Chitosan Oligosaccharide Reduces Intestinal Inflammation That

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Chitosan oligosaccharide reduces intestinal inflammation that involves CaSR activation in LPS challenged-piglets Bo Huang, Dingfu Xiao, Bi E Tan, Hao Xiao, Jing Wang, Jie Yin, Jielin Duan, Ruilin Huang, Chenbo Yang, and Yulong Yin J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05195 • Publication Date (Web): 10 Dec 2015 Downloaded from http://pubs.acs.org on December 15, 2015

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Journal of Agricultural and Food Chemistry

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Chitosan oligosaccharide reduces intestinal inflammation that involves CaSR activation in

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LPS challenged-piglets

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Bo Huang,†,‡, §Dingfu Xiao,§,#,

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Duan,†,‡Ruilin Huang,†Chenbo Yang,†and Yulong Yin†, *

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†

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South-Central China, Ministry of Agriculture, Hunan Provincial Engineering Research Center of

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Healthy Livestock Key Laboratory of Agro-ecological Processes in Subtropical Region, Institute

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of Subtropical Agriculture, Chinese Academy of Scienses, Changsha, Hunan, China

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‡

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§

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China

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#

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Hunan Collaborative Innovation Center of Animal Production Safety, Changsha Hunan, 410128,

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China

§

Bie Tan,†,#,

*

Hao Xiao,†,‡ Jing Wang,†,‡ Jie Yin,†,‡Jielin

Scientific Observing and Experimental Station of Animal Nutrition and Feed Science in

University of Chinese Academy of Sciences, Beijing, China

College of Animal Science and Technology, Hunan Agricultural University, Changsha , Hunan,

Hunan Collaborative Innovation Center for Utilization of Botanical Functional Ingredients;

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ABSTRACT: Chitosan oligosaccharide (COS) is a degradation product of chitosan with

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anti-oxidative, anti-inflammatory and anti-bacterial effects. This study was conducted to

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investigate the effects of dietary COS on the intestinal inflammatory response and the CaSR and

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NF-κB signaling pathways that may be involved using a lipopolysaccharide (LPS)-challenged

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piglet model. A total of 40 weaned piglets were used in a 2 × 2 factorial design; the main factors

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were dietary treatment (basal or 300 µg/kg COS) and inflammatory challenge (LPS or saline). On

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the morning of days 14 and 21 after the initiation of treatment, the piglets were injected

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intraperitoneally with Escherichia coli LPS at 60 and 80 µg/kg BW or the same amount of

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sterilized saline, respectively. Blood and small intestine samples were collected on day 14 or 21,

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respectively. The results showed that piglets challenged with LPS has a significant decrease in

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average daily gain and gain:feed and histopathological injury in the jejunum and ileum while

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dietary supplementation with COS significantly alleviated intestinal injury induced by LPS.

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Piglets fed the COS diet had lower serum concentrations of TNF-α, IL-6 and IL-8 as well as lower

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intestinal abundances of proinflammatory cytokinesmRNA but higher anti-inflammatory cytokines

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mRNA compared with piglets fed the basal diet among LPS-challenged piglets (p < 0.05). Dietary

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COS increased intestinal CaSR and PLCβ2 protein expressions both in saline- and LPS-treated

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piglets, but decreased p-NF-κB p65, IKKα/β and IκB protein expressions in LPS-challenged

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piglets (p < 0.05). These findings indicate that COS has the potential to reduce the intestinal

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inflammatory response, which is concomitant with the activation of CaSR and the inhibition of

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NF-κB signaling pathways under an inflammatory stimulus.

