) were subsequently purified using AMPure XP (Beckman Coulter, Brea, CA, USA), and quantified using a Quant-iT Picogreen dsDNA assay kit (Invitrogen, Waltham, MA, USA) and a TBS-380 Mini-Fluorometer (Turner Biosystems, Waltham, MA, USA). The 16S sequencing was performed using MiSeq in accordance with the Illumina protocol (Illumina, San Diego, CA). The paired-end reads were merged using the fastq-join program based on overlapping sequences. Reads with an average quality value of <25 and inexact matches to both universal primers were filtered out.

Filter-passed reads were used for further analysis after trimming off both primer sequences. For each sample, the quality filter-passed reads were rearranged in descending order in accordance with the quality value and then clustered into OTUs with a 97% pairwise-identity cutoff using the UCLUST program version 5.2.32 (https://www.drive5.com). Taxonomic assignment of each OTU was made by a similarity search of the Ribosomal Database Project (RDP) and the National Center for Biotechnology Information (NCBI) genome database using the GLSEARCH program. For calculation of microbial abundance, taxa with a relative abundance of >0.1% were considered as positive. For assignment at the phylum, genus, and species levels, the sequence similarity thresholds of 70%, 94%, and 97%, were applied, respectively [26, 28, 29].

The UniFrac distance was used for the assessment of the dissimilarity (distance) between any pair of the samples [30]. We performed a principal coordinate analysis (PCoA) to visualize the similarities or dissimilarities in the microbiome structure in the UniFrac analysis using skbio.stats.ordination module of Python. We conducted Permutational multivariate analysis of variance (PERMANOVA, adonis in the R-vegan package) to compare the overall microbiome structure, and the p-values were adjusted for multiple testing by the Benjamin–Hochberg procedure. We used the observed and Chao 1-estimated OTU numbers and Shannon’s index to evaluate the species richness and diversity of the overall microbial community. Chao1 and Shannon’s index were calculated using Scikit-Bio’s diversity function.

The similarity of the relative abundance at the phylum, family, genus, and species levels was assessed using the Kruskal–Wallis test followed by the Steel–Dwass test for multiple comparisons.

Statistical analysis

The data are presented as the mean ± standard error of the mean (S.E.M.). The Mann–Whitney U test was used to evaluate the between-group differences. The differences among multiple groups were first analyzed by the Kruskal–Wallis test, and when a statistical significance was detected, The Mann–Whitney U test with Bonferroni correction was used to determine the statistical significance between multiple testing groups. Statistical significance was set at p<0.05.

Results

The effects of transplantation of rebamipide-modulated small intestinal microbiota on indomethacin-induced small intestinal damage

We determined whether the small intestinal microbiota derived from the mice treated with rebamipide exert inhibitory effects on the indomethacin-induced small intestinal damage. As shown in Fig 2, the lesion index of the mice transplanted with the small intestinal microbiota obtained from rebamipide-treated mice was smaller than that of the mice transplanted with the small intestinal microbiota obtained from vehicle-treated mice (Fig 2B). The histological scores of the vehicle and rebamipide groups treated with indomethacin showed a similar tendency with respect to the lesion indices (Fig 2C).

Fig 2 — A. Typical macroscopic and histological image of indomethacin-induced small intestinal damage. Damaged mucosa (arrows) was stained dark blue with 1% Evans blue. B. Lesion indices of indomethacin-induced small intestinal damage. C. Histological evaluation of indomethacin-induced small intestinal damage. Ct: control microbiota group Reb: rebamipide-modulated microbiota group. Each column represents the mean ± standard error of the mean (S.E.M.). N = 5–6. *p<0.05 and **p< 0.01 vs. control microbiota group.

The influence of omeprazole on small intestinal damage and the inhibitory effect of transplantation of the rebamipide-modulated small intestinal microbiota on exacerbation of indomethacin-induced small intestinal damage by omeprazole

Next, we examined whether omeprazole exacerbate indomethacin-induced small intestinal damage in mice and whether it is suppressed by modulation of the small intestinal microbiota by rebamipide. In mice transplanted with the small intestinal microbiota obtained from vehicle-treated mice, the administration of omeprazole increased the lesion index (Fig 3B). In mice administered with omeprazole, the lesion index in mice transplanted with the small intestinal microbiota obtained from rebamipide-treated mice was smaller than that in mice transplanted with the small intestinal microbiota obtained from vehicle-treated mice (Fig 3B). The histological scores of these groups treated with indomethacin showed a similar tendency with respect to the lesion indices (Fig 3C).

Fig 3 — A. Typical macroscopic and histological image of indomethacin-induced small intestinal damage. Damaged mucosa (arrows) was stained dark blue with 1% Evans blue. B. Lesion indices of indomethacin-induced small intestinal damage. C. Histological score of indomethacin-induced small intestinal damage. OPZ: omeprazole Each column represents the mean ± standard error of the mean (S.E.M.). N = 6–7. *p<0.05 and **p< 0.01 vs. vehicle-administered mice transplanted with control microbiota.

The effect of rebamipide on the composition of the small intestinal microbiota in mice

There was no difference in OTU number in the small intestinal microbiota between the control- and rebamipide-treated groups. Shannon’s index, ACE and the Chao1 index were also similar between the control- and rebamipide-treated groups (Table 1).

Table 1. α-diversity, measured by operational taxonomic unit (OTU) number, Chao1 index, ACE index, and Shannon’s diversity index of small intestinal lumen contents in mice given rebamipide or vehicle.

Index	control microbiota	rebamipide-modulated microbiota
Number of OTU	107.62 ± 15.94	108.88 ±14.33
Chao1	242.70 ± 36.55	252.25 ± 41.51
ACE	238.74 ± 39.63	265.61 ± 43.72
Shannon	2.47 ± 0.31	2.52 ± 0.23

Open in a new tab

N = 8. Values are expressed as mean ± standard error of the mean (S.E.M.).

UniFrac distances between the small intestinal microbial communities of the mice were visualized by a scatter plot created by PCoA to assess the β-diversity (Fig 4). PERMANOVA of the unweighted UniFrac distances showed significant differences in the composition of the small intestinal microbiota between the control- and rebamipide-treated groups. PERMANOVA of the weighted UniFrac distances showed the trend in the differences in the composition of the small intestinal microbiota between the control- and rebamipide-treated groups (Table 2).

Table 2. PERMANOVA of UniFrac distance in small intestine.

Category	Weigthed UniFrac		Unweigthed UniFrac
Category	R²	P value	R²	P value
control microbiota vs rebamipide-modulated microbiota	0.14909	0.06494	0.12584	0.000999
control microbiota+vehicle vs control microbiota+omeprazole	0.3884	0.01698	0.28218	0.002997
control microbiota+omeprazole vs rebamipide-modulated microbiota + omeprazole	0.33983	0.005994	0.20049	0.00999
control microbiota+vehicle vs rebamipide-modulated microbiota + omeprazole	0.2165	0.06993	0.1494	0.01499

Open in a new tab

At the phylum level, the majority of the small intestinal microbiota in the mice was dominated by Firmicutes followed by Bacteroidetes and Proteobacteria. There was no significant difference in taxonomic assignment performed at the phylum level between the control- and rebamipide-treated groups (Fig 5A).

Fig 5 — The major bacterial composition of small intestine in mice given rebamipide or vehicle at (A) phylum level (B) genus level (C) species level. (D) *Bacteroidetes* to *Firmicutes* ratio. Cont: control group Reb: rebamipide-treated group. Each column represents the mean ± standard error of the mean (S.E.M.). N = 6–7.

Taxonomic assignment performed at the genus level showed that the major constituent of the small intestinal microbiota was the genus Lactobacillus. There was a statistically significant difference in Dubosiella and Enterococcus between the control- and rebamipide-treated groups; however, these bacteria constituted a minor proportion of the population of the small intestinal microbiota (Fig 5B).

At the species level, the dominant species in small intestinal microbiota was L. taiwanensis followed by L. reuteri and L. murinus. The administration of rebamipide decreased the proportion of L. taiwanensis, but increased the proportion of L. murinus (Fig 5C).

Bacteroidetes to Firmicutes ratio (B/F ratio) is a parameter of dysbiosis in the large intestine and it is indicated that the ratio is associated with some pathophysiological conditions such as obesity [31], diabetes [32], and multiple sclerosis [33]. In the present study there was not significant difference in B/F ratio in the small intestinal microbiota between the control- and rebamipide-treated groups (p = 0.17) (Fig 5D).

Influence of omeprazole on the composition of small intestinal microbiota in mice transplanted with the control small intestinal microbiota and mice transplanted with rebamipide-modulated small intestinal microbiota

In mice transplanted with small intestinal microbiota from vehicle-treated animals, the administration of omeprazole significantly decreased Chao1 index, ACE index, and Shannon’s index of the small intestinal microbiome. Transplantation with small intestinal microbiota from rebamipide-treated mice reversed these parameters observed in omeprazole-treated mice transplanted with small intestinal microbiota from vehicle-treated mice, up to the values observed in the vehicle-treated mice (Table 3).

Table 3. α-diversity, measured by operational taxonomic unit (OTU) number, Chao1 index, ACE index, and Shannon’s diversity index of small intestinal lumen contents in microbiota-transplanted mice administered with omeprazole.

Index	control microbiota + vehicle	control microbiota + omeprazole	rebamipide-modulated microbiota + omeprazole
Number of OTUs	92.86 ± 13.68	51.33 ± 7.77	91.00 ± 16.69
Chao1	208.63 ± 37.48	97.68 ± 22.10^*	190.68 ± 31.10^#
ACE	199.66 ± 27.74	98.60 ± 20.40^*	180.41 ± 38.84
Shannon	2.69 ± 0.20	1.53 ± 0.21^**	2.65 ± 0.23^##

Open in a new tab

N = 6–7. Values are expressed as mean ± standard error of the mean (S.E.M.).

*p<0.05 and

**p <0.01 vs control microbiota + vehicle group.

^#p <0.05 and

^##p <0.01 vs control microbiota + omeprazole group.

