Ingestion of Low Dose Pyroglutamyl Leucine Improves Dextran Sulfate

A dose of 0.1 mg/kg body weight/day showed the most significant improvement. The pyroGlu-Leu concentration was significantly increased 24 h after oral...
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Ingestion of Low Dose Pyroglutamyl Leucine Improves Dextran Sulfate Sodium-Induced Colitis and Intestinal Microbiota in Mice Sayori Wada, Kenji Sato, Ryoko Ohta, Eri Wada, Yukiho Bou, Miki Fujiwara, Tamami Kiyono, Eun Young Park, Wataru Aoi, Tomohisa Takagi, Yuji Naito, and Toshikazu Yoshikawa J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf402515a • Publication Date (Web): 21 Aug 2013 Downloaded from http://pubs.acs.org on August 26, 2013

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

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Ingestion of Low Dose Pyroglutamyl Leucine Improves Dextran Sulfate

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SodiumSodium-Induced Colitis and Intestinal Microbiota in Mice

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Running title: Improvement of colitis by pyroGlu-Leu

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Sayori Wada†, Kenji Sato†*, Ryoko Ohta†, Eri Wada†, Yukiho Bou†, Miki

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Fujiwara†, Tamami Kiyono†, Eun Young Park†, Wataru Aoi†, Tomohisa

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Takagi¶, Yuji Naito¶, Toshikazu Yoshikawa¶

9

†Division

of

Applied

Life

Sciences, Graduate

School

of

Life

and

10

Environmental Sciences, Kyoto Prefectural University, Shimogamo, Kyoto,

11

606 8522, Japan,

12

¶Molecular

13

of Medicine, Kajii-cho, Kyoto, 602 8566, Japan

Gastroenterology and Hepatology, Kyoto Prefectural University

14 15

* Corresponding author. TEL/FAX: 81 75 723 3503; E-mail:[email protected]

16 17 18 19

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ABSTRACT

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Inflammatory bowel diseases (IBD) are based on chronic inflammation in

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the gastrointestinal tract. We previously found anti-inflammatory peptide

23

pyroGlu-Leu in the enzymatic hydrolysate of wheat gluten. The objective of

24

present study is to elucidate improvement of colitis by oral administration of

25

pyroGlu-Leu in animal model. Acute colitis was induced by dextran sulfate

26

sodium

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administrated by oral gavage for 7 days. A dose of 0.1 mg/kg body weight/day

28

showed the most significant improvement. The pyroGlu-Leu concentration

29

was significantly increased 24 h after oral administration both in the small

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intestine and the colon compared with the baseline. It was 20-fold higher in

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the small intestine than the colon. Administration of pyroGlu-Leu

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normalized population of Bacteroidetes and Firmicutes in the colon. These

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results indicate that pyroGlu-Leu has a potential therapeutic effect against

34

IBD at a practical dose.

(DSS),

and

various

concentrations

of

pyroGlu-Leu

35 36

Keywords; IBD, pyroGlu-Leu, gluten peptide, microbiota, colitis.

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INTRODUCTION

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Inflammatory bowel diseases (IBD) are relapsing-emitting conditions characterized

40

by chronic inflammation in the gastrointestinal tract, which result in diarrhea and

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abdominal pain. The major types of IBD are Crohn’s disease (CD) and ulcerative colitis

42

(UC). UC is limited to the colon. On the other hand, CD can occur in any parts of the

43

gastrointestinal tract.1, 2 IBD affects people of all ages, but usually begins before age

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30.3 In the U.S.A., the mean annual costs for CD and UC are estimated as $8,265 and

45

$5,066 per patient, respectively.4

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It has been suggested that early appendectomy5 reduces the risk of IBD, and the use

47

of some non-steroidal anti-inflammatory drugs (NSAIDs)6 increases the risk. A smoking

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habit also increases the risk of CD, but has a protective effect on the development of

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UC.7-9 The incidence of IBD in Asia is far less than in Western Europe and North

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America.10 However, the incidence and prevalence of IBD in Asia has been increasing

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in the last two decades.11, 12 In the same period, eating habits in Asia have changed. For

52

example, the consumption of traditional fermented food, such as soy sauce and soy

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paste, have decreased in Japan.13 These facts suggest that in addition to genetic and

54

environmental factors, life-style might affect the development of IBD. In fact, it has

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been reported that a high consumption of sweets increases the risk of both UC and

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CD.14 On the other hand, increasing numbers of studies have suggested that some