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KEYWORDS: chitosan oligosaccharide; CaSR; NF-κB; intestinal inflammation; weanling piglets

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INTRODUCTION

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Chitosan oligosaccharides (COS)are hydrolyzed productsfromchitosan with a mixture of

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oligomers of β-1,4-linked d-glucosamine residues.1 Due to its water-solubility, biocompatibility,

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intestinal absorbability and bioactivity, COS has received considerable interest for potential

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application as a dietary supplement or nutraceutical.1 The intestinal epithelium is continuously

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exposed to potentially harmful antigens, pathogens, toxins and air pollutants, and premature

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enterocytes are vulnerable to these exogenous and endogenous stimuli.2, 3 An immature mucosal

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barrier function and immune response are thought to make premature neonates particularly

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susceptible to intestinal inflammation, which can lead to mucosal damage and dysfunction such as

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necrotizing enterocolitis.

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fatty acid and chitosan may play an active role in maintaining the barrier function of the intestine

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as well as in down-regulating inflammation.6-8 COSalso may be a good source material for the

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development of a potent therapeutic agent against inflammatory responses that has been shown to

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have beneficial effects on inflammatory diseases in animal models and clinical trials.9, 10

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Certain nutrients such as glutamine, arginine, (n-3) polyunsaturated

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Some studies have identified the direct target and molecular mechanism of the

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anti-inflammatory effect of COS.6, 11 Nuclear transcription factor kappa B (NF-κB) is one of the

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major signal transduction pathways that is activated in response to inflammation and regulates the

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expression of a variety of genes involved in the inflammatory response.12-14 It has been indicated

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that COS reduces inflammation through the suppression of NF-κB activation in some cell and

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animal models.6, 11, 15, 16 A recent study revealed that COS inhibited NF-κB transcriptional activity

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and the NF-κB-mediated inflammatory response and barrier disruption via adenosine

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monophosphate-activated protein kinase (AMPK)-independent mechanisms.17 The mechanism of


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COS-induced AMPK activation involved calcium-sensing receptor (CaSR)-phospholipase C

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(PLC)-inositol triphosphate (IP3) receptor channel-mediated calcium release from the

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endoplasmic reticulum.17 CaSR is also involved in the ability of γ-glutamyl cysteine(γ-EC) and

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γ-glutamyl valine (γ-EV) to prevent tumor necrosis factor-alpha (TNF-α)-induced inflammatory

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responses in intestinal epithelial cells , and the activation ofCaSR can aid in maintaining intestinal

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homeostasis and reducing inflammation in chronic inflammatory conditions.18

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Therefore, the present study was conducted to investigate the effects of dietary COS on the

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intestinal inflammatory response and the CaSR and NF-κB signaling pathways that may be

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involved using a LPS-challenged piglet model.

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MATERIALS AND METHODS

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Animals and Experimental Design. The animal experiments were approved by the

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Institutional Animal Care and Use Committee of the Institute of Subtropical Agriculture, Chinese

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Academy of Sciences (2013020).

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A total of 40 piglets (Duroc × Landrace × Large Yorkshire) weaned at 28 days with similar

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body weight were randomly assigned to 4 groups (10 piglets/group). Piglets were fed the basal

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diet or the basal diet plus 300 µg/kg COS; the basal diet was formulated to meet the nutrient

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requirements for weanling piglets (NRC, 1998) and has been described in a previous study.19 The

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piglets were housed individually in an environmentally controlled nursery with hard plastic slatted

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flooring. All animals had free access to drinking water. The experiment was arranged as a 2×2

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factorial design. The main factors were dietary treatment (piglets were fed the basal diet or the 300



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µg/kg COS-supplemented diet) and LPS challenge (piglets were challenged with LPS or treated

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with sterile saline). After an adaptation period of 7 days, piglets were fed their respective diets 3

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times per day at 8:00, 13:00 and 18:00 for a 21-d period. Weight gain and feed consumption were

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calculated.

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On the morning of days 14 and 21 after the initiation of treatment, the challenged piglets

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were received an intraperitoneal injection of Escherichia coli LPS (E. coli serotype 055: B5,

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Sigma Chemical) at 60 and 80 µg/kg BW, respectively, based on the previous study and mental

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status in the preliminary test.