PERMANOVA of both unweighted and weight UniFrac distances showed significant differences between the vehicle-administered group transplanted with small intestinal microbiota obtained from control mice, the omeprazole-administered group transplanted with small intestinal microbiota obtained from vehicle-treated mice, and the omeprazole-administered group transplanted with small intestinal microbiota obtained from rebamipide-treated mice (Table 2).

At the phylum level, the administration of omeprazole decreased the population of Bacteroidetes, whereas the influence of omeprazole on Bacteroidetes was ameliorated in mice transplanted with the small intestinal microbiota from rebamipide-treated mice. The administration of omeprazole decreased the population of Actinobacteria, however, Actinobacteria was a minor population (less than 1%) (Fig 6A).

Fig 6 — The major bacterial composition of small intestine in microbiota-transplanted mice administered with omeprazole or vehicle at (A) phylum level (B) genus level (C) species level. (D) *Bacteroidetes* to *Firmicutes* ratio. Cont + Ve: vehicle-administered group transplanted with control microbiota, Cont + OPZ: omeprazole-administered group transplanted with control microbiota, Reb + OPZ: omeprazole-administered group transplanted with rebamipide-modulatd microbiota. Each column represents the mean ± standard error of the mean (S.E.M.). N = 6–7.

At the genus level, the administration of omeprazole tended to increase the population of Lactobacillus and significantly decreased the population of Robinsoniella (Fig 6B).

At the species level, the administration of omeprazole increased the population of L. taiwanensis, whereas the influence of omeprazole on the population of L. taiwanensis was ameliorated in mice transplanted with the small intestinal microbiota from rebamipide-treated mice. The population of L. murinus was not significantly affected by administration of omeprazole, but was significantly increased by rebamipide in mice transplanted with the small intestinal microbiota from rebamipide-treated mice (Fig 6C).

Administration of omeprazole decreased the B/F ratio, while the influence of omeprazole on B/F ratio was ameliorated in mice transplanted with the small intestinal microbiota from rebamipide-treated mice (Fig 6D).

Discussion

In the present study, we demonstrated that rebamipide ameliorated indomethacin-induced small intestinal damage, as previously reported [22]. Moreover, by using transplantation with the small intestinal microbiota, we demonstrated that the effect of rebamipide on indomethacin-induced small intestinal damage was, at least in part, due to the modulation of the small intestinal microbiota. Finally, we revealed that small intestinal microbiota from mice treated with rebamipide exerted inhibitory effect against the exacerbation of indomethacin-induced small intestinal damage by omeprazole. These results suggest that the small intestinal microbiota modulated by rebamipide results in resistance to exacerbation of NSAID-induced small intestinal damage by PPI.

Recently, several clinical studies, including our previous study, suggest that PPI is a risk factor for NSAID-induced small intestinal damage [8, 10, 11]. It is speculated that the possible mechanism through which PPI exacerbate NSAID-induced small intestinal damage is the induction of dysbiosis in the small intestine. Several studies suggest that the loss of microbial diversity in the gut microbiota is associated with some gastrointestinal diseases, such as Crohn’s disease [34], ulcerative colitis [35], irritable bowel syndrome [36], graft-versus-host disease in allogeneic hematopoietic-cell transplantation [37–40]. Moreover, it is reported that lower microbial diversity correlated with increased small intestinal permeability in chronic liver diseases [41]. Interestingly, we recently reported that PPI significantly enhanced the stress-induced pathogenic increase in small intestinal permeability accompanied by reduction of α-diversity [42]. The present study showed that omeprazole tended to decrease the parameters of microbial diversity, OTU number, Chao1 index, and Shannon index. The result was similar to a previous clinical study, in that PPI use was associated with a significant decrease in Shannon’s diversity in the gut microbiome [43]. These results suggest that PPI induce the loss of small intestinal microbial diversity, which may be a crucial factor for the exacerbation of indomethacin-induced small intestinal damage by PPI. Importantly, mice transplanted with small intestinal microbiota obtained from mice treated with rebamipide showed resistance to the omeprazole-induced loss of microbial diversity, which may account for the mechanism through which rebamipide ameliorate the exacerbation of NSAID-induced small intestinal damage by PPI via modulation of the small intestinal microbiota.

In terms of β-diversity metrics, PCoA analysis and PERMANOVA analysis showed that there was a significant difference in β-diversity of small intestinal microbiota between control mice and rebamipide-treated mice. Moreover, in the present study, in omeprazole-administered animals, there was significant difference in β-diversity between animals transplanted with control microbiota and animals transplanted with rebamipide-modulated microbiota. From the viewpoint of β-diversity, these results suggest that rebamipide has the potential to modulate small intestinal microbiota resistant to NSAID-induced small intestinal microbiota with or without PPI.

Firmicutes/Bacteroidetes (F/B) or B/F ratio is a relevant marker of dysbiosis. Accumulating evidence suggest the significance of F/B or B/F ratio as a biomarker of dysbiosis in the pathophysiology of various diseases such as obesity [31], diabetes [32], and multiple sclerosis [33]. In gastrointestinal mucosal damage, it is reported that Huai hua san, a traditional Chinese herbal formula, alleviated dextran sulphate sodium (DSS)-induced large intestinal mucosal damage with modulation of F/B ratio [44]. Our present study showed that omeprazole markedly decreased B/F ratio and transplantation of rebamipide-modulated microbiota recovered it. In the pathophysiology of NSAID-induced small intestinal damage, it is possible that F/B or B/F is useful marker for the assessment of NSAID-induced small intestinal damage-associated dysbiosis.

In the present study, we performed a comprehensive analysis of the alteration of the microbial composition of the small intestine by rebamipide and omeprazole at the species level using 16S rRNA gene sequencing analysis. The issue to be clarified is the determination of the responsible alterations of microbial composition, which modifies the pathophysiology of NSAID-induced small intestinal damage in the administration of rebamipide and PPI. In this study, in control microbiota-transplanted mice, omeprazole increased the proportion of L. taiwanensis, while transplantation of rebamipide-modulated microbiota significantly reduced the proportion of L. taiwanensis. Recently it is reported that L. taiwanensis produce bacteriocins, an antimicrobial peptide [45]. In the present study, increase in concentration of bacteriocins produced by excessive amount of L. taiwanensis may modulate small intestinal microbiota. However, there is little information about L. taiwanensis-derived bacteriocin in physiological and pathophysiological condition the small intestine, and impact of L. taiwanensis-derived bacteriocin on the component of microbiota remains unknown. In literature, accumulating studies indicate that Lactobacillus-derived bacteriocin exerts inhibitory effect against dysbiosis [46–48]. Considering these reports, it is possible that the result of reduction of proportion of L. taiwanensis is just the accompanying phenomenon and L. taiwanensis-derived bacteriocin is irrelevant to the effect of rebamipide on PPI-induced exacerbation of NSAID-induced small intestinal damage.

In our present study, rebamipide increased the proportion of L. murinus, even in the presence of the administration of omeprazole. The accumulated evidence suggested that L. murinus protected rats from necrotizing enterocolitis [49] and DSS-induced colitis in mice [50], and that L. murinus had anti-inflammatory properties in a human intestinal epithelial cell lines (Caco-2 cells) and it also improved gut barrier function in a mice model [51]. Our previous study revealed that supplementation of L. murinus ameliorated indomethacin-induced small intestinal damage [14]. These results indicate that it is possible that rebamipide increased the percentage of L. murinus in the small intestinal microbiota, which resulted in a suppressive effect on indomethacin-induced small intestinal damage and its exacerbation by omeprazole. This issue warrants further investigation.

Other statistically significant alteration of microbial composition in the small intestine by omeprazole and rebamipide was observed in the present study, such as the reduction of Bacteroidetes and Actinobacteria by omeprazole and the attenuation of the alteration by rebamipide; however, these were minor populations of microbial compositions of small intestine, and the pathophysiological impact on NSAID-induced small intestinal damage remains unknown.

There are several limitations to the present study. First, we cannot precisely evaluate the impact of each of alteration of the microbial component; even alteration of minor microbial component may have great impact on the pathophysiology of NSAID-induced small intestinal damage. To evaluate them, usage of gnotobiotic mice is necessary.

Second, we cannot exclude the possibility of the significance of residual rebamipide in the transplanted ileal contents. To minimize the possibility, we put 24 hours of washout period before transplantation and 4 days period before indomethacin challenge in the experiment protocol. Even though, it is possible that there may be small amount of residual rebamipide in the ileal content transferred with donor microbiota to the recipient mice, and the residual rebamipide may exerts direct pharmacological effect on the intestinal mucosa of the recipient mice. To clear the possibility, the analysis of the detection of residual rebamipide in the ileal contents obtained from donor mice may be useful, however, it is impossible for us because of technical problem; there is no commercial assay kit and hand-made laboratory method to determine the amount of rebamipide in the ileal contents is not established. Third, accumulating evidence is establishing the dysbiosis in the large intestine, while it remains unknown about the dysbiosis in the pathophysiology of NSAID-induced small intestinal damage. For example, B/F ratio is originally determined in the large intestinal microbiota and is well studied in the field of the pathophysiology of obesity [31]. However, it remains unclear about the significance of B/F or F/B ratio in the pathophysiology of NSAID-induced small intestinal damage.

In conclusion, rebamipide has potential to modulate small intestinal microbiota resistant to indomethacin-induced small intestinal damage. The suppressive effect extends to the suppression of exacerbation of indomethacin-induced small intestinal damage by proton pump inhibitors.

Supporting information

S1 Table. The major bacterial composition of small intestine in mice given rebamipide or vehicle at phylum level.

(DOCX)

Click here for additional data file.^{(13KB, docx)}

S2 Table. The major bacterial composition of small intestine in mice given rebamipide or vehicle at genus level.

(DOCX)

Click here for additional data file.^{(13.2KB, docx)}

S3 Table. The major bacterial composition of small intestine in mice given rebamipide or vehicle at species level.

(DOCX)

Click here for additional data file.^{(13.9KB, docx)}

S4 Table. The major bacterial composition of small intestine in microbiota-transplanted mice administered with omeprazole at phylum level.

