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Bioactive Constituents, Metabolites, and Functions

Dietary intake of whole strawberry inhibited colonic inflammation in dextran sulfate sodium-treated mice via restoring immune homeostasis and alleviating gut microbiota dysbiosis Yanhui Han, Mingyue Song, Min Gu, Daoyuan Ren, Xiaoai Zhu, Xiaoqiong Cao, Fang Li, Weicang Wang, Xiaokun Cai, Biao Yuan, Timothy Goulette, Guodong Zhang, and Hang Xiao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05581 • Publication Date (Web): 27 Feb 2019 Downloaded from http://pubs.acs.org on February 27, 2019

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Journal of Agricultural and Food Chemistry

Dietary intake of whole strawberry inhibited colonic inflammation in dextran sulfate sodium-treated mice via restoring immune homeostasis and alleviating gut microbiota dysbiosis Yanhui Han1, Mingyue Song1,2, Min Gu1, Daoyuan Ren1,3, Xiaoai Zhu1,2, Xiaoqiong Cao1, Fang Li1, Weicang Wang1, Xiaokun Cai1, Biao Yuan1, Timothy Goulette1, Guodong Zhang1, Hang Xiao 1* 1

Department of Food Science, University of Massachusetts, Amherst, MA 01003, USA College of Food Science, South China Agricultural University, Guangzhou 510642, China 3 College of Food Engineering and Nutritional Science, Shannxi Normal University, Xi’an 710062, China 2

Corresponding Author: * Hang Xiao Tel: (413) 545-2281; Tax: (413) 545-1262; Email: [email protected]

ACS Paragon Plus Environment 1


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Abstract Strawberry (Fragaria chiloensis) is a major edible berry with various potential health

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benefits. This study determined the protective effects of whole strawberry (WS) against dextran

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sulfate sodium (DSS)-induced colitis in mice. In colitic mice, dietary WS reduced the disease

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activity index (DAI), prevented the colon shortening and spleen enlargement, and alleviated the

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colonic tissue damages. The abundance of pro-inflammatory immune cells was reduced by

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dietary WS in the colonic mucosa, which was accompanied by the suppression of overproduction

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of pro-inflammatory cytokines. Western blotting and immunohistochemical analysis revealed

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that dietary WS decreased the expression of pro-inflammatory proteins in the colonic mucosa.

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Moreover, dietary WS partially reversed the alteration of gut microbiota in the colitic mice by

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increasing the abundance of potential beneficial bacteria, e.g. Bifidobacterium and Lactobacillus,

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and decreasing the abundance of potential harmful bacteria, e.g. Dorea and Bilophila. Dietary

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WS also restored the decreased production of short chain fatty acids (SCFAs) in the cecum of the

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colitic mice. The results revealed the anti-inflammatory effects and mechanisms of dietary WS in

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the colon, which is critical for the rational utilization of strawberry for the prevention of

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inflammation-driven diseases.

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Key Words: strawberry, DSS, colitis, immune, gut microbiota dysbiosis


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Introduction Inflammatory bowel disease (IBD) has posed serious threats to the human health,1 which lead

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to chronic malabsorption of nutrients, abnormal pain and rectal bleeding. Both genetic

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predisposition and environmental factors contribute to the onset of IBD.2 Pathological immune

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response against exotic antigen and bacterial drives the progression of IBD.3 Multiple studies

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suggested that dysbiosis in colon plays an important role in the development of colitis, and gut

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microbiota composition are different between patients with IBD and healthy individuals.4 The

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incidence of colonic inflammation has also been associated with dietary patterns through the

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modification of gut microbiota.5 The diet with high fat, high sugar, high protein and low fiber

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has been proposed to increase the risk of IBD. In contrast, dietary consumption of fruits and

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vegetables has been associated with lowered risk of IBD.6

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Accumulating evidence suggests that colonic inflammation may be prevented by increased

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consumption of certain fruits and vegetables such as berries.6,7 Berries (e.g. chokeberry, red

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raspberry, strawberry, cowberry) and their extracts have been shown to have anti-inflammatory

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and anticancer effects in intestine in various animal models such as azoxymethane-induced rat

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colon cancer model, Apc 1638+/- mouse and Muc2-/- mouse model, and DSS-induced colitis

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models.8-10 Strawberry is one of the most popular berry fruits, which is abundant in polyphenols

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that have been associated with anti-oxidation, anti-inflammation and anti-cancer bioactivities.