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nutrients or food components exert beneficial effects on IBD beyond their conventional

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nutritional values. Epidemiological studies have demonstrated that the consumption of

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foods rich in vitamin C decreases the risk of IBD.14 It has been also demonstrated that

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the supplementation of some amino acids, such as Gln, Cys, Ser, Thr, and Pro attenuate

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IBD in animal models.15, 16 In addition, the supplementation of cheese whey protein 16,

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casein macropeptide17, lysozyme18, and soy protein hydrolysate19 also attenuate IBD in

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animal models. These data indicate that specific food proteins or peptides have a

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potential therapeutic effect against IBD, possibly due to the provision of protective

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intestinal amino acids or the direct modulation of pro-inflammatory cytokines. However,

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a relatively large dose of these protein/peptides is required to exert any beneficial

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activity against IBD in animal models. In addition, some human trials revealed that a

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Gln-enriched polymeric diet offers no advantage over a standard low-Gln diet in the

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treatment of active CD.20

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Alternatively, the present authors reported that the ingestion of an enzymatic

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hydrolysate of wheat gluten can attenuate chronic and acute hepatitis in rat models21, 22

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and also that it can decrease serum amino transferase activity in patients suffering from

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hepatitis.23 Very recently, a peptide, pyroglutamyl leucine (pyroGlu-Leu), was identified

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as one of the hepatoprotective peptides in the wheat gluten hydrolysate.23 Pyroglutamyl

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peptides are induced from peptides with glutaminyl residue at the amino terminal

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position in the water phase during food processing and are resist to digestion by amino

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peptidase, due to the lack of an amino-group.24 As well as IBD, inflammation plays a

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significant role in the development of hepatitis. Therefore, we hypothesized that

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pyroGlu-Leu might have a positive effect on colitis.

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The objective of the present study was to elucidate the potential of pyroGlu-Leu to control IBD by using dextran sulfate sodium-induced colitis in mice.

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MATERIALS AND METHODS

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Reagents. Dextran sulfate sodium (DSS, average mol. wt. 8,000) was purchased from

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Seikagaku (Tokyo, Japan). Pyroglutamic acid (pyroGlu) and acetonitorile (HPLC grade)

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were obtained from Wako Pure Chemicals (Osaka, Japan). The resin which was linked

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to

88

Laboratories (Kyoto, Japan).

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Preparation of PyroGlu-Leu. PyroGlu-Leu was synthesized by a manual liquid-phase

90

method as described previously.22

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automatic solid-phase method employing a 9-Fluorenylmethoxycarbonyl (FMOC)

9-Fluorenylmethoxycarbonyl

(FMOC)-leucine

was

obtained

from

HiPep

In some cases, it was also synthesized by an

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strategy using a PSSM-8 peptide synthesizer (Shimadzu, Kyoto, Japan). Free pyroGlu

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was reacted to FMOC-Leu-resin.

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DSS-induced Colitis in Mice. Seven-week-old male C57BL/6 mice were purchased

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from Shimizu Laboratory Supplies (Osaka, Japan). The mice were caged, 6-7 animals in

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each cage, in a room kept at 18-24ºC and 40–70% relative humidity, with a 12-h

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light/dark cycle. The mice were allowed free access to food and drinking water, and fed

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a certified CRF-1 diet (Oriental Yeast, Kyoto, Japan) during a one-week acclimatization

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period. All animals were treated and cared for in accordance with the National Institutes

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of Health's (NIH) guidelines for the use of experimental animals. All experimental

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procedures were approved by the Animal Care Committee of Kyoto Prefectural

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University of Medicine (M23-37, M24-25, Kyoto, Japan).

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Acute colitis was induced by oral administration of 2.5 or 3.0% (w/v) DSS dissolved

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into drinking water, for 7 days according to the method employed in previous studies.25,

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26

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experiments for the induction of the same grade of inflammation in each individual

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experiment, as the severity of DSS-induced colitis in mice depended on the animal lots.

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The mice were divided into 8 groups: a sham group (without DSS, no pyroGlu-Leu;

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sham 0), a sham group treated with 0.1 mg/kg pyroGlu-Leu (without DSS; sham 0.1), a

The concentration of DSS (2.5% or 3.0%) was determined by preliminary

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DSS-induced colitis group as a control, and a DSS-induced colitis group treated with

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0.01, 0.05, 0.1, 0.5 and 1.0 mg/kg pyroGlu-Leu (Table 1). Synthetic pyroGlu-Leu

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dissolved in PBS was administered to the mice by oral gavage during the entire colitis

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induction period. For the DSS-induced colitis groups, same experiments were carried

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out duplicate to 6 times.