(DOCX)

Click here for additional data file.^{(13.5KB, docx)}

S5 Table. The major bacterial composition of small intestine in microbiota-transplanted mice administered with omeprazole at genus level.

(DOCX)

Click here for additional data file.^{(13.8KB, docx)}

S6 Table. The major bacterial composition of small intestine in microbiota-transplanted mice administered with omeprazole at species level.

(DOCX)

Click here for additional data file.^{(14.8KB, docx)}

Acknowledgments

We thank Emi Suzuki-Yoshioka for her technical assistance.

Data Availability

All relevant data are available in figshare (https://figshare.com/articles/dataset/Rebamipide_Suppress_Indomethacin-Induced_Small_Intestinal_Damage_by_Modulation_of_Small_Intestinal_Microbiota/13241267), DOI: 10.6084/m9.figshare.13241267.

Funding Statement

The authors received no specific funding for this work.

References

1.Iddan G, Meron G, Glukhovsky A, Swain P. Wireless capsule endoscopy. Nature. 2000;405(6785):417 Epub 2000/06/06. 10.1038/35013140 . [DOI] [PubMed] [Google Scholar]
2.Watanabe T, Fujiwara Y, Chan FKL. Current knowledge on non-steroidal anti-inflammatory drug-induced small-bowel damage: a comprehensive review. Journal of gastroenterology. 2020;55(5):481–95. Epub 2019/12/23. 10.1007/s00535-019-01657-8 . [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Graham DY, Opekun AR, Willingham FF, Qureshi WA. Visible small-intestinal mucosal injury in chronic NSAID users. Clinical gastroenterology and hepatology: the official clinical practice journal of the American Gastroenterological Association. 2005;3(1):55–9. Epub 2005/01/13. 10.1016/s1542-3565(04)00603-2 . [DOI] [PubMed] [Google Scholar]
4.Goldstein JL, Eisen GM, Lewis B, Gralnek IM, Aisenberg J, Bhadra P, et al. Small bowel mucosal injury is reduced in healthy subjects treated with celecoxib compared with ibuprofen plus omeprazole, as assessed by video capsule endoscopy. Alimentary pharmacology & therapeutics. 2007;25(10):1211–22. Epub 2007/04/25. 10.1111/j.1365-2036.2007.03312.x . [DOI] [PubMed] [Google Scholar]
5.Maiden L, Thjodleifsson B, Theodors A, Gonzalez J, Bjarnason I. A quantitative analysis of NSAID-induced small bowel pathology by capsule enteroscopy. Gastroenterology. 2005;128(5):1172–8. Epub 2005/05/12. 10.1053/j.gastro.2005.03.020 . [DOI] [PubMed] [Google Scholar]
6.Sugimori S, Watanabe T, Tabuchi M, Kameda N, Machida H, Okazaki H, et al. Evaluation of small bowel injury in patients with rheumatoid arthritis by capsule endoscopy: effects of anti-rheumatoid arthritis drugs. Digestion. 2008;78(4):208–13. Epub 2009/01/15. 10.1159/000190403 . [DOI] [PubMed] [Google Scholar]
7.Shimada S, Watanabe T, Nadatani Y, Otani K, Taira K, Hosomi S, et al. Clinical factors associated with positive capsule endoscopy findings in patients with obscure gastrointestinal bleeding: a single-center study. Scandinavian journal of gastroenterology. 2017;52(11):1219–23. Epub 2017/07/12. 10.1080/00365521.2017.1349174 . [DOI] [PubMed] [Google Scholar]
8.Watanabe T, Tanigawa T, Nadatani Y, Nagami Y, Sugimori S, Okazaki H, et al. Risk factors for severe nonsteroidal anti-inflammatory drug-induced small intestinal damage. Digestive and liver disease: official journal of the Italian Society of Gastroenterology and the Italian Association for the Study of the Liver. 2013;45(5):390–5. Epub 2013/01/22. 10.1016/j.dld.2012.12.005 . [DOI] [PubMed] [Google Scholar]
9.Lanza FL, Chan FK, Quigley EM. Guidelines for prevention of NSAID-related ulcer complications. Am J Gastroenterol. 2009;104(3):728–38. Epub 2009/02/26. 10.1038/ajg.2009.115 . [DOI] [PubMed] [Google Scholar]
10.Endo H, Sakai E, Taniguchi L, Kessoku T, Komiya Y, Ezuka A, et al. Risk factors for small-bowel mucosal breaks in chronic low-dose aspirin users: data from a prospective multicenter capsule endoscopy registry. Gastrointestinal endoscopy. 2014;80(5):826–34. Epub 2014/05/17. 10.1016/j.gie.2014.03.024 . [DOI] [PubMed] [Google Scholar]
11.Washio E, Esaki M, Maehata Y, Miyazaki M, Kobayashi H, Ishikawa H, et al. Proton Pump Inhibitors Increase Incidence of Nonsteroidal Anti-Inflammatory Drug-Induced Small Bowel Injury: A Randomized, Placebo-Controlled Trial. Clinical gastroenterology and hepatology: the official clinical practice journal of the American Gastroenterological Association. 2016;14(6):809–15.e1. Epub 2015/11/06. 10.1016/j.cgh.2015.10.022 . [DOI] [PubMed] [Google Scholar]
12.Satoh H, Amagase K, Takeuchi K. Exacerbation of nonsteroidal anti-inflammatory drug-induced small intestinal lesions by antisecretory drugs in rats: the role of intestinal motility. The Journal of pharmacology and experimental therapeutics. 2012;343(2):270–7. Epub 2012/08/03. 10.1124/jpet.112.197475 . [DOI] [PubMed] [Google Scholar]
13.Wallace JL, Syer S, Denou E, de Palma G, Vong L, McKnight W, et al. Proton pump inhibitors exacerbate NSAID-induced small intestinal injury by inducing dysbiosis. Gastroenterology. 2011;141(4):1314–22, 22.e1–5. Epub 2011/07/13. 10.1053/j.gastro.2011.06.075 . [DOI] [PubMed] [Google Scholar]
14.Nadatani Y, Watanabe T, Suda W, Nakata A, Matsumoto Y, Kosaka S, et al. Gastric acid inhibitor aggravates indomethacin-induced small intestinal injury via reducing Lactobacillus johnsonii. Scientific reports. 2019;9(1):17490 Epub 2019/11/27. 10.1038/s41598-019-53559-7 . [DOI] [PMC free article] [PubMed] [Google Scholar]
15.DeGruttola AK, Low D, Mizoguchi A, Mizoguchi E. Current Understanding of Dysbiosis in Disease in Human and Animal Models. Inflammatory bowel diseases. 2016;22(5):1137–50. Epub 2016/04/14. 10.1097/MIB.0000000000000750 . [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Blackler RW, De Palma G, Manko A, Da Silva GJ, Flannigan KL, Bercik P, et al. Deciphering the pathogenesis of NSAID enteropathy using proton pump inhibitors and a hydrogen sulfide-releasing NSAID. American journal of physiology Gastrointestinal and liver physiology. 2015;308(12):G994–1003. Epub 2015/04/18. 10.1152/ajpgi.00066.2015 . [DOI] [PubMed] [Google Scholar]
17.Zhang S, Qing Q, Bai Y, Mao H, Zhu W, Chen Q, et al. Rebamipide helps defend against nonsteroidal anti-inflammatory drugs induced gastroenteropathy: a systematic review and meta-analysis. Digestive diseases and sciences. 2013;58(7):1991–2000. Epub 2013/03/05. 10.1007/s10620-013-2606-0 . [DOI] [PubMed] [Google Scholar]
18.Watanabe T, Takeuchi T, Handa O, Sakata Y, Tanigawa T, Shiba M, et al. A multicenter, randomized, double-blind, placebo-controlled trial of high-dose rebamipide treatment for low-dose aspirin-induced moderate-to-severe small intestinal damage. PloS one. 2015;10(4):e0122330 Epub 2015/04/16. 10.1371/journal.pone.0122330 Pharmaceutical Co., Ltd and Takeda Pharmaceutical Co., Ltd. KH has received research grants and speaker’s fee from Otsuka Pharmaceutical Co., Ltd. TA has received research grants from Eisai Co., Ltd. and Otsuka Pharmaceutical Co., Ltd. Otsuka Pharmaceutical Co., Ltd provided rebamipide and identical placebo. There are no further patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Kurokawa S, Katsuki S, Fujita T, Saitoh Y, Ohta H, Nishikawa K, et al. A randomized, double-blinded, placebo-controlled, multicenter trial, healing effect of rebamipide in patients with low-dose aspirin and/or non-steroidal anti-inflammatory drug induced small bowel injury. Journal of gastroenterology. 2014;49(2):239–44. Epub 2013/04/19. 10.1007/s00535-013-0805-2 . [DOI] [PubMed] [Google Scholar]
20.Mizukami K, Murakami K, Abe T, Inoue K, Uchida M, Okimoto T, et al. Aspirin-induced small bowel injuries and the preventive effect of rebamipide. World journal of gastroenterology. 2011;17(46):5117–22. Epub 2011/12/16. 10.3748/wjg.v17.i46.5117 . [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Fujimori S, Takahashi Y, Gudis K, Seo T, Ehara A, Kobayashi T, et al. Rebamipide has the potential to reduce the intensity of NSAID-induced small intestinal injury: a double-blind, randomized, controlled trial evaluated by capsule endoscopy. Journal of gastroenterology. 2011;46(1):57–64. Epub 2010/10/07. 10.1007/s00535-010-0332-3 . [DOI] [PubMed] [Google Scholar]
22.Tanigawa T, Watanabe T, Otani K, Nadatani Y, Ohkawa F, Sogawa M, et al. Rebamipide inhibits indomethacin-induced small intestinal injury: possible involvement of intestinal microbiota modulation by upregulation of alpha-defensin 5. European journal of pharmacology. 2013;704(1–3):64–9. Epub 2013/02/23. 10.1016/j.ejphar.2013.02.010 . [DOI] [PubMed] [Google Scholar]
23.Imaeda H, Fujimoto T, Takahashi K, Kasumi E, Fujiyama Y, Andoh A. Terminal-restriction fragment length polymorphism (T-RFLP) analysis for changes in the gut microbiota profiles of indomethacin- and rebamipide-treated mice. Digestion. 2012;86(3):250–7. Epub 2012/09/12. 10.1159/000341508 . [DOI] [PubMed] [Google Scholar]
