Effects of synbiotics on intestinal mucosal barrier in rat model

Accepted Manuscript Effects of Synbiotics on Intestinal Mucosal Barrier in Rat Model Zhigang Xue, Jianchun Yu, Mingli Zhao, Weiming Kang, Zhiqiang Ma

PII:

S2352-9393(16)30055-0

DOI:

10.1016/j.yclnex.2017.02.001

Reference:

YCLNEX 31

To appear in:

Clinical Nutrition Experimental

Received Date: 11 December 2016 Accepted Date: 9 February 2017

Please cite this article as: Xue Z, Yu J, Zhao M, Kang W, Ma Z, Effects of Synbiotics on Intestinal Mucosal Barrier in Rat Model, Clinical Nutrition Experimental (2017), doi: 10.1016/j.yclnex.2017.02.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Effects of Synbiotics on Intestinal Mucosal Barrier in Rat Model Zhigang Xue1, Jianchun Yu1*, Mingli Zhao2, Weiming Kang1, Zhiqiang Ma1 1. Department of General Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing, China 2. Department of General Surgery, Nanfang Hospital, Southern Medical University,

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Guangzhou, China

*Corresponding author: Jianchun Yu, M.D. 1# Shuaifuyuan, Dongcheng District, 100730, Beijing, China Email address: [email protected]

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Tel number: (+86) 010-69152213

ACCEPTED MANUSCRIPT Effects（of（Synbiotics（on（Intestinal（Mucosal（Barrier（in（Rat（Model（

Abstract Objective: To（investigate（the（protective（effects（of（synbiotics（on（intestinal（barrier（function, and its

priority to probotics and prebiotics on intestinal barrier function in rat models. Methods:（We（randomly（divided（32（rats（into（four（groups:（control（group n=8

,（prebiotics（group

n=8

（ or（synbiotics（group n=8

n=8

,（probiotics（group

.（All（rats（received（cecectomy（and（

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gastrostomy, and tube feeding amoxicillin 50mg and metronidazole 20mg on operation day and

the first operative day (POD1).（After（the（establishment（of（impaired（intestinal（barrier（model,（rats（ in（the（4（groups（received（intact（protein（enteral（nutrition,（intact（protein（enteral（

nutrition+（bifid-triple（viable,（intact（protein（enteral（nutrition（with（dietary（fiber,（intact（protein（

enteral（nutrition（with（dietary（fiber+（bifid-triple（viable（respectively.（Samples（of（blood,（urine（and（ tissues（of（colonic（mucosae（were（collected（on（POD6.（Thereafter（we（measured（lactulose/mannitol（ of（portal（vein（serum（and（thickness（of（colonic（mucosae.（

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(L/M)（ratio,（concentration（of（certain（gut（microflora,（bacterial（translocation（rate,（endotoxin（level（ Results: Concentration（of（lactobacilli（in（probiotics（group（[(0.828±0.366)（×10^7（CFU/g]（and（

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synbiotics（group（[(1.424±0.928)（×10^7（CFU/g]（were（significantly（higher（than（those（in（control（ group（[(0.046±0.037)（×10^7（CFU/g]（and（prebiotics（group（[(0.074±0.046)（×10^7（CFU/g],（P<0.05.（ Concentration（of（bifidobacteria（in（probiotics（group（[(0.876±0.701)（×10^7（CFU/g]（and（synbiotics（ group（[(2.246±1.112)（×10^7（CFU/g]（were（significantly（higher（than（those（in（control（group（[(0.072 ±0.051)（×10^7（CFU/g]（and（prebiotics（group（[(0.072±0.051)（×10^7（CFU/g],（P<0.05,（meanwhile（ concentration（of（bifidobacteria（in（synbiotics（group（was（significantly（higher（than（that（in（the（ probiotics（group,（P<0.05.（

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L/M（ratio（in（synbiotics（group（(0.78±0.43)（was（significantly（lower（than（those（in（control（group（(1.45 ±0.40),（probiotics（group（(1.08±0.22)（and（prebiotics（group（(1.35±0.38),（P<0.05.（Colonic（mucosa（ thickness（in（synbiotics（group（(228.2±13.9 m)（was（significantly（higher（than（those（in（control（(198.5 ±10.3 m),（ probiotics（ (199.8±11.8 m)（ and（ prebiotics（ group（ (206.2±11.7 m),（ P<0.01.（ Bacterial（ translocation（rate（in（synbiotics（group（is（significantly（lower（than（that（in（control（group（(34.38%（vs（

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80%,（P<0.05,（but（no（significant（difference（compared（to（probiotics（group（(40.63%)（and（prebiotics（ group（(53.13%)（was（found.（

Conclusions: Probiotics（can（improve（the（concentration（of（colonic（probiotics,（while（synbiotics（can（

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improve（probiotics（concentration（and（mucosa（thickness（in（colon,（decrease（L/M（ratio（and（ bacterial（translocation.（Synbiotics（shows（more（protective（effects（on（intestinal（mucosal（barrier（in（ rats（after（cecectomy（and（gastrostomy（and（the（intervention（of（specific（antibiotics.（ （ （