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The body weight of each mouse was measured on day 0, 3, 5 and 7. The mice were

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sacrificed on day 7 and the entire colon was removed from the cecum to the anus. The

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colon length was measured as an indirect marker of inflammation, immediately after

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colon resection. The inner contents of the colon from 3 mice in each group were

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collected by washing with 500 µL of ice-cold PBS per mice, and 3 samples in the same

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group were mixed together for microbiota analysis. Then, colon tissue samples were

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fixed in 10% buffered formalin for histological examination.

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Evaluation of Colitis Severity. We evaluated the colitis severity on the basis of body

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weight, colon length, and the macroscopic and microscopic observations of stool and

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colon, respectively. The disease activity index (DAI) score was determined by the

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method employed in previous studies

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or weight gain; 1, 1–5% loss; 2, 5–10% loss; 3, 10–20% loss; and 4, more than 20%

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loss), stool consistency (0, normal; 1, mild loose; 2, loose; 3, mild diarrhea; and 4,

26, 27

, using 5 grades of weight change (0, no loss

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diarrhea), and stool bleeding (0, negative; 1, light bleeding; 2, mild bleeding; 3, severe

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bleeding; and 4, entire bleeding). The DAI score was obtained using an average of each

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criterion. Colon sections were prepared and stained with hematoxylin and eosin (H&E).

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Determination of PyroGlu-Leu.

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and colon were collected day 7 from the normal mice and DSS-induced colitis mice that

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received vehicle or 0.1 mg/kg pyroGlu-Leu (n=3 for each group). To examine time

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course of absorption of pyroGlu-Leu, the normal mice were administered with 0.1

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mg/kg of synthetic pyroGlu-Leu dissolved in PBS by oral gavage after one night fasting,

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and then sacrificed before and 0.5, 1, 3, 6, and 24 h after the administration, respectively

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(n=3 except baseline which were n=4). The small intestine and colon were

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thoroughly washed 3-5 times by PBS, and added to the same amounts of PBS and

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homogenized. Then, a two-fold amount of 10% TCA was added, and the samples were

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re-homogenized, followed by centrifugation at 13,000 rpm. The supernatants were

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collected. PyroGlu-Leu in the TCA extract was separated from the amino acids and

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other peptides having an amino group by solid phase extraction using a strong cation

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exchanger (AG50W-×8, hydrogen form, 100-200 mesh, Bio-Rad Laboratories, Hercules,

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CA)22. The non-absorbed fraction was collected. Before injection to LC-MS/MS,

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samples were clarified by passing through a filter (0.45 µm, column guard, Millipor,

For detemination of pyroGlu-Leu, small intestine

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Billerica, MA). PyroGlu-Leu was determined by LC-MS/MS in multiple reaction

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monitoring (MRM) mode using the Q-TRAP 3200 (AB SCIEX, Foster City, CA) as

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described previously.22

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Microbiota Analysis. The inner contents of the colon were collected from 3 mice in

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each group and mixed them together for microbiota analysis. Population of Bacteroiditis

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and Firmicutes were quantified on the basis of amplification of genome DNA coding

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16S ribosomal RNA by polymerase chain reaction by using group-specific primers.28-30

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For detection of Bacteroidetes, Bact934F

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and Bact1060R

(AGCTGACGACAACCATGCAG) were used.

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Firm934F

(GGAGYATGTGGTTTAATTCGAAGCA)

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(AGCTGACGACAACCATGCAC) were used. DNA was extracted using by QIAamp

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DNA StoolMini Kit (Qiagen, Venlo, Netherland) according to the instruction manual.

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Real-time PCR was done using a LightCycler 480 (Roche Applied Science, Manheim

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German) according to SYBR Green I Master Protocol. DNA extraction and PCR

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analysis were carried out in Primary Cell Division of Cosmo Bio (Sapporo, Japan).

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Statistical Analysis. After assignment to the 8 groups, the homogeneity of variances

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and the mean of the body weight were confirmed by Bartlett’s test and one-way

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ANOVA, respectively. Results were presented as mean ± SEM. For body weight change

(GGARCATGTGGTTTAATTCGATGAT)

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and DAI score, we compared the values between groups using Kruskal-Wallis analysis.

