Exopolysaccharides from Lactobacillus plantarum NCU116

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Bioactive Constituents, Metabolites, and Functions

Exopolysaccharides from Lactobacillus plantarum NCU116 regulate intestinal epithelial barrier function via STAT3 signaling pathway Xingtao Zhou, Wucheng Qi, Tao Hong, Tao Xiong, Deming Gong, M. Y. Xie, and Shao-Ping Nie J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03340 • Publication Date (Web): 23 Aug 2018 Downloaded from http://pubs.acs.org on August 27, 2018

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Journal of Agricultural and Food Chemistry

Exopolysaccharides from Lactobacillus plantarum NCU116 regulate intestinal barrier function via STAT3 signaling pathway Xingtao Zhou †, Wucheng Qi†, Tao Hong†, Tao Xiong†, Deming Gong †, ‡, Mingyong Xie†, Shaoping Nie*, † †

State Key Laboratory of Food Science and Technology, Nanchang University, 235

Nanjing East Road, Nanchang, Jiangxi 330047, China ‡

New Zealand Institute of Natural Medicine Research, 8 Ha Crescent, Auckland 2104,

New Zealand Correspondence to Professor Shaoping Nie *Phone and fax: +86 791-88304452. E-mail: [email protected].

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ACS Paragon Plus Environment


1

Abstract

2

Lactic acid bacteria (LAB) and their exopolysaccharides (EPS) are recognized to

3

promote intestinal barrier function by mechanisms that remain incompletely

4

understood. Herein, we sought to identify the roles of exopolysaccharides from

5

Lactobacillus plantarum NCU116 (EPS116) in intestinal barrier function. Our data

6

showed that EPS116 attenuated dextran sodium sulfate (DSS) induced colitis,

7

promoted epithelial barrier function and the expression of tight junction (TJ) proteins

8

in vivo and in vitro. Moreover, Chromatin immunoprecipitation data showed that

9

EPS116 facilitated STAT3 (Signal transducer and activator of transcription 3)

10

binding to the promoter of Occludin and ZO-1. Furthermore, knockdown of STAT3

11

in Caco-2 cell with EPS116 treatment led to decreased expression of Occludin and

12

ZO-1 ， and increased intestinal permeability, suggesting that the regulation of

13

epithelial barrier function by EPS116 should be STAT3 dependent. Thus, our data

14

revealed a novel mechanism that EPS116 inhibited intestinal inflammation via

15

regulating intestinal epithelial barrier function.

16

17

Keywords: exopolysaccharides, intestinal epithelial barrier, STAT3 signaling

18

pathway.

19 20 21

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Introduction

23

It has been reported that the microbiota influences the function of intestinal

24

epithelial cells, including gene expression, cell growth and proliferation [1, 2]. The

25

symbiotic microbiota is indispensable for the maintenance of gut homeostasis.

26

Importantly, microbial dysbiosis, such as specific reduction of lactic acid bacteria, has

27

been linked to many diseases. LAB have been linked to a number of health-promoting

28

activities, including diarrhea prevention, immunomodulation and anti-tumor activity [3,

29

4]

30

effects by LAB remain largely unknown, although recently one of the components,

31

exopolysaccharide, has been shown to play a vital role in modulating protective

32

effects of the intestinal epithelium [5-7].

. Despite these benefits, the molecular mechanisms underlying these protective

33

As a physical barrier, the intestinal epithelium insulates intestinal flora from

34

tissues of submucosa, averting bacterial invasion and succedent inflammation, while

35

maintaining absorption of nutrients and ions[8]. The intestinal epithelial barrier

36

includes epithelial cells, intercellular junctions (including tight junctions, subjacent

37

adherens junction, and desmosomes between epithelial cells), mucus layer, and

38

associated intestinal immune cells [9, 10] .