Introduction

The（intestinal（mucosal（barrier（is（a（single（contiguous（layer（of（cells（lining（the（gastrointestinal（tract（ [1].（This（intestinal（epithelium（allows（transcellular（transport（of（dietary（nutrients,（electrolytes,（and（ water（from（the（intestinal（lumen（into（the（circulation,（as（well（as（plays（a（critical（role（in（preventing（ access（of（harmful（agents（including（endotoxin,（microorganisms,（and（hydrolytic（enzymes（from（ entering（the（internal（environment,（which（is（termed（as（barrier（function（[2-4].（The（barrier（function（ mainly（reflects（in（three（levels:（(1)（mechanical（barrier（consists（of（enterocyte（membranes（and（tight（ junctions（between（enterocytes;（(2)（interaction（between（commensal（bacteria（and（host（forms（

ACCEPTED MANUSCRIPT microbial（ecosystem（as（the（ecological（barrier;（(3)（gut-associated（lymphoid（tissue（and（ immunological（molecules（establish（the（immunological（barrier（[5].（ Dysregulation（of（the（intestinal（barrier（may（be（caused（by（stress,（invasion（of（pathogenic（organisms,（ infection（and（immunological（challenges.（Among（these（factors,（major（abdominal（surgery（as（a（ traumatic（stress（could（often（interrupt（the（intestinal（microbial（balance,（decrease（the（intestinal（ barrier（function,（irritate（systemic（inflammatory（reaction,（and（increase（the（risk（of（postoperative（ infections,（which（is（closely（related（to（bacterial（translocation（[6,（7].（Generally,（mechanism（of（these（

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disturbances（includes:（(1)（Impairment（and（failure（of（the（structural（mucosal（barrier（leading（to（

increased（gut（permeability;（(2)（imbalanced（gut（flora,（bacterial（overgrowth（and（translocation;（(3)（ dysregulation（of（the（pro-（and（anti-inflammatory（balance（of（the（immune（system（[8].（

Bacteria（can（be（used（to（improve（human（health（and（even（save（lives（in（many（cases.（A（bacterium（ that（provides（specific（health（benefits（when（consumed（as（a（food（component（or（supplement（is（

termed（as（a（probiotic.（And（a（prebiotic（is（defined（as（a（non-digestible（food（ingredient（that（benefits（

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the（host（by（selectively（stimulating（the（growth（and/or（activity（of（some（bacteria（in（the（colon.（The（ term（synbiotic（is（a（combination（of（probiotics（and（prebiotics（[9,（10].（Studies（showed（many（ positive（effects（of（probiotics, including to prevent bacterial translocation and overgrowth, induce

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colonization resistance against pathogenic bacteria, help to maintain epithelial integrity through feeding of enterocytes, production of omega-3-fatty acids, and inhibit pathogenic-induced alterations of epithelial permeability [11-14]. Prebiotics as non-absorbable food components like inulin or pectin benefit to stimulate certain specific probiotic species leading to a positive effect on human health [15]. We（hypothesize（that（the（positive（effects（of（synbiotics（on（intestinal（barrier（ function（are（superior（to（the（use（of（probiotics（or（prebiotics（alone.（Current（trials（are（generally（ designed（to（compare（synbiotics（with（probiotics,（probiotics（or（placebo（[16-18].（Therefore,（we（

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designed（the（animal（trial（aiming（to（investigate（the（effects（of（synbiotics（on（intestinal（barrier（ function（and（whether（there（is（superiority（over（single（use（of（probiotics（and（prebiotics.（

Methods Rats and materials

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Sprague（Dawley（male（rats（with（weight（from（240g（to（260g（(Beijing（Vital（River（Laboratory（Animal（ Technology（Co.,（Ltd) were（raised（in（metabolism（cages（in（a（room（kept（at（22±1

（with（a（12-h（

light-dark（cycle（at（Peking（Union（Medical（College（Hospital（Animal（House.（And（urine（can（be（

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collected（from（the（cages.（The（antibiotics（used（on（operation（day（and（POD1（are（amoxicillin（capsule（ (Batch（No.（080409)（and（metronidazole（(Batch（No.（080503).（The（probiotic（formulations（are（live（ combined（bifidobacterium,（lactobacillus（and（enterococcus（named（Bifico（(Sine（Pharm（Batch（No.（ 20080909)（available（as（a（capsule（with（live（bacteria（no（less（than（1.0×107（cfu.（The（intact protein

enteral nutrition emulsion named Fresubin (Sino-Swed Pharm. Corp.,（Ltd. Batch No. 80CB404) contained 100 kcal and 4 g protein per 100 ml. The（intact protein-fiber enteral nutrition emulsion named Nutrison (Nutricia Pharmaceutical (Wuxi) Co., Ltd. Batch No.0812029) contained 100 kcal, 4 g protein and 1500 mg dietary fiber per 100 ml. The fiber in Nutrison can be used as prebiotics, and when used with probiotics, the formulation is regarded as synbiotics [19, 20], since there is no synbiotic production in Chinese Market when the trial was set. Randomization We（marked（all（rats（with（numbers（1（to（32（respectively.（Then（we（used（Microsoft（Excel（(Office（2010)（ to（generate（randomized（numbers.（Numbers（1（to（32（were（input（into（A1（to（A32（respectively（in（