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Strawberry juice exhibited anti-inflammatory effects in lipopolysaccharide-treated macrophage

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by decreasing the production of pro-inflammatory cytokines,11 and inhibited the cell proliferation

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of human colon cancer cell lines.12 A human study showed that consumption of strawberry could

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increase the serum antioxidant capacity.13 The consumption of strawberry beverage was

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associated with the amelioration of postprandial inflammatory response.14 These evidences



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suggested the promising bioactivity of berry fruits against the colonic inflammation. Whole

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strawberry contains vitamins, flavonoids, phenolic acids, anthocyanins, polysaccharides and

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fiber,12 which may exhibit stronger anti-inflammatory effects when compared to the extracts.

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However, there is no detailed mechanistic information available on the potential protective

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effects of whole strawberry against IBD. Therefore, this study for the first time determined anti-

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inflammatory effect of whole strawberry in DSS-induced colitis in mice and elucidated the

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potential mechanisms involved.

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MATERIALS AND METHODS

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Animals, diet and experiment design

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Male CD-1 mice were purchased from Charles River Laboratories (Wilmington, MA, USA). All

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animal use was in compliance with regulations of Institutional Animal Care and Use Committee

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of University of Massachusetts Amherst. The mice were randomly divided into six groups

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(n=10) after one-week acclimation. Three groups were treated with 1.5% (w/v) DSS in drinking

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water for 4 cycles (4 days/cycle, with 7-day recovery after each of the first three DSS cycles) to

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induce colitis. These three colitic groups were treated with AIN93G diet containing 0%, 2.5% or

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5% (w/w) freeze-dried whole strawberry powder, respectively, throughout the entire experiment.

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The other three noncolitic groups were treated with regular drinking water plus AIN93G diet

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containing 0%, 2.5% or 5% (w/w) WS, respectively, throughout the entire experiment.

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LC-MS analyses of polyphenols in whole strawberry powder

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Polyphenols in whole strawberry powder were extracted with 80% acetone with 0.1% acetic

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acid for three times. Each time, the extraction mixture was sonicated for 30 min at room

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temperature, and then the supernatant was collected by centrifuging at 3,500 g for 5 min. The


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pooled supernatant was dried under vacuum and then re-dissolved in 50% methanol aqueous

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before analysis by LC-MS. Identification and quantification of polyphenols in strawberry were

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conducted using by LC-MS technique. Chromatographic separation of polyphenols was carried

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out on Kinetex XB-C18 column (100 mm ×4.6 mm, 2.6 μm, Phenomenex, Torrance, CA, USA).

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The mobile phase consisted of 0.1% formic acid in 95% water mixed with 5% ACN (solvent A),

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and 0.1% formic acid in ACN (solvent B). The elution program was as follows: 0-2 min,

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isocratic elution with 5% B; 2-37 min, 5-95% B; 37-40 min, 95-100% B; 40-46 min, 100-5% B;

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46-52 min, isocratic elution with 5% B. The flow rate was 0.4 mL/min, and injection volume was

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10 µL. Pelargonidin 3-glucoside, pelargonidin 3-rutinoside, proanthocyanidins B1, catechin,

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myricetin, kaempferol were used as external standards, and pinostilbene was used as an internal

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standard. All chemicals were from Sigma Aldrich (St. Louis, MO, USA).

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Disease activity index (DAI), histological and immunohistochemistry evaluation

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DAI was evaluated by the scores of bodies weight loss (the weight before each DSS treatment

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compared to that after the DSS treatment, scored as: 0, no change; 1, 1-5% loss; 2, 5-10% loss; 3,

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10-20% loss), stool consistency (0, normal; 1, soft but firm; 2, soft; 3, diarrhea), and fecal blood

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(0, none; 1-2, blood; 3, gross bleeding).15 Once the mice were sacrificed, colons were harvested

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and processed for histological analysis as we previously described.16,17 The score system was as

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follows: epithelium: 0, normal; 1, loss of goblet cells; 2, loss of goblet cells in large areas; 3, loss

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of crypts; 4, loss of crypts in large areas. Infiltration: 0, none; 1, infiltrate around crypt basis; 2,

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infiltrate reaching to mucosae; 3: extensive infiltration to mucosae; 4: infiltration of

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submucosa.18 Immunohistochemistry analysis of the colon was performed as we previously

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described.16,17 Processed tissue sections were treated with a primary antibody against inducible



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nitric oxide synthase (iNOS) and visualized after the treatment with a biotin-conjugated

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secondary antibody (Dako, Santa Clara, CA, USA).