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Statistical significance (P < 0.05) was analyzed by one-way analysis of variance

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followed by Steel’s test for multiple comparisons. For the concentration of pyroGlu-Leu,

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data were subjected to one-way ANOVA with Dunnett’s multiple comparison of means

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test. Differences showing P < 0.05 were considered significant. Statistical analyses were

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performed using Excel 2010 (Microsoft, Redmond, WA) with the add-in software

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Ekuseru-Toukei 2010 (Social Survey Research Information, Tokyo Japan).

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RESULTS

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Improvement of DSS-Induced Colitis by PyroGlu-Leu. There were no significant

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differences in the consumption of the water containing DSS among the groups. DSS

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treatment induced significant weight loss, shortened colon length, diarrhea, and rectal

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bleeding, and an increase in the DAI score, compared with the non-DSS treatment

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group (Figure 1A, B). As shown in Figure 2, DSS treatment induced extensive

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infiltration of inflammatory cells reaching the muscularis mucosa and thickening of the

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mucosa with edema. Severe inflammation was then induced by the DSS treatment. The

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supplementation of pyroGlu-Leu at a dose of 0.1 mg/kg body weight significantly

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attenuated the DSS-induced weight loss and improved the DAI score. In addition, as

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shown in Figure 2C, the supplementation of pyroGlu-Leu in this dose improved colonic

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inflammation. On the other hand, pyroGlu-Leu treatment at higher doses, such as 0.5

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mg/kg or 1.0 mg/kg did not show the positive effects on the DSS-induced colitis. The

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administration of pyroGlu-Leu to normal mice (sham 0.1) did not show any significant

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effect on the parameters examined in the present study, compared with sham 0 mice

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(Figure 1B).

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PyroGlu-Leu in Intestines. The time course of absorption of pyroGlu-Leu into the

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intestines was first examined in the normal mice.

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pyroGlu-Leu was detected in the TCA extract of the washed small intestine even before

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the administration. In all cases, intestines were washed thoroughly with PBS. The final

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washing effluents contained pyroGlu-Leu less than 10% of that in the TCA extracts of

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the washed organs (data not shown), which indicates most of free pyroGlu-Leu in the

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intestinal tract could be washed away. Therefore, the pyroGlu-Leu in the TCA extract

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can be considered as pyroGlu-Leu in the cell and matrix of intestines. The contents of

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pyroGlu-Leu in small intestine tended to increase 30 and 60 min after the administration

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of pyroGlu-Leu at 0.1 mg/kg body weight, but no significant differences were shown.

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With administration at a higher dose (10 mg/kg body weight), pyroGlu-Leu content in

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the small intestinal tissue significantly increased at 30 and 60 min, compared to the

As shown in Figure 3A,

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baseline level (data not shown). Unexpectedly, 24 h after the administration at a dose of

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0.1 mg/kg body weight, a significantly higher level of pyroGlu-Leu (2.416 ± 0.143

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nmol/g) was observed in the small intestine, compared to the baseline (Figure 3B). The

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concentrations of pyroGlu-Leu in colon were approximately 5% of those shown in the

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small intestinal tissue.

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As shown in Figure 3C, similar results were obtained after the 7 days feeding

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experiment using normal and DSS-induced colitis mice. PyroGlu-Leu was also detected

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in the intestines from the normal and DSS-induced colitis mice that received vehicle.

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The content of pyroGlu-Leu in small intestine tended to increase by the administration

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of pyroGlu-Leu at 0.1 mg/kg body weight for 7 days, while the small intestine was

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collected 24 h after the final administration of pyroGlu-Leu. The contents in the colon

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were considerably lower than those in the small intestine even after 7 days

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administration. Administration of pyroGlu-Leu did not affect on the contents in the

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colon.

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Effects of PyroGlu-Leu on Microbiota. In normal colon (sham 0 group), the

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population of Bacteroidetes was much higher than that of Firmicutes, whereas the

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population of Firmicutes increased in mice with colitis. Administration of pyroGlu-Leu

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increased and decreased the Bacteroidetes and Firmicutes, respectively, in a dose

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dependent manner up to 0.1 mg/kg.

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(Figure 4).

Thereafter, these changes reached a plateau

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DISCUSSION

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PyroGlu-Leu was first identified in wheat gluten hydrolysate and it was noted that it

223

attenuated D-galactosamine-induced hepatitis at 20 mg/kg body weight.22 The present

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study demonstrated that pyroGlu-Leu significantly improved body weight loss and the

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DAI score of mice with DSS-induced colitis. The effects of pyroGlu-Leu showed a

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reverse J-curve in body weight change and a J-curve in the DAI score. These results

227

indicated that 0.1 mg/kg is the optimum dose for the moderation of colitis in the present

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animal model. Improvement of colitis by oral administration of pyroGlu-Leu in this

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dose was not so high (less than 1 DAI score). However, decrease of 1 DAI score implies

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significant improvement of quality of life (see criteria of DAI score in Materials and

231

Methods section).

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considered, since a higher dose not show improving effects in the present animal model.

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Ingestion of high dose (10 mg/kg body weight) of pyroGlu-Leu tentatively increased its

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content in the small intestine to approximately 40 nmol/g of wet tissue 30 and 60 min

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after the ingestion (data not shown). On the other hand, ingestion of low dose (0.1

For human application, the dose of pyroGlu-Leu must be carefully

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mg/kg) increased to approximately 2.5 nmol/g unexpectedly 24 h after ingestion by

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single and daily administrations (Figure 3 B and C). Tentative large increase of

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pyroGlu-Leu by the high dose might be involved in adverse effect. Mechanism for

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increase of pyroGlu-Leu in small intestinal tissues 24 h after the ingestion of the small

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dose is unclear. To explain these phenomena, we propose two possibilities. First,

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pyroGlu-Leu might be absorbed into blood and then accumulated in small intestine by

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24 h. However, daily administration of pyroGlu-Leu did not increase small intestine

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level. Then it is unlikely that food-derived pyroGlu-Leu is specifically accumulated in

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small intestine. Second, the food-derived pyroGlu-Leu might induce endogenous

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pyroGlu-Leu in small intestine. PyroGlu-Leu can be produced from parent specific

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peptides and proteins by protease digestion. In this case, endogenous pyroGlu-Leu

247

might be involved in communication between cells.

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mice small intestine before the administration and difference in its level between organs

249

support this speculation. To address these problems, comprehensive metabolome

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analysis on intestines and other organs using stable isotope-labeled pyroGlu-Leu is

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necessary.

Occurrence of pyroGlu-Leu in the

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While the mechanism of IBD remains to be solved, IBD could be induced by a

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combination of 1) persistent specific infection, 2) dysbiosis (abnormal ratio of

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beneficial and detrimental commensal microbial agents), 3) defective mucosal barrier

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function, 4) defective microbial clearance, or 5) aberrant immunoregulation.31 In the

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present animal model, severe inflammation of the colon and colonal microbiota change

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occurred. The administration of pyroGlu-Leu moderated these pathological changes.

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However, the pyroGlu-Leu level in colonic tissue was low level even by daily

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administration for 7 days, which was less than values in small intestinal tissue before

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administration. Then, it is unlikely that the ingested pyroGlu-Leu directly suppresses

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inflammation in colon. Several studies have suggested that the Frimicutes/Bacteroidetes

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ratio change by progress of CD.32-34 In the present study, supplementation of

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pyroGlu-Leu normalized colonic microbiota. Ratio of Bacteroidetes to Firmicutes in the

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inner content in colon was highest by the supplementation of pyroGlu-Leu at 0.1 mg/kg

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body weight/day in the DSS-induced mice, which was closest to that in the normal mice

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(sham 0).

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inflammation of colonic cells. PyroGlu-Leu level in the inter content of colon was also

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low; less than 5 nmol/g of dry matter even after ingestion of higher dose of

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pyroGlu-Leu (10 mg/kg body weight, data not shown). It is, therefore, also unlikely

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such low dose of peptide can directly affect growth of bacteria.

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of pyroGlu-Leu, in the small intestinal tissue were much higher than those shown in the

Then it could be speculated that normalizing microbiota might suppress

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colon. Therefore, we hypothesized that the pyroGlu-Leu might act on small intestinal

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cells to induce some biologically active compounds, which might modulate colonic

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microbiota. In fact, it has been demonstrated that intestinal cells produce bactericidal

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peptides including defensins35,

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microbiota.

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production of bioactive compounds including defensin by small intestinal cells are now

278

under progress.

36

and/or mucin16,

37

, which can modulate colonic

To confirm the hypothesis, further study on effect of pyroGlu-Leu on

279

It has been demonstrated that some modified peptides derived from bio-active

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proteins, such as acetyl-Gln-Ala-Trp from annexin A1 and Lys-D-Pro-Thr from

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melanocortin-related peptide, improved DSS-induced colitis by oral administration at 5

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mg/kg body weight.38, 39 However, these peptides cannot be derived from food protein.