ACCEPTED MANUSCRIPT column（A.（Randomized（numbers（were（generated（in（column（B（and（then（we（put（these（numbers（in（ an（ascending（order（so（that（numbers（in（column（A（changed（in（correspondence.（Rats（with（numbers（ now（in（A1（to（A8（were（in（control（group,（A9（to（A16（in（probiotics（group,（A17（to（A24（in（prebiotics（ group（and（A25（to（A32（in（synbiotics（group.（ Damaged intestinal barrier model and interventions After（12-hour（fasting,（all（rats（were（carried（on（cecectomy（and（gastrostomy（and（fed（with（ amoxicillin（50mg（and（metronidazole（20mg（once（a（day（through（gastrostomy（tube（on（the（ the（following（enteral（nutrition（programs（(Table（1（and（Table（2).（ （

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operation（day（and（the（1st（postoperative（day（(POD1).（All（rats（were（raised（in（metabolism（cages（with（

Interventions (Qd through gastrostomy tube)

Control(n=8) Probiotics(n=8) Prebiotics(n=8) synbiotics(n=8)

Intact protein EN+1.0ml normal saline Intact protein EN+（bifid-triple（viable 210mg+0.8ml normal saline Intact protein-fiber EN+1.0ml normal saline Intact protein-fiber EN+（bifid-triple（viable 210mg+0.8ml normal saline

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Group(n=32)

Table（1（shows（rats（in（different（groups（treated（with（different（formulation.（

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（

POD n

1

2

3

4

5

Daily amount(ml) Total energy(kcal) Total protein(g) Intervals(hours) Amount each time(ml)

10 10 0.4 1 1

10 10 0.4 1 2

20 20 0.8 1 2

30 30 1.2 1 3

40 40 1.6 1 4

with（the（same（project.（ Sample collection

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Table（2（shows（the（stepwise（amounts（of（nutrition（on（postoperative（days,（rats（in（the（four（groups（

On（POD（5,（all（rats（were（fed（with（lactulose-mannitol（1.6ml（respectively（through（gastrostomy（tube.（

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24-hour（urines（were（collected（and（10ml（urine（kept（in（routine（tube（was（preserved（in（-20

（

refrigerator.（On（POD（6,（portal（vein（blood（were（collected（to（test（endotoxin（concentration.（Portal（ vein（blood,（liver（tissues,（lung（tissues（and（mesenteric（lymph（nodes（were（obtained（to（test（bacterial（

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translocation.（Colonic（contents（were（diluted（to（test（concentration（of（probiotics（and（ gram-negative（bacterium.（The（starting（part（of（colon（tissues（was（used（to（measure（colonic（mucosa（ thickness.（The（study（design（is（shown（in（figure（1.（ （

High（performance（liquid（chromatography（with（pulse（electrochemical（detector（(HPLC-PED) Dionex（China（Limited（type（DX-500 chromogenic（substrate（Limulus（Kit（

（ was（used（to（test（lactulose/mannitol（ratio[21].（The（ Shanghai（Yihua（Medical（Tech（Co.,（Ltd. and（

spectrophotometer（ Shanghai（Techcomp were（used（to（test（endotoxin（concentration.（Portal（vein（ blood,（liver（tissues,（lung（tissues（and（mesenteric（lymph（node（tissues（were（homogenated（and（then（ inoculated（onto（Columbia（blood（agar（ Themo（Fisher（Biochemical（Product(Beijing)（Co.,（Ltd.（Batch（ No.（XW09163

preserved（in（37

（ refrigerator.（Culture（results（were（interpreted（in（48（hours.（To（

culture（gram-negative（bacterium（in（colonic（contents,（we（inoculated（its（dilution（onto（Rosolic（acid（ Chinese（blue（blood（agar（plate

Themo（Fisher（Biochemical（Product(Beijing)（Co.,（Ltd.（Batch（No.（

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and（uniform（coated（it（with（burning（sterilized（oese（on（the（plate（surface.（The（culture（

media（were（preserved（in（37 （ refrigerator（and（results（were（interpreted（in（48（hours.（For（samples（ from（portal（vein,（colony（forming（unit（(CFU)（ 1（is（recorded（as（positive（culture.（And（For（samples（ from（liver,（lung（and（mesenteric（lymph（nodes（tissues（CFU（ 5（is（recorded（as（positive（culture（[22].（ Colon（tissue（samples（were（embedded,（sliced（up（and（HE（stained.（Then（we（used（NDP（ (NanoZoomer（Digital（Pathology,（Hamamatsu（Photonics（K.K.)（imaging（process（system（to（measure（ colonic（mucosa（thickness.（