24.Kurata S, Nakashima T, Osaki T, Uematsu N, Shibamori M, Sakurai K, et al. Rebamipide protects small intestinal mucosal injuries caused by indomethacin by modulating intestinal microbiota and the gene expression in intestinal mucosa in a rat model. Journal of clinical biochemistry and nutrition. 2015;56(1):20–7. Epub 2015/04/04. 10.3164/jcbn.14-67 . [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Shimada S, Tanigawa T, Watanabe T, Nakata A, Sugimura N, Itani S, et al. Involvement of gliadin, a component of wheat gluten, in increased intestinal permeability leading to non-steroidal anti-inflammatory drug-induced small-intestinal damage. PloS one. 2019;14(2):e0211436 Epub 2019/02/21. 10.1371/journal.pone.0211436 . [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Kim SW, Suda W, Kim S, Oshima K, Fukuda S, Ohno H, et al. Robustness of gut microbiota of healthy adults in response to probiotic intervention revealed by high-throughput pyrosequencing. DNA research: an international journal for rapid publication of reports on genes and genomes. 2013;20(3):241–53. Epub 2013/04/11. 10.1093/dnares/dst006 . [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Suzuki Y, Nishijima S, Furuta Y, Yoshimura J, Suda W, Oshima K, et al. Long-read metagenomic exploration of extrachromosomal mobile genetic elements in the human gut. Microbiome. 2019;7(1):119 Epub 2019/08/29. 10.1186/s40168-019-0737-z . [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Said HS, Suda W, Nakagome S, Chinen H, Oshima K, Kim S, et al. Dysbiosis of salivary microbiota in inflammatory bowel disease and its association with oral immunological biomarkers. DNA research: an international journal for rapid publication of reports on genes and genomes. 2014;21(1):15–25. Epub 2013/09/10. 10.1093/dnares/dst037 . [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Iwasawa K, Suda W, Tsunoda T, Oikawa-Kawamoto M, Umetsu S, Takayasu L, et al. Dysbiosis of the salivary microbiota in pediatric-onset primary sclerosing cholangitis and its potential as a biomarker. Scientific reports. 2018;8(1):5480 Epub 2018/04/05. 10.1038/s41598-018-23870-w . [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Lozupone C, Lladser ME, Knights D, Stombaugh J, Knight R. UniFrac: an effective distance metric for microbial community comparison. The ISME journal. 2011;5(2):169–72. Epub 2010/09/10. 10.1038/ismej.2010.133 . [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Magne F, Gotteland M, Gauthier L, Zazueta A, Pesoa S, Navarrete P, et al. The Firmicutes/Bacteroidetes Ratio: A Relevant Marker of Gut Dysbiosis in Obese Patients? Nutrients. 2020;12(5). Epub 2020/05/23. 10.3390/nu12051474 . [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Pellegrini S, Sordi V, Bolla AM, Saita D, Ferrarese R, Canducci F, et al. Duodenal Mucosa of Patients With Type 1 Diabetes Shows Distinctive Inflammatory Profile and Microbiota. The Journal of clinical endocrinology and metabolism. 2017;102(5):1468–77. Epub 2017/03/23. 10.1210/jc.2016-3222 . [DOI] [PubMed] [Google Scholar]
33.Cosorich I, Dalla-Costa G, Sorini C, Ferrarese R, Messina MJ, Dolpady J, et al. High frequency of intestinal T(H)17 cells correlates with microbiota alterations and disease activity in multiple sclerosis. Science advances. 2017;3(7):e1700492 Epub 2017/07/15. 10.1126/sciadv.1700492 . [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Mosca A, Leclerc M, Hugot JP. Gut Microbiota Diversity and Human Diseases: Should We Reintroduce Key Predators in Our Ecosystem? Frontiers in microbiology. 2016;7:455 Epub 2016/04/12. 10.3389/fmicb.2016.00455 . [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Alipour M, Zaidi D, Valcheva R, Jovel J, Martinez I, Sergi C, et al. Mucosal Barrier Depletion and Loss of Bacterial Diversity are Primary Abnormalities in Paediatric Ulcerative Colitis. Journal of Crohn’s & colitis. 2016;10(4):462–71. Epub 2015/12/15. 10.1093/ecco-jcc/jjv223 . [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Lo Presti A, Zorzi F, Del Chierico F, Altomare A, Cocca S, Avola A, et al. Fecal and Mucosal Microbiota Profiling in Irritable Bowel Syndrome and Inflammatory Bowel Disease. Frontiers in microbiology. 2019;10:1655 Epub 2019/08/06. 10.3389/fmicb.2019.01655 . [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Holler E, Butzhammer P, Schmid K, Hundsrucker C, Koestler J, Peter K, et al. Metagenomic analysis of the stool microbiome in patients receiving allogeneic stem cell transplantation: loss of diversity is associated with use of systemic antibiotics and more pronounced in gastrointestinal graft-versus-host disease. Biology of blood and marrow transplantation: journal of the American Society for Blood and Marrow Transplantation. 2014;20(5):640–5. Epub 2014/02/05. 10.1016/j.bbmt.2014.01.030 . [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Kumari R, Palaniyandi S, Hildebrandt GC. Microbiome: An Emerging New Frontier in Graft-Versus-Host Disease. Digestive diseases and sciences. 2019;64(3):669–77. Epub 2018/12/14. 10.1007/s10620-018-5369-9 . [DOI] [PubMed] [Google Scholar]
39.Peled JU, Gomes ALC, Devlin SM, Littmann ER, Taur Y, Sung AD, et al. Microbiota as Predictor of Mortality in Allogeneic Hematopoietic-Cell Transplantation. The New England journal of medicine. 2020;382(9):822–34. Epub 2020/02/27. 10.1056/NEJMoa1900623 . [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Staffas A, Burgos da Silva M, van den Brink MR. The intestinal microbiota in allogeneic hematopoietic cell transplant and graft-versus-host disease. Blood. 2017;129(8):927–33. Epub 2016/12/13. 10.1182/blood-2016-09-691394 . [DOI] [PMC free article] [PubMed] [Google Scholar]
41.Raj AS, Shanahan ER, Tran CD, Bhat P, Fletcher LM, Vesey DA, et al. Dysbiosis of the Duodenal Mucosal Microbiota Is Associated With Increased Small Intestinal Permeability in Chronic Liver Disease. Clinical and translational gastroenterology. 2019;10(8):e00068 Epub 2019/08/03. 10.14309/ctg.0000000000000068 . [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Takashima S, Tanaka F, Kawaguchi Y, Usui Y, Fujimoto K, Nadatani Y, et al. Proton pump inhibitors enhance intestinal permeability via dysbiosis of gut microbiota under stressed conditions in mice. Neurogastroenterology and motility: the official journal of the European Gastrointestinal Motility Society. 2020;32(7):e13841 Epub 2020/04/23. 10.1111/nmo.13841 . [DOI] [PubMed] [Google Scholar]
43.Imhann F, Bonder MJ, Vich Vila A, Fu J, Mujagic Z, Vork L, et al. Proton pump inhibitors affect the gut microbiome. Gut. 2016;65(5):740–8. Epub 2015/12/15. 10.1136/gutjnl-2015-310376 . [DOI] [PMC free article] [PubMed] [Google Scholar]
44.Liu P, Bian Y, Liu T, Zhong J, Zhong Y, Zhuang S, et al. Huai hua san alleviates dextran sulphate sodium-induced colitis and modulates colonic microbiota. Journal of ethnopharmacology. 2020;259:112944 Epub 2020/05/11. 10.1016/j.jep.2020.112944 . [DOI] [PubMed] [Google Scholar]
45.Kim SW, Ha YJ, Bang KH, Lee S, Yeo JH, Yang HS, et al. Potential of Bacteriocins from Lactobacillus taiwanensis for Producing Bacterial Ghosts as a Next Generation Vaccine. Toxins. 2020;12(7). Epub 2020/07/08. 10.3390/toxins12070432 . [DOI] [PMC free article] [PubMed] [Google Scholar]
46.Lopetuso LR, Giorgio ME, Saviano A, Scaldaferri F, Gasbarrini A, Cammarota G. Bacteriocins and Bacteriophages: Therapeutic Weapons for Gastrointestinal Diseases? International journal of molecular sciences. 2019;20(1). Epub 2019/01/10. 10.3390/ijms20010183 . [DOI] [PMC free article] [PubMed] [Google Scholar]
47.Nilsen T, Swedek I, Lagenaur LA, Parks TP. Novel Selective Inhibition of Lactobacillus iners by Lactobacillus-Derived Bacteriocins. Applied and environmental microbiology. 2020;86(20). Epub 2020/08/18. 10.1128/AEM.01594-20 . [DOI] [PMC free article] [PubMed] [Google Scholar]
48.Todorov SD, Kang HJ, Ivanova IV, Holzapfel WH. Bacteriocins From LAB and Other Alternative Approaches for the Control of Clostridium and Clostridiodes Related Gastrointestinal Colitis. Frontiers in bioengineering and biotechnology. 2020;8:581778 Epub 2020/10/13. 10.3389/fbioe.2020.581778 . [DOI] [PMC free article] [PubMed] [Google Scholar]
49.Isani M, Bell BA, Delaplain PT, Bowling JD, Golden JM, Elizee M, et al. Lactobacillus murinus HF12 colonizes neonatal gut and protects rats from necrotizing enterocolitis. PloS one. 2018;13(6):e0196710 Epub 2018/06/23. 10.1371/journal.pone.0196710 . [DOI] [PMC free article] [PubMed] [Google Scholar]
50.Tang C, Kamiya T, Liu Y, Kadoki M, Kakuta S, Oshima K, et al. Inhibition of Dectin-1 Signaling Ameliorates Colitis by Inducing Lactobacillus-Mediated Regulatory T Cell Expansion in the Intestine. Cell host & microbe. 2015;18(2):183–97. Epub 2015/08/14. 10.1016/j.chom.2015.07.003 . [DOI] [PubMed] [Google Scholar]
51.Pan F, Zhang L, Li M, Hu Y, Zeng B, Yuan H, et al. Predominant gut Lactobacillus murinus strain mediates anti-inflammaging effects in calorie-restricted mice. Microbiome. 2018;6(1):54 Epub 2018/03/23. 10.1186/s40168-018-0440-5 . [DOI] [PMC free article] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0245995.r001

Decision Letter 0

Jan S Suchodolski

10 Sep 2020

PONE-D-20-23408

Rebamipide Ameliorates Indomethacin-Induced Small Intestinal Damage and Proton Pump Inhibitor-Induced Exacerbation of this Damage by Modulation of Small Intestinal Microbiota

PLOS ONE

Dear Dr. Tanigawa,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

As you will see below, the reviewers highlighted several deficiencies in the methods that need to be addressed. Furthermore, the manuscript requires attention to address the grammatical and spelling concerns highlighted by the reviewers.

