Rhubarb Supplementation Promotes Intestinal Mucosal Innate

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


Rhubarb supplementation promotes intestinal mucosal Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any


innate immune homeostasis



through modulating intestinal epithelial microbiota in goat kids Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any


Jinzhen Jiao, Jian Wu, Min Wang, Chuanshe Zhou, Rong-Zhen Zhong, and Zhi-Liang Tan



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Page 1 of 32

Journal of Agricultural and Food Chemistry

ACS Paragon Plus Environment


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Running title: rhubarb intervention in young goats

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Rhubarb supplementation promotes intestinal mucosal innate immune homeostasis through

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modulating intestinal epithelial microbiota in goat kids

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Jinzhen Jiao†,‡, Jian Wu†,§, Min Wang†, ‡, Chuanshe Zhou†,‡ , Rongzhen Zhong#

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and Zhiliang Tan†,‡,*

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†

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

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Subtropical Agriculture, The Chinese Academy of Sciences; National Engineering Laboratory

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for Pollution Control and Waste Utilization in Livestock and Poultry Production; Hunan

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Provincial Engineering Research Center for Healthy Livestock and Poultry Production;

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

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South-Central, Ministry of Agriculture, Changsha, Hunan 410125, P. R. China.

14 15

‡

Hunan Co-Innovation Center of Animal Production Safety, CICAPS, Changsha, Hunan

410128, P. R. China.

16

§

University of Chinese Academy of Sciences, Beijing, China.

17

#

Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences,

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Changchun, Jilin 130102, P. R. China. *

Corresponding author. Address: Institute of Subtropical Agriculture, the Chinese

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Academy of Sciences, Changsha, Hunan 410125, P.R. China; Email: [email protected]; Tel:

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+8673184619702; Fax: +8673184612685.

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ABSTRACT

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The abuse and misuse of antibiotics in livestock production pose a potential health risk

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globally. Rhubarb can serve as a potential alternative to antibiotics, and several studies have

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looked into its anti-cancer, anti-tumor and anti-inflammatory properties. The aim of this study

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was to test the effects of rhubarb supplementation to the diet of young ruminants on innate

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immune function and epithelial microbiota in the small intestine. Goat kids were fed with a

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control diet supplemented with or without rhubarb (1.25% DM), and were slaughtered at d 50

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and 60 of age. Results showed that the supplementation of rhubarb increased ileal villus

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height (P = 0.036), increased jejujal and ileal anti-inflammatory IL-10 production (P < 0.05),

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increased jejunal and ileal Claudin-1 expression at both mRNA and protein levels (P < 0.05),

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and decreased ileal pro-inflammatory IL-1β production (P < 0.05). These changes in innate

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immune function were accompanied by shifts in ileal epithelial bacterial ecosystem in favor

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of Blautia, Clostridium, Lactobacillus and Pseudomonas, and with a decline in the relative

35

abundance of Staphylococcus (P < 0.001) when rhubarb was supplemented. Additionally, age

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also affected (P < 0.05) crypt depth, cytokine production, Claudin-1 expression and relative

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abundances of specific genera in epithelial bacteria. Collectively, the supplementation of

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rhubarb could enhance host mucosal innate immune homeostasis by modulating intestinal

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epithelial microbiota during early stages of animal development.

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KEY WORDS: rhubarb, innate immune homeostasis, epithelial bacteria, small intestine,

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young ruminants.

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INTRODUCTION

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The constituency of a mammal’s body includes a diverse gut microbiota in which the

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host benefits from the mutual relationship 1. Mounting evidence are emerging that the

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microbial community perform various tasks that are indispensable to the host, such as

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modulating host immune function, providing nutrients to the gut, maintaining metabolic

47

function, and defending against pathogens

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intestinal lumen, their epithelial counterparts locate at the border of the tissue, and exert

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significant roles in modulating host innate immune homeostasis 5. Toll like receptors (TLRs)

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on the gut mucosa have the capacity to recognize microbial structures, thereby triggering

51

innate immune responses 6. Additionally, microbial products, such as acetate, propionate and

52

butyrate, have been proved exert anti-inflammatory effects 7.