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sterilized saline.Two hours following the injection with LPS or saline, 10 mL of blood was

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collected from a jugular vein and serum samples were obtained by centrifugation at 2000 × g for

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10 min at 4 oC and then immediately stored at -80 oC for further analysis.Piglets did not have

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access to feed during the course of the 2-h LPS challengeand then were electrically stunned and

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killedon day 21. The small intestine was rinsed thoroughly with ice-cold physiological saline

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solution, and samples of the jejunum and ileum were immediately snap-frozen in liquid nitrogen

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and stored at -80 oC for RNA extraction and western blot analysis.

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And the unchallenged piglets injected with the same amount of

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Histopathological Grading. Histopathological grading of the jejunum and ileum was

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performed as described previously. Macroscopic evaluation was based on criteria that reflected

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inflammation, including bowel wall thickening, luminal bleeding. After a macroscopic evaluation,

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jejunal and ileal tissues were fixed in 10% buffered formalin solution and embedded in paraffin.

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The embedded specimens were then sectioned (6 µm), stained with hematoxylin-eosin, and

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examined by light microscopy. The slides were assigned a histological grading score that ranged

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from 0 (minimal injury) to 15 (maximal injury) according to 4 grade that included mononuclear or


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polymorphonuclear cell infiltration, histological injury and erosion or epithelial hyperplasia, as

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previously reported (grade 1, normal , histological score 1 to 4; grade 2, mild, histological score 5

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to7; grade 3, moderate, histological score 8 to 10; grade 4, severe, histological score 11 to 14).21-23

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A veterinary pathologist who was blind to the treatment carried out the histological scoring.

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Determination of Serum Concentrations of TNF-α, IL-1β, IL-6, and IL-8. Serum

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concentrations of TNF-α, IL-1β, IL-6, d IL-8 were determined using ELISA kits (Cell Biolabs,

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San Diego, CA, USA) in accordance with the manufacturer's instructions.

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Real-time Quantitative RT-PCR. The abundances of mRNA for thecytokines TNF-α, interleukin

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(IL)-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, interferon-gamma (IFN-γ),

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transforming growth factor-beta 1 (TGF-β1), monocyte chemotactic protein 1 (MCP1) and

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granulocyte macrophage colony stimulating factor (GM-CSF) in the jejunum and ileum were

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determined by real-time quantitative RT-PCR as described previously.24 The primers used to

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amplify genes are shown in Table 1. The relative expression was expressed as a ratio of the target

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gene to β-actin and data are expressed relative to those in basal diet-treated piglets injected with

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saline.

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Western Blot Analysis. The expression of CaSR, PLCβ2, NF-κB p65, p-NF-κB p65, inhibitor of

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nuclear factor kappa-B kinase (IKK)α/β and inhibitor of nuclear factor kappa-B (IκB) protein in

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the jejunum and ileum was determined by western blot analysis as described previously.19

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following antibodies were used for protein quantification: CaSR (1:1000; Bioss Inc, Woburn, MA,

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USA), PLCβ2 (1:1000; Antibodies-online, Atlanta, GA, USA), NF-κB p65 (1:1000; Cell

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Signaling Technology, Danvers, MA, USA), p-NF-κB p65 (Ser536) (1:1000; Cell Signaling


The


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Technology, Danvers, MA, USA), IKKα/β (1:400; Santa Cruz Biotechnology, Dallas, TX, USA),

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IκB (1:1000; Cell Signaling Technology, Danvers, MA, USA) and β-actin (1:1000; Cell Signaling

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Technology, Danvers, MA, USA)and secondary antibody horseradish peroxidase-conjugated goat

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anti-rabbit IgG (1:4000; Boster Biological Technology, Wuhan, China). All protein measurements

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were normalized to β-actin and data are expressed relative to the values in control piglets.

