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Sodium Butyrate Improves High-Concentrate Diet-Induced Impairment of Ruminal Epithelium Barrier Function in Goats Kai Zhang, Meijuan Meng, Lipeng Gao, Yuanyu Tu, and Yunfeng Bai J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 16 Jul 2018 Downloaded from http://pubs.acs.org on July 16, 2018

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Journal of Agricultural and Food Chemistry

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Sodium Butyrate Improves High-Concentrate Diet-Induced Impairment of

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Ruminal Epithelium Barrier Function in Goats

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Kai Zhang†,#, Meijuan Meng†, Lipeng Gao†, Yuanlu Tu†, and Yunfeng Bai*,†

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†

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Nanjing, 210014, China

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#

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Circular Agriculture Research Center, Jiangsu Academy of Agricultural Sciences,

Key Laboratory of Crop and Livestock Integrated Farming, Ministry of Agriculture,

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*Corresponding author:

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Yunfeng Bai,

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Circular Agriculture Research Center,

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Jiangsu Academy of Agricultural Sciences,

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Tel: +86-025-84390204,

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Fax: +86-025-84390204,

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E-mail: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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We investigated the effect of sodium butyrate feeding on the disruption of ruminal

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epithelium barrier function in goats fed a high-concentrate diet. Eighteen male Boer

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goats (live weight 31.75 ± 1.35 kg, aged 1 year) were randomly assigned into three

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groups, which were fed a low-concentrate diet (LC), a high-concentrate diet (HC), or

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a high-concentrate diet with 1% sodium butyrate by weight (SH) for 9 weeks. We

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found that the pH of rumen fluid in the SH and LC groups was higher than that in the

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HC group. The activity of protein kinase C (PKC) kinase in the rumen epithelium was

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higher in the HC group than in the LC and SH groups. The mRNA expression and

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phosphorylated protein levels of mitogen-activated protein kinases (MAPKs) in the

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rumen epithelium were lower in the SH and LC groups than in the HC group. The

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DNA methylation rate of occludin was higher in the HC group than in the SH and LC

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groups. The mRNA and protein expression of claudin-1, claudin-4, occludin, and zona

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occludin-1 was greater in the SH and LC groups than in the HC group. In addition,

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sodium butyrate mitigated damage to the rumen epithelium caused by the HC diet.

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Together, our results suggest that the supply of sodium butyrate reverses the damage

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of rumen epithelium tight-junction by inhibiting PKC and MAPK signaling pathways

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and is protective to the rumen epithelium during SARA.

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KEYWORDS: goat, subacute rumen acidosis, rumen epithelium, sodium butyrate,

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tight junction proteins

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INTRODUCTION

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To stimulate rapid growth rates or high milk production, ruminants are usually

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fed a high-concentrate diet. However, high-concentrate diets result in metabolic

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disorders and systemic disease 1, 2. The most economically important severe metabolic

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disorder of ruminants is subacute rumen acidosis (SARA), which is characterized by a

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lower ruminal below 5.8 for more than 3 hours a day 3. Decreased ruminal pH can

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impair rumen epithelial barrier function

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endotoxin release in the gastrointestinal tract. These bacteria can then enter the

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bloodstream, increasing the risk of low dry matter intake, laminitis, liver abscesses,

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and other diseases, which can cause significant economic losses in the ruminant

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industry 6-8.

4, 5

and stimulate lipopolysaccharide (LPS)

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The rumen epithelium serves multiple functions; it is a semipermeable

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paracellular diffusion barrier affecting nutrient absorption and metabolism and can

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counteract decreases in the ruminal pH and immune responses. In the normal

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physiologic state, penetration of the gastrointestinal epithelial barrier by endotoxins

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and antigens is limited 9. However, SARA induced by a high-grain diet impairs

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ruminal epithelial barrier function. The low pH and hyperosmolarity caused by SARA

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prompt the translocation of LPS and other microbial components 10, 11.

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Tight junctions (TJs) are the most important cell-cell interactions in forming

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epithelial barrier. Them are located in the spinosum and stratum granulosum and

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composed of the integral transmembrane proteins claudins and occludin as well as the 3



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adaptor proteins zonula occludin (ZO) 1, 2, and 3, which form the scaffold of the

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cytoplasmic plaque 12-14. Occludin is the most reliable immunohistochemical marker

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for TJs because its expression correlates with various junctional functions

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Although overexpression of occludin increases transepithelial electrical resistance and

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barrier function in mammalian epithelial cells 18, 19, increased or decreased expression

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of occludin does not affect TJ morphology or structure in most conditions

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Occludin-knockout mice do not display disrupted barrier function

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suggest that occludin may not be a critical structural component in TJs and that other

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TJ proteins, such as the claudins

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expression.