2-4

. Compared to the microbes residing in the

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Several factors have been demonstrated to shape host-microbiota symbiosis, with diet as

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the main determinant. In the colon of goats, when compared to a hay diet, the high grain diet

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increased the relative abundance of Blautia and decreased the relative abundances of Bacillus,

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Enterococcus, and Lactococcus. And these were associated with intercellular tight junction

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erosion, and up-regulation of gene expression of IL-2 and IFN-γ in the colonic mucosa 8.

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Secondly, in both monogastric animals and ruminants, gastrointestinal bacterial diversity is

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age-related, which at least partly, accounts for the elevated carbohydrate digestion capacity

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9-12

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Moreover, several studies indicated that early dietary manipulation of animals had a much

62

more lasting effect than those happening later in the lifetime

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promising to regulate gastrointestinal microbial programming at early stages of animal

, highlighting the significance of age as another factor influencing host-microbe symbiosis.

3


13, 14

. On this premise, it is

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development.

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The anti-cancer and anti-tumor properties of rhubarb have promoted a range of research

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questions regarding the effect of rhubarb as a feed additive in shaping gastrointestinal

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bacterial diversity of young animals 15. The anthraquinone of rhubarb shows a wide array of

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pharmacological activities, including anti-inflammatory, antifungal and antibacterial effects 16.

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Supplementation with 0.3% rhubarb extract in mice altered bacterial ecosystem in favor of

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Akkermansia muciniphila and Parabacteroides goldsteinii in the gut digesta. And this was

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coupled with improved hepatic injury, down-regulated inflammatory and oxidative stresses in

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the liver

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increased the relative abundances of Lachnospiraceae NK3A20 group, Ruminococcaceae

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NK4A214 group and Christensenellaceae R-7 group, and in the ruminal mucosa of goats 18.

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However, scanty knowledge is available regarding the effect of rhubarb supplementation on

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interaction between epithelial bacteria and local innate immune function in the small

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intestine.

17

. Another study conducted in our team revealed that rhubarb supplementation

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Therefore, we aimed to explore the effects of rhubarb supplementation to the diet of

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young goats on epithelial bacterial diversity and mucosal immune function in the jejunum

80

and ileum (d 50 and d 60). The DNA-based 16S rRNA amplicon sequencing was used to

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explore epithelial bacterial diversity, together with mRNA and protein level expression of

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genes was used to investigate mucosal innate immune function.

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MATERIALS AND METHODS

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Ethics

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All management and experimental protocols were in line with the animal care protocol 4



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approved by the Animal Care Committee, Institute of Subtropical Agriculture, Chinese

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Academy of Sciences, Changsha, China, with protocol ISA-201603.

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Animal management

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Sixteen newborn Xiangdong black goats (native breed) were housed in a well-ventilated

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room with controlled humidity and temperature. The goats were randomly assigned to two

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diets: the control diet (Control, n = 8) and the diet supplemented with rhubarb (Rhubarb, n =

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8). The rhubarb was obtained from a local herbalist retailer in Changsha, Hunan, China, and

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contained the dried and milled rhizomes of Rheum offcinale Baill. The main bioactive

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constituents of rhubarb root powder (% dry matter) are anthraquinone derivatives, including

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rhein (3.12%), emodin (1.15%), aloe-emodin (1.42%), sennoside A (0.51%), sennoside B

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(0.22%), physcion (0.08%) and chrysophano (0.06%). Goat kids were left with their mothers

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until weaning at d 40, and reared separately from their mothers after weaning. For the

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Rhubarb treatment, goats were gradually habituated to the rhubarb intervention from one

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week before weaning. After weaning, goats in the Control group were supplied with a

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mixture of 105 g starter concentrate (60% of total dry matter) and 70 g fresh grass

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(Miscanthus sinensis, 40% of total dry matter) per meal. The diet was formulated to meet 1.3

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times of maintenance metabolisable energy requirements on the basis of the feeding standard

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of Chinese goats. Two goats in the Rhubarb group were removed due to the reason irrelevant

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to the experiment, and the remaining goat kids received 175 g control diet plus 2.1875 g (1.25%

105

DM) rhubarb per meal. Four goats in the Control group, and three goats in the Rhubarb group

106

were slaughtered at each of the following days: d 50 and 60.