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Immunohistochemical Analysis.The activity of NF-κB p65 in the jejunal and ileal mucosa of

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piglets was determined using immunohistochemical analysis. Firstly, tissue pieces were fixed with

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4% paraformaldehyde and then serial paraffin-embedded sections were made. After dewaxing in

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xylene and re-hydration via gradient ethyl alcohol, the sections were heat by microwave in 0.01

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mol/L citral acid solution for antigen retrieval and were blocked with 4.5 % hydrogen peroxide in

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phosphate-saline buffer for 15 minutes. The sections were incubated with the anti NF-κB p65

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antibody (1:1000; Cell Signaling Technology, USA) overnight at 4°C and then washed 3 × 5 min

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in PBS and incubated with SV Rabbit hypersensitivity two-step immunohistochemical Kit (Boster

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Biological Technology, Wuhan, China) overnight at 4°C according to the manufacturer’s

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instructions. The sections were washed 3 × 3 min with PBS, followed by the addition of

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diaminobenzidine (Boster Biological Technology, Wuhan, China) as a chromogen for 3 to 5 min,

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which was strictly controlled under a microscope. Before staining, the PBS was substituted for

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primary antibodies as a negative control. After rinsing under cold tap water for 5 min and

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counterstaining with hematoxylin (Boster Biological Technology, Wuhan, China), sections were

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dehydrated through an alcohol gradient and covered by general clarity gum. The stained sections

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were reviewed and scored independently by 2 investigators using a microscope at 400-fold

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magnification (Olympus, Tokyo, Japan).


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Statistical Analysis. All statistical analyses were performed by ANOVA using the general linear

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model procedures of SAS appropriate for a 2 × 2 factorial design (SAS Inc., Cary, NC). The

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statistical model included the effects of challenge (saline or LPS), diet (basal or COS), and their

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interactions. The differences among treatments were evaluated using Tukey’s test. Probability

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values < 0.05 were taken to indicate statistical significance.

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RESULTS

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Growth performance. The average daily gain, average daily feed intake and gain:feed are shown

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in the Table 2. Piglets challenged with LPS showed a significant decrease in average daily gain

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from day 0 to 21 as well as gain:feed from day 14 to 21 and day 0 to 21 (p < 0.05). COS diet had

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no effect on the average daily gain, average daily feed intake and gain:feed and there also was no

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LPS challenge × diet interaction for growth performance (p > 0.05).

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Histopathological Grading. Representative hematoxylin-eosin-stained images of the jejunum and

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ileum are shown in Figure 1. The macroscopic appearance of the jejunum and ileum in piglets

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injected with saline showed minimal mononuclear cell and polymorphonuclear cell infiltration,

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minimal histological injury, erosion and epithelial hyperplasia. In contrast, LPS-challenged piglets

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fed the basal diet showed pronounced mononuclear or polymorphonuclear cell infiltration,

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histological injury and epithelial erosion. Dietary supplementation with COS significantly reduced

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the histopathological injury induced by LPS.While piglets challenged with LPS had a higher

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histological injury score in the jejunum (p < 0.05) , diet did not affect the injury score in either the

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jejunum or ileum (p > 0.05).

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injury score was observed: among LPS-treated piglets, those fed the COS diet had a lower injury

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score than piglets fed the basal diet, while there was no difference in the injury score among

A LPS challenge × diet interaction (p < 0.05) for the histological



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saline-treated piglets (Table 3).

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Serum Concentrations of TNF-α, IL-1β, IL-6, and IL-8. On day 14, piglets challenged with

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LPS had higher serum concentrations of TNF-α, IL-1β, IL-6 and IL-8 than piglets injected with

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saline (p < 0.05). However, piglets fed the COS diet had lower serum TNF-α and IL-8 contents

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than those fed the basal diet (p < 0.05). There was a LPS challenge × diet interaction (p < 0.05)

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for the serum concentrations of TNF-α, IL-6 and IL-8, in that the response of these variables to

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LPS challenge was lower in piglets fed the COS diet compared with the basal diet (Table 4). On

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day 21, LPS challenge increased serum concentrations of TNF-α, IL-6 and IL-8 compared with

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saline injection (p < 0.05) , and the COS diet had no effect (p > 0.05) on the serum contents of the

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cytokines examined, except for TNF-α. There was a LPS challenge × diet interaction (p < 0.05)

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for serum TNF-α; piglets fed the COS diet had a lower serum TNF-α content than piglets fed the

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basal diet among LPS-challenged piglets (Table 4).