.

20, 21

.

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. These findings

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, might compensate for defects in occludin

Overexpression of claudin-1 enhances barrier function

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15, 16

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. In addition, claudins

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are important in the maintenance of junctional ion permeability and barrier function 24,

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occludin

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kinase C (PKC) and mitogen-activated protein kinase (MAPK) signaling pathways. In

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vitro, activators of PKC can downregulate the expression of occludin, claudin-1, and

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ZO-1

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epithelial barrier function through alteration of TJ protein expression 28, 29.

. Zona occludin-1 (ZO-1), an essential TJ component, can interact with claudins and 26

. The expression of these TJ proteins is mainly regulated by the protein

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. We have also found that MAPK signaling pathway activity can disrupt

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Sodium butyrate (SB) has been used as a histone deacetylase inhibitor (HDACi).

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SB inhibits HDACs class I and II, alter the chromatin interactions with DNA and

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thereby regulate gene expression

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. Furthermore, SB plays a pivotal role in 4


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anti-inflammatory activities, energy metabolism and the maintenance of epithelial

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junctions. SB promotes the growth of rumen epithelia and increases growth

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performance in dairy cows 31, 32. SB can also protect against beta-cell damage through

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the p38/MAPK signaling pathway

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ruminal epithelial barrier dysfunction when grain-induced SARA occurs via

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modulation of the p38/MAPK pathway is unknown. Therefore, the present study was

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conducted to confirm whether SB feeding increases the TJs expression in rumen

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epithelial through modification and regulation of the PKC and MAPK signaling

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pathways and if it can be used to treat impaired barrier function.

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. However, whether SB feeding can prevent

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MATERIALS AND METHODS

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Ethical approval

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Experimental samples were collected in strict accordance with the guidelines of

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Jiangsu Province Animal Regulations (Government Decree No. 45) and the

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Committee on the Ethics of Animal Experiments of Jiangsu Academy of Agricultural

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Science.

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Chemicals

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Dietary sodium butyrate was purchased from Dongying Degao Biotechnology,

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Jinan, China. Analytical grade chloroform, isopropanol and other reagents were of

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analytical grade (Lingfeng Chemical, Shanghai, China).

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Animals, diet, and experimental design

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Eighteen male Boer goats (live weight 31.75 ± 1.35 kg, aged 1 year) were

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randomly were randomly assigned into the three groups (n = 6), which were fed a

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low-concentrate diet (LC; concentrate:forage = 3:7), a high-concentrate diet (HC;

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concentrate:forage = 7:3), or a high-concentrate diet with 1% SB by weight (SH). The

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goats were cannulated 3 weeks prior to the start of the study. The nutrient composition

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of the diets are listed in Table 1. The goats were freely fed at 8:00 A.M. and 6:00 P.M.,

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and had free access to fresh water for 9 weeks.

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Sample collection and analysis

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On the last day of weeks 7, 8, and 9, approximately 20 mL of rumen fluid was 6


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collected through the cannula at 0, 1, 2, 4, 6, 8, and 10 hours after feeding. 2 mL of

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the ruminal fluid were used to measure the pH by a portable pH meter (Sartorius,

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Göttingen, Germany). The remaining fluid from each sample was stored at −20°C for

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later analysis.

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The goats were slaughtered 4 to 5 hours after the last feeding according to the

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law of the Jiangsu Provincial People’s Government, China, and rumen tissue samples

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were collected after washing with phosphate-buffered saline. Rumen tissues from the

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ventral sac were cut into 1cm × 1cm pieces and fixed in 4% paraformaldehyde

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solution for histologic analysis. Ten-gram samples of the rumen epithelium were also

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collected from the ventral sac, transferred to liquid nitrogen and kept at −80°C for

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later analysis.

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Histologic analysis

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Briefly, fixed tissues were immersed in a 4% paraformaldehyde solution for 72

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hours, dehydrated by ethanol and embedded in paraffin. 5 µm sections of rumen tissue

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were cut and mounted on slides, and hematoxylin and eosin were used for dyeing 34.