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Western blot and enzyme-linked immunosorbent analysis (ELISA)

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Colonic mucosa was scrapped and lysed in RIPA buffer (Boston BioProducts, MA, USA)

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containing a protease inhibitor cocktail (Boston BioProducts, MA, USA), and phosphatase

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inhibitor. Contents of protein were determined by BCA protein assay kit (Boston BioProducts,

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MA, USA). Samples were subjected to SDS-PAGE, and proteins were transferred to

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nitrocellulose membranes. Membranes were then processed and visualized by Odyssey CLx

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system (LI-COR Biosciences, Lincoln, NE, USA).17,19

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Colonic mucosa was scrapped and homogenized in MSD Tris Lysis buffer (Rockville,

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Maryland, USA) containing protease inhibitor cocktail and phosphatase inhibitor. The protein

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content was quantified by BCA kit (Boston BioProducts, MA, USA), and then cytokines were

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quantified by U-PLEX ELISA kit (Meso Scale Discovery, Rockville, Maryland, USA) following

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the manufacture’s procedure.

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Quantification of immune cells in the colonic mucosa and GSH/GSSG in the liver Quantification of immune cells in the colonic mucosa was conducted as the study previously

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described.20 Briefly, distal colons were lysed with Hanks’s balanced salt solution (1mM DTT

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and 5 mM EDTA) for 2 h at 4 ℃ after washing with cold PBS. Seventy μm sorters (BD

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Biosciences, San Jose, CS, USA) were used to obtain single-cell suspensions, followed by the

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staining with APC-conjugated CD11c antibody, FITC-conjugated CD45 antibody, PE/Cy7-

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conjugated Ly-6G/Ly-6C(Gr-1) antibody, PerCP/Cy5.5-conjugated F4/80 antibody, and isotype

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control antibody (Biolegend, San Diego, CA, USA). BD LSRFortessa cell analyzer (BD


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Biosciences, San Jose, CA, USA) was used to quantify the stained cells, and data were analyzed

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by FlowJo software.

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Livers were rinsed in cold PBS before homogenization with PBS (1 mM EDTA), followed by

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centrifugation at 10,000 g for 15 min at 4 ℃. Supernatants were deproteinated with MPA reagent

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(Cayman Chemical, Ann Arbor, MI, USA). GSH and GSSG were quantified by a glutathione

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assay kit (Cayman Chemical, Ann Arbor, MI, USA) with the help of SpectraMax i3 (Molecular

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Devices, CA, USA).

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Quantification of SCFAs by gas chromatography

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SCFAs were extracted from cecal digesta with 0.05% phosphoric acid, followed by the vortex

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and centrifugation for 20 min at 14,000 rpm at 4 ℃. Supernatants were analyzed by gas

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chromatography with a flame ionization detector (Shimadzu GC-QP2010 SE, Tokyo, Japan).

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Identification of SCFAs was achieved by comparing with the chemical standards (acetic acid,

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propionate acid, butyric acid, isovaleric acid, and valeric acid (Sigma-Aldrich, St. Louis, MO,

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USA)), and 2-ethylbutyric acid was used as the internal reference.

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Microbial analysis

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Fecal pellets were store at -80 ℃ once being collected from mouse colon until analysis.

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Bacterial DNA was extracted from feces using PowerFecal DNA isolation kit (MoBio

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Laboratories, Inc., Carlsbad, CA, USA) following the manufacturer’s instructions. The

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concentration of DNA was measured by NanoDrop Spectrophotometer (ThermoScientific,

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Waltham, MA, USA). The primer sequences used to amplify V3-V4 regions of 16S rRNA gene

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was:16S Amplicon PCR Forward Primer =

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5'TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG and



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16S Amplicon PCR Reverse Primer =

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5’GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC.

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The second PCR was conducted to attach dual indices and Illumina sequencing adapters using

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Nextera XT Index Kit (Illumina, San Diego, CA, USA). PCR products were quantified using

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Qubit dsDNA BR Assay kit (Life Technology, Carlsbad, CA, USA), and the size of PCR

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products was verified by DNA analysis ScreenTape Assay on Tape Station 2200 (Agilent

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Technologies, Santa Clara, CA, USA). PCR final products were pooled in equimolar

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concentration and diluted to 4nM and denatured by NaOH. After combining the amplicon library

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and PhiX control, the samples were loaded onto the 600-cycle MiSeq Reagent kit v3 cartridge

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and sequenced on an Illumina MiSeq platform (Illumina Inc, San Diego, CA, USA).