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Abundance of Cytokine mRNA in the Jejunum and Iileum. In the jejunum, LPS challenge

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increased the abundance of IL-1β, IL-6, IL-8 and IL-12 mRNA in piglets fed the basal diet and

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increased IL-4 mRNA but decreased IL-18 and IFN-γ mRNA in piglets fed both the basal diet and

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COS diet (p < 0.05). Treatment with the COS diet decreased the abundance of IL-1β and IL-6

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mRNA in LPS-challenged piglets and decreased IL-8 mRNA but increased the abundance of

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IL-18, TGF-β1 and MCP1 mRNA in piglets injected with saline or LPS (p < 0.05). There was a

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LPS challenge × diet interaction effect on the abundance of IL-1β, IL-6, IFN-γ and TGF-β1

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mRNA (p < 0.05) (Table 5).In the ileum, the abundance of TNF-α, IL-1α, IL-2 and IL-8 mRNA in

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piglets fed the basal diet as well as the abundance of IL-18 and IFN-γ mRNA in piglets fed both


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the basal diet and COS diet were increased in response to LPS challenge (p < 0.05). Treatment

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with the COS diet increased the abundance of IL-1α and IL-8 mRNA in saline-treated piglets as

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well as the abundance of IL-4 and TGF-β1 mRNA in both saline- and LPS-treated piglets , but

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decreased the abundance of TNF-α, IL-1α, IL-1β, IL-2, IL-8, IL-18, IFN-γ and GM-CSF mRNA

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in LPS-challenged piglets (p < 0.05). A LPS challenge × diet interaction (p < 0.05) was observed

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for the abundance of TNF-α, IL-1α, IL-2, IL-8, IL-18 and IFN-γ mRNA (Table 5).

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Expression of CaSR and NF-κB Pathway Proteins in the Jejunum and Ileum. LPS challenge

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increased (p < 0.05) the expression of CaSR protein in piglets fed the basal diet and COS diet as

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well as the expression of NF-κB and p-NF-κB p65 proteins in the jejunum and ileum of piglets fed

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the basal diet. Dietary supplementation with COS increased the expression of CaSR and PLCβ2

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proteins in the jejunum and ileum of both saline- and LPS-treated piglets but decreased the

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expression of p-NF-κB p65, IKKα/β and IκB proteins in the jejunum and ileum of LPS-challenged

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piglets (p < 0.05). There was a LPS challenge × diet interaction (p < 0.05) for the expression of

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NF-κB p65 and IKKα/β proteins in the jejunum as well as PLCβ2 and IKKα/β proteins in the

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ileum; LPS-challenged piglets, but not saline-injected piglets, fed the COS diet had lower levels of

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jejunal NF-κB p65 and IKKα/β and ileal IKKα/β proteins compared with piglets fed the basal diet

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(Figure 2, Table 6).The immunohistochemical staining for NF-κB p65 showed that there was

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increased NF-κB nuclear expression in the jejunum and ileum of LPS-challenged piglets

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compared to the other three groups.The dramatic increase in the translocation of p65 into the

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nucleus induced by LPS was markedly suppressed by dietary supplementation with COS that there

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were less NF-κB antibody binding in the nucleus of jejunum and ileum in COS + LPS treated

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piglets (Figure 3).