39

The relationship between intestinal disease and intestinal barrier dysfunction was

40

recognized in active inflammatory bowel disease (IBD). Composition and function of

41

tight junction protein have been proven to alter in IBD[11]. The expression of MLCK

42

and claudin-2 have been also demonstrated to increase in IBD, indicating that tight

43

junction disorder might closely associate with IBD. 3



44

STAT3 , as a transcription activator, can mediate the expression of various genes

45

in respond to external stimulations, and thus plays a critical part in many

46

physiological functions, including cell growth, apoptosis, self-renewal of embryonic

47

stem cells , and maintenance of intestinal mucosal barrier[12-15]. It can be activated via

48

phosphorylation in answer to a variety of growth factors and cytokines, like

49

epidermal growth factor and interferons, and translocate to the cell nucleus where it

50

acts as a transcription activator[16].

51

Our study aimed to examine the impacts of EPS116 on regulation of epithelial

52

barrier function and to investigate the molecular mechanisms of EPS116-driven

53

alleviation of colitis.

54

MATERIALS AND METHODS

55

Materials

56

Cell culture products were from Hyclone (Logan, UT). Penicillin, streptomycin

57

and Puromycin were from Life Technologies, Inc. (Gaithersburg, MD). DSS (MW

58

40–50 kD) was provided by MP Biomedicals (Santa Ana, California). Reverse

59

transcription PCR and RT-qPCR kits were provided by Takara Bio (Dalian, China).

60

Lactobacillus plantarum NCU116, Stabl3 and pLKO.pig vector were stored at - 80ºC.

61

Preparation of EPS116

62

The exopolysaccharides from Lactobacillus plantarum NCU116 (EPS116)

63

were extracted as previously described[17]. The polysaccharide of EPS116 was

64

determined to be 83.7% and it contained protein (15.1%). Furthermore, EPS116 was 4


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made up of mannose, glucose, glucuronic acid, glucosamine, and galactosamine with

66

a molar ratio of 9.6:4:2:1.4:1, as shown by monosaccharide composition analysis[17].

67

Animal model of colitis

68

All animal experiments were conducted in approved protocol and license

69

(SYKX-2015-0001) by the Animal Care Review Committee of Nanchang University,

70

China. C57BL/6 mice (male, 18–22 g, aged 8 weeks) were from University of

71

Chinese Medicine, Jiangxi, China. Mice were randomly divided into different

72

treatment groups according to their weight. The mice were weighed daily after a week

73

of acclimation. Healthy control mice received standard chow, while the other groups

74

(n = 12 mice) received 4% DSS (IBD model

75

EPS116 (0/80/160 mg EPS116/kg mouse/day) for consecutive 7 days.

76

Assessment of intestinal inflammation

[18]

) in drinking water, and treated with

77

Body weight, stool consistency, blood in the stool and on the anus of mice were

78

assessed daily. Fecal samples were acquired from each mouse and checked by fecal

79

occult blood test kit (Nanjing jiancheng Inc., Nanjing, China). Disease activity index

80

(DAI) was evaluated as mentioned before [19]. Mice were culled on Day 8 or 9.

81

Histological assessment of colitis

82

Fixed colons were embedded in paraffin, sectioned at 4 µm, and stained with

83

hematoxylin and eosin. All sections were assessed for the severity of tissue damage

84

with an optical microscope (Nikon TE2000, Nikon Corporation, Japan) in a blinded

85

manner. The scores of colitis were obtained in line with the criteria illustrated 5



86

previously [20] .

87

Cytokine measurement

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Cytokines were measured by commercial ELISAs kits (Boster-Bio, Wuhan,

89

China) according to the protocols supplied by the manufacturer, IFN-γ, TNF-α and

90

IL-6 were measured in serum.

91

Analysis of intestinal barrier function

92

FITC-dextran

tracer

(in

vivo

analysis):

Mice

received

93

intragastric administration of FITC-dextran (MW 4kd, Sigma-Aldrich) to trace

94

intestinal permeability at 500 mg/kg body weight. Serum was harvested after 4 h

95

gavage, and FITC-dextran was detected by a multifunctional spectrophotometer

96

(Varioskan, Thermo Fisher Scientific,).

97

Transepithelial electrical resistance (TER, In vitro analysis): Human epithelial

98

colorectal cell line Caco-2 (American Type Culture Collection) were seeded in the

99

upper chamber of a transwell filter within medium (DMEM+10% FBS+100 µg/ml

100

streptomycin and penicillin). Three weeks late, Caco-2 monolayers barrier model was

101

established. TER was used as an epithelial voltohmmeter.