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Data Analysis

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Microbial data were analyzed by Quantitative insights into microbial ecology (QIIME)

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software pipeline v1.9.1. Taxonomic abundances analysis based on 16S rRNA gene data set was

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performed by LEfSe.21 Comparison of two or more groups was analyzed by either one-way

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parametric ANOVA followed by Tukey’s post hoc test or one-way nonparametric ANOVA

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(Kruskal- Wallis test) followed by Dunn’s post hoc test.

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RESULTS AND DISCUSSION

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Dietary whole strawberry inhibited colitic symptoms

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The doses of WS used in the experiment were 2.5% and 5% (w/w) in the mouse diet, which were equivalent to about 167 g (1.1 cup) and 334 g (2.2 cup) of fresh strawberry per day per


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person (60 kg), respectively. These doses are within the achievable range for humans. As shown

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in Figure 1A, during the experimental period, the body weight of the control mice kept

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increasing (at a decreasing rate) and reached plateau at the fourth DSS cycle. In contrast, DSS

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group (positive control) showed decreased body weight during the third DSS cycle and

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continued body weight loss during the third recovery period and the last DSS cycle. Dietary WS

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treatments slowed down the body weight loss of the DSS-treated mice, especially those fed with

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5% WS. The DAI of the DSS group was much higher than that of the control group, and WS at

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both 2.5% and 5.0% effectively decreased DAI (Fig. 1A). The shortening of the colon and

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increase of spleen weight are indicators of severity of colitis. Our results showed WS reversed

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the shortening of the colon and increase in spleen weight in colitic mice (Fig. 1B and C). These

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results demonstrated that dietary WS effectively inhibited the severity of colitis in DSS-treated

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mice. Strawberry is a rich source of bioactive components such as various polyphenols. Using

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LC-MS, the abundance of pelargonidin 3-glucoside, pelargonidin 3-rutinoside,

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proanthocyanidins B1, catechin, myricetin, and kaempferol were quantified in the whole

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strawberry powder. The results were shown in the Table 1. These polyphenolic compounds have

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been previously reported to have different bioactivities.9-13,22 Another important component in

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strawberry is dietary fiber (about 20% w/w dry weight).23,24 All of these components are

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potential contributors of the observed protective effects against colonic inflammation in mice. It

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is important to note that there might be synergistic interactions among different strawberry

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components, which might produce enhanced anti-inflammatory effects. The exact mechanism of

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actions of each component and their interactions are important focuses of future investigations.

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Dietary whole strawberry ameliorated colonic injuries, reduced infiltration of immune

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cells, and improved cellular anti-oxidative status.



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Hematoxylin and Eosin (H&E) staining indicated that DSS treatment caused severe colonic

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injuries, evidenced by the epithelium distortion, large areas of depletion of goblet cells, damage

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of crypts and the infiltration of inflammatory cells, resulting in much higher histopathological

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scores compared to the control group (Fig. 1D and E). In contrast, WS treatments (2.5% and 5%)

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significantly reduced the abnormal histopathological changes in the colon of the DSS-treated

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mice. The preserved colon architecture suggested the protective effect of dietary WS (2.5% and

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5%) against DSS-induced colonic injuries.

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In response to DSS treatment, infiltration of immune cells increased in the mouse colonic

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mucosa, which is similar to the situation of IBD patients.25 Increased infiltration of neutrophils

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was reported in DSS treated mice, along with the high oxidative stress via production of ROS in

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the colon.26 The flow cytometry results indicated that DSS treatment significantly elevated the

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infiltration of total immune cells, macrophage, neutrophils and dendritic cells by 2.7-folds, 8.1-

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folds, 6.4-folds and 10.9-folds, respectively, in the mucosa of the distal colons when compared to

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the control group (Fig. 1G). Dietary WS decreased the infiltration of these immune cells in the

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colon, especially, dietary WS almost reversed the infiltration of macrophage, neutrophils and

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dendritic cells. Similar to these results, polyphenol-rich apple extracts and red raspberries were

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shown to reduce the immune cell infiltration in a rat model of colitis.27

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Innate immunity is activated when special cells recognize pathogens, and this process triggers

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the production of ROS and cytokines that amplifies the host response.28 Increased oxidative

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stress has been associated with extensive infiltration of immune cells in DSS-induced colitis.29

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Antioxidants have been found effective in attenuating colitis. Glutathione is the most important

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intracellular ROS scavenger, and its depletion can be observed under inflammatory conditions.30

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The ratio of GSH/GSSG in the liver was determined, and the data showed that GSH/GSSG was


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markedly reduced in DSS-treated mice than the control mice, indicating the depletion of GSH

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and higher level of ROS in the DSS group than that in the control (Fig. 1F). Dietary WS (2.5%

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and 5%) increased the GSH/GSSG ratio to the levels similar to that of the control mice, thus

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suggesting the protective effects of WS against ROS in colitic mice.