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DISCUSSION

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COS is a potent therapeutic agent against inflammatory responses and has potential for

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application as a dietary supplement or nutraceutical. The anti-inflammatory activity of COS has

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been demonstrated both in vitro and in vivo in previous studies.10, 13 COS has been shown to exert

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anti-inflammatory effects by stimulating TNF-α under LPS-stimulated inflammation in RAW

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264.7 cells,10 and markedly attenuated both the LPS- and TNF-α-stimulated production of TNF-α

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and IL-6 in human colonic epithelial cell-like T84 cells.2 COS also reduced the symptoms of

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asthma via anti-inflammatory activity by down-regulating Th2 cytokines and proinflammatory

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cytokines in IgE-antigen complex-stimulated rat basophilic leukemia RBL-2H3 cells and in

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OVA-sensitized/challenged allergic asthma model mice.9 Nonetheless, the present results in piglets

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showed the anti-inflammatory effect of COS only under an inflammatory stimulus induced by

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LPS.

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LPS has been widely used to mimic features of inflammatory diseases.25, 26 LPS can interact

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with their respective receptors (toll-like receptor (TLR4), TNF receptors 1 and 2) and elicit the

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NF-κB-mediated production of proinflammatory cytokines such as TNF-α, IL-1β and IL-6 and

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induce extensive intestinal inflammation.12, 13 In the present study, LPS induced higher serum

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concentrations of TNF-α, IL-1β, IL-6 and IL-8, and this was accompanied by significant growth

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impairment and intestinal histopathological injury. And the LPS-induced production of these

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proinflammatory cytokines was suppressed by COS but was not observed in the normal piglets.

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Consistent with the reduction in circulating proinflammatory cytokines, COS down-regulated the

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expression of intestinal proinflammatory cytokines including TNF-α, IL-1α, IL-1β, IL-2, IL-8,

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IFN-γand GM-CS in LPS-challenged piglets. This may reflect an improvement in intestinal barrier


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function and the prevention of subsequent infiltration of immune cells.21 Increased intestinal

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permeability has been shown to be closely associated with elevated proinflammatory cytokines in

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inflammatory bowel disease (IBD),27 and this is consistent with the results of histopathological

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grading in the present study.

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With regard to the major target of the inflammatory response, the NF-κB signal transduction

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pathway was inhibited by COS in LPS-challenged piglets. This inhibition may involve

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interference with the binding of bacterial products to its membrane receptors.2 Qiao et al.

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demonstrated that COS significantly inhibited the binding of LPS to the TLR4/MD-2 receptor

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complex, and thus attenuated the activation of mitogen-activated protein kinases (MAPKs) and

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decreased the nuclear translocation of NF-κB.11 COS also decreased O-GlcNAc transferase

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(OGT)-dependent O-GlcNAcylation of NF-κB and thereby attenuated the LPS-induced

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inflammatory response.6 A recent study demonstrated that COS inhibited NF-κB transcriptional

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activity and the NF-κB-mediated inflammatory response in an AMPK-independent manner.17

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Recently, a novel target, CaSR , has been studied to prevent chronic inflammation and tumor

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progression.18, 28, 29 It has been demonstrated that the activation of CaSR mediated the inhibition

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of NF-κB activity and subsequently decreased LPS-stimulated proinflammatory cytokine

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production.30 CaSR was functionally expressed in T cells, and activated CaSR contributed to the

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secretion of both anti-inflammatory and pro-inflammatory cytokines through the partial MAPK

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and NF-κB pathways.31, 32 In the intestine, the activation of CaSR promoted the differentiation of

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colonic myofibroblasts and stimulated regeneration of the intestinal barrier.33 In the present study,

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dietary supplementation with COS increased intestinal expression of CaSR and PLCβ2 protein,

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suggesting that CaSR activation plays a role in the regulation of COS in the intestinal



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inflammatory response. These results are consistent with the finding that γ-EC and γ-EV activate

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CaSR and block NF-κB transposition in intestinal epithelial cells and a mouse model of colitis.18

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COS has also been shown to be a CaSR agonist and to induce CaSR-PLC-IP3 receptor

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channel-mediated calcium release and active the AMPK pathway.2 While both LPS and COS

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stimulated CaSR activation, they regulated the NF-κB signal transduction pathway differently.