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Sample collection 5


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Immediately after the goats were slaughtered, the jejunal and ileal tissue were rinsed

109

three times with ice-cold sterile phosphate-buffered saline (pH = 7.4). The tissue samples

110

were fixed in 10% formalin solution for anatomic analysis of villus and crypt. The mucosa

111

(approximately 2 g) were scraped from the underlying tissue with the help of a germ-free

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glass slide, divided into four equal proportions, transferred into liquid nitrogen in a moment,

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and then stored at -80°C. The four proportions were used for microbial DNA extraction,

114

mucosal RNA extraction, mucosal protein extraction, and mucosal cytokine detection,

115

respectively.

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Morphology assessment

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The jejunal and ileal tissue samples were removed from buffered formalin, processed in

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low-melt paraffin, and then stained with eosin and hematoxylin (Sinopharm Chemical

119

Reagent Co., Ltd, Shanghai, China)

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accompanying crypts were measured using a fluorescence microscope (Olympus, Tokyo,

121

Japan). The villus height was measured from the tip to the base, and the crypt depth was

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measured from the base of the villus to the base of the crypt. The VCR (ratio of villus height

123

to crypt depth) was calculated.

124

Real-time PCR analysis

11

. Twenty random straightest villi and their

125

Total RNA was extracted from the mucosal samples using TRIzol reagent (Invitrogen,

126

Carlsbad, CA) in accordance with the manufacturer's instructions. The genomic DNA was

127

removed by DNase I digestion (Thermo Scientific, Waltham, MA, USA). The quantity and

128

quality of extracted RNA was evaluated using an ND1000 spectrophotometer (NanoDrop

129

Technologies Inc., Wilmington, DE), and the RNA integrity was further verified through gel 6



130

electrophoresis. Afterwards, cDNA was synthesized using a commercial PrimeScript RT

131

reagent Kit (Takara, Dalian, China). The synthesized cDNA samples were stored at -20°C

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until subsequent real-time PCR analysis.

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Real-time PCR for expression of genes encoding innate immune homeostasis was

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conducted on an ABI-7900HT qPCR system (Applied Biosystems, Foster City, CA, USA),

135

using SYBR Premix Ex TaqII (Tli RnaseH Plus, TaKaRa, Dalian, China) with default option.

136

All the primers are detailed in Supplemental Table S1 (β-actin and GADPH as the internal

137

references), and the 2−∆∆Ct method was used to analyze the relative gene expression 19.

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Western blot analysis

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The mucosa were ground in liquid nitrogen, and homogenized in RIPA lysis buffer

140

(Beyotime, Shanghai, China), with 1% protease inhibitor cocktail (Roche Diagnostics GmbH,

141

Mannheim, Germany). Cell lysis was performed on ice for 30 min. Afterwards, the samples

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were centrifuged at 12,000 g for 15 min at 4 °C, and the supernatant was then taken as total

143

soluble protein (membrane and cytosol). The concentration of extracted protein was

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determined using a commercially available enhanced BCA protein assay kit (Beyotime,

145

Shanghai, China). The extracted proteins (80 µL, 200 µg) were mixed with 5× loading buffer

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(20 µL), incubated at 95 °C for 5 min, and stored at -20 °C.

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Sample proteins (40 µg) and pre-stained standards were separated with SDS-PAGE in 12%

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polyacrylamide gels and transferred onto PVDF membranes (Millipore, 0.45 µM). After

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blocking in 5% nonfat milk diluted in Western wash buffer (Beyotime, Shanghai, China) for 2

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h at room temperature, the membranes were then incubated with primary antibodies for

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overnight at 4°C. The primary antibodies consisted of mouse anti-β actin monoclonal 7


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antibody (Proteintech, Rosemont, USA), mouse anti-occludin polyclonal antibody, and rabbit

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anti-claudin-1 polyclonal antibody (Invitrogen, Carlsbad, CA), and. After several washes with

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TBST, membranes were incubated with an anti-mouse or anti-rabbit HRP-conjugated

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secondary antibody (Proteintech, Rosemont, USA) at 1/4000 dillution for 1.5 h at room

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temperature. Then, the membranes were washed four times for 10 min with wash buffer, and

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visualized using ImmobilonTM Western Chemiluminescent HRP Substrate (Merck Millipore,

158

Beijing, China). The densities of the blotting bands were analyzed using the AlphaImager

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2200 digital imaging system (Digital Imaging System, Kirchheim, Germany).