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Statistical analysis

The（statistical（analyses（were（performed（with（SPSS（software（version（16.0.（The（data（are（presented（ as（the（means（±（standard（deviation.（A（normality（test（and（a（homogeneity（test（for（variance（were（ performed（first.（If（the（data（were（in（compliance（with（a（normal（distribution（and（homogeneity（of（ variance,（an（ANOVA（with（Bonferroni s（post-test（or（a（Student s（t-test（was（performed;（otherwise,（ a（rank（sum（test（was（used.（The（categorical（data（were（analyzed（using（the（Chi-squared（test.（P<0.05（

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was（considered（to（be（statistically（significant.（

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（

（

Figure（1（shows（the（flow（chart（of（this（study（design（ （ Results Basal（body（weights（of rats in control（group n=8

（ and（synbiotics（group

n=8

n=8

,（probiotics（group

n=8

,（prebiotics（group

were（256.25±6.76（g,（252.63±5.85（g,（254.88±7.10（g（and（

ACCEPTED MANUSCRIPT 251.50±7.03（g（respectively.（After cecectomy（and（gastrostomy,（all（rats（were（weighted（immediately,（ the（weights（rats（in（four groups were 199.63±14.17（g,（194.00±11.49（g,（200.75±3.28（g（and（192.75 ±8.76（g（respectively.（Body（weights（in（four（groups（were（comparable（before（and（after（cecectomy（ and（gastrostomy（(Figure（2).（Weight（loss（in（four（groups（were（comparable（as（shown（in（Figure（3.（ preoperative

postoperative

300.00 254.88

252.63

251.50

194.00

200.75

150.00 100.00

192.75

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body weight(g)

199.63 200.00

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256.25 250.00

0.00 control

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50.00

probiotics

prebiotics

synbiotics

（ Figure（2（shows（body（weights（of（rats（in（control（group n=8 ,（probiotics（group n=8 ,（prebiotics（ group

n=8

（ and（synbiotics（group

25.00

23.40

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21.20

20.00 15.00

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10.00 5.00

（ before（and（after（cecectomy（and（gastrostomy.（

23.20

22.20

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percentage of weight loss(%)

30.00

n=8

0.00

control

probiotics

prebiotics

synbiotics

（

Figure（3（shows（percentage（of（weight（loss（in（four（groups,（P=0.577.（ （

Base-10（logarithm（of（lactobacilli（concentration（of（colonic（contents（in（control（group n=8 probiotics（group

n=8

,（prebiotics（group

n=8

（ and（synbiotics（group

n=8

,（

postoperatively（

were（6.49±0.45,（7.85±0.28,（6.96±0.55（and（8.08±0.27（respectively.（Log（value（of（lactobacilli（ concentration（in（probiotics（group（and（synbiotics（group（were（significantly（higher（compared（to（ that（in（the（control（group（and（prebiotics（group.（But（no（significant（difference（was（shown（between（ probiotics（group（and（synbiotics（group.（Base-10（logarithm（of（bifidobacteria（concentration（of（ colonic（contents（in（four（groups（were（5.73±0.40,（6.78±0.45,（5.76±0.53（and（7.29±0.27（respectively.（ Log（value（of（bifidobacteria（concentration（in（probiotics（group（was（significantly（higher（compared（

ACCEPTED MANUSCRIPT to（that（in（the（control（group（and（prebiotics（group.（While（Log（value（of（bifidobacteria（ concentration（in（synbiotics（group（was（significantly（higher（compared（to（that（in（the（probiotics（ group,（P<0.05（(Figure（4（and（5).（ （ 9.00 8.08 *

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7.85 *

8.00 7.50

6.96 7.00 6.49 6.50 6.00 5.50

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Base-10 logarithm of lactobacilli concentration 108CFU/g

8.50

5.00 4.50

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4.00 control

probiotics

prebiotics

synbiotics

（ Figure（4（shows（lactobacilli（concentration（of（colonic（contents（in（four（groups（postoperatively（ *（means（significant（difference（when（compared（to（control（group,（P<0.05.（ （

7.50

6.00 5.50 5.00 4.50 4.00

7.29

6.78

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6.50

（

5.73

5.76

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7.00

3.50

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Base-10 logarithm of bifidobacteria concentration 107CFU/g

8.00

3.00

control

probiotics

prebiotics

synbiotics

Figure（5（shows（bifidobacteria（concentration（of（colonic（contents（in（four（groups（postoperatively（ *（means（significant（difference（when（compared（to（control（group,（P<0.05.（ **means（significant（difference（when（compared（to（probiotics（group,（P<0.05.（ （ （ Base-10（logarithm（of（gram-negative（bacteria（concentration（of（colonic（contents（in（control（group n=8

,（probiotics（group n=8

,（prebiotics（group

n=8 （ and（synbiotics（group n=8

postoperatively（were（9.27±0.47,（9.28±0.66,（9.13±0.60（and（9.12±0.67（respectively.（There（was（no（ significance（of（gram-negative（bacteria（concentration（in（four（groups.（But（10-base（logarithm（of（