102

Reverse Transcription-quantitative PCR (RT-qPCR)

103

RNA preparation and RT-qPCR was performed as previously described[17]. In

104

brief, the integrity, concentration and purity of isolated total RNA was checked before

105

reverse transcription PCR.

106

The mRNA level were determined by RT-qPCR with the SYBR Premix Ex 6


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107

Taq™ II (Takara Bio, Dalian, China). The specific RT-qPCR primers for target genes

108

were showed in Table 1. Changes in mRNA levels of target genes were expressed as

109

the fold of the negative control.

110

Western blot analysis

111

Protein samples were prepared and subjected to 8%-12% SDS-PAGE, then

112

transferred to PVDF membrane. The membranes were probed overnight at 4oC with

113

the corresponding antibodies as below: anti-ZO-1, anti-Occludin (Cell Signaling,

114

Danvers, MA), anti-Phospho-STAT3 (Thermo Fisher Scientific, Waltham, MA),

115

anti-β-actin (ZSGB-Bio, Beijing, China), and anti-lamin A (Boster-Bio, Wuhan,

116

China). The signals of western blots were checked and quantified by Gel Doc

117

XR+ system (Bio-Rad Laboratories, Hercules, CA).

118

Chromatin immunoprecipitation (ChIP) assay

119

ChIP was performed using the method by Euskirchen, G. M. et al

[21]

with

120

minor modifications. In brief, Caco-2 cells were cross-linked with 1% formaldehyde

121

for 15 min at RT, then this reaction was quenched by adding glycine to 0.125M final

122

concentration. Cells were lysed with lysis buffer, sonicated to generate DNA

123

fragments (about 300 bp). Clarified lysates were incubated with anti-STAT3 (Santa

124

Cruz Biotechnology, Santa Cruz, CA) or IgG control overnight at 4°C. Protein–DNA

125

complexes were reversed cross-links with 5 M NaCl. All samples were treated with

126

proteinase K, then followed by extraction with phenol–chloroform. ChIP-DNA was

127

analyzed by qPCR with primers as follow: occludin promoter (Potential promoter

7



were

predicted

by

the

web

site

http://www.ensembl.org/

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sequences

or

129

https://epd.vital-it.ch/): occluding promoter forward: 5’-AGATGCCTTTTTCCAGCA

130

AC-3’, reverse: 5’-AGGTCCAGAGGGGACTGTTT-3’; ZO-1 promoter: forward:

131

5’-GGGAAGTTACGTGGCGAAG-3’, reverse: 5’-GGGAATTCAACTCGGACAAA

132

-3’.

133

Construction of STAT3-shRNA vector

134

pLKO.pig plasmid was digested by restriction enzymes EcoRI-HF and

135

AgeI-HF (New England Biolabs, Ipswich, Ma), and ligated with STAT3-targeting

136

shRNA to produce recombinant plasmids. shRNA primers for STAT3: Primer 1:

137

Forward:5'-CCGGGCACAATCTACGAAGAATCAACTCGAGTTGATTCTTCGTA

138

GATTGTGCTTTTTG-3',Reverse:5'-AATTCAAAAAGCACAATCTACGAAGAATC

139

AACTCGAGTTGATTCTTCGTAGATTGTGC-3'; Primer 2: Forward: 5'-CCGGG

140

GCGTCCAGTTCACTACTAAACTCGAGTTTAGTAGTGAACTGGACGCCTTTT

141

TG-3',Reverse:5'-AATTCAAAAAGGCGTCCAGTTCACTACTAAACTCGAGTTT

142

AGTAGTGAACTGGACGCC-3'. The recombinant plasmids were verified by DNA

143

sequencing.

144

Establishment of STAT3-deficient cells

145

To generate STAT3-deficient Caco-2 cells, we used the method described

146

before [17]with minor modification. In brief, recombinant STAT3 targeting shRNA-

147

pLKO.pig plasmids were transfected into 293t cells with Lipofectamine® 3000

148

transfection reagent (Thermo Fisher Scientific, Waltham, MA). After that lentiviral

8


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particles were produced and collected, and infected with Caco-2 cells. Stable

150

integration were achieved by culture within DMEM media plus 5 µg/mL puromycin

151

for 72 h. The knockdown efficiency of STAT3 was detected by RT-qPCR or Western

152

blots.