160

Cytokine levels for intestinal mucosal samples

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Three milliliters of 4°C PBS (pH = 7.4) was added to intestinal mucosa (1 g), and then

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homogenizated on ice using a Vibra CellTM sonicator (Bertin technoloies, Montigny le

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Bretonneux, France) for 5 times for 15 s with 10-s intervals. The homogenates were

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centrifuged (10,000 × g for 8 min at 4°C), and the supernatant fluids were used for

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determining cytokine concentrations (TNF-α, IL-1β and IL-10), following the user's

166

instructions of commercially available ELISA kits (Cusabio, Wuhan, China). All the

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measurements were done in triplicate.

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Microbial DNA extraction

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Total genomic DNA extraction was carried out on the mucosal samples using the 20

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bead-beating method as suggested by Chen and her colleagues

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measured using 1% agarose gel, and DNA quantity was determined based on absorbance at

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260 and 280 nm, respectively, with a NanoDrop ND-1000 spectrophotometer (NanoDrop

173

Technologies Inc., Wilmington, DE, USA). 8


. The quality of DNA was


174

Page 10 of 32

Amplicon sequencing

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The amplicon sequencing was conducted using the method modified from Kozich, et al.

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(2013). Specifically, the V4 region of the bacterial 16S rRNA gene was targeted using

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specific

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GGACTACNNGGGTATCTAAT-3'), with each sample had its unique barcode. For each DNA

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sample, 30 µL of reaction mix was prepared which contained 15 µL of Phusion Master Mix

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(New England Biolabs, Beijing, China), 3 µL of each barcoded primer (2 µM ), 2 µL of dd

181

H2O and 10 µL of DNA (3 ng/µL). The PCR conditions were as follows: initial denaturation

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at 98◦C for 3 min; 30 cycles of denaturation (95◦C, 20 s), annealing (50◦C, 30 s) and

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elongation (72◦C, 30 s); and a final 5-min extension at 72◦C for 5 min. Afterwards,

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amplification products were verified through agarose gel electrophoresis. Product quantities

185

were determined and equal molar amount of each product was pooled. The pooled products

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were run in a 2% agarose gel, and bands were purified with QIAquick Gel extraction Kit

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(Qiagen, Hilden, Germany). The amplicon library was constructed using TruSeq® DNA

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PCR-Free Sample Preparation Kit (Illumina Inc., San Diego, USA) prior to submission on an

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Illumina HiSeq 2500 sequencing system generating 250 bp paired-end reads.

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Bioinformatics analysis

primers

515F

(5'-GTGCCAGCMGCCGCGGTAA-3')

and

806R

(5'-

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The raw data were deconvoluted according to their barcodes, filtered through the quality

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control pipeline using the Quantitative Insight into Microbial Ecology (QIIME) software 22,

193

and the primers were trimmed. Two jejunal samples in the Control group that failed quality

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control (one sample at d 50 and one sample at d 60, respectively) were excluded for

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taxonomic classification; therefore, 26 samples were analyzed. The pair-end reads were 9


Page 11 of 32


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assembled into tags using FLASH 23. Chimeric sequences were removed using UCHIME 24.

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Sequences were clustered into operational taxonomic units (OTUs) of 97% sequence identity

198

using UPARSE

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performed against the latest Greengenes database (May, 2013 release) using the RDP

200

classifier with a 0.80 confidence threshold. Sequences were aligned using PyNAST software

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(Version 1.2), and the phylogenetic tree was constructed using FastTree

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(Observed species, Chao1, Shannon, Simpson, ACE and Good’s coverage) and beta diversity

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(UniFrac distance) were calculated using QIIME

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was conducted using vegan, ade4 and ggplot2 packages implemented in the R software

205

(http://www.R-project.org/). The sequences obtained in the current paper have been deposited

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in the NCBI Sequence Read Archive (SRA) under accession number PRJNA407285.