ACCEPTED MANUSCRIPT gram-negative（bacteria（(marked（as（log1)（to（10-base（logarithm（of（bifidobacteria（concentration（ (marked（as（log2)（ratio（value（in（probiotics（group（and（synbiotics（group（(1.39±0.14（and（1.39±0.21)（ were（significantly（lower（compared（to（that（in（the（control（group（and（prebiotics（group（(1.63±0.16（ and（1.59±0.12),（P<0.05.（No（significant（difference（was（shown（between（probiotics（group（and（ synbiotics（group（(Figure（6).（

1.63

1.59

1.5

1.39 *

1.39 *

1.3 1.1

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log1 to log2 ratio value

1.7

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1.9

0.9

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0.7 0.5 control

probiotics

prebiotics

synbiotics

（

Figure（6（shows（10-base（logarithm（of（gram-negative（bacteria（(log1)（to（10-base（logarithm（of（ bifidobacteria（concentration（(log2)（ratio（value（

*（means（significant（difference（when（compared（to（control（group,（P<0.05.（ （ n=8

n=8

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Lactulose（to（mannitol（ratio（in（control（group （ and（synbiotics（group

n=8

,（probiotics（group

n=8

,（prebiotics（group

postoperatively（were（1.45±0.40,（1.08±0.22,（1.35±0.38（and（

0.78±0.43（respectively.（Lactulose（to（mannitol（ratio（in（synbiotics（group（were（significantly（lower（ compared（to（that（in（the（control（group（and（prebiotics（group,（P<0.05.（Lactulose（to（mannitol（ratio（ in（probiotics（group（were（not（significantly（lower（compared（to（that（in（the（control（group（and（

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prebiotics（group（(Figure（7).（

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1.6

1.45 1.35

1.4 1.2

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1.08

1

0.78 *

0.8 0.6 0.4

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Lactulose to mannitol ratio value

1.8

0.2

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0 control

probiotics

prebiotics

synbiotics

（

Figure（7（shows（Lactulose（to（mannitol（ratio（in（four（groups（

*（means（significant（difference（when（compared（to（control（group,（P<0.05.（ （

Colon（mucosa（thickness（in（control（group n=8 ,（probiotics（group n=8 ,（prebiotics（group n=8

（

and（synbiotics（group n=8 postoperatively（were（198.5±10.3（ m,（199.8±11.8 m,（206.2±11.7（ m（

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and（228.2±13.9（ m（respectively.（Colon（mucosa（thickness（in（synbiotics（group（was（significantly（ higher（compared（to（that（in（the（control（group,（probiotics（group（and（prebiotics（group,（P<0.01（ (Figure（8).（ （ （

220 200

198.5

228.2 *

199.8

206.2

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colon mucosa thickness (μm)

240

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260

180 160 140 120 100

control

probiotics

prebiotics

synbiotics

Figure（8（shows（colon（mucosa（thickness（in（four（groups（on（POD6（ *（means（significant（difference（when（compared（to（control（group,（P<0.01.（ （

（

ACCEPTED MANUSCRIPT Bacterial（translocation（rate（in（control（group n=8

（ and（synbiotics（group

n=8

n=8

,（probiotics（group

n=8

,（prebiotics（group

postoperatively（were（20/32(62.5%),（13/32(40.6%),（

17/32(53.1%)（and（11/32(34.4%)（respectively.（Bacterial（translocation（rate（in（synbiotics（group（were（ significantly（lower（compared（to（that（in（the（control（group,（P<0.05.（No（significance（was（found（ among（the（other（groups.（When（the（probiotics（group（and（synbiotics（group（joined（as（PROBIOTIC（ group,（the（control（group（and（prebiotics（group（joined（as（non-PROBIOTIC（group,（bacterial（ translocation（rate（in（PROBIOTIC（group（37.5%（(24/64)（is（significantly（lower（than（that（in（the（

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non-PROBIOTIC（group（57.8%（(37/64),（P<0.05.（Similarly,（when（compare（the（PREBIOTICS（(joined（ by（prebiotics（and（synbiotics（group)（with（the（non-PREBIOTICS（group（(joined（by（probiotics（and（

control（group),（we（did（not（find（any（significant（difference（in（aspect（of（bacterial（translocation（rate（ (43.8%（vs（51.6%（respectively,（P=0.376).（Endotoxin（concentration（in（portal（vein（in（control（group n=8

and（synbiotics（group

n=8

were（0.0217±0.0028（and（0.0203±0.0043.（We（did（not（find（

significant（difference（of（endotoxin（concentration（in（portal（vein（between（the（two（groups.（

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Discussions

From（the（results（of（our（study,（it（is（obvious（that（the（application（of（probiotics（(probiotic（s（group（ and（synbiotics（group)（can（increase（gut（probiotics（as（evidence（shown（in（the（following:（lactobacilli（