153

Statistical analysis

154

Statistics were obtained by software Graphpad prism 7. ANOVA was used to

155

compare data among different groups. Tukey's multiple comparisons test and

156

Dunnett's multiple comparisons test were applied. Statistical significance of result

157

was set to a p value < 0.05,*. Means ± SEM were shown to present results from at

158

least three in vitro experiments or from two independent animal experiments.

159

Results:

160

EPS116 relieved DSS-induced colitis in mice

161

The main external symptoms of DSS-induced colitis in mice are weight loss,

162

severe diarrhea, and blood in the stool. Our data showed that EPS1116 obviously

163

relieved loss of body weight correlated to DSS administration, while the model group

164

(treatment with DSS only) continuously lost body weight (Figure. 1a). At the same

165

time, EPS116 distinctly improved colon length, colon weight, DAI score, and

166

histopathological damage of DSS colitis in mice (Figure. 1b-1f).

167

EPS116 reversed intestinal permeability under DSS-induced experimental colitis

168

conditions

169

Intestinal barrier dysfunction results in increased serum level of FITC-dextran (4 9



[22]

170

kDa) in vivo

. Our study showed that the serum level of FITC-dextran was

171

remarkably elevated in the colitis mice compared with the healthy control. While

172

gavage of EPS116 markedly reduced FITC-dextran level in serum (Figure 2a).

173

EPS116 up-regulated expression of tight junction protein in intestinal GC

174

It has been shown that TJ proteins play a vital part in preserving epithelial

175

barrier function. Hence, we checked the expression of tight junction protein genes,

176

such as Claudins, Occludin, and ZO-1. Compared with DSS group, the expression of

177

Claudin-1, Occludin and ZO-1 was distinctly increased in the 160 mg/kg EPS116

178

treatment group, while Claudin-2 was obviously decreased (Figure 2b, 2c and 2d).

179

EPS116 down-regulated the serum levels of pro-inflammatory cytokines in colitis

180

mice

181

To further investigate the molecular mechanisms underlying the inhibition of

182

colitis by EPS116, the impacts of EPS116 on the expression of pro-inflammatory

183

cytokines were assessed. DSS administration resulted in significantly elevated serum

184

levels of the pro-inflammatory cytokines TNF-α, IFN-γ, and IL-6, compared with the

185

healthy control group (Figure. 3). Our data showed that pro-inflammatory cytokine

186

levels were significantly decreased in the 160 mg/kg EPS116 treatment group and

187

was similar to the expression of healthy control group.

188

EPS116 promoted epithelial barrier function and the expression of tight junction

189

proteins in vitro

190

To investigate the molecular mechanisms underlying the promotion of epithelial 10


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barrier function by EPS116, we examined whether EPS116 were able to increase the

192

transepithelial electrical resistance of Caco-2 cell monolayers. Our results showed

193

that value of TEER was markedly raised in 160 µg/ml EPS116-treated Caco-2

194

monolayers (Figure 4a). Moreover, treatment with EPS116 at a high does obviously

195

increased the expression of occludin and ZO-1 in Caco-2 cells (Figure 4b, 4c and 4d).

196

EPS116 promoted the expression and activation of STAT3 in Caco-2 cells

197

As transcriptional factors, ap-1 and STATs can be activated and subsequently

198

transactivate special genes to involve in many cellular processes, like cell growth,

199

proliferation and apoptosis. To find out which transcription factor was activated by

200

EPS116 and subsequently promoted epithelial barrier function, we measured the

201

expression of C-FOS, c-Jun, STAT3, STAT5 and P65. It was found that the expression

202

of STAT3 was obviously increased by exposure of EPS116 (Figure. 5a and 5b). In

203

order to detect the phosphorylation level of STAT3 in Caco-2 cells after EPS116

204

treatment, Western blots with anti-phospho-STAT3 antibody were performed in this

205

work. As shown in Figure 5b, the phosphorylation of STAT3 was markedly raised

206

after exposure of EPS116.