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Tissue morphology was observed by light microscope and the images were captured.

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Histological damage was determined with a scoring criteria described previously

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Briefly, the damage score graded 0 to 3: mild superficial epithelial injury; moderate

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accompanying focal erosions and severe accompanying multifocal erosions.

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Quantitative real-time polymerase chain reaction

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35

.


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100 mg of rumen epithelium samples were used to extract total RNA via RNAiso

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Plus (Takara, Dalian, China). The A260/A280 ratio was assessed using a Nano Drop

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2000 (Thermo Fisher Scientific Inc., Waltham, MA) to evaluate mRNA quality.

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Genomic DNA was removed from RNA with DNase using RNeasy Mini Kit columns

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(Qiagen, Duesseldorf, Germany), and a pool of RNA samples without reverse

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transcriptase to exclude the contamination of genomic DNA. Only samples with a

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ratio between 1.8 and 2.1 were subsequently employed for cDNA analysis. Then,

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cDNA was synthesized using 400 ng of the total RNA template through reverse

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transcription using PrimeScript RT Master Mix Kit (Cat. RR036A; Takara). Primers

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were designed to fall across exon–exon junctions using Primer Premier Software 5.0

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(Premier Biosoft, Palo Alto, CA) and aligned against publicly available databases

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using BLASTN at NCBI (https://www.ncbi.nlm.nih.gov/). The Primers are presented

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in Table 2. RT-PCR was performed in the ABI 7300 system (Applied Biosystems,

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Foster City, CA) using a SYBR Premix EX Taqkit (Cat. DRR420A; Takara). PCR

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amplification protocol including 30 seconds at 95°C, 40 cycles of 5 seconds at 95°C,

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and 31 seconds at the annealing temperature of 60°C. the possible contamination of

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genomic DNA in water and regents was monitored by Non-Template Controls (NTC).

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The PCR products was evaluated via electrophoresis in 3% agarose gels and

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sequencing. The quantitative PCR data was analyzed using the 2-∆∆Ct method 36. PCR

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amplification

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Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was stable across

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treatments and deemed an appropriate internal reference gene in qPCR.

efficiency

of

each

gene

were

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between

91%-110%.

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Methylation status analysis of occludin gene promotor

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Genomic DNA was isolated from rumen tissues using the SQ Tissue DNA

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Extraction Kit (Omega Bio-Tek, Norcross, GA). Bisulfite treatment and recovery of

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DNA samples were carried out using 1 ug of genomic DNA by the DNA

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Methylation-Fast/Simple Kit (Qiagen, Frederick, MD), following the manufacturer’s

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instructions. CpG-rich segments were selected from the promoter sequence by using

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UCSC online sequence retrieval software. The primers were designed in MethPrimer

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(http://www.urogene.org/cgi-bin/methprimer/methprimer.cgi)

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NC_030827.1. The primer sequences are listed in Table 3. PCR analysis was

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conducted using four microliters bisulfite-modified DNA in a 50-µL volume. The

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amplification conditions were as follows: preheating at 95°C for 10 minutes, 40

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cycles of 30 seconds at 95°C, 30 seconds at the annealing temperature of 55°C, and

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10 minutes at 72°C for a final extension. SanPre Column PCR Purification Kit

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(Sangon, Shanghai, China) was used to purify and recover the amplified bisulfate

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PCR products. The products were subcloned into the pUCm-T vector system. DNA

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sequencing was performed on at least 10 clones that showed only > 99% cytosine

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conversions (Sangon, Shanghai, China). The PCR products were confirmed via

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agarose gel electrophoresis and sequencing. The methylation rate of every sample was

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determined with BiQ Analyzer software 37.

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PKC kinase activity assay

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based

on

file

The ruminal epithelium tissue homogenate was used to measure PKC kinase 9



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activity. The PKC kinase activity assay was carried out according to the

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manufacturer’s protocol (Cat. ADI-EKS-420A, Enzo Life Sciences, New York, NY,

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USA). Briefly, the pre-treated samples were diluted until their PKC kinase

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concentrations were in the range of 0.1 to 40 ug/mL. The absorbance was observed at

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450 nm on an Thermo Scientific Microplate Reader, and coefficient of variation for

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replicates in the assay were less than 5%.