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concentration（and（bifidobacteria（concentration（of（colonic（contents（in（probiotics（group（and（ synbiotics（group（were（significantly（higher（compared（to（that（in（the（control（group（and（prebiotics（ group,（P<0.05（respectively.（Furthermore,（bifidobacteria（concentration（in（synbiotics（group（was（ significantly（higher（compared（to（that（in（the（probiotics（group,（P<0.05,（which（shows（the（effect（that（ synbiotics（increase（the（amounts（of（bifidobacteria（in（colon（is（better（than（that（of（single（use（of（ probiotics.（The（use（of（probiotics,（prebiotics（or（synbiotics（did（not（decrease（the（amounts（of（ gram-negative（bacteria（in（colon.（But（with（the（increase（of（probiotics（in（probiotics（group（and（

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synbiotics（group,（the（gram-negative（bacteria（to（bifidobacteria（concentration（ratio（value（ (log1/log2（as（described（in（the（Results)（were（significantly（lower（in（probiotics（group（and（synbiotics（ group（(1.39±0.14（and（1.39±0.21)（compared（to（that（in（the（control（group（and（prebiotics（group（ (1.63±0.16（and（1.59±0.12),（P<0.05,（which（demonstrates（the（improvement（of（gut（microflora（ composition.（

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The（use（of（probiotics（can（decrease（bacterial（translocation（significantly.（Bacterial（translocation（ rate（in（the（joined（PROBIOTIC（group（(probiotics（and（synbiotics（group)（is（significantly（lower（than（ that（in（the（joined（non-PROBIOTIC（group（(control（and（prebiotics（group),（37.5%（vs（57.8%,（P<0.05.（

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But（the（use（of（dietary（fiber（(the（joined（PREBIOTICS（by（prebiotics（and（synbiotics（group)（did（not（ show（the（benefit（compared（to（the（joined（non-PREBIOTICS（group（(control（and（probiotics（group).（ The（single（use（of（probiotics（or（prebiotics（did（not（increase（colon（mucosa（thickness（compared（to（ the（control（group（significantly,（neither（show（certain（improvement（in（aspect（of（intestinal（ permeability（(lactulose（to（mannitol（ratio). Colon（mucosa（thickness（in（synbiotics（group（(228.2± 13.9（ m)（was（significantly（higher（compared（to（that（in（the（control（group,（probiotics（group（and（ prebiotics（group（(198.5±10.3（ m,（199.8±11.8 m（and（206.2±11.7（ m（respectively),（P<0.01.（And（ lactulose（to（mannitol（ratio（in（synbiotics（group（(0.78±0.43)（was（significantly（lower（compared（to（ that（in（the（control（group,（probiotics（group（and（prebiotics（group（(1.45±0.40,（1.08±0.22（and（1.35 ±0.38（respectively),（P<0.05.（These（results（show（the（superiority（of（synbiotics（over（single（use（of（ probiotics（or（prebiotics.（ （ （ Colon（contains（the（largest（quantity（of（symbiotic（bacteria,（and（colonic（bacteria（is（an（important（

ACCEPTED MANUSCRIPT pathogen（of（intestinal（infection（[23].（Therefore,（the（colonic（mucosa（barrier（may（play（a（more（ important（role（than（small（intestinal（mucosa（barrier（in（reducing（intestine-derived（infection.（Major（ energy（substance（of（colonic（mucosal（cells（is（short（chain（fatty（acids（(SCFAs)（in（the（intestinal（lumen（ [24,（25].（SCFAs（are（mainly（composed（of（dietary（fiber（not（digested（and（absorbed（in（small（ intestinal（and（fermented（by（probiotics,（including（acetic（acid,（propionic（acid（and（butyric（acid（ produced（by（bifidobacteria.（Lactobacilli-produced（lactic（acid（does（not（interact（with（intestinal（ mucosa（directly,（but（the（acidic（environment（formed（by（lactic（acid（is（beneficial（to（the（growth（of（

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other（probiotics（[24,（26].（Therefore,（the（probiotics（and（non-digestible（dietary（fiber（components（ in（the（colon（are（indispensable（to（the（nutrition（support（and（function（protection（of（the（colon（ mucosa（[9].（ （

Colonic（mucosa（thickness（and（intestinal（permeability（(L/M（ratio)（are（important（indicators（

reflecting（structure（and（function（of（the（colon（mucosa（[1,（27]. Normal（thickness（of（the（colon（

mucosa（represents（sufficient（quantity（and（fine（morphology（of（the（intestinal（epithelium,（and（

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alteration（in（intestinal（permeability（reflects（the（decrease（of（selective（barrier（function（caused（by（ the（damage（of（intestinal（barrier.（The（results（of（our（study（show（that（the（protective（effect（of（ synbiotics（is（better（than（the（single（use（of（probiotics（or（prebiotics,（consistent（with（prediction（and（

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the（results（from（previous（studies（[19,（26,（28].（ （

Since（synbiotics（can（improve（the（nutritional（status（of（colonic（epithelial（cells（and（reduce（the（ permeability（of（the（intestinal（mucosa,（it（should（help（to（reduce（the（incidence（of（intestine-derived（ infection（theoretically.（And（indeed,（several（studies（have（proved（that（synbiotics（helps（to（reduce（ bacterial（translocation（and（postoperative（infection（[29-31]. Our（study（did（have（such（tendency（as（ bacterial（translocation（rate（in（synbiotics（group（(34.4%)（lower（than（that（in（probiotics（group（ (40.6%),（but（without（statistical（significance,（P=0.606.（Meanwhile（some（studies（did（not（show（