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This may be because CaSR stimulation by COS generates a negative feedback inhibition of

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Toll-like receptor-mediated NF-κB activation.30

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In conclusion, we have shown that COS can act as an agonist of CaSR to exhibit intestinal

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anti-inflammatory effects. These findings indicate that COS may be able to reduce the intestinal

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inflammatory response, concomitant with the activation of CaSR and the inhibition of NF-κB

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signaling pathways under an inflammatory stimulus. Further studies will be needed to elucidate

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the mechanisms that regulate homeostasis and cross-talk between CaSR and the NF-κB-mediated

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inflammatory pathway in health and disease.

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AUTHOR INFORMATION

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§

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Corresponding Author

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*

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Phone: +86 84619756. Fax: +86 84615285. E-mail:[email protected]

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Funding

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This project was funded by the National Natural Science Foundation of China (31330075,

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31301985, 31301989, 31372326), the National Key Basic Research Program of China

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(2013CB127302), the China Postdoctoral Science Foundation（2014M562111, 2015T80871）and

These authors contributed equally to this work.

(B.T.) Phone: +86 84619706. Fax: +86 84615285. E-mail:[email protected] or (Y.Y.)


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the Science and Technology Department of Hunan province (2015RS4035).

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Notes

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All of the authors declare no conflicts of interest.

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ACKNOWLEDGMENTS

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We thank Changsha Lvye Biotechnology Limited Company Academician Expert Workstation,

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Guangdong Wangda Group Academician Workstation for Clean Feed Technology Research and

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Development in Swine, Guangdong Hinapharm Group Academician Workstation for Biological

284

Feed and Feed Additives and Animal Intestinal Health for providing technical assistance.

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385

Figure legends

386

Figure 1.The representive hematoxylin-eosin stained image (×200) of the jejunum (A) and ileum

387

(B) in the LPS-challenged piglets. Basal-S, piglets fed the basal diet and injected with saline;

388

Basal-LPS, piglets fed the basal diet and challenged with LPS; COS-S, piglets fed the COS diet

389

and injected with saline; COS-LPS, piglets fed the COS diet and challenged with LPS.

390

Figure 2.The representative Western blot images ofCaSR, PLCβ, NF-κB, IKKα/β,IκBandβ-Actin

391

in the jejenum (A) and ileum (B) of LPS-challenged piglets. Column Basal-S, piglets fed the basal

392

diet and injected with saline; column Basal-LPS, piglets fed the basal diet and challenged with

393

LPS; column COS-S, piglets fed the COS diet and injected with saline; column COS-LPS, piglets

394

fed the COS diet and challenged with LPS.

395

Figure 3.The representiveimmunohistochemical stain image (×400) of the jejunum (A) and ileum

396

(B) in the LPS-challenged piglets. Basal-S, piglets fed the basal diet and injected with saline;

397

Basal-LPS, piglets fed the basal diet and challenged with LPS; COS-S, piglets fed the COS diet

398

and injected with saline; COS-LPS, piglets fed the COS diet and challenged with LPS.

399



400

Table 1. Primers used for quantitative reverse transcription-PCR Gene

Accession No.