207

EPS116 induced STAT3 binding to TJ genes’ promoter in Caco-2 cells

208

To further check whether EPS116-driven activated STAT3 bonded to TJ genes’

209

promoter, we performed ChIP assay to examine the efficiency of STAT3 binding to

210

the promoters of occludin and ZO-1. By comparison with negative control, STAT3

211

binding to the promoters of occludin and ZO-1 was markedly increased with EPS116

11



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treatment in Caco-2 cells, as shown by ChIP assays (Figure. 5d and 5e).

213

EPS116 regulated intestinal function via STAT3 in vitro

214

Up-regulation of STAT3 and its binding to the promoters of TJ genes in Caco-2

215

cells treated with EPS116 indicted that STAT3 might be involved in regulation of

216

intestinal permeability by EPS116. To further validate this hypothesis, we used

217

shRNA to knockdown STAT3 in Caco-2 cells and verified it by RT-qPCR and

218

Western blots (Figure 6a and 6b). We then measured the transepithelial electrical

219

resistance (TER) of EPS116 on STAT3 knockdown cells. We found that

220

STAT3-deficient Caco-2 cells were insensitive to EPS116 treatment (Figure 6c), and

221

the expression of Occludin and ZO-1 showed indistinctive change in these cells.

222

However, it was reversed in Caco-2 cells with empty vector (Figure 6d, 6e and 6f).

223

Discussion

224

Lactobacillus is among the most abundant microbes in the human

225

gastrointestinal tract and related to gut health[23]. Numerous studies have shown that

226

EPS from commensals can also regulate intestinal barrier function

227

indicated that EPS116 might alleviate IBD via stimulating the intestinal barrier

228

function. In DSS induced IBD mode, EPS116 inhibited colitis development via

229

improving colonic mucosal inflammation and less disrupting of intestinal barrier

230

function.

[24-27]

. Our data

231

Pro-inflammatory cytokines (such as IL-1b, IL-6, and TNF-α) have been

232

proved to be involved in the pathogenesis of IBD [28-30]. Previous researches indicated

233

that down-regulation of TJ protein induced by TNF-α contributes to the intestinal

234

epithelial barrier dysfunction in IBD

235

reduced the serum levels of pro-inflammatory cytokine IFN-γ, IL-6 and TNF-α in

[31]

. Our data showed that EPS116 significantly

12


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236

mice with IBD, suggesting that EPS116 might regulate intestinal epithelial barrier

237

function by inhibiting the expression of pro-inflammatory cytokines. NF-κB played a

238

vital part in the release of pro-inflammatory cytokines based on its transcriptional

239

activation ability of these pro-inflammatory cytokine genes [32], implying that EPS116

240

might be involved in regulating the expression of pro-inflammatory cytokines via

241

NF-κB.

242

Enterocyte can express TJ proteins, including claudins, occludin and ZO-1,

243

which shape a natural gut barrier, preventing toxins and microbial antigens through

244

the lamina propria

245

contributed to the development of experimental colitis

246

epithelial permeability, claudins can be divided into tight claudins (such as claudin 1),

247

which improve the tightness of the barrier, and leaky claudins (like claudin 2), which

248

help to enhance intercellular permeability

249

up-regulated expression of tight junction protein occludin and ZO-1 in vivo and in

250

vitro, and repressed the expression of leaky claudin 2 in colon. These results

251

suggested that EPS116 promoted intestinal barrier function via regulation of the

252

expression of TJ proteins.

[33]

. It was proved that decrease of TJ protein expression

[8, 35]

[34]

. Due to the impacts on

. Our data showed that EPS116

253

Next, we showed potential mechanism for this relation between EPS116 and TJ

254

proteins in vitro. EPS116 markedly up-regulated the expression of occluded and ZO-1

255

in Caco-2 cells, this relationship was further validated by ChIP and RNAi results.

