Undernutrition Shapes the Gut Microbiota and Bile Acid Profile in

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Undernutrition shapes the gut microbiota and bile acid profile in association with altered gut-liver FXR signaling in weaning pigs SEN LIN, Xiaomin Yang, Peiqiang Yuan, Jiameng Yang, Peng Wang, Heju Zhong, Xiaoling Zhang, Lianqiang Che, BIN FENG, JIAN LI, YONG ZHUO, YAN LIN, Shengyu Xu, DE WU, DOUGLAS BURRIN, and Zhengfeng Fang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01332 • Publication Date (Web): 13 Mar 2019 Downloaded from http://pubs.acs.org on March 14, 2019

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Journal of Agricultural and Food Chemistry 1

Undernutrition shapes the gut microbiota and bile acid profile in association with altered gut-liver FXR signaling in weaning pigs †#

†#

†#

†

†

†

Sen Lin , Xiaomin Yang , Peiqiang Yuan , Jiameng Yang , Peng Wang , Heju Zhong , Xiaoling †

†

†

†

†

†

†

†

Zhang , Lianqiang Che , Bin Feng , Jian Li , Yong zhuo , Yan Lin , Shengyu Xu , De Wu , Douglas ‡ † G Burrin , Zhengfeng Fang* †

Key Laboratory for Animal Disease Resistance Nutrition of the Ministry of Education, Animal

Nutrition Institute, Sichuan Agricultural University, Wenjiang 611130, People’s Republic of China. ‡

Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, 1100

Bates Street, Houston, TX 77030, USA. #

These authors contributed equally to this work.

*Corresponding

author:

E-mail:

[email protected];

+86-28-86290922;

ACS Paragon Plus Environment

Tel:

+86-28-86290920;

Fax:

Journal of Agricultural and Food Chemistry

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Abstract

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Bile acids, synthesized in liver and metabolized by microbiota, have emerged as important signaling

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molecules regulating immune responses and cell proliferation. However, the crosstalk among

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nutrition, microbiota and bile acids remains unclear. Our study indicated undernutrition in weaning

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piglets led to intestinal atrophy, increased colonic production and systemic accumulation of

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lithocholic acid (LCA), deoxycholic acid (DCA) or their conjugated forms, which might be

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associated with decreased Lactobacillus abundance. Moreover, undernutrition led to increased portal

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fibroblast growth factor 19 (FGF19) level, up-regulated hepatic heterodimer partner (SHP) while

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down-regulated Cholesterol 7a-hydroxylase (CYP7A1) expression. The detrimental effects of DCA

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and LCA on proliferation and barrier function were confirmed in porcine enterocytes, whereas their

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roles in weaning piglets warrant further research. In summary, undernutrition in weaning piglets led

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to increased secondary bile acids production, which might be related to altered gut microbiome, and

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enhanced farnesoid X receptor (FXR) signaling while suppressed CYP7A1 expression.

14

Key words: bile acid metabolism, intestinal microbiome, undernutrition, FXR signaling, piglets.


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Introduction

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Bile acids are synthesized in the liver and secreted into the gut in bile after ingestion of food.

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Bile acids have well established metabolic functions in cholesterol homeostasis and solubilization

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and digestion of lipids-soluble nutrients1. In recent decades, bile acids have been shown to function

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as signaling molecules2-5. A variety of nuclear and membrane receptors have been identified

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including farnesoid X receptor (FXR) and G protein-coupled bile acid receptor 1 (TGR5)6, Bile acid

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signaling regulated by FXR and TGR5 have been shown to regulate not only their own synthesis and

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enterohepatic recirculation, but also energy and glucose homeostasis, inflammation and cell

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proliferation1, 7-9.

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FXR is the principal regulator of bile acid homeostasis10 and deficiency in FXR function may

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lead to cholestasis11. Activation of FXR induces a series of target genes in both the liver and

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intestine, including hepatic Small heterodimer partner (SHP) and intestinal fibroblast growth factor

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19 (FGF19), which in turn inhibit the transcription of Cholesterol 7a-hydroxylase (CYP7A1), the

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rate-limiting enzyme in bile acid synthesis, and thus maintain bile acid homeostasis12. Notably,

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different bile acids have different potency to bind bile acid receptors. The potency of bile acids to

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activate FXR is chenodeoxycholic acid (CDCA) > deoxycholic acid (DCA) > lithocholic acid

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(LCA) > cholic acid (CA)13. Secondary bile acids, especially DCA and LCA, appear to induce cell

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apoptosis more potent than primary bile acids14 and may contribute to inflammatory bowel

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diseases15. High levels of DCA has been correlated with ileal damage in experimental necrotizing

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enterocolitis and sequestration of ileal bile acids by cholestyramine has been shown to decrease the

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incidence of necrotizing enterocolitis16. Therefore, the bile acid homeostasis is crucial in maintaining

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cell functions regulated by FXR and/or TGR5 signaling and its variation may subsequently affect

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host inflammation, cell proliferation and cell barrier function1, 7.