Sequence (5'-3') F: CTGCGGCATCCACGAAACT

β-Actin

XM_0031242803 R: AGGGCCGTGATCTCCTTCTG F: TCCAGCATCATTGCATCATC

IL-6

NM_214403.1 R: GGCTCCACTCACTCCACAAG F: ATCTCGGTTGGTGTTGTTCC

IL-12

NM_214097.2 R: GGGTATCTCGTCCTCTGTCC F: CCCGAGTGTCAAGTGGCTTA

IL-4

NM_214340.1 R: TGATGATGCCGAAATAGCAG F: CGGCTGTGATGAATGAAACC

GM-CSF

NM_2141182 R: GTGCTGCTCATAGTGCTTGG F: TTCAGCTTTGCGTGACTTTG

IFN-γ

NM_2139481 R: GGTCCACCATTAGGTACATCTG F: ACAGGCCAGCTCCCTCTTAT

TNFα

NM_214022.1 R: CCTCGCCCTCCTGAATAAAT F:GGGCTATTTGTCCTGACTGC

IL-10

NM_214041.1 R:GGGCTCCCTAGTTTCTCTTCC F:CCTCATCCTCCAGCATGAAGGTCTCTGC

MCP1

NM_2142141 R:GGTGGAGTCAGGCTTCAAGGCTTCGG F: GCTAACTACGGTGACAACAA

IL-1β

NM_214055.1 R: TCTTCATCGGCTTCTCCACT


Page 22 of 33

Page 23 of 33


F: TGCACTAACCCTTGCACTCA IL-2

NM_213861.1 R: CAACTGTAAATCCAGCAGCAA F:TGAGAAGCAACAACAACAGCA

IL-8

NM_213867.1 R: CAGCACAGGAATGAGGCATA F: ACGATGAAGACCTGGAATCG

IL-18

NM_213997.1 R: GGCTTGATGTCCCTGGTTAAT F: ACCCGACTGTTTGTGAGTGC

IL-1α

NM_214029.1 R: TTCCCAGAAGAAGAGGAGACTG F: AAGCGGCAACCAAATCTATG

TGF-β1

NM_214015.1 R: CCCGAGAGAGCAATACAGGT

401



402

Page 24 of 33

Table 2. Growth performance of piglets Basal diet

COS diet

P-Value

Item Saline

LPS

Saline

LPS

Diet

LPS

Diet×LPS

Average daily gain, kg/d day 0 to 14

0.29±0.02

0.23±0.01

0.28±0.01

0.28±0.01

0.169

0.060

0.072

day 14 to 21

0.29±0.02

0.27±0.01

0.33±0.03

0.26±0.02

0.576

0.074

0.333

day 0 to 21

0.29±0.01

0.25±0.01

0.29±0.01

0.27±0.01

0.129

0.010

0.304

Average daily feed intake, kg/d day 0 to 14

0.40±0.01

0.39±0.02

0.41±0.02

0.4±0.01

0.371

0.738

0.933

day 14 to 21

0.40±0.01

0.43±0.02

0.42±0.02

0.44±0.01

0.291

0.058

0.828

day 0 to 21

0.40±0.01

0.4±0.02

0.41±0.02

0.42±0.01

0.337

0.693

0.895

Gain:feed, kg/kg day 0 to 14

0.72±0.03

0.61±0.04

0.69±0.06

0.69±0.03

0.474

0.144

0.174

day 14 to 21

0.75±0.06

0.64±0.04

0.81±0.01

0.59±0.04

0.945

0.022

0.405

day 0 to 21

0.73±0.02

0.62±0.04

0.73±0.06

0.66±0.02

0.598

0.025

0.584

403

Values are mean ± SEM, n = 8 per treatment group.


Page 25 of 33


404

Table 3. Histopathological gradingof the jejenum and ileum in piglets Basal diet

COS diet

P-Value

Item Saline

405

LPS

Saline

LPS

Diet

LPS

Diet×LPS

Jejenum

3.33±1.67 14.67±1.33 6.33±1.20 6.67±1.33

0.111

0.003

0.004

Ileum

3.33±1.76 14.00±2.00 6.67±1.33 4.00±2.31

0.115

0.067

0.008

Values are mean ± SEM, n = 8 per treatment group.



406

Page 26 of 33

Table 4. Serum concentrations ofTNF-α, IL-1β, IL-6 and IL-8in piglets Basal diet

COS diet

P-Value

Item Saline

LPS

Saline

TNF-α (ng/ml)

0.08±0.01

0.69±0.06

0.07±0.01

IL-1β (pg/ml)

9.61±1.23

22.61±1.50

IL-6 (pg/ml)

13.62±4.01

IL-8 (pg/ml)

LPS

Diet

LPS

Diet×LPS

0.33±0.03

Chitosan Oligosaccharide Reduces Intestinal Inflammation That

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