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Western blotting

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Approximately100 mg of frozen grated rumen tissue was homogenized in 1 mL

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ice-cold RIPA protein isolation buffer (Cat. No. SN338; Sunshine Biotechnology Co.,

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Nanjing, China) for the total protein extraction. The protein concentration was

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measured via the BCA Protein Assay kit (No. 23225, Thermo Fisher, USA). 50 µg of

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total protein was loaded onto 12% sodium dodecyl sulfate-polyacrylamide gel

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electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (Bio Trace;

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Pall Co., Port Washington, NY). The membranes were incubated in the appropriate

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primary antibodies: Jun N-terminal kinase (JNK) (No. 9252S; Cell Signaling

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Technology, Danvers, MA), p-JNK (No. 9255S; Cell Signaling Technology), p38 (No.

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8690S; Cell Signaling Technology), p-p38 (No. 4511S; Cell Signaling Technology),

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Erk1/2 (No. 4695S; Cell Signaling Technology), p-Erk1/2 (No. 4370S; Cell Signaling

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Technology), PKC-α (No. 2056S; Cell Signaling Technology), occludin (No.

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ab167161; Abcam, Cambridge, United Kingdom), claudin-1 (No. ab15098; Abcam),

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and ZO-1 (No. ab214228; Abcam) at 4°C overnight, then washed and incubated in 10


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corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies for 45

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minutes at room temperature. β-actin (Cat. No. SC130656; Santa Cruz Biotechnology,

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Dallas, TX) was employed as a reference protein. Finally, the membranes were

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washed and visualized using an enhanced chemiluminescence (ECL) Kit (Pierce,

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Rockford, IL). The signals were recorded using an Bio-Rad imaging system (Bio-Rad,

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Hercules, CA), and the results were analyzed using Quantity One software (Bio-Rad)

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38

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Statistical analysis

.

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The ruminal pH was analyzed by performing general linear model repeated

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measures using IBM SPSS 21.0 statistics for Windows (IBM Inc., New York, NY,

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USA). For each goat, pH data for the last day of consecutive weeks 7, 8 and 9 were

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averaged before analysis. The results of DNA methylation rate, enzyme activity, and

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mRNA and protein expression are expressed as the mean fold change relative to the

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LC group set as 1 and were analyzed using one-way ANOVA with Dunnett's posttest

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using SPSS 21.0 software (IBM). P < 0.05 was considered statistically significant.

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RESULTS

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Rumen pH analysis

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The HC group had a significantly lower pH value than the LC and SH group in 7,

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8, 9 weeks (P < 0.05; Fig. 1). There was no significant difference in ruminal pH

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between the LC and SH groups. 11



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Morphologic analysis of the rumen epithelium

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The results of rumen epithelium histologic analysis, which were confirmed based

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on the epithelial injury score, are shown in Fig. 2. The stratum corneum of the

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epithelium was severely damaged in the HC group, whereas the rumen epithelium

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remained intact in the LC and SH groups. The histologic damage score was

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significantly lower in the LC and SH groups than in the HC group (P < 0.05).

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Relative expression of genes related to barrier function in the rumen epithelium

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The relative mRNA expression of MAPKs involved in TJs, such as extracellular

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regulated protein kinase (ERK) 1, ERK2, and MAPK, was greater in the HC group

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than the LC and SH group (Fig. 3. P < 0.01), while there was no significant difference

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between the LC and SH groups. c-JNK was also significantly upregulated in the HC

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group (P < 0.05), whereas JNK expression in the SH and LC groups was similar.

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Rumen epithelium mRNA expression of the TJ proteins claudin-1, claudin-4,

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occludin, and ZO-1 was determined. The expression of these genes was found to be

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significantly lower in the HC group than the LC and SH group (P < 0.05), while their

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expression was similar in the LC and SH groups.

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Phosphorylation levels of Erk1/2, JNK, and p38 in the rumen epithelium

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The phosphorylation levels of Erk1/2, p38, and JNK were determined via

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Western blot analysis (Fig. 4). JNK and p38 MAPK phosphorylation levels were

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significantly greater in the HC group than in the LC group (P < 0.05), whlie their 12


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phosphorylation levels in the SH group were similar to the LC group. Although the

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expression of Erk1/2 was not significantly different between the HC and SH groups,

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the phosphorylation levels of Erk1/2 were decreased in the SH group.