Please submit your revised manuscript by Oct 25 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Jan S Suchodolski, DVM, PhD

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please ensure that all statements are justified and/or referenced appropriately. For example, in the Introduction section, there are numerous statements which appear to be currently unsubstantiated and require a reference. For example “The emergence of video capsule endoscopy and balloon-assisted enteroscopy have enabled the precise diagnosis of diseases involved in small intestinal damage, and the clinical features of nonsteroidal anti-inflammatory drug (NSAID)-induced small intestinal damage have been clarified” and “NSAIDs frequently induce small intestinal damage, which cause overt and occult bleeding, perforation, and stenosis of the small intestine".

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Interesting and well written manuscript. The study elucidates one mechanism through which rebapimide has a protective effect against indomethacin-induced small intestinal damage, even in the presence of omeprazole. One limitation of the study is the method of DNA extraction, which included a number of filtering and washing steps, followed by enzymatic lysis. While individually each one of those steps may have a minor impact, combined they introduce a significant bias which may have impacted the detection of microbial diversity. However, given that the protocol is thoroughly described and was equally applied to all samples, the results are still valid and applicable.

Specific comments:

Table numbers: There is no table 3, please renumber the tables accordingly.

Tables 4 and 8: It would be interesting to calculate the Firmicutes:Bacteroidetes ratio, an indicator of dysbiosis commonly used in the study of obesity and neurologic diseases. Based on your means in table 8 I believe it will be significantly different between treatments, and it is an additional indicator that will strengthen your discussion.

At the beginning of page 22 (paragraph before table 10) you mention that omeprazole decreased L. murinus. Based on table 10 that is not correct, as the change is of only 0.5% and non-significant. Please correct. Accordingly, correct that L. murinus was non ameliorated but significantly increased by rebamipide.

Page 23:

On the sentence after reference 29, please italicize L. taiwanensis.

It would be interesting to discuss the bacteriocin production further – that is a potential explanation for the reduced diversity in the omeprazole-treated mice.

Reviewer #2: This study aims to determine the ability of rebamipide conditioned microbiota to protect against NSAID-induced GI injury and omeprazole exacerbated NSAID-induced GI injury. The study design is simple (although many important details are not included) and involves ileal microbiota transplant of rebamipide treated mice to recipients that are then treated with indomethacin and indomethacin plus omeprazole. The limited readouts include histology, macroscopic injury scores, and basic microbiota analysis. There are numerous grammatical errors but overall it is well-written.

There is minimal new information in this simple study and the lack of details in M and M, lack of better control groups, and minimal readouts prevent robust evaluation.

where are the sequecing data available?

Please include more information in M and M section regarding animal studies. What were mice fed? How were they housed (all groups separately from shipping through study? all treatment groups in individual cages, etc...)?

Use of isoflurane is uncecessarily repeated.

What is deep euthanasia?

How was indomethacin administered? "Orally" is not clear. Gavage?

How were sections of SI that "that exhibited typical

mucosal damage" selected? Was the person who selected these blinded to treatment group?

Was the ileal contents from the donor mice stored or given fresh. If stored, how was it stored?

It is stated that the ileal contents were dissolved. This seems incorrect as I do not believe lumenal contents are soluble.

Suggest: ...intestinal damage was evaluated after 24 h.

In the second series of experiments it is stated that "the mice were treated with antibiotics for 7 d" same antibiotics as first study? If so this should be clearly stated.

Figure 1 is helpful. Typo in figure 1 (anpicillin)

How many mice in each group?

How was omep administered?

what do you mean "The content of the small intestinal lumen was collected immediately after sampling

from the mice."?

typo - In brief, prior to DNA extraction, each sample suspended in 15 mL PBS buffer

(Thermo Fisher Scientific K.K., Tokyo, Japan) was filtered ...

In this sentence...twice using a glass or plastic bar with PBS

buffer..How much PBS? what is a bar? why switch between glass and plastic?

The complete computational analysis of 16S data is lacking. For example, how was PCoA generated?

How was mm^2 area of evans blue staining generated (how was area of evans blue staining quantified?

Table 2 could be made much more visually appealing and easier to follow.

Numerous grammatical errors throughout, far too many to correct. One example, "There is no difference in OTU number in"... present tense here. past tense elsewhere in results.

There are many limitations in this manuscript that should be discussed.

How can you be sure that the positive effects seen are not due to residual rebemipide in the ileal contents?

Inclusion of a control group not being treated with indomethacin would greatly aid in interpretation of these data. For example, perhaps rebamipide had no effect on the microbiota but instead reduced GI injury via another mechanism and the modest differences in the microbiota were related to less inflammation and not rebamipides influences on the microbiota.

Please discuss beta diversity findings in more detail.

The comparisons and where/which significant differiences exist is not clear to me in tables 4-6 and 8-10

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2021 Jan 28;16(1):e0245995. doi: 10.1371/journal.pone.0245995.r002

Author response to Decision Letter 0

16 Nov 2020

PONE-D-20-23408

PLOS ONE Decision: Major Revision

“Rebamipide Ameliorates Indomethacin-Induced Small Intestinal Damage and Proton Pump Inhibitor-Induced Exacerbation of this Damage by Modulation of Small Intestinal Microbiota”

Response to Reviewers and Editors

Editor’s comment

(Authors’ response)

We feel deeply sorry about the lack of appropriate references supporting the statement. We added them in the revised manuscript.

3. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: No

Reviewer #2: No

(Authors’ response)

We put the raw dataset on the “figshare”. Now all dataset underlying the findings in our revised manuscript fully available in figshare (https://figshare.com, DOI: 10.6084/m9.figshare.13241267).

5. Review Comments to the Author

Specific comments:

(1) Table numbers: There is no table 3, please renumber the tables accordingly.

(Authors’ response)

We feel terribly sorry about our careless mistake. We renumbered the tables accordingly.

(2) Tables 4 and 8: It would be interesting to calculate the Firmicutes:Bacteroidetes ratio, an indicator of dysbiosis commonly used in the study of obesity and neurologic diseases. Based on your means in table 8 I believe it will be significantly different between treatments, and it is an additional indicator that will strengthen your discussion.

(Authors’ response)

We really appreciate the reviewer’s suggestion. Because of the mathematical problem (“division by zero” problem, because in some mice the percentage of Bacteroidetes was “zero”), alternatively we calculated Bacteroidetes/Firmicutes ratio. We added the data and discussion about the data with relevant references.

(3) At the beginning of page 22 (paragraph before table 10) you mention that omeprazole decreased L. murinus. Based on table 10 that is not correct, as the change is of only 0.5% and non-significant. Please correct. Accordingly, correct that L. murinus was non ameliorated but significantly increased by rebamipide.

(Authors’ response)

We appreciate the reviewer’s advice and we agree to it. It is our careless misunderstanding. We revised the sentences accordingly.

(4) Page 23: On the sentence after reference 29, please italicize L. taiwanensis.

(Authors’ response)

We appreciate the reviewer’s advice. We italicized the word, “L. taiwanensis.” We checked again the manuscript and italicized all of the bacterial names.

(5) It would be interesting to discuss the bacteriocin production further – that is a potential explanation for the reduced diversity in the omeprazole-treated mice.

(Authors’ response)

We appreciate the reviewer’s advice. In accordance with the reviewer’s advice, we discuss the bacteriocin production more profoundly.

(1) There is minimal new information in this simple study and the lack of details in M and M, lack of better control groups, and minimal readouts prevent robust evaluation.

(Authors’ response)

We deeply appreciate the editors’ and the reviewers’ patience to check my grammatical errors, typos, the lack of details in Materials and Methods, the lack of necessary references, and other careless mistakes. We would like to apologize all of them, and we corrected them.

(2) Where are the sequencing data available?

(Authors’ response)

We put the raw dataset on the “figshare”. Now all dataset underlying the findings in our revised manuscript fully available in figshare (https://figshare.com, DOI: 10.6084/m9.figshare.13241267).

(3) Please include more information in M and M section regarding animal studies. What were mice fed? How were they housed (all groups separately from shipping through study? all treatment groups in individual cages, etc...)?

(Authors’ response)

We are terribly sorry for the reviewer’s confusion about the animal experiments of the present study. We added more information about the animal experiment in Materials and Methods section.

(4) Use of isoflurane is uncecessarily repeated.

(Authors’ response)

We appreciate the reviewer’s suggestion. We revised the portion of the manuscript.

(5) What is deep euthanasia?

(Authors’ response)

We appreciate the reviewer’s suggestion. We feel sorry for simple careless mistake which confuse the reviewers. That should be “deep sedation”. We revised the portion of the manuscript.

(6) How was indomethacin administered? "Orally" is not clear. Gavage?

(Authors’ response)

We administered indomethacin to mice by gavage tube. We revised the portion.

(7) How were sections of SI that "that exhibited typical mucosal damage" selected? Was the person who selected these blinded to treatment group?

(Authors’ response)

We appreciate the reviewer’s suggestion. We added the additional information about the Materials and Methods in the revised manuscript.

(8) Were the ileal contents from the donor mice stored or given fresh? If stored, how was it stored?

(Authors’ response)

The ileal contents from the donor mice were given fresh to the recipient mice.