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positive（effects（of（synbiotics（on（bacterial（translocation（or（postoperative（infection（[32,（33].（ Possible（reasons（for（the（lack（of（effectiveness（may（be（short（postoperative（period（of（administration（ (median（time（4（days),（and（the（oral（(instead（of（jejunal)（route（of（administration（with（unclear（ survival（of（probiotics（in（the（stomach（due（to（low（pH（[8].（Further（investigation（may（expand（sample（ size（or（focus（on（comparison（of（synbiotics（and（probiotics,（and（explore（possible（mechanism（that（

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protective（effect（of（synbiotics（on（intestinal（mucosa（barrier.（ We（compared（the（levels（of（portal（endotoxin（in（synbiotics（group（and（control（group,（endotoxin（ concentration（in（synbiotics（group（was（slightly（lower（than（that（in（the（control（group,（but（with（no（

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statistical（significance.（As（endotoxin（is（a（component（of（the（outer（membrane（of（gram-negative（ bacteria,（one（possible（explanation（may（be（related（to（no（significant（differences（among（the（groups（ regarding（the（number（of（intestinal（gram-negative（bacteria.（Also,（the（sample（size（may（be（not（ enough（to（reveal（significant（differences.（Further（studies（may（focus（on（the（relation（between（ number（of（intestinal（gram-negative（bacteria（and（portal（endotoxin（concentration（and（expansion（ of（sample（size.

In（addition,（there（was（no（difference（in（the（number（or（the（rate（of（body（weight（alteration（among（ the（groups（in（this（study.（In（the（condition（of（enteral（nutrition（support（with（isonitrogen（and（ isocalories,（synbiotics（failed（to（improve（the（nutritional（status（of（the（body.（While（alterations（in（ weight（is（only（one（general（index（to（reflect（nutritional（status（of（the（body,（we（should（investigate（ albumin,（prealbumin,（hemoglobin（and（other（indicators（to（have（a（comprehensive（understanding（ of（the（nutritional（status.（Furthermore,（with（the（potential（benefit（on（reducing（risk（of（infection,（ synbiotics（may（stop（further（deterioration（of（the（nutritional（status（caused（by（infection（when（

ACCEPTED MANUSCRIPT energy（consumption（increases（inevitably（[31].（ Conclusions The（use（of（synbiotics（can（increase（the（number（of（colonic（bifidobacteria（and（lactobacilli,（optimize（ the（composition（of（colonic（flora（and（repair（the（damaged（biological（barrier（in（damaged（mucosa（ barrier（rat（model.（Meanwhile（it（can（increase（mucosa（thickness（in（colon,（decrease（intestinal（ permeability,（and（repair（the（damaged（mechanical（barrier,（thereby（reducing（bacterial（ translocation（in（the（colon.（The（use（of（probiotics（can（increase（the（number（of（colonic（probiotics（as（

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well,（but（the（effect（is（not（as（good（as（synbiotics（demonstrates.（Probiotics（may（reduce（bacterial（

translocation,（but（showed（no（effect（on（colonic（mucosa（thickness（and（intestinal（permeability.（The（ use（of（prebiotics（alone（did（not（show（protective（effect（on（intestinal（mucosal（barrier.（ （

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References:

[1]. Arrieta, M.C., L. Bistritz and J.B. Meddings, Alterations in intestinal permeability. Gut, 2006. 55(10): p. 1512-20.

[2]. Artis, D., Epithelial-cell recognition of commensal bacteria and maintenance of immune

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homeostasis in the gut. Nat Rev Immunol, 2008. 8(6): p. 411-20.

[3]. Jacobi, S.K. and J. Odle, Nutritional factors influencing intestinal health of the neonate. Adv Nutr, 2012. 3(5): p. 687-96.

[4]. Ruth, M.R. and C.J. Field, The immune modifying effects of amino acids on gut-associated lymphoid tissue. J Anim Sci Biotechnol, 2013. 4(1): p. 27.

[5]. Baumgart, D.C. and A.U. Dignass, Intestinal barrier function. Curr Opin Clin Nutr Metab Care, 2002. 5(6): p. 685-94.

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[6]. Wacha, H., et al., Risk factors associated with intraabdominal infections: a prospective multicenter study. Peritonitis Study Group. Langenbecks Arch Surg, 1999. 384(1): p. 24-32. [7]. Diepenhorst, G.M., et al., Influence of prophylactic probiotics and selective decontamination on bacterial translocation in patients undergoing pancreatic surgery: a randomized controlled 2011. 35(1): p. 9-16.

trial. Shock,

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[8]. van Santvoort, H.C., et al., Probiotics in surgery. Surgery, 2008. 143(1): p. 1-7. [9].Schrezenmeir, J. and M. de Vrese, Probiotics, prebiotics, and synbiotics--approaching a definition. Am J Clin Nutr, 2001. 73(2 Suppl): p. 361S-364S.