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Protein levels of PKC-α, claudin-1, occludin, and ZO-1 in the rumen epithelium

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Western blotting detection of claudin-1, occludin, and ZO-1 showed significantly

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lower protein levels in the HC group than the LC group (P < 0.05), whereas the levels

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in the SH and LC groups were similar (Fig. 5). The protein level of PKC-α was

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greater in the HC and SH groups than in the LC group (P < 0.05).

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PKC kinase activity

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PKC kinase activity was increased more than threefold in the HC group but was

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significantly decreased in the SH group (P < 0.01, Fig. 6). There was no significant

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difference in PKC kinase activity between the LC and SH groups.

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DNA methylation status of occludin

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As shown in Fig. 7, the DNA methylation rate of occludin was higher in the HC

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group than in the LC group, whereas it was lower in the SH group than in the HC

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group.

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DISCUSSION

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An HC diet in ruminants can increase growth and milk production over the short

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term but can have a negative impact on the health of animals as a result of

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accumulation of volatile fatty acids, which decrease the ruminal pH, leading to SARA

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39, 40

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dysfunction. Impairment of ruminal epithelial barrier function can lead to LPS

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translocation, which can cause systemic inflammation and disease and result in

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economic losses 41, 42.

. A decrease in the ruminal pH also results in ruminal epithelial barrier

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In this study, goats fed the HC diet for 9 weeks exhibited a low ruminal pH,

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between 5.6 and 5.8, for more than 3 hours, which meets the current definition of

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SARA, indicating that SARA was successfully induced by the HC diet. We found that

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supplementation of the HC diet with SB (SH group) led to a higher ruminal pH than

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that in the HC group. The aqueous solution of SB is alkaline, which may be the cause

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of the SH diet-induced increase in ruminal pH.

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Our study demonstrates that an HC diet can disrupt the barrier function of the

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rumen epithelium. Although Penner et al. have indicated that a mild case of SARA

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does not alter ruminal barrier function in the short term 43, in our study, the cellular

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structure of the rumen epithelium was damaged by 9 weeks of feeding on the HC diet.

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We also found that the SH diet neutralized the damage to the ruminal epithelial barrier

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function induced by the HC diet through histologic alterations.

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mRNA expression of claudin-1, claudin-4, occludin, and ZO-1 was significantly 14


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decreased in the rumen epithelium of the goats in the HC group. However, in the SH

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group, the expression of genes involved in TJs was similar to that in the LC group.

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DNA methylation is an effective approach to silence TJ proteins expression and alter

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TJ function

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intestinal epithelial cells can increase transepithelial electrical resistance and disrupt

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barrier function, suggesting that occludin is important for TJ barrier function 46. Thus,

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the occludin gene was chosen for methylation analysis of genomic DNA in our study.

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Occludin in the rumen epithelium in the HC group had a higher DNA methylation rate

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than that in the LC and SH groups. Sodium butyrate inhibited the transcriptional

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activation of occludin, as shown by our RT-qPCR results. In addition, claudin-1,

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claudin-4, occludin, and ZO-1 protein levels in the rumen epithelium were lower in

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the HC group than in the LC and SH groups.

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44

. Occludin influences TJ function

45

, and knockout of occludin in

Phosphorylation via the PKC, MAPK, JNK, and NF-κB pathways can be applied 47

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to control transient downregulation of TJ proteins

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serine-threonine kinases are directly linked to TJ assembly

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regulates the phosphorylation and transcription of several TJ proteins 49. In line with

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these findings, we showed that in the SH group, PKC activity was significantly

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decreased in the rumen epithelium, which might affect the activation of the

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TJ-associated genes. The PKC isoform whose role in the regulation of junction

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assembly is best understood is PKC-α. A recent study showed that overexpression of

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PKC-α increased the permeability of the rumen epithelium 15


. The PKC family of 48

50

, and PKC activation

. In the present study,


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PKC-α protein expression was increased in both the HC and SH groups; thus, TJ

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function may be regulated by another isoform of PKC.