(9) It is stated that the ileal contents were dissolved. This seems incorrect as I do not believe luminal contents are soluble.

(Authors’ response)

We appreciate the reviewer’s suggestion. It is just our careless mistake. We revised it (dissolved>>>suspended).

(10) Suggest: ...intestinal damage was evaluated after 24 h.

(Authors’ response)

We feel terribly sorry for the grammatical error. We revised it.

(11) In the second series of experiments it is stated that "the mice were treated with antibiotics for 7 d" same antibiotics as first study? If so this should be clearly stated.

(Authors’ response)

We used the same antibiotics as first study. We revised the manuscript to describe it clearly in the Materials and Methods section.

(12) Figure 1 is helpful. Typo in figure 1 (anpicillin)

(Authors’ response)

We really appreciate the reviewer’s point out and we are terribly sorry for the typo. We revised it.

(13) How many mice in each group?

(Authors’ response)

For molecular biological analysis, we used 6-8 animals in each group and for assessment of macroscopic and microscopic experiments we used 5-7 animals. It is described in the Materials and Methods and Figure Legends.

(14) How was omep administered?

(Authors’ response)

We are terribly sorry for our immature description. Omeprazole was orally administered with gavage tube. We revised the Materials and Methods section.

(15) What do you mean "The content of the small intestinal lumen was collected immediately after sampling from the mice."?

(Authors’ response)

We are terribly sorry for our immature description. We revised it as “The content of the small intestinal lumen was collected immediately after removing the intestine from the mice”.

(16) typo - In brief, prior to DNA extraction, each sample suspended in 15 mL PBS buffer (Thermo Fisher Scientific K.K., Tokyo, Japan) was filtered ...

(Authors’ response)

We appreciate the reviewer’s indication. We revised the manuscript.

(17) In this sentence...twice using a glass or plastic bar with PBS buffer..How much PBS? what is a bar? why switch between glass and plastic?

(Authors’ response)

We feel sorry for the reviewer’s confusion. We comfirmed the truth to the technical assistant. Actually we used a plastic bar. We revised it.

(18) The complete computational analysis of 16S data is lacking. For example, how was PCoA generated?

(Authors’ response)

We really appreciate the reviewer’s suggestion. We revised the manuscript to make it clear.

(19) How was mm^2 area of evans blue staining generated (how was area of Evans blue staining quantified?

(Authors’ response)

The shape of macroscopic mucosal break was round or elliptical shape, therefore we measured the major axis and minor axis with a digital precision caliper, summed for the small intestine, and used the value as the lesion index. In accordance with the reviewer’s query, we revised the portion to describe the detail.

(20) Table 2 could be made much more visually appealing and easier to follow.

(Authors’ response)

We really appreciate the reviewer's advice. We considered the data again and we omitted the unnecessary comparison in the Table.

(21) Numerous grammatical errors throughout, far too many to correct. One example, "There is no difference in OTU number in"... present tense here. past tense elsewhere in results.

(Authors’ response)

We feel very sorry for your check of our manuscript with numerous grammatical errors. We checked the manuscript again extensively and we revised it.

(22) There are many limitations in this manuscript that should be discussed.

(Authors’ response)

We agree to the reviewer’s suggestion and all of the reviewer’s suggestion is very helpful to consider the limitations in the present study. We discuss the limitation to the present study in Discussion section.

(23) How can you be sure that the positive effects seen are not due to residual rebemipide in the ileal contents?

(Authors’ response)

We understand the intention of the reviewer’s comment. To avoid the effect of residual rebamipide in the ileal contents, we put 24 hours of washout period before transplantation and 4 days period before indomethacin challenge in the experiment protocol. Even though, it is possible that there may be small amount of residual rebamipide in the ileal content transferred with donor microbiota to the recipient mice and the residual rebamipide may exerts direct pharmacological effect on the small intestine of the recipient mice independent of modulation of small intestinal microbiota. To clear the possibility, the detection assay of residual rebamipide in the ileal content obtained from donor mice may be useful, however, it is impossible for us because of technical problem; there is no commercial assay kit and hand-made laboratory method to determine the amount of rebamipide in the ileal contents is not established. We describe the issue as the limitation to the present study in the Discussion session.

(24) Inclusion of a control group not being treated with indomethacin would greatly aid in interpretation of these data. For example, perhaps rebamipide had no effect on the microbiota but instead reduced GI injury via another mechanism and the modest differences in the microbiota were related to less inflammation and not rebamipides influences on the microbiota.

(Authors’ response)

In the analysis of the microbial composition, all animals were not treated with indomethacin to avoid the influence of indomethacin-induced mucosal damage.

(25) Please discuss beta diversity findings in more detail.

(Authors’ response)

We discuss beta diversity findings in more detail.

(26) The comparisons and where/which significant differences exist is not clear to me in tables 4-6 and 8-10.

(Authors’ response)

To make the comparisons and significant differences clear, we made the figures for the main results. Tables are also submitted as the supplementary data.

Attachment

Submitted filename: PLOS ONE Response to reviewers.docx

Click here for additional data file.^{(27.5KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0245995.r003

Decision Letter 1

Jan S Suchodolski

12 Jan 2021

Rebamipide Ameliorates Indomethacin-Induced Small Intestinal Damage and Proton Pump Inhibitor-Induced Exacerbation of this Damage by Modulation of Small Intestinal Microbiota

PONE-D-20-23408R1

Dear Dr. Tanigawa,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Jan S Suchodolski, DVM, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0245995.r004

Acceptance letter

Jan S Suchodolski

20 Jan 2021

PONE-D-20-23408R1

Rebamipide Ameliorates Indomethacin-Induced Small Intestinal Damage and Proton Pump Inhibitor-Induced Exacerbation of this Damage by Modulation of Small Intestinal Microbiota

Dear Dr. Tanigawa:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Jan S Suchodolski

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. The major bacterial composition of small intestine in mice given rebamipide or vehicle at phylum level.

(DOCX)

Click here for additional data file.^{(13KB, docx)}

S2 Table. The major bacterial composition of small intestine in mice given rebamipide or vehicle at genus level.

(DOCX)

Click here for additional data file.^{(13.2KB, docx)}

S3 Table. The major bacterial composition of small intestine in mice given rebamipide or vehicle at species level.

(DOCX)

Click here for additional data file.^{(13.9KB, docx)}

S4 Table. The major bacterial composition of small intestine in microbiota-transplanted mice administered with omeprazole at phylum level.

(DOCX)

Click here for additional data file.^{(13.5KB, docx)}

S5 Table. The major bacterial composition of small intestine in microbiota-transplanted mice administered with omeprazole at genus level.

(DOCX)

Click here for additional data file.^{(13.8KB, docx)}

S6 Table. The major bacterial composition of small intestine in microbiota-transplanted mice administered with omeprazole at species level.

(DOCX)

Click here for additional data file.^{(14.8KB, docx)}

Attachment

Submitted filename: PLOS ONE Response to reviewers.docx

Click here for additional data file.^{(27.5KB, docx)}

Data Availability Statement

[pone.0245995.ref001] 1.Iddan G, Meron G, Glukhovsky A, Swain P. Wireless capsule endoscopy. Nature. 2000;405(6785):417 Epub 2000/06/06. 10.1038/35013140 . [DOI] [PubMed] [Google Scholar]

[pone.0245995.ref002] 2.Watanabe T, Fujiwara Y, Chan FKL. Current knowledge on non-steroidal anti-inflammatory drug-induced small-bowel damage: a comprehensive review. Journal of gastroenterology. 2020;55(5):481–95. Epub 2019/12/23. 10.1007/s00535-019-01657-8 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0245995.ref003] 3.Graham DY, Opekun AR, Willingham FF, Qureshi WA. Visible small-intestinal mucosal injury in chronic NSAID users. Clinical gastroenterology and hepatology: the official clinical practice journal of the American Gastroenterological Association. 2005;3(1):55–9. Epub 2005/01/13. 10.1016/s1542-3565(04)00603-2 . [DOI] [PubMed] [Google Scholar]

[pone.0245995.ref004] 4.Goldstein JL, Eisen GM, Lewis B, Gralnek IM, Aisenberg J, Bhadra P, et al. Small bowel mucosal injury is reduced in healthy subjects treated with celecoxib compared with ibuprofen plus omeprazole, as assessed by video capsule endoscopy. Alimentary pharmacology & therapeutics. 2007;25(10):1211–22. Epub 2007/04/25. 10.1111/j.1365-2036.2007.03312.x . [DOI] [PubMed] [Google Scholar]

[pone.0245995.ref005] 5.Maiden L, Thjodleifsson B, Theodors A, Gonzalez J, Bjarnason I. A quantitative analysis of NSAID-induced small bowel pathology by capsule enteroscopy. Gastroenterology. 2005;128(5):1172–8. Epub 2005/05/12. 10.1053/j.gastro.2005.03.020 . [DOI] [PubMed] [Google Scholar]

[pone.0245995.ref006] 6.Sugimori S, Watanabe T, Tabuchi M, Kameda N, Machida H, Okazaki H, et al. Evaluation of small bowel injury in patients with rheumatoid arthritis by capsule endoscopy: effects of anti-rheumatoid arthritis drugs. Digestion. 2008;78(4):208–13. Epub 2009/01/15. 10.1159/000190403 . [DOI] [PubMed] [Google Scholar]

[pone.0245995.ref007] 7.Shimada S, Watanabe T, Nadatani Y, Otani K, Taira K, Hosomi S, et al. Clinical factors associated with positive capsule endoscopy findings in patients with obscure gastrointestinal bleeding: a single-center study. Scandinavian journal of gastroenterology. 2017;52(11):1219–23. Epub 2017/07/12. 10.1080/00365521.2017.1349174 . [DOI] [PubMed] [Google Scholar]

[pone.0245995.ref008] 8.Watanabe T, Tanigawa T, Nadatani Y, Nagami Y, Sugimori S, Okazaki H, et al. Risk factors for severe nonsteroidal anti-inflammatory drug-induced small intestinal damage. Digestive and liver disease: official journal of the Italian Society of Gastroenterology and the Italian Association for the Study of the Liver. 2013;45(5):390–5. Epub 2013/01/22. 10.1016/j.dld.2012.12.005 . [DOI] [PubMed] [Google Scholar]