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Statistics analysis

25

. Taxonomic assignment of the representative sequences of OTUs was

26

. Alpha diversity

22

. Principle coordinate analysis (PCoA)

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Data were analyzed according to the MIXED procedure of SAS (SAS Inst. Inc., Cary,

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NC). The model included the fixed effects of rhubarb supplementation, age and their

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interaction, together with the random effect of animal nested within rhubarb supplementation

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× age. Each animal was used as the experiment unit, and Tukey’s test was used to compare

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least squares means. An α-level of P ≤ 0.05 was taken for indication of statistical significance,

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and the α-level of P ≤ 0.10 was used to indicate a statistical tendency.

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RESULTS

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Small intestinal morphology

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Diet and age unaffected (P > 0.10) villus height, crypt depth and CVR in the jejunum

10



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(Table 1). In the ileum, the supplementation of rhubarb increased (P = 0.036) villus height,

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and crypt depth increased (P = 0.044) with age.

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Gene expression pattern

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As illustrated in Table 2, for toll like receptors, in the jejunum, TLR-2 expression tended

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to decrease (P = 0.075), while TLR-4 expression increased (P < 0.001) as age increased. In

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the ileum, TLR-2 expression decreased (P = 0.029) with age. Diet × age interaction (P =

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0.033) was observed for TLR-4 expression. The supplementation of rhubarb decreased (P =

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0.006) TLR-4 expression, while age elevated (P < 0.001) TLR-4 expression.

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For cytokines, in the jejunum, diet and age unaffected (P > 0.10) expression of TNF-α

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and IFN-γ. IL-1β expression increased with age (P < 0.001). The supplementation of rhubarb

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tended to elevate (P = 0.057) IL-10 expression. In the ileum, diet and age unaffected (P >

228

0.10) TNF-α expression. Diet × age interaction was significant for IL-1β expression (P =

229

0.020), and tended to be significant for IFN-γ expression (P = 0.099). The supplementation of

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rhubarb decreased (P = 0.036) IL-1β expression, whilst increased (P = 0.011) IL-10

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expression. Expression of both IL-1β and IL-10 increased (P < 0.01) with age.

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For tight junction proteins, in the jejunum, diet and age unaffected (P > 0.10) expression

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of Occludin and Claudin-2. Diet × age interaction (P = 0.005) was observed for Claudin-1

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expression. The supplementation of rhubarb elevated (P = 0.005) Claudin-1 expression and

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age affected (P = 0.039) its values. Claudin-4 expression tended to decrease (P = 0.063) with

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age. In the ileum, diet × age interaction (P = 0.002) was observed for Claudin-1 expression.

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Its value was enhanced by the supplementation of rhubarb (P = 0.008), while was decreased

238

by age (P = 0.002). 11


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Functional protein expression pattern

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As presented in Figure 1, in the jejunum, the supplementation of rhubarb enhanced (P =

241

0.032) Claudin-1 protein expression. Greater Claudin-1 (P = 0.025), while lower Occudin (P

242

= 0.009) protein expression was noted on d 60 when compared with d 50. In the ileum, diet

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and age unaffected (P > 0.10) Occludin protein expression. Furthermore, there was diet × age

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interaction (P = 0.018) on Claudin-1 protein expression. Both rhubarb supplementation (P =

245

0.047) and age (P = 0.015) up-regulated Occludin protein expression on d 60.

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Mucosal cytokine secretion

247

As reflected by ELISA, in the jejunum, diet and age unaffected mucosal TNF-α

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concentration (Table 3). The supplementation of rhubarb tended to decrease (P = 0.088)

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mucosal IL-1β concentration. Mucosal IL-1β concentration decreased (P = 0.004), while

250

IL-10 concentration increased (P < 0.001) with age. Moreover, in the ileum, mucosal TNF-α

251

concentration increased (P = 0.004) with age. Diet × age interactions (P < 0.01) were

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observed for mucosal concentrations of IL-1β and IL-10. The supplementation of rhubarb

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decreased (P = 0.012) mucosal IL-1β concentration, whilst increased (P < 0.001) IL-10

254

concentration. Mucosal IL-1β concentration tended to decrease (P = 0.071), whilst IL-10

255

concentration increased (P < 0.001) from d 50 to 60.