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ERK/MAPK activation can be suppressed by overexpression of the junctional

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membrane protein occludin 51. Activation of the p38 MAPK pathway can regulate the

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expression of claudin-1 and -2 in vitro. Rat hepatocytes treated with the p38 MAPK

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activator anisomycin show downregulated expression of claudin-1

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MAPK inhibitor upregulates claudin-1 in the regenerating rat liver

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expression is also decreased by inhibition of p38 MAPK activation in primary

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cultures of occludin-knockout mouse hepatocytes. Furthermore, increased activation

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of p38 MAPK and Akt can downregulate the expression of claudin-4 in hepatic cell

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lines 54. In our study, SH treatment ameliorated the decreases in the phosphorylation

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levels of ERK and p38 induced by the HC diet, which is consistent with the

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expression of ERK and p38.

52

, whereas a p38 53

. Claudin-4

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The JNK signaling pathway is also essential for the regulation of TJ expression.

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Recently, epithelial barrier function was shown to be enhanced through differential

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modulation of claudin expression via inhibition of JNK activity in murine mammary

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epithelial cells

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proinflammatory cytokines was previously performed to determine the effect on the

309

expression of TJ proteins through the JNK pathway in human pancreatic cancer cells

310

56

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TJ proteins, whereas these changes were reversed by the JNK inhibitor. HC diet

55

. Stimulation with the JNK activator anisomycin and the

; treatment with proinflammatory cytokines was found to induce the expression of

16


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feeding has been shown to increase immune gene expression and to activate the JNK

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signaling pathway in the liver of dairy cows 57. These findings suggest that activation

314

of the JNK pathway could disrupt the epithelial barrier in the presence of

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inflammation or other disorders. In our study, the phosphorylation level of JNK in the

316

HC group was increased, consistent with the mRNA expression of the corresponding

317

gene. The addition of SH reversed this effect. Hence, the evidence suggests that TJ

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expression is regulated by the MAPK signaling pathway in the rumen epithelium of

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goats.

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In conclusion, 9 weeks of feeding on the HC diet severely compromised the

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rumen epithelium and impaired ruminal epithelial barrier function through the PKC

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and MAPK signaling pathways. However, these adverse effects were attenuated by

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adding SB to the diet. These findings provide a foundation for new treatments for

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SARA.

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FUNDING SOURCES

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This study was supported by the Project of Jiang Su Independent Innovation

327

(CX(15)1003) and Postdoctoral Foundation of Jiangsu Province (Grant number

328

1701031A).

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Disclosures

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The authors declare that they have no conflicts of interest to disclose.

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beta-cell proliferation, function and glucose homeostasis through modulation of

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macromolecule flux across the intestinal epithelial tight junction barrier. Am J Physiol

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cell monolayer. J. Biol. Chem. 2007, 282, 11487-11498.

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49. Banan, A.; Zhang, L. J.; Shaikh, M.; Fields, J. Z.; Choudhary, S.; Forsyth, C. B.;

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mechanism for regulation of permeability. J. Pharmacol. Exp. Ther. 2005, 313,

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50. Clark, H.; Ginanni, N.; Laughlin, K. V.; Smith, J. B.; Pettit, G. R.; Mullin, J. M.

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The transient increase of tight junction permeability induced by bryostatin 1 correlates

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with rapid downregulation of protein kinase C? Exp. Cell Res. 2000, 261, 239-249.

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51. Li, D.; Mrsny, R. J. Oncogenic Raf-1 disrupts epithelial tight junctions via

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downregulation of occludin. J. Cell Biol. 2000, 148, 791-800.

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52. Kojima, T.; Yamamoto, T.; Murata, M.; Chiba, H.; Kokai, Y.; Sawada, N.

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Regulation of the blood-biliary barrier: Interaction between gap and tight junctions in

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53. Yamamoto, T.; Kojima, T.; Murata, M.; Takano, K.; Go, M.; Hatakeyama, N.;

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during regeneration of rat hepatocytes. J. Hepatol. 2005, 42, 707-718.

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54. Saadat, I.; Higashi, H.; Obuse, C.; Umeda, M.; Murata-Kamiya, N.; Saito, Y.; Lu,

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H.; Ohnishi, N.; Azuma, T.; Suzuki, A.; Ohno, S.; Hatakeyama, M. Helicobacter

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pylori CagA targets PAR1/MARK kinase to disrupt epithelial cell polarity. Nature

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2007, 447, 330-333.

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55. Carrozzino, F.; Pugnale, P.; Féraille, E.; Montesano, R. Inhibition of basal p38 or

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JNK activity enhances epithelial barrier function through differential modulation of

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claudin expression. Am. J. Physiol. Cell Physiol. 2009, 297, C775-787.