[pone.0245995.ref009] 9.Lanza FL, Chan FK, Quigley EM. Guidelines for prevention of NSAID-related ulcer complications. Am J Gastroenterol. 2009;104(3):728–38. Epub 2009/02/26. 10.1038/ajg.2009.115 . [DOI] [PubMed] [Google Scholar]

[pone.0245995.ref010] 10.Endo H, Sakai E, Taniguchi L, Kessoku T, Komiya Y, Ezuka A, et al. Risk factors for small-bowel mucosal breaks in chronic low-dose aspirin users: data from a prospective multicenter capsule endoscopy registry. Gastrointestinal endoscopy. 2014;80(5):826–34. Epub 2014/05/17. 10.1016/j.gie.2014.03.024 . [DOI] [PubMed] [Google Scholar]

[pone.0245995.ref011] 11.Washio E, Esaki M, Maehata Y, Miyazaki M, Kobayashi H, Ishikawa H, et al. Proton Pump Inhibitors Increase Incidence of Nonsteroidal Anti-Inflammatory Drug-Induced Small Bowel Injury: A Randomized, Placebo-Controlled Trial. Clinical gastroenterology and hepatology: the official clinical practice journal of the American Gastroenterological Association. 2016;14(6):809–15.e1. Epub 2015/11/06. 10.1016/j.cgh.2015.10.022 . [DOI] [PubMed] [Google Scholar]

[pone.0245995.ref012] 12.Satoh H, Amagase K, Takeuchi K. Exacerbation of nonsteroidal anti-inflammatory drug-induced small intestinal lesions by antisecretory drugs in rats: the role of intestinal motility. The Journal of pharmacology and experimental therapeutics. 2012;343(2):270–7. Epub 2012/08/03. 10.1124/jpet.112.197475 . [DOI] [PubMed] [Google Scholar]

[pone.0245995.ref013] 13.Wallace JL, Syer S, Denou E, de Palma G, Vong L, McKnight W, et al. Proton pump inhibitors exacerbate NSAID-induced small intestinal injury by inducing dysbiosis. Gastroenterology. 2011;141(4):1314–22, 22.e1–5. Epub 2011/07/13. 10.1053/j.gastro.2011.06.075 . [DOI] [PubMed] [Google Scholar]

[pone.0245995.ref014] 14.Nadatani Y, Watanabe T, Suda W, Nakata A, Matsumoto Y, Kosaka S, et al. Gastric acid inhibitor aggravates indomethacin-induced small intestinal injury via reducing Lactobacillus johnsonii. Scientific reports. 2019;9(1):17490 Epub 2019/11/27. 10.1038/s41598-019-53559-7 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0245995.ref015] 15.DeGruttola AK, Low D, Mizoguchi A, Mizoguchi E. Current Understanding of Dysbiosis in Disease in Human and Animal Models. Inflammatory bowel diseases. 2016;22(5):1137–50. Epub 2016/04/14. 10.1097/MIB.0000000000000750 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0245995.ref016] 16.Blackler RW, De Palma G, Manko A, Da Silva GJ, Flannigan KL, Bercik P, et al. Deciphering the pathogenesis of NSAID enteropathy using proton pump inhibitors and a hydrogen sulfide-releasing NSAID. American journal of physiology Gastrointestinal and liver physiology. 2015;308(12):G994–1003. Epub 2015/04/18. 10.1152/ajpgi.00066.2015 . [DOI] [PubMed] [Google Scholar]

[pone.0245995.ref017] 17.Zhang S, Qing Q, Bai Y, Mao H, Zhu W, Chen Q, et al. Rebamipide helps defend against nonsteroidal anti-inflammatory drugs induced gastroenteropathy: a systematic review and meta-analysis. Digestive diseases and sciences. 2013;58(7):1991–2000. Epub 2013/03/05. 10.1007/s10620-013-2606-0 . [DOI] [PubMed] [Google Scholar]

[pone.0245995.ref018] 18.Watanabe T, Takeuchi T, Handa O, Sakata Y, Tanigawa T, Shiba M, et al. A multicenter, randomized, double-blind, placebo-controlled trial of high-dose rebamipide treatment for low-dose aspirin-induced moderate-to-severe small intestinal damage. PloS one. 2015;10(4):e0122330 Epub 2015/04/16. 10.1371/journal.pone.0122330 Pharmaceutical Co., Ltd and Takeda Pharmaceutical Co., Ltd. KH has received research grants and speaker’s fee from Otsuka Pharmaceutical Co., Ltd. TA has received research grants from Eisai Co., Ltd. and Otsuka Pharmaceutical Co., Ltd. Otsuka Pharmaceutical Co., Ltd provided rebamipide and identical placebo. There are no further patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0245995.ref019] 19.Kurokawa S, Katsuki S, Fujita T, Saitoh Y, Ohta H, Nishikawa K, et al. A randomized, double-blinded, placebo-controlled, multicenter trial, healing effect of rebamipide in patients with low-dose aspirin and/or non-steroidal anti-inflammatory drug induced small bowel injury. Journal of gastroenterology. 2014;49(2):239–44. Epub 2013/04/19. 10.1007/s00535-013-0805-2 . [DOI] [PubMed] [Google Scholar]

[pone.0245995.ref020] 20.Mizukami K, Murakami K, Abe T, Inoue K, Uchida M, Okimoto T, et al. Aspirin-induced small bowel injuries and the preventive effect of rebamipide. World journal of gastroenterology. 2011;17(46):5117–22. Epub 2011/12/16. 10.3748/wjg.v17.i46.5117 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0245995.ref021] 21.Fujimori S, Takahashi Y, Gudis K, Seo T, Ehara A, Kobayashi T, et al. Rebamipide has the potential to reduce the intensity of NSAID-induced small intestinal injury: a double-blind, randomized, controlled trial evaluated by capsule endoscopy. Journal of gastroenterology. 2011;46(1):57–64. Epub 2010/10/07. 10.1007/s00535-010-0332-3 . [DOI] [PubMed] [Google Scholar]

[pone.0245995.ref022] 22.Tanigawa T, Watanabe T, Otani K, Nadatani Y, Ohkawa F, Sogawa M, et al. Rebamipide inhibits indomethacin-induced small intestinal injury: possible involvement of intestinal microbiota modulation by upregulation of alpha-defensin 5. European journal of pharmacology. 2013;704(1–3):64–9. Epub 2013/02/23. 10.1016/j.ejphar.2013.02.010 . [DOI] [PubMed] [Google Scholar]

[pone.0245995.ref023] 23.Imaeda H, Fujimoto T, Takahashi K, Kasumi E, Fujiyama Y, Andoh A. Terminal-restriction fragment length polymorphism (T-RFLP) analysis for changes in the gut microbiota profiles of indomethacin- and rebamipide-treated mice. Digestion. 2012;86(3):250–7. Epub 2012/09/12. 10.1159/000341508 . [DOI] [PubMed] [Google Scholar]

[pone.0245995.ref024] 24.Kurata S, Nakashima T, Osaki T, Uematsu N, Shibamori M, Sakurai K, et al. Rebamipide protects small intestinal mucosal injuries caused by indomethacin by modulating intestinal microbiota and the gene expression in intestinal mucosa in a rat model. Journal of clinical biochemistry and nutrition. 2015;56(1):20–7. Epub 2015/04/04. 10.3164/jcbn.14-67 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0245995.ref025] 25.Shimada S, Tanigawa T, Watanabe T, Nakata A, Sugimura N, Itani S, et al. Involvement of gliadin, a component of wheat gluten, in increased intestinal permeability leading to non-steroidal anti-inflammatory drug-induced small-intestinal damage. PloS one. 2019;14(2):e0211436 Epub 2019/02/21. 10.1371/journal.pone.0211436 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0245995.ref026] 26.Kim SW, Suda W, Kim S, Oshima K, Fukuda S, Ohno H, et al. Robustness of gut microbiota of healthy adults in response to probiotic intervention revealed by high-throughput pyrosequencing. DNA research: an international journal for rapid publication of reports on genes and genomes. 2013;20(3):241–53. Epub 2013/04/11. 10.1093/dnares/dst006 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0245995.ref027] 27.Suzuki Y, Nishijima S, Furuta Y, Yoshimura J, Suda W, Oshima K, et al. Long-read metagenomic exploration of extrachromosomal mobile genetic elements in the human gut. Microbiome. 2019;7(1):119 Epub 2019/08/29. 10.1186/s40168-019-0737-z . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0245995.ref028] 28.Said HS, Suda W, Nakagome S, Chinen H, Oshima K, Kim S, et al. Dysbiosis of salivary microbiota in inflammatory bowel disease and its association with oral immunological biomarkers. DNA research: an international journal for rapid publication of reports on genes and genomes. 2014;21(1):15–25. Epub 2013/09/10. 10.1093/dnares/dst037 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0245995.ref029] 29.Iwasawa K, Suda W, Tsunoda T, Oikawa-Kawamoto M, Umetsu S, Takayasu L, et al. Dysbiosis of the salivary microbiota in pediatric-onset primary sclerosing cholangitis and its potential as a biomarker. Scientific reports. 2018;8(1):5480 Epub 2018/04/05. 10.1038/s41598-018-23870-w . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0245995.ref030] 30.Lozupone C, Lladser ME, Knights D, Stombaugh J, Knight R. UniFrac: an effective distance metric for microbial community comparison. The ISME journal. 2011;5(2):169–72. Epub 2010/09/10. 10.1038/ismej.2010.133 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0245995.ref031] 31.Magne F, Gotteland M, Gauthier L, Zazueta A, Pesoa S, Navarrete P, et al. The Firmicutes/Bacteroidetes Ratio: A Relevant Marker of Gut Dysbiosis in Obese Patients? Nutrients. 2020;12(5). Epub 2020/05/23. 10.3390/nu12051474 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0245995.ref032] 32.Pellegrini S, Sordi V, Bolla AM, Saita D, Ferrarese R, Canducci F, et al. Duodenal Mucosa of Patients With Type 1 Diabetes Shows Distinctive Inflammatory Profile and Microbiota. The Journal of clinical endocrinology and metabolism. 2017;102(5):1468–77. Epub 2017/03/23. 10.1210/jc.2016-3222 . [DOI] [PubMed] [Google Scholar]