256

Epithelial bacterial community diversity and composition

257

All samples were rarefied at 47, 062 reads per sample, and the Good' coverage (from

258

0.988 to 0.997) indicated that sampling depth was adequate (Table 4). In the jejunum, most

259

alpha diversity indices were unaffected (P > 0.10) by age, while observed species, Chao1 and

260

ACE indices were increased by the supplementation of rhubarb (P < 0.01). In the ileum, diet 12



261

× age interactions were observed for Shannon (P = 0.018) and Simpson (P = 0.011) indices.

262

They increased with age (P < 0.05), and were elevated by the supplementation of rhubarb (P

263

< 0.01). Moreover, the supplementation of rhubarb elevated observed species, Chao1, and

264

ACE indices (P < 0.01), and observed species increased from d 50 to 60 (P = 0.044).

265

As presented in Figure 2, PCoA analysis revealed that a clear separation between

266

Control and Rhubarb groups in the jejunum and ileum at d 50, while did not show a clear

267

separation of Control and Rhubarb groups in the jejunum and ileum at d 60. Meanwhile,

268

except Rhubarb group in the ileum, clear separations between d 50 and 60 in both groups

269

were observed in the jejunum and ileum.

270

Analysis of epithelial microbiome showed shifts in bacterial relative abundances at both

271

the phylum and genus levels when rhubarb was supplemented at d 50 and 60. As illustrated in

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Table 5, at the phylum level, in the jejunum, an increase in the sequences assigned to

273

Chloroflexi was found when rhubarb was supplemented (P = 0.008), and relative abundances

274

of Actinobacteria and Cyanobacteria increased with age (P < 0.05). In the ileum, diet × age

275

interactions were observed for relative abundances of Acidobacteria (P = 0.038) and

276

Actinobacteria (P = 0.005). Increases in relative abundances of Actinobacteria (P = 0.026),

277

Chloroflexi (P = 0.022) and a decline in relative abundance of Firmicutes (P = 0.018) were

278

observed when rhubarb was supplemented. Moreover, relative abundance of Proteobacteria

279

increased (P = 0.017), relative abundance of Cyanobacteria tended to increase (P = 0.096),

280

whilst relative abundance of Firmicutes (P = 0.001) decreased from d 50 to 60.

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Bacterial composition at the genus level revealed that in the jejunum, relative abundance

282

of Veillonella tended to increase by the supplementation of rhubarb (P = 0.084, Table 6). 13


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283

Relative abundances of Actinomyces (P = 0.054) and Rothia (P = 0.060) tended to increase,

284

and relative abundances of Staphylococcus, Streptococcus and Veillonella increased from d

285

50 to 60 (P < 0.05). In the ileum, diet × age interactions were significant for relative

286

abundances of Blautia, Butyrivibrio and Staphylococcus, Pseudomonas (P < 0.05), and

287

tended to be significant for relative abundance of Bifidobacterium (P = 0.079). An increase in

288

relative abundance of Blautia (P = 0.002), increase trends in relative abundances of

289

Clostridium (P = 0.089), Lactobacillus (P = 0.060), Pseudomonas (P = 0.087), and a

290

decrease in relative abundance of Staphylococcus (P < 0.001) were observed when rhubarb

291

was supplemented. Furthermore, relative abundances of Actinomyces (P = 0.048), Rothia (P

292

= 0.020), Staphylococcus (P < 0.001) and Veillonella (P = 0.048) decreased, while relative

293

abundance of Pseudomonas (P = 0.025) increased from d 50 to 60.

294

DISCUSION

295

The phytochemicals present in the rhubarb, especially anthraquinone derivatives such as

296

rhein, emodin and aloe-emodin, have been proposed to exert beneficial effects as

297

pharmacological drugs to inflammation, liver injury, and cancer

298

conducted in pathological condition of adult rodents and humans. Therapeutic and toxic

299

effects of rhubarb were reported to be dose dependent, with hepatotoxicity occurred at doses >

300

3 g/ kg body weight per day 27, whilst hepatoprotective effects occurred at doses around 0.35

301

to 0.50 g/kg body weight per day 17, 28. In the present study, we for the first time, used a dose

302

of 0.47 to 0.49 g/kg body weight per day in young goats at early ages after weaning (d 50 and

303

60). In order to accustom the sucking ruminants to concentrate and forage based solid diets,

304

early weaning (d 40) and the related nutritional interventions have been regarded as effective 14


16

. Most of the studies are


Page 16 of 32

29

305

approaches to improve feed efficiency and immune function

. As anticipated, rhubarb

306

presented a potential candidate for supplementation to benefit intestinal homeostasis.