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[10]. Guarner, F. and J.R. Malagelada, Gut flora in health and disease. Lancet, 2003. 361(9356): p. 512-9.

[11]. Servin, A.L., Antagonistic activities of lactobacilli and bifidobacteria against microbial pathogens. FEMS Microbiol Rev, 2004. 28(4): p. 405-40. [12]. Marco, M.L., S. Pavan and M. Kleerebezem, Towards understanding molecular modes of probiotic action. Curr Opin Biotechnol, 2006. 17(2): p. 204-10. [13]. Bengmark, S., Bioecologic control of the gastrointestinal tract: the role of flora and supplemented probiotics and synbiotics. Gastroenterol Clin North Am, 2005. 34(3): p. 413-36, viii. [14]. Otte, J.M. and D.K. Podolsky, Functional modulation of enterocytes by gram-positive and gram-negative microorganisms. Am J Physiol Gastrointest Liver Physiol, 2004. 286(4): p. G613-26. [15]. Salminen, S., et al., Functional food science and gastrointestinal physiology and function. Br J Nutr, 1998. 80 Suppl 1: p. S147-71. [16]. McNaught, C.E., et al., A prospective randomised study of the probiotic Lactobacillus plantarum

ACCEPTED MANUSCRIPT 299V on indices of gut barrier function in elective surgical patients. Gut, 2002. 51(6): p. 827-31. [17]. Anderson, A.D., et al., Randomised clinical trial of synbiotic therapy in elective surgical patients. Gut, 2004. 53(2): p. 241-5. [18]. Rayes, N., et al., Early enteral supply of fiber and Lactobacilli versus conventional nutrition: a controlled trial in patients with major abdominal surgery. Nutrition, 2002. 18(7-8): p. 609-15. [19]. Spindler-Vesel, A., et al., Synbiotics, prebiotics, glutamine, or peptide in early enteral nutrition: a randomized study in trauma patients. JPEN J Parenter Enteral Nutr, 2007. 31(2): p. 119-26.

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[20]. Sugawara, G., et al., Perioperative synbiotic treatment to prevent postoperative infectious complications in biliary cancer surgery: a randomized controlled trial. Ann Surg, 2006. 244(5): p. 706-14.

[21]. Fleming, S.C., et al., Rapid and simultaneous determination of lactulose and mannitol in urine, by with pulsed amperometric detection, for use in studies of intestinal permeability. Clin Chem,

1990. 36(5): p. 797-9.

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HPLC

[22]. Chiva, M., et al., Effect of Lactobacillus johnsonii La1 and antioxidants on intestinal flora and bacterial translocation in rats with experimental cirrhosis. J Hepatol, 2002. 37(4): p. 456-62. [23]. Thorlacius, H., et al., Lactobacilli attenuate bacteremia and endotoxemia associated with severe

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intra-abdominal infection. Surgery, 2003. 134(3): p. 467-73.

[24]. Roy, C.C., et al., Short-chain fatty acids: ready for prime time? Nutr Clin Pract, 2006. 21(4): p. 351-66.

[25]. Nakao, M., et al., Usefulness of soluble dietary fiber for the treatment of diarrhea during enteral nutrition in elderly patients. Nutrition, 2002. 18(1): p. 35-9.

[26]. Roberfroid, M.B., Prebiotics and probiotics: are they functional foods? Am J Clin Nutr, 2000. 71(6 Suppl): p. 1682S-7S; discussion 1688S-90S.

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[27]. Meddings, J.B. and M.G. Swain, Environmental stress-induced gastrointestinal permeability is mediated by endogenous glucocorticoids in the rat. Gastroenterology, 2000. 119(4): p. 1019-28. [28]. Rayes, N., D. Seehofer and P. Neuhaus, Prebiotics, probiotics, synbiotics in surgery--are they only trendy, truly effective or even dangerous? Langenbecks Arch Surg, 2009. 394(3): p. 547-55. [29]. Seehofer, D., et al., Probiotics partly reverse increased bacterial translocation after simultaneous

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liver resection and colonic anastomosis in rats. J Surg Res, 2004. 117(2): p. 262-71. [30]. Kanazawa, H., et al., Synbiotics reduce postoperative infectious complications: a randomized controlled trial in biliary cancer patients undergoing hepatectomy. Langenbecks Arch Surg, 2005.

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390(2): p. 104-13.

[31]. Sugawara, G., et al., Perioperative synbiotic treatment to prevent postoperative infectious complications in biliary cancer surgery: a randomized controlled trial. Ann Surg, 2006. 244(5): p. 706-14.

[32]. McNaught, C.E., et al., A prospective randomised study of the probiotic Lactobacillus plantarum 299V on indices of gut barrier function in elective surgical patients. Gut, 2002. 51(6): p. 827-31. [33]. Anderson, A.D., et al., Randomised clinical trial of synbiotic therapy in elective surgical patients. Gut, 2004. 53(2): p. 241-5.