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Dietary whole strawberry decreased the production of proinflammatory cytokines

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Improper upregulation of proinflammatory cytokines is a marker of inflammation. IL-10 is

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considered an anti-inflammatory cytokine, the expression of IL-10 in colitic mice was higher

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than that in healthy mice (Fig. 2A), this may due to the self-defeating response, indicating more

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severe colitis in DSS-treated mice. Also, as shown in Fig. 2A, DSS-treated mice showed

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drastically enhanced production of proinflammatory cytokines in the colonic mucosa than the

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control, such as TNF-α, IL-1β, IFN-γ and IL-17. Dietary WS effectively decreased the

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production of all five cytokines mentioned above in the DSS-treated mice. Considering the data

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on immune cells and cytokines, the trend of cytokine production in different groups was similar

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to that of the infiltration of the immune cells (Fig. 1G and Fig. 2A), which was consistent with

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the notion that infiltrated immune cells are the major producers of the various cytokines. These

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data suggest that anti-inflammatory effect of WS were at least partially due to maintenance of

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immune system homeostasis by reducing immune cell influx and overproduction of

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proinflammatory cytokines in the colon.

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Dietary whole strawberry modulated inflammation-related signaling proteins

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To investigate the mechanism by which dietary WS inhibits the production of inflammation

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mediators, the expression of COX-2 and iNOS were determined. We found that levels of COX-2

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and iNOS were elevated in colitic mice, whereas WS successfully decreased their expressions



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(Fig. 2B and C). The NF-κB signaling pathway strongly influences the pathogenesis of colitis,

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and dictates the expression of proinflammatory proteins, such as COX-2 and iNOS, which

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markedly increased in the DSS-treated mice.31 The results showed that NF-κB pathway was

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activated in the colon of the colitic mice, evidenced by the elevated expression of p50, a vital

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subunit of NF-κB (Fig. 2B). Dietary WS effectively suppressed the activation of NF-κB

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signaling. Similar to these findings, polyphenol-rich fruits or extracts were found to suppress

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NF-κB signaling pathway in DSS-treated rodent models.7,10 AP-1 is another critical pathway that

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involved in the proinflammatory response. The DSS-induced colitis was associated with

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increased expression levels of c-JUN, the key component in AP-1 pathway.32 The results showed

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the colonic level of c-JUN was increased by 5.6-fold in the colitic mice than the control.

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Whereas, dietary WS (2.5% and 5%) treatments decreased the expression of c-JUN by 73% and

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91%, respectively in colitic mice.

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The p53, an important tumor suppressor, can be activated and its expression level has been

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found increased in colitis,33 the elevated p53 expression in colitic mice is likely to be adaptive

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tissue response to facilitate the damaged cells. We found that the expression of p53 was

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increased in the colitic mice, but it was decreased by dietary WS treatments, suggesting the less

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severe colitis with dietary WS treatment. The activity of cleaved caspase-3 (C-C 3) was found

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increased in both colon cancer cells treated with LPS and the colon of the DSS-treated mice,34,35

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which may be related with tissue injuries caused by the colonic inflammation. Consistent with

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these findings, Figure 2B showed that DSS treatment increased expression levels of cleaved

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caspase 3 by 4.8-folds compared to the control. Dietary WS (2.5% and 5%) suppressed the

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expression of cleaved caspase 3 by 76% and 98% compared to the DSS group, indicating that

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WS protected the colonic tissues against DSS-induced tissue injuries.


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Dietary whole strawberry alleviated gut microbiota dysbiosis Accumulating evidence suggested that gut microbiota play a vital role in colonic

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inflammation, and the change from “disease-free” to “disease” microbiota composition is an

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important characteristic of IBD.36 In the disease-free (noncolitic) mice, i.e. the control and WS

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(2.5% or 5%) groups, 16S rRNA sequencing showed that 5% WS treatment significantly

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increased α diversity of the microbiota, which was assessed by PD whole tree (Fig. 3A), and

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modulated β diversity of the microbiota, which was assessed by principal coordinate analysis of

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the weighted UniFrac Index(P

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