256

EPS116 indirectly activated STAT3, which in turn bound to the promoter of occludin

257

and ZO-1. Knockdown of STAT3 in Caco-2 with EPS116 treatment increased

258

intestinal permeability and decreased expression of occludin and ZO-1. Collectively,

259

EPS116 maintained the intestinal barrier function via upregulating expressions of

260

ZO-1 and occludin; STAT3 acted as a vital signaling molecule in this process. It has 13



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been shown that activated STAT3 played an impotent role in enterocyte for

262

recovering the intestinal barrier and regaining intestinal homeostasis in mice with

263

colitis or in human patients with IBD

264

pro-proliferative genes and anti-apoptotic, along with genes that can significantly

265

enhance migration and restitution of epithelial barrier

266

consistent with those researches. Moreover, we found that EPS116 activated STAT3

267

which led to upregulation of occludin and ZO-1 in the intestinal epithelial cells.

[36]

. STAT3 induces the transcription of

[37, 38]

.

Our data were in

268

EPS116 was demonstrated as a new health care products for intestinal health

269

that displayed the intestinal barrier function regulation activities (Figure 1, 2 and 4).

270

To further develop EPS116 as a health care products for regulating intestinal mucosal

271

barrier, we should better understand the mechanisms by which EPS116 protected the

272

intestines colon from inflammation and promoted intestinal epithelium cell barrier

273

integrity. Based on the results from this study, a model of the mechanisms that

274

EPS116 regulated the intestinal barrier function was proposed (Figure 7). This model

275

suggested that upregulation of tight junction protein ZO-1 and Occludin (Figure 2 and

276

4) through STAT3 phosphorylation and activation (Figure 5) play a vital role in

277

EPS116-driven regulation of the intestinal barrier function. Firstly EPS116 indirectly

278

upregulated, phosphorylated and activated STAT3, then STAT3 translocated into

279

nucleus and bound to the promoter of tight junction protein genes ZO-1 and Occludin

280

(Figure 5), subsequently promoted the expression of ZO-1 and Occludin, which

281

improved the tightness of the barrier(Figure 2 and 4). However, knockdown of STAT3

282

in Caco-2 with EPS116 treatment reversed this phenomenon (Figure 6). Moreover,

283

EPS116 might regulate intestinal epithelial barrier function by inhibiting the

284

expression of pro-inflammatory cytokines (Figure 3). Herein, our data revealed that

285

EPS116 facilitated the gut barrier function in vitro and in vivo, and increased the 14


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286

expression of tight junction proteins via STAT3 signaling pathway.

287

In summary, we have demonstrated that treatment with EPS116 protected the

288

intestines colon from inflammation and promoted intestinal epithelium cell barrier

289

integrity, which might be caused by the activation of STAT3 signaling pathway. Thus,

290

EPS116 may be a preventive therapeutic agent for IBD and a new health care

291

products for intestinal health.

292

Abbreviations

293

(LAB), lactic acid bacteria; (EPS), exopolysaccharides

294

(EPS116), exopolysaccharides from Lactobacillus plantarum NCU116;

295

(DSS), dextran sodium sulfate; (TJ), tight junction;

296

(ChIP), Chromatin immunoprecipitation;

297

(STAT3), signal transducer and activator of transcription 3;

298

(IBD), inflammatory bowel disease; (DAI), disease activity index.

299

Author Contributions

300

X.T.Z., M.Y.X., S.P.N. designed the study; X.T.Z., W.C.Q., T.H., T.X. conducted the

301

experiments; X.T.Z., D.M.G. wrote and revised the manuscript. All authors read and

302

approved the final manuscript.

303

Funding

304

This study was funded by the National Natural Science Foundation of China for

305

Excellent Young Scholars (31422042), the National Key Technology R & D Program

306

of China (2012BAD33B06), the Outstanding Science and Technology Innovation

307

Team Project in Jiangxi Province (2016RCYTB0030). 15



308

Notes

309

The authors declare no competing financial interest.

310

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Figure Captions

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Figure 1. EPS116 protected mice from DSS-induced colitis. Body weight loss (a),

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changes in colon length (b) , colon weight (c) , disease activity index (DAI) score (d)

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in the mice after administration of 4% DSS with different EPS116 concentrations

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(0,80,160

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photomicrographs of colon (H&E; magnifcations: 100×; n = 8). (f) Histological

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scores got from (e). Values represent mean ± SD of the mean; *, P < 0.05; **, P

Exopolysaccharides from Lactobacillus plantarum NCU116

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