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For food-derived peptides, Val-Pro-Tyr found in soy protein hydrolysate improved

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DSS-induced colitis by oral administration at 100 mg/kg/day.19 Compared to these

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peptides, pyroGlu-Leu attenuates colitis at an extremely low dose. This is the first report

286

demonstrating that oral ingestion of food-derived short chain peptide can moderate

287

colitis in such low dose. Therefore, pyroGlu-Leu is a potential therapeutic agent against

288

IBD, while its mechanism in action is not clear, which might be different from

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conventional mechanisms; direct anti-inflammatory and/or anti-microbial activities. The

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present study also encourages us to examine occurrence of other short-chain

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food-derived peptides, which can moderate colitis by oral ingestion at low dose without

292

adverse effect.

293 294

ACKNOWLEDGMENTS

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This work was supported by a JSPS Grant-in-Aid for Young Scientists (B) to

296

Sayori Wada (Grant no. 23700913).

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glycomacropeptide

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Faecalibacterium prausnitzii in colitis microbiota. Inflamm. Bowel. Dis. 2009,

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(37) Faure, M.; Mettraux, C.; Moennoz, D.; Godin, J. P.; Vuichoud, J.;

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179, 1230-1242.

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FIGURE LEGEND

450 451

Figure 1.

452

Effects of pyroGlu-Leu (pEL) on body weight change (A) and disease activity

453

index (DAI) score (B) of mice with dextran sulfate sodium (DSS)-induced

454

colitis. Values are presented as mean ± SEM. Open squares indicate the

455

non-colitis inducing groups, and the solid squares indicate the groups

456

induced colitis by DSS. The numbers 0, 0.01, 0.05, 0.1, 0.5, and 1.0 represent

457

the dose of pyroGlu-Leu (mg/kg/day), respectively. Sham: no DSS treatment.

458

The number of experimental animals in each group is listed in Table 1.

459

* and ** represent P < 0.05, P < 0.01 when compared with control mice

460

receiving DSS solution alone, respectively.

461 462

Figure 2.

463

The histological appearance of colonic tissue in sham mice without DSS (A),

464

mice with DSS-induced colitis (B), and mice with DSS-induced colitis treated

465

with 0.1 mg/kg pyroGlu-Leu. Hematoxylin and eosin staining. Magnification,

466

×400. In B, extensive infiltration reaching the muscularis mucosa and

467

thickening of the mucosa with abundant edema was observed, whereas

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improvement was shown in C.

469 470

Figure 3. Representative MRM chromatograms of pyroGlu-Leu (m/z 243.1 >

471

86.1 and m/z 243.1 > 84.0) (A) and pyroGlu-Leu contents in small intestine

472

and colon (B, C).

473

A: Std: authentic pyroGlu-Leu (1 µM), TCA extract of the washed intestine

474

before (0), 1, 24 h after the ingestion of pyroGlu-Leu (0.1 mg/kg body weight).

475

B: Time course of contents of pyroGlu-Leu in the small intestine and colon

476

after oral administration of 0.1 mg/kg body weight of pyroGlu-Leu. Values

477

are presented as mean ± SEM (n=3 except baseline which were n=4).

478

** represents P < 0.01 when compared with the baseline.

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C: Contents of pyroGlu-Leu in the small intestine and colon after the 7 days

480

feeding experiment (n=3 for each group). Normal and colitis mice received

481

vehicle (0) or pyroGlu-Leu at 0.1 mg/kg/day for 7 days.

482 483

Figure 4.

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Population of Firmicutes and Bacteroidetes in the inner content of the colon

485

in each group. The inner contents of the colon were collected from 3 mice in each

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Journal of Agricultural and Food Chemistry

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group and mixed them together for microbiota analysis. Closed squares indicate

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Firmicutes and open squares indicate Bacteroidetes.

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pEL reperesnts dose of pyroGlu-Leu

489

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Table 1. Treated condition of DSS and pyroGlu-Leu in each group

group

pyroGlu-Leu

DSS

(mg/kg/day)

n

sham 0

-

0

17

sham 0.1

-

0.1

6

0

+

0

43

0.01

+

0.01

13

0.05

+

0.05

19

0.1

+

0.1

44

0.5

+

0.5

32

1.0

+

1.0

18

DSS: dextran sulfate sodium

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Journal of Agricultural and Food Chemistry

A

B

Figure 1. 31

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C

B

Figure 2.

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Journal of Agricultural and Food Chemistry

Figure 3. 33

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