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56. Kojima, T.; Fuchimoto, J.; Yamaguchi, H.; Ito, T.; Takasawa, A.; Ninomiya, T.;

500

Kikuchi, S.; Ogasawara, N.; Ohkuni, T.; Masaki, T.; Hirata, K.; Himi, T.; Sawada, N.

501

c-Jun N-terminal kinase is largely involved in the regulation of tricellular tight

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225, 720-733.

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57. Guo, J.; Chang, G.; Zhang, K.; Xu, L.; Jin, D.; Bilal, M. S.; Shen, X.

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Rumen-derived lipopolysaccharide provoked inflammatory injury in the liver of dairy

506

cows fed a high-concentrate diet. Oncotarget. 2017, 8(29), 46769-46780.

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507

Table 1. Ingredients and calculated nutrient composition of experimental diets. Ingredient, % DM

Percentage (%) of ingredients in different diets (air dry matter)

LC

HC

SH

Maize

12

40

40

Soybean meal

11.92

6.36

6.36

Wheat bran

1.00

8.98

8.98

Wheat

2.09

12.00

12.00

Straw

70

30

30

Calcium hydrophosphate

1.40

0.55

0.55

Limestone

0.59

1.10

0.10

Salt

0.50

0.50

0.50

Premix*

0.50

0.50

0.50

Sodium butyrate

0.00

0.00

1.00

DE, MJ/kg

8.65

11.43

11.43

CP %

11.77

11.76

11.76

NDF %

44.81

26.53

26.53

ADF %

24.63

13.45

13.45

Ash %

10.26

8.13

8.13

Nutrient composition

508

*The premix consisted of the following ingredients per kg of diet: 6.60 × 104 IU of vitamin A, 8.00 × 105 IU of vitamin D3, 1.49 × 103

509

of vitamin E, 35.2 mg of Cu, 120 mg of Fe, 115 mg of Zn, 80 mg of Mn, 0.35 mg of Co, and 19.5 mg of Se.

27



510

Page 28 of 40

Table 2.The primer sequences of target and internal reference genes used in qPCR. Reference/Gene Gene

Forward primer

Reverse primer

PCR products (bp) bank accession

Erk1

CTCAGCTTACGACCATGTGC

TCAGGTCCTGCACGATGTAG

203

XM_018040780.1

Erk2

CTCAGCAACGACCACATCTG

CCAGGCCAAAGTCACAGATC

151

NM_001314202.1

p38

ACAACATCGTCAAGTGCCAG

CACGTAGCCAGTCATCTCCT

209

XM_018048951.1

JNK

TCAGTCAGTTGAGCACCAGT

ACTTATGCCTGCTCTGCTCA

229

XM_018051618.1

Claudin-1

CACCCTTGGCATGAAGTGTA

AGCCAATGAAGAGAGCCTGA

216

XM_005675123.3

Claudin-4

AAGGTGTACGACTCGCTGCT

GACGTTGTTAGCCGTCCAG

238

XM_005697785.2

Occludin

GTTCGACCAATGCTCTCTCAG

CAGCTCCCATTAAGGTTCCA

200

XM_018065677.1

ZO-1

CGACCAGATCCTCAGGGTAA

AATCACCCACATCGGATTCT

163

XM_018066114.1

GAPDH

GGGTCATCATCTCTGCACCT

GGTCATAAGTCCCTCCACGA

180

XM_005680968.3

511

28


Page 29 of 40


512

Table 3. Primers for bisulfite-treated DNA Primer

OCCLUDIN-m1-F

Sequence (5'-3')

Length

GGTTAAAAGTGATGTTTGGAATTTT 265 bp

OCCLUDIN-m1-R

ATCCATAAATCCAATCCCTAATACA

OCCLUDIN-m2-F

GGTTAAAAGTGATGTTTGGAATTTT 201 bp

OCCLUDIN-m2-R

AATCCATAAATCCAATCCCTAATAC

513

29



514

FIGURE LEGENDS

515

Figure 1: Ruminal pH values of the low concentrate diet (LC), high concentrate

516

diet (HC) and high concentrate diet with sodium butyrate (SH) groups at

517

different time points after feeding. The pH was markedly lower in the HC group

518

than the LC and SH group throughout the time of 10 h after feeding. A low pH value,

519

between 5.6 and 5.8, was observed for approximately 4 hours a day in the HC group.