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Importantly, the intestinal microbiota has profound effects on bile acid metabolism by promoting

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deconjugation, dehydrogenation and dihydroxylation of primary bile acids in the distal small



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intestine and colon17. In the intestine, glyco- and tauro- conjugated CA and CDCA are deconjugated

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by bacteria with bile salt hydrolase activity, and then the 7α-hydroxy group is removed by bacterial

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dehydroxylase activity to form the secondary bile acids, DCA and LCA17-18, which have been linked

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to cell injury19 and gallstones20. Therefore, changes in the gut microbiota may impact host health via

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bile acid signaling. Weaned piglets are regarded as a good model for studying the gut microbiota21,

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which can be used to investigate the interaction between microbiota and bile acid metabolism.

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Undernutrition usually occurs in piglets suffered from weaning stresses which is characterized

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by low feed intake, intestinal atrophy, body weight loss22-24, gut inflammation25, and alteration of

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microbiome composition26-28. With the emergence of antimicrobial-resistance bacteria, minimizing

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the use of antibiotics in feeds has become the consensus worldwide. In post-antibiotic age, it is a

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great challenge in pork industry to guarantee the growth of weaning piglets, which requires more

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deeply understanding of the mechanisms of weaning stresses. Investigating the effects of

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undernutrition on bile acids profile and signaling will provide basic understanding of the potential

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role of bile acids in weaning stress of piglets. In this study, pigs with different nutrition status after

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weaning were used as a model to determine digesta and plasma bile acid metabolomics, tissue bile

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acid signaling, and the gut microbiome, which is aimed to address the crosstalk mechanisms among

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nutrition status, intestinal microbiota and bile acid metabolism.

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Materials and Methods

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Animals and study design.

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Use of animals in the current study was approved by the animal care and use committee of

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Sichuan

Agricultural

University.

All

piglets

with

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(Duroc×Landrace×Yorkshire) were obtained from Research farm of Sichuan Agricultural University.

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According to our recent study24, undernutrition in pigs mainly occurs during the first week

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post-weaning. Therefore, we designed this one-week study. To highlight the effect of weaning on pig

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nutrition and health status, piglets reared by sows for 21 or 28 days of age were used as the control


similar

genetic

background

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groups. In contrast, piglets weaned from sows and immediately received solid feed at 21 days of age

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till 28 days of age were used to select for the treatment groups. At day 21 of lactation, piglets were

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weighed from three LY (Landrace×Yorkshire) sows (parity 3) with litter size around 13. From each

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litter, 9 to 10 medium sized piglets were selected and randomly assigned to either suckling or weaned

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groups. To minimize the effect of litter source, it was ensured that every two of the six pigs (n=6) in

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either the suckling 21 (S21) or suckling 28 (S28) group was from the same litter, and,

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simultaneously, each litter provided 5 or 6 piglets to be weaned at 21 days of age and housed

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individually in metabolism cages located in the same room with controlled temperature (30 ± 1 °C).

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To minimize the effect of initial body weight, the average body weight at the start of the experiment

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(21-day of age) was similar among groups (Supplemental Table 2). Piglets in S21 group were

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directly euthanized for sampling. Piglets in S28 group were respectively put back to their mothers

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and mixed with other piglets with similar age to adjust the litter size to 12. After another 7-day

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sucking, S28 piglets were euthanized for sampling. A total of 16 piglets weaned from the three litters

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were allowed to have free access to feeds and water for 7 days and fresh feeds were given to piglets 4

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times each day. During this period, some piglets had very low feed intake and gained little or no

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weight, which is spontaneously developed and usually happens in early weaned piglets. From these

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pigs, healthy piglets without severe diarrhea were selected for sample collection. According to the

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growth performance in this week, piglets were assigned to two groups and followed with euthanasia.

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Piglets with feed intake lower than 52 g/day (32.36 ± 3.87 g/day) and thus showing zero or negative

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increase in body weight (Average daily gain = -0.04 ± 0.02kg/day) were selected and regarded as the

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undernutrition (UN28, n = 6) group while those with feed intake at least twice of that in UN28

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piglets (higher than 104g/day,168.57 ± 17.83g/day) and thus showing positive increase in body

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weight (Average daily gain = 0.15 ± 0.01kg/day) were selected and considered as the normal

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nutrition (NN28, n = 6) group. As a result, every two of the six piglets in either the UN28 or NN28

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group were from the same litter. Dietary ingredients and composition are shown in Table S1. Initial



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and final body weight, weight gain and feed intake are shown in Table S2.

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Sampling.

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Blood samples were collected via jugular vein or portal vein at the morning of 21 and 28 days of

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age following fasting overnight, centrifuged at 3000rpm for 10 minutes and stored at -20 °C until

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analysis. Liver, colon and cecum were separated and weighed after removing of contents

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respectively. Bile and colonic content were snap-frozen in liquid nitrogen and stored at -80 °C. Small

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intestine was removed and separated as duodenum, jejunum and ileum as previously described29 and

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weighed respectively. For measurement of morphometry, approximately 2-cm length of duodenum,

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jejunum and ileum samples were fixed in 4% paraformaldehyde. Small intestine samples for gene

99

expression and western blotting analysis were snap-frozen in liquid nitrogen and stored at -80 °C.

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Intestinal morphology

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Intestinal morphology was measured as previously described30. Briefly, intestinal segments were

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taken out from 4% paraformaldehyde and then dehydrated with increasing concentrations of ethanol

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and chloroform. The segments were processed with paraffin and cross-sections were cut with a

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microtome. Then, segments were stained with hematoxylin and eosin. Two transverse sections of

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each sample (duodenum, jejunum, and ileum) were prepared on one slide for morphometric analysis.

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Ten intact, well-oriented crypt-villi units per sample were selected randomly and measured. Villi

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height was measured from the tip of the villi to the base between individual villi, and crypt-depth

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measurements were taken from the valley between individual villi to the basal membrane.

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Morphometric measurements were performed using an Olympus BX51 microscope equipped with a

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DP70 digital camera (Olympus, Tokyo, Japan) and JD801 morphologic image analysis software.

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Determination of bile acid composition

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Bile acid profile analysis was performed as previously described31. Bile standard were obtained

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from Sigma-Aldrich (Saint Louis, MO, US). HPLC grade chemicals were obtained from fisher

114

scientific (Waltham, MA, US). The mix reference standards were prepared by dissolving each bile


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115

acid reference standard respectively in methanol. The calibration standards were prepared from

116

0.01to10 mg/mL. Bile acids were extracted from blood samples, bile and colonic content by adding

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methanol and then vortexed and centrifuged at 20000×g for 10 minutes. The supernatants were dried

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with nitrogen. The residuals were dissolved in methanol, vortexed and centrifuged at 20000×g for 10

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minutes. Then the supernatant (100uL) was loaded to the UPLS-MS system for analysis. The bile

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acids were quantified by a Waters Acquity UPLC System coupled with a ZQ 2000 quadtruple

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spectrometry (Waters, MA, US).

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FGF19 concentration analysis

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Concentration of FGF19 in portal plasma was measured using a commercial ELISA kit

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(ELP-FGF-19, RayBiotech Inc, GA, US) according to the manufacturer’s instructions.

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DNA extraction, 16S rRNA gene sequencing

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Total genome DNA from colon contents was extracted using CTAB/SDS method. DNA

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concentration and purity were monitored on 1% agarose gels. According to the concentration, DNA

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was diluted to 1ng/μL using sterile water. For PCR amplification of the V4 region of the 16S rRNA

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gene,

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806R:5′-GGACTACHVGGGTWT CTAAT-3′) was used. All PCR reactions were carried out with

131

Phusion®High-Fidelity PCR Master Mix (New England Biolabs). Mix same volume of 1X loading

132

buffer (contained SYB green) with PCR products and operate electrophoresis on 2% agarose gel for

133

detection. Samples with bright main strip between 400-450bp were chosen for further analysis. PCR

134

products was mixed in equi-density ratios. Then, mixture PCR products was purified with Qiagen

135

Gel Extraction Kit (Qiagen, Germany). Sequencing libraries were generated using TruSeq®DNA

136

PCR-Free Sample Preparation Kit (Illumina, USA) following manufacturer's recommendations and

137

index codes were added. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo

138

Scientific) and Agilent Bioanalyzer 2100 system. At last, the library was sequenced on an

139

IlluminaHiSeq2500 platform and 250 bp paired-end reads were generated.

the

515F/806R

primer

set

(515F:

5′-GTGCCAGCMGCCGCGGTAA-3′;



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140

A total of 2,143,509 reads were obtained for our 24 samples. Paired-end reads was assigned to

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samples based on their unique barcode and truncated by cutting off the barcode and primer sequence.

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Paired-end reads were merged using FLASH. Quality filtering on the raw tags were performed under

143

specific filtering conditions to obtain the high-quality clean tags according to the QIIME(V1.7.0)

144

quality-controlled process32. The tags were compared with the reference database (Gold database,)

145

using UCHIME algorithm (UCHIME Algorithm,)33 to detect chimera sequences, and then the

146

chimera sequences were removed34. Then the Effective Tags were finally obtained. Sequences

147

analysis were performed by Uparse software (Uparse v7.0.1001). Sequences with ≥97% similarity

148

were assigned to the same OTUs. Representative sequence for each OTU was screened for further

149

annotation.

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Determination of the number of Clostridium scindens gene copies in colon contents

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Primers (forward: GCAACCTGCCTTGCACT reverse: ACCGAATGGCCTTGCCA) for

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Clostridium Scindens were according to previous research35. Genomic DNA was extracted using

153

QIAamp DNA Stool Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions.

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Standard curve was set up by serially diluting plasmid of a pMD18-T vector with the appropriate

155

insert from 107 to 102 target gene copies ∙ µl-1. Quantitative PCR amplification was performed on

156

FTC-3000 system (Funglyn Biotech Inc., Toronto, Canada). Quantitation was done by using standard

157

curve made from known concentrations of plasmid DNA.

158

Determination of proliferation and gene expression in IPEC-J2 cells

159

IPEC-J2 cells were maintained in DMEM/F12 medium containing 5% fetal bovine serum,

160

50U/mL penicillin, 50μg/mL streptomycin and 2mM Glutamax (all from Gibco, US) at 37 °C and

161

5% CO2. LCA (Sigma-Aldrich, US) and DCA (Sigma-Aldrich, US) were dissolved in DMSO

162

(Sigma-Aldrich, US) before they were used to treat cells. Cells were seeded in 96 well plate at a

163

density of 10000 cells/ well and allowed to attach for 24 h prior to treatment with different levels of

164

LCA, DCA or DMSO for 24h in serum free medium, at 37 °C and 5%CO2. Cell proliferation was


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determined by using CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega, US)

166

according to manufacturer’s instructions. Next, cells were seeded in 12 well plate at a density of 105

167

cells/well and allowed to attach for 24 h prior to treatment with LCA (5μM), DCA (25μM) or DMSO

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in serum free medium for 24h. Finally, cells were washed with PBS and harvested for further

169

extraction of RNA.

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Real-time PCR

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Total RNA from ileum and liver tissues or cells was extracted using TRIZOL Reagent

172

(Invitrogen, Carlsbad, CA). The cDNA was synthesized using a reverse transcription (RT) kit

173

(TAKARA, Japan) following the manufacturer’s instructions. Primers (Table S3) were derived from

174

Gene bank and synthesized by a commercial company (Invitrogen, Chengdu, China). Quantitative

175

real-time RT-PCR analysis was carried out using a CFX96 Real-Time PCR Detection System

176

(Bio-Rad, Hercules, CA, USA) with commercial SYBR Green kits (TaKaRa, Japan). The specificity

177

of PCR products was examined with melting curve analysis. Relative mRNA expression was

178

calculated with 2−ΔΔCt method using GAPDH as the internal control where ΔΔCt = (Ct target gene

179

unknown sample − Ct GAPDH unknown sample) − (Ct target gene calibrator sample − Ct GAPDH

180

calibrator sample).

181

Western blot

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After treated with either 5μM of LCA or 25μM of DCA for 24 hours as described above,

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IPEC-J2 cells were washed with cold PBS for three times. Then RIPA lysis buffer (Beyotime

184

Biotechnology) was added to each well and plate was set on ice for 30 minutes followed by

185

centrifugation at 12000g for 30 minutes. The concentration of protein was determined by Pierce

186

BCA protein Assay kit (Thermo Scientific). Approximately 20μg of protein was separated on

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SDS-PAGE gel and then transferred to PVDF membrane (Bio-Rad). After blocking in 5% skimmed

188

milk at room temperature for 1 hour, PVDF membrane were incubated with primary antibodies at

189

4 °C over night. Primary anti-ZO-1 (1:1000, 617300) and anti-Claudin-1 (1:1000, 519000) antibody



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were purchased from Invitrogen. Anti-occludin (1:1000, ab31721) antibody was purchased from

191

Abcam while anti- βactin (1:1000, 4967S) antibody were purchased from cell signaling.

192

Visualization was carried out using the ECL kit (Beyotime Biotechnology) according to

193

manufacturer’s instructions.

194

Statistical analysis

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All data were analyzed using GraphPad prism 6.0. Normally distributed data from different

196

groups were compared by One-way ANOVA and Tukey’s post hoc test. Other data were analyzed by

197

the Kruskal-Wallis test with Dunn’s multiple comparison test. Values are expressed as means ±

198

SEMs. It was considered significant when P

Undernutrition Shapes the Gut Microbiota and Bile Acid Profile in

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