307

The supplementation of rhubarb drastically improved morphology of ileal mucosa with

308

higher villus length, implying better absorptive capacity for nutrients. It has been reported

309

that gut barrier is primarily regulated by well-organized tight junction proteins between

310

intestinal epithelial cells, which are constituted of trans-membrane proteins such as occludin,

311

claudin and junctional adhesion molecular, together with adaptor proteins such as zonula

312

occluden protein

313

remarkably up-regulated in the Rhubarb group at both mRNA and protein levels. We thereby

314

speculated that gut barrier function was elevated by rhubarb intervention to prevent the

315

passage of harmful intra-luminal permeations, including foreign microbes and their toxins 31.

30

. In the current study, the trans-membrane Claudin-1 protein was

316

Weaning has been suggested to be related to up-regulation of pro-inflammatory

317

cytokines in the small intestine, leading to increased epithelial permeability and inflammation

318

in piglets and calves

319

intestine through rhubarb intervention in goat kids during early life. Within the

320

pro-inflammatory cytokines examined, expression of IL-1β at mRNA level and its mucosal

321

concentration was down-regulated by the supplementation of rhubarb. Toll like receptor

322

(TLR) signaling pathway can provide insights into the mechanism by which inflammation is

323

prevented. Strikingly, TLR-4 gene expression was inhibited in the ileum. This is in line with

324

previous observation that supplementation of rhubarb extract inhibited TLR-4 signaling in the

325

liver of mice 17. In fact, TLR signaling can be activated by recognizing microbial-associated

326

molecular patterns such as lipopeptides, LPS, glycolipids and flagellin, and thereafter elicits a

32, 33

. Herein, we aimed to prevent local inflammation in the small

15


Page 17 of 32


327

pro-inflammatory signaling pathway dependent on NF-κ B 6. Moreover, anti-inflammatory

328

cytokine IL-10 was enhanced at both gene expression and mucosa levels at d 50. The IL-10 is

329

produced preferentially by mononuclear phagocytes and lymphocytes, and this cytokine has

330

the capacity to inhibit secretion of pro-inflammatory cytokines and chemokines

331

Collectively, rhubarb intervention inhibited TLR-4-dependent inflammation and promoted

332

anti-inflammatory cytokine secretion.

34

.

333

The link between mucosal innate immune function and gut microbiota is well

334

established 35, and it is mostly bidirectional; changes in gut microbiota may not only occur as

335

a result of dysbiosis in gut barrier, but also contribute to maintain intestinal immune

336

homeostasis. The proximity that epithelial bacteria are in intimate contact with the gut

337

mucosa provides them with a higher potential in exerting effects on host mucosal immunity

338

than their digesta counterparts

339

examined in this study. As manifested by PCoA, Control and Rhubarb groups harbored their

340

unique epithelial bacteria at d 50, while there were no clear separations between them at d 60.

341

These suggest that the effect of rhubarb intervention on epithelial bacteria in the small

342

intestine is not consistent at different developmental stages of young ruminants.

36

. On this premise, epithelial bacterial community was

343

Further evaluation on taxonomic composition revealed that members of Proteobacteria

344

phylum were predominant in the epithelia of jejunum and ileum. It is well demonstrated that

345

the small intestine is characterized by relatively high levels of oxygen, and the steep oxygen

346

gradient in the mucosa creates a microenvironment for survival of these oxygen-tolerant

347

communities 5. A shift in favor of Akkermansia muciniphila and Parabacteroides goldsteinii

348

in the microbial ecosystem of cecal digesta was observed when mice was fed with a diet 16



Page 18 of 32

349

supplemented with 0.3% rhubarb 17. Further study revealed that ethanol exposure diminished

350

abundance of A. muciniphila in the intestine of both humans and mice, which could be

351

recovered in experimental alcoholic liver disease by oral supplementation

352

suggest that A. muciniphila can promote intestinal barrier function and ameliorate

353

experimental ALD. However, in the present study, relative abundance of A. muciniphila in

354

epithelial community was relatively low (< 0.5%), and was unaffected by the

355

supplementation

356

supplementation of rhubarb did not result from colonization of this species in the epithelium.

357

In contrast, in the ileum, relative abundance of Staphylococcus genus at d 50 was drastically

358

decreased during rhubarb intervention. Some species of Staphylococcus, such as S. aureus,

359

can produce S. aureus enterotoxin B (SEB), which is potent activator of the immune system,

360

and has the capacity to disturb barrier function 38. The decreased Staphylococcus abundance

361

can, at least partly, explain enhanced barrier function by the supplementation of rhubarb.

362

Another reason lies in the noticeable increase in relative abundances of Clostridium and

363

Lactobacillus in the ileal epithelium during rhubarb intervention. Similar observation of

364

elevated relative abundance of Lactobacillus after the treatment of rhubarb was found in the

365

rumen of steers

366

indispensible roles in host resistance against intestinal pathogens and maintaining host

367

immune homeostasis 40, 41. All these confirm the hypothesis that the enhanced mucosal innate

368

immune homeostasis by rhubarb supplementation is, at least partly, mediated by altered

369

epithelial bacterial community in the small intestine.

370

of

rhubarb,

suggesting

that enhanced

37

. All these

barrier function

by

the

39

. Most species of these two genera are beneficial bacteria, and play

In conclusion, the supplementation of rhubarb during early life improved host mucosal 17


Page 19 of 32


371

innate immune homeostasis in the small intestine, as characterized by increased villus height

372

and elevated barrier function. These were associated with shifts in ileal epithelial bacteria in

373

favor of Blautia, Clostridium, Lactobacillus and Pseudomonas. The present study aids in

374

enhancing knowledge regarding to the crosstalk among microbiota, nutrition and immunity,

375

and highlights the prospect of rhubarb as a feed additive to improve health status during early

376

life of ruminants.

377

378

ACKNOWLEDGEMENT

379

This work was supported by grants from the National Natural Science Foundation of

380

China (grants 31601967, 31320103917), the Open Foundation of Key Laboratory of

381

Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture,

382

Chinese Academy of Sciences (Grant No. ISA2016301), Youth Innovation Team Project of

383

ISA, CAS (2017QNCXTD_ZCS), and the project from Mr. Sheng Yang, Mr. Genhuo Shao,

384

and DA BEI NONG Group (No. B2016009).

385

386

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Table 1. Small intestinal morphology in the Control and Rhubarb groups of goat kids at d 50 and 60

Item

Diet

Jejunum Villus height, µm Crypt depth, µm VCR Ileum Villus height, µm Crypt depth, µm VCR 509 510 511 512

Age (d)

Diet

P value Age Diet × Age

23.15

0.682

0.436

0.625

2.74

0.230

0.549

0.645

0.188

0.544

0.640

0.813

11.76

0.036

0.222

0.737

3.87

0.378

0.044

0.383

0.191

0.486

0.198

0.581

SEM

50

60

C R C R C R

567.2 545.7 99.4 94.5 5.71 5.78

574.3 576.3 99.8 97.5 5.75 5.92

C R C R C R

488.3 512.7 97.1 97.1 5.03 5.28

499.6 532.2 102.5 109.6 4.88 4.91

VCR, villus crypt ratio; C, Control group; R, Rhubarb group.

22



513 514 515 516

Page 24 of 32

Table 2. Expression patterns of mucosal innate immune function related genes in the Control and Rhubarb groups of goat kids at d 50 and 60

Item

Diet

50

60

1.27 1.53 1.09 1.35

0.62 0.77 2.61 3.03

6.80 6.09 2.85 2.63

C R C R C R C R C R C R C R C R

Toll like receptors Jejunum TLR-2 C R TLR-4 C R Ileum TLR-2 C R TLR-4 C R Cytokines Jejunum IL-1β TNF-α IFN-γ IL-10 Ileum IL-1β TNF-α IFN-γ IL-10

Age (d)

Tight junction proteins Jejunum Occludin C R Claudin-1 C R

SEM

Diet

P value Age Diet × Age

0.355

0.580

0.075

0.888

0.255

0.216

Rhubarb Supplementation Promotes Intestinal Mucosal Innate

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