520

The error bars indicate the standard error of the mean (SEM).

521

Figure 2: Histologic alteration of the rumen epithelium in goats from the low

522

concentrate diet (LC), high concentrate diet (HC) and high concentrate diet with

523

sodium butyrate (SH) groups. Representative photomicrographs with hematoxylin

524

and eosin staining are shown. The rumen epithelia of the LC group were intact and

525

showed no disruption (A), whereas the stratum corneum of the epithelium was

526

severely damaged in the HC group (B). The epithelium showed slight damage in the

527

SH group (C). (D) Epithelial injury score. All sections were stained with H&E and

528

examined at 400X magnification. *P < 0.05 compared with LC; #P < 0.05 compared

529

with HC.

530

Figure 3: Gene expression related to barrier function in the rumen epithelium of

531

goats in the low concentrate diet (LC), high concentrate diet (HC) and high

532

concentrate diet with sodium butyrate (SH) groups. Relative gene expression of

533

Erk1, Erk2, p38 MAPK, and JNK was significantly greater in the HC group than in

534

the LC and SH group, whereas there was no significant difference between the SH 30


Page 30 of 40

Page 31 of 40


535

and LC groups.Relative gene expression of claudin-1, claudin-4, occludin, and ZO-1

536

in the rumen epithelium was significantly decreased in the HC group compared with

537

the LC and HC group, whereas no significant difference was observed between the

538

SH and LC groups. The results are expressed as fold changes relative to the LC group

539

(means ± SEM). *P < 0.05; **P < 0.01 compared with LC; #P < 0.05 compared with

540

HC.

541

Figure 4: Phosphorylation levels of Erk1/2, JNK, and p38 MAPK in the rumen

542

epithelium of goats in the low concentrate diet (LC), high concentrate diet (HC)

543

and high concentrate diet with sodium butyrate (SH) groups. Erk1/2, JNK, and

544

p38 MAPK phosphorylation levels were significantly greater in the HC group than in

545

the LC and HC group, whereas the levels in the SH group were similar to those in the

546

LC group. The results are expressed as fold changes relative to the LC group (means

547

± SEM). *P < 0.05; **P < 0.01 compared with LC; #P < 0.05 compared with HC.

548

Figure 5: Protein levels of PKC-α, claudin-1, occludin, and ZO-1 in the rumen

549

epithelium of goats in the low concentrate diet (LC), high concentrate diet (HC)

550

and high concentrate diet with sodium butyrate (SH) groups. Claudin-1, occludin,

551

and ZO-1 protein levels were significantly lower in the HC group than in the LC and

552

SH group, whereas the levels in the SH group were not significantly changed. The

553

protein level of PKC-α was greater in the HC and SH groups than in the LC group.

554

The results are expressed as fold changes relative to the LC group (means ± SEM). *P

555

< 0.05; **P < 0.01 compared with LC; #P < 0.05 compared with HC. 31



556

Figure 6: PKC activity in the rumen epithelium of goats in the low concentrate

557

diet (LC), high concentrate diet (HC) and high concentrate diet with sodium

558

butyrate (SH) groups. PKC kinase activity was significantly greater in the HC group

559

than in the LC and SH group, whereas it was lower in the SH group than in the HC

560

group. The results are expressed as relative PKC activity and fold change relative to

561

LC group (mean ± SEM). *P < 0.05; **P < 0.01 compared with LC; #P < 0.05

562

compared with HC.

563

Figure 7: DNA methylation status of occludin in the rumen epithelium of goats in

564

the low concentrate diet (LC), high concentrate diet (HC) and high concentrate

565

diet with sodium butyrate (SH) groups. The DNA methylation rate of occludin was

566

higher in the HC group than in the LC and SH groups. The solid circles represent

567

CpG-segments of occludin promotor. The solid circles indicate sites where DNA

568

methylation occurs, and the blank circles indicate sites where DNA unmethylated. *P

569

< 0.05 compared with LC; #P < 0.05 compared with HC.

32


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571 572

Figure 1

33



573

574

575

Figure 2

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577 578

Figure 3

35



579

580 581

Figure 4

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582

583 584

Figure 5

37



585

586 587

Figure 6

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589

590

Figure 7

39



591

592 593

TOC Graphic

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Sodium Butyrate Improves High-Concentrate Diet ... - ACS Publications

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