[pone.0245995.ref033] 33.Cosorich I, Dalla-Costa G, Sorini C, Ferrarese R, Messina MJ, Dolpady J, et al. High frequency of intestinal T(H)17 cells correlates with microbiota alterations and disease activity in multiple sclerosis. Science advances. 2017;3(7):e1700492 Epub 2017/07/15. 10.1126/sciadv.1700492 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0245995.ref034] 34.Mosca A, Leclerc M, Hugot JP. Gut Microbiota Diversity and Human Diseases: Should We Reintroduce Key Predators in Our Ecosystem? Frontiers in microbiology. 2016;7:455 Epub 2016/04/12. 10.3389/fmicb.2016.00455 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0245995.ref035] 35.Alipour M, Zaidi D, Valcheva R, Jovel J, Martinez I, Sergi C, et al. Mucosal Barrier Depletion and Loss of Bacterial Diversity are Primary Abnormalities in Paediatric Ulcerative Colitis. Journal of Crohn’s & colitis. 2016;10(4):462–71. Epub 2015/12/15. 10.1093/ecco-jcc/jjv223 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0245995.ref036] 36.Lo Presti A, Zorzi F, Del Chierico F, Altomare A, Cocca S, Avola A, et al. Fecal and Mucosal Microbiota Profiling in Irritable Bowel Syndrome and Inflammatory Bowel Disease. Frontiers in microbiology. 2019;10:1655 Epub 2019/08/06. 10.3389/fmicb.2019.01655 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0245995.ref037] 37.Holler E, Butzhammer P, Schmid K, Hundsrucker C, Koestler J, Peter K, et al. Metagenomic analysis of the stool microbiome in patients receiving allogeneic stem cell transplantation: loss of diversity is associated with use of systemic antibiotics and more pronounced in gastrointestinal graft-versus-host disease. Biology of blood and marrow transplantation: journal of the American Society for Blood and Marrow Transplantation. 2014;20(5):640–5. Epub 2014/02/05. 10.1016/j.bbmt.2014.01.030 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0245995.ref038] 38.Kumari R, Palaniyandi S, Hildebrandt GC. Microbiome: An Emerging New Frontier in Graft-Versus-Host Disease. Digestive diseases and sciences. 2019;64(3):669–77. Epub 2018/12/14. 10.1007/s10620-018-5369-9 . [DOI] [PubMed] [Google Scholar]

[pone.0245995.ref039] 39.Peled JU, Gomes ALC, Devlin SM, Littmann ER, Taur Y, Sung AD, et al. Microbiota as Predictor of Mortality in Allogeneic Hematopoietic-Cell Transplantation. The New England journal of medicine. 2020;382(9):822–34. Epub 2020/02/27. 10.1056/NEJMoa1900623 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0245995.ref040] 40.Staffas A, Burgos da Silva M, van den Brink MR. The intestinal microbiota in allogeneic hematopoietic cell transplant and graft-versus-host disease. Blood. 2017;129(8):927–33. Epub 2016/12/13. 10.1182/blood-2016-09-691394 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0245995.ref041] 41.Raj AS, Shanahan ER, Tran CD, Bhat P, Fletcher LM, Vesey DA, et al. Dysbiosis of the Duodenal Mucosal Microbiota Is Associated With Increased Small Intestinal Permeability in Chronic Liver Disease. Clinical and translational gastroenterology. 2019;10(8):e00068 Epub 2019/08/03. 10.14309/ctg.0000000000000068 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0245995.ref042] 42.Takashima S, Tanaka F, Kawaguchi Y, Usui Y, Fujimoto K, Nadatani Y, et al. Proton pump inhibitors enhance intestinal permeability via dysbiosis of gut microbiota under stressed conditions in mice. Neurogastroenterology and motility: the official journal of the European Gastrointestinal Motility Society. 2020;32(7):e13841 Epub 2020/04/23. 10.1111/nmo.13841 . [DOI] [PubMed] [Google Scholar]

[pone.0245995.ref043] 43.Imhann F, Bonder MJ, Vich Vila A, Fu J, Mujagic Z, Vork L, et al. Proton pump inhibitors affect the gut microbiome. Gut. 2016;65(5):740–8. Epub 2015/12/15. 10.1136/gutjnl-2015-310376 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0245995.ref044] 44.Liu P, Bian Y, Liu T, Zhong J, Zhong Y, Zhuang S, et al. Huai hua san alleviates dextran sulphate sodium-induced colitis and modulates colonic microbiota. Journal of ethnopharmacology. 2020;259:112944 Epub 2020/05/11. 10.1016/j.jep.2020.112944 . [DOI] [PubMed] [Google Scholar]

[pone.0245995.ref045] 45.Kim SW, Ha YJ, Bang KH, Lee S, Yeo JH, Yang HS, et al. Potential of Bacteriocins from Lactobacillus taiwanensis for Producing Bacterial Ghosts as a Next Generation Vaccine. Toxins. 2020;12(7). Epub 2020/07/08. 10.3390/toxins12070432 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0245995.ref046] 46.Lopetuso LR, Giorgio ME, Saviano A, Scaldaferri F, Gasbarrini A, Cammarota G. Bacteriocins and Bacteriophages: Therapeutic Weapons for Gastrointestinal Diseases? International journal of molecular sciences. 2019;20(1). Epub 2019/01/10. 10.3390/ijms20010183 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0245995.ref047] 47.Nilsen T, Swedek I, Lagenaur LA, Parks TP. Novel Selective Inhibition of Lactobacillus iners by Lactobacillus-Derived Bacteriocins. Applied and environmental microbiology. 2020;86(20). Epub 2020/08/18. 10.1128/AEM.01594-20 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0245995.ref048] 48.Todorov SD, Kang HJ, Ivanova IV, Holzapfel WH. Bacteriocins From LAB and Other Alternative Approaches for the Control of Clostridium and Clostridiodes Related Gastrointestinal Colitis. Frontiers in bioengineering and biotechnology. 2020;8:581778 Epub 2020/10/13. 10.3389/fbioe.2020.581778 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0245995.ref049] 49.Isani M, Bell BA, Delaplain PT, Bowling JD, Golden JM, Elizee M, et al. Lactobacillus murinus HF12 colonizes neonatal gut and protects rats from necrotizing enterocolitis. PloS one. 2018;13(6):e0196710 Epub 2018/06/23. 10.1371/journal.pone.0196710 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0245995.ref050] 50.Tang C, Kamiya T, Liu Y, Kadoki M, Kakuta S, Oshima K, et al. Inhibition of Dectin-1 Signaling Ameliorates Colitis by Inducing Lactobacillus-Mediated Regulatory T Cell Expansion in the Intestine. Cell host & microbe. 2015;18(2):183–97. Epub 2015/08/14. 10.1016/j.chom.2015.07.003 . [DOI] [PubMed] [Google Scholar]

[pone.0245995.ref051] 51.Pan F, Zhang L, Li M, Hu Y, Zeng B, Yuan H, et al. Predominant gut Lactobacillus murinus strain mediates anti-inflammaging effects in calorie-restricted mice. Microbiome. 2018;6(1):54 Epub 2018/03/23. 10.1186/s40168-018-0440-5 . [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Rebamipide ameliorates indomethacin-induced small intestinal damage and proton pump inhibitor-induced exacerbation of this damage by modulation of small intestinal microbiota

Tetsuya Tanigawa

Toshio Watanabe

Akira Higashimori

Sunao Shimada

Hiroyuki Kitamura

Takuya Kuzumoto

Yuji Nadatani

Koji Otani

Shusei Fukunaga

Shuhei Hosomi

Fumio Tanaka

Noriko Kamata

Yasuaki Nagami

Koichi Taira

Masatsugu Shiba

Wataru Suda

Masahira Hattori

Yasuhiro Fujiwara

Roles

Abstract

Introduction

Materials and methods

Induction of small intestinal damage by indomethacin in mice

Histological evaluation of small intestinal damage

Experimental groups and transplantation of small intestinal microbiota

Fig 1. Schemas of design of experimental protocol.

Sample collection and DNA extraction

16S rRNA gene sequencing

Statistical analysis

Results

The effects of transplantation of rebamipide-modulated small intestinal microbiota on indomethacin-induced small intestinal damage

Fig 2. Small intestinal damage after indomethacin administration and the inhibitory effects of rebamipide-modulated small intestinal microbiota on indomethacin-induced small intestinal damage.

The influence of omeprazole on small intestinal damage and the inhibitory effect of transplantation of the rebamipide-modulated small intestinal microbiota on exacerbation of indomethacin-induced small intestinal damage by omeprazole

Fig 3. The deleterious effect of omeprazole and the inhibitory effects of rebamipide-modulated small intestinal microbiota on indomethacin-induced small intestinal damage.

The effect of rebamipide on the composition of the small intestinal microbiota in mice

Table 1. α-diversity, measured by operational taxonomic unit (OTU) number, Chao1 index, ACE index, and Shannon’s diversity index of small intestinal lumen contents in mice given rebamipide or vehicle.

Fig 4. Principal coordinate analysis (PCoA) of unweighted and weighted UniFrac distances of small intestinal microbiota.

Table 2. PERMANOVA of UniFrac distance in small intestine.

Fig 5.

Influence of omeprazole on the composition of small intestinal microbiota in mice transplanted with the control small intestinal microbiota and mice transplanted with rebamipide-modulated small intestinal microbiota

Table 3. α-diversity, measured by operational taxonomic unit (OTU) number, Chao1 index, ACE index, and Shannon’s diversity index of small intestinal lumen contents in microbiota-transplanted mice administered with omeprazole.

Fig 6.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Jan S Suchodolski

Roles

Author response to Decision Letter 0

Decision Letter 1

Jan S Suchodolski

Roles

Acceptance letter

Jan S Suchodolski

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases