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Sodium Butyrate Ameliorates High-Concentrate DietInduced Inflammation in the Rumen Epithelium of Dairy Goats Hongyu Dai, Xinxin Liu, Jinyu Yan, Zain ul Aabdin, Muhammad Shaid Bilal, and Xiangzhen Shen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04447 • Publication Date (Web): 29 Dec 2016 Downloaded from http://pubs.acs.org on January 7, 2017

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Journal of Agricultural and Food Chemistry

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Sodium Butyrate Ameliorates High-Concentrate Diet-Induced Inflammation in the

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Rumen Epithelium of Dairy Goats

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Hongyu Dai, Xinxin Liu, Jinyu Yan, Zain ul Aabdin, Muhammad Shaid Bilal,

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Xiangzhen Shen*

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College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, P. R. China

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*Corresponding author:

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Xiangzhen Shen,

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Department of Veterinary Clinical Science,

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College of Veterinary Medicine, Nanjing Agricultural University,

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Nanjing, 210095, P. R. China;

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Phone: +86 25 84395505;

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Fax: +86 25 84398669

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E-mail: [email protected]

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Title running header: Sodium butyrate attenuates HC-induced rumen inflammation

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ACS Paragon Plus Environment


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Abstract

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To investigate the effect of sodium butyrate on high-concentratediet-induced local

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inflammation of the rumen epithelium, 18 mid-lactating dairy goats were randomly

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assigned to 3 groups: a low-concentrate diet group as the control (concentrate:

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forage=4:6), a high-concentrate (HC) diet group (concentrate: forage=6:4), and a

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sodium butyrate (SB) group (concentrate: forage=6:4, with 1% SB by weight). The

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results showed that, with the addition of sodium butyrate, the concentration of

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lipopolysaccharide(LPS) in rumen fluid (2.62 × 104±2.90 × 103 EU/mL) was

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significantly lower than that in the HC group (4.03×104±2.77×103 EU/mL). The

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protein abundance of pp65, gene expression of pro-inflammatory cytokines, and

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activity of myeloperoxidase (MPO) and matrix metalloproteinase (MMP)-2,9 in the

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rumen epithelium were significantly down-regulated by SB compared with those in

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the HC group. With sodium butyrate administration, the concentration of NH3-N

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(19.2±0.890 mM) in the rumen fluid was significantly higher than that for the HC

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group (12.7±1.38 mM). Severe disruption of the rumen epithelium induced by HC

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was also ameliorated by dietary SB. Therefore, local inflammation and disruption of

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the rumen epithelium induced by HC were alleviated with SB administration.

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Keywords: Dairy goat; High-concentrate diet; Rumen epithelium; Inflammation;

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Sodium butyrate


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Introduction

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With the increasing global demand for dairy products, dairy cows and dairy goats are

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increasingly being fed a high-concentrate diet (some are up to 75%1 concentrate) to

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improve their milk yield. Although a high-concentrate diet can increase milk yield in

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the short term, these diets can be harmful to the health of the animal when

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administered for long periods. A high-concentrate diet contains large amounts of

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rapidly fermentable non-structural carbohydrates, producing plenty of volatile fatty

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acids and other organic acids that accumulate in the rumen 2, 3, and this effect plays a

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key role in the depression of the rumen pH. The rumen is the first organ affected by a

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low pH, and many studies have found that low pH can compromise the integrity and

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permeability of the rumen epithelium 4-6. Additionally, the long-term administration of

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a high-concentrate diet results in ruminal parakeratosis and hyperkeratosis 7. A

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decrease in rumen pH leads to defects in the rumen epithelium 8, thereby increasing

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the risk of pathogenic invasion into the portal vein drainage (PVD), resulting in liver

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abscess and other more severe diseases 9, decreasing productivity and possibly leading

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to death.

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Additionally, a decrease in the pH can also increase the level of free

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lipopolysaccharide (LPS) released from dead Gram-negative bacteria

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strong inflammatory inducer that can elicit inflammatory responses

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receptor 4 (TLR4) is the specific receptor of LPS; upon the binding of LPS to TLR4,

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nuclear factor kappa B (p65) signaling is activated, and the expression of genes (i.e.,

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Interleukin (IL)-6, IL-8, tumor necrosis factor (TNF)-α et al.) involved in


10, 11

. LPS is a

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. Toll-like


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inflammatory responses 13 is induced. Cytokines, such as TNF-α and interleukins, are

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commonly observed at inflammatory sites and can increase the inflammatory response

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by initiating the infiltration of innate immune cells, such as neutrophils and

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macrophages, into inflamed tissues

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neutrophil cell marker

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neutrophil infiltration. Tight junctions in the stratum granulosum confer the

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mechanical strength of the rumen epithelium

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junction proteins are substrates of matrix metalloproteinase (MMPs) 18. An increase in

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the activity of these enzymes in the epithelium results in the degradation of tight

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junction proteins and further disruption of the epithelium.

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Butyric acid, one of the volatile fatty acids, is a natural substance that exists in the

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fore-stomachs and colons of ruminants and colons of mono-gastric species. A

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previous study revealed that butyrate can suppress the inflammatory response and that

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this suppression is associated with the reduction of NF-κB activation 19 and abolition

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of LPS-induced cytokine expression

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salts (i.e., sodium butyrate) are often used instead of butyric acid itself because they

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are solid, stable and much less odorous.

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Previous studies have found that sodium butyrate (SB) promoted the growth of rumen

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epithelia when orally administered 21, 22; when supplied as a milk supplement to calves

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in the first 4 weeks after birth, SB improves the development of the rumen papillae

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and increases the production performance and health status of the animals

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addition, the oral administration of SB attenuates inflammation and mucosal lesions in

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. Myeloperoxidase(MPO) has been used as a

15, 16

, and the activity of this enzyme can be used to determine

4, 17

. Studies have revealed that tight

20

. In animal studies and in practice, butyrate


23

. In

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experimental acute ulcerative colitis 24.

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The above observations demonstrate that SB has great potential in modulating the

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inflammatory response. Therefore, we propose that SB may also play a role in

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attenuating the adverse effects of a high-concentrate diet on the rumen epithelium.

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Thus, 18 pre-cannulated mid-lactating goats were randomly divided into 3 groups and

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were challenged with different diets— low-concentrate (LC; concentrate: forage=4:6),

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high-concentrate (HC; concentrate: forage=6:4), and HC diet with SB (concentrate:

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forage=6:4, with 1% SB by weight)—to characterize the local inflammation in the

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rumen epithelium in response to long-term high-concentrate diet feeding, to examine

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the mechanisms underlying this condition by evaluating the expression of genes and

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protein involved in this process, and to evaluate the role of SB in ameliorating these

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processes.

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Materials and methods

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Ethics statement

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The Animal Care and Use Committee of Nanjing Agricultural University approved

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the experimental protocol, which was performed in accordance with the Guidelines

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for Experimental Animals of the Ministry of Science and Technology (2006, Beijing,

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China).

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Chemicals

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Dietary SB was obtained from Dongying Degao Biotechnology, Jinan, China.

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Analytical grade chloroform and isopropanol were purchased from Shanghai

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Lingfeng Chemical, Shanghai, China, and other reagents were of analytical grade.



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Ultrapure water was prepared from a Milli-Q system (Bedford, MA, USA). H2

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(99.999% purity), N2 (99.999% purity) and helium (99.999% purity) were acquired

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from Jiangsu Tianhong Chemical, Nanjing, China.

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Animals and experimental design

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Eighteen (average body weight, 38.9± 2.06 kg) mid-lactating (milk yield, 1.13 ±

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0.0200kg/day) Saanen dairy goats in parity 1 or 2 with a rumen fistula were randomly

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divided into three groups (n=6)—high-concentrate diet (HC; concentrate: forage=6:4),

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high-concentrate diet with SB (SHC; concentrate: forage=6:4 with 1% SB by weight)

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and low-concentrate diet (LC; concentrate: forage=4:6). The goats were fed these

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diets for 20 weeks. The formulations of the diets for each group are listed in Table 1.

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The goats were maintained in tie stalls individually and were provided fresh water ad

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libitum. The goats were fed twice a day (8:00 a.m. and 6:00 p.m.) after milking. Prior

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to the start of the experiment, the goats were adapted to the experimental diet for two

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weeks. The experimental period was 20 weeks, and the animals were slaughtered at

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the end of the experiment.

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Sample collection and analysis

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Rumen fluid was sampled at the last day of weeks 17, 18 and 19through a cannula

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from the ventral sac of the rumen after mixing the content at 0 h, 1 h, 2 h, 4 h, 6 h, 8 h

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and 10 h after feeding. Simultaneously, blood samples were collected from the jugular

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vein using 5-mL vacuum tubes containing sodium heparin together with samples of

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the rumen fluid. The rumen fluid was filtered through a 4-layer cheesecloth. The pH

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was immediately measured using a basic pH meter (Sartorius, Goettingen, Germany).


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Thereafter, the rumen fluid samples were stored at -20°C. Plasma was prepared by

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centrifugation (3000×g) at 4°C for 15 min and was stored at -20°C for later analysis.

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The goats were slaughtered at the end of the experiment, and the rumens were

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exteriorized and washed with PBS. Approximately 10 g of rumen epithelium was

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obtained from the ventral sac, was transferred into liquid nitrogen after segregation

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from the muscle layer and was stored at -80°C for later analysis. Whole-thickness

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samples of rumen tissues (approximately 1 cm2) from the ventral sac were fixed in 4%

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paraformaldehyde solution for histological analysis.

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Rumen fluid analysis

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For LPS detection, the rumen fluid was preprocessed according to a previous report25.

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Briefly, the rumen fluid was centrifuged at 10,000×g for 45 min. The supernatant was

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aspirated and subsequently passed through a disposable 0.22-µm pyrogen-free filter

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(Pall Gelman Laboratory, Ann Arbor, MI, USA). The filtrate was collected in a

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depyrogenated tube (heated at 180°C for 4 h) and boiled at 100°C for 30 min. The

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rumen fluid was subsequently cooled at room temperature(25°C)for 10 min. The

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pretreated rumen fluid was diluted 100,000-fold, and the LPS concentration was

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detected using the Chromogenic End-point Tachypleus Amebocyte Lysate Assay Kit

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(Chinese Horseshoe Crab Reagent Manufactory Co. Ltd，China) according to the

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manufacturer’s instructions, with a minimum detection limit of 0.01 EU/mL.

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For the determination of volatile fatty acid (VFA) and ammonia nitrogen, the rumen

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fluid was centrifuged at 1900×g for 15 min, and the supernatant was stored at -20°C.

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The ammonia nitrogen concentration in the rumen fluid was determined using a



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colorimetric assay as described previously26. The VFA concentration was measured by

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gas chromatography using an FFAP 123-3233, 30-m×0.32-mm×0.5-µm, capillary

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column (Agilent Technologies, Stevens Creek Blvd, Santa Clara, CA, USA) in an

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Agilent 7890A system (Agilent Technologies) as previously described 27.

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RNA analysis

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Total RNA was isolated from ground rumen epithelium samples (approximately 100

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mg) using RNAiso Plus (Takara Co., Otsu, Japan) according to the manufacturer’s

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instructions. The concentration of isolated RNA was detected using a Nano Drop

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ND-2000 spectrophotometer (Thermo Fisher Scientific Inc.,Waltham, MA, USA).

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RNA purity was evaluated by assessing the ratio of A260/A230 and A260/A280 using

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a Nano Drop 2000 (Thermo Fisher Scientific Inc.); only A260/280 ratios between 1.8

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and 2.0 were used for later cDNA analysis. The ribosomal RNA integrity was

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assessed using agarose gel electrophoresis (1%).

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In each group, 2 ng of RNA was reverse transcribed to cDNA using the Prime Script®

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RT Master Mix Perfect Real Time Kit (Takara Co.) according to the manufacturer’s

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instructions. The relative gene expression of IL-1β, IL-6, IL-8, IL-10, TNF-α, p65,

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TLR4, MPO and CD68 was assessed using real-time PCR. For each transcript, a

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standard curve was constructed using the purified PCR product generated from each

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specific primer pair. The cDNA was diluted 2-fold and was subjected to real-time

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PCR using the SYBR® Premix Ex Taq™ Kit (Takara Co.) according to the

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manufacturer’s instructions. The PCR was performed using a 7300 real-time PCR

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system (Applied Biosystems, Foster City, CA, USA) and the following conditions:


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denaturation at 95°C for 15 s, followed by 40 cycles of 95°C for 5 s and 60°C for 31 s,

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β-Actin was selected as a reference gene as a control for normalization. The primers

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were commercially synthesized (Sangon, Shanghai, China), and the information is

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displayed in Table 2. The specificity of the products of different primers was

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evaluated by electrophoresis on 3% agarose gels. The real-time PCR results were

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analyzed using the 2-∆∆Ct method28, and the results were expressed as fold changes.

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Western blot analysis

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Approximately 100 mg of ground rumen epithelium was homogenized in 1 mL of

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ice-cold RIPA protein isolation buffer (Beyotime, Shanghai, China) for 15 s,

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incubated on ice for 20 min and then centrifuged at 12000×g for 30 min at 4°C. The

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supernatant was transferred to another tube without touching the pellet at the bottom.

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The protein concentration was determined using the BCA Protein Assay kit (Thermo

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Fisher Scientific Inc.). Sixty micrograms of isolated protein was separated after

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loading into 5% and 10% SDS-PAGE gels and was transferred onto nitrocellulose

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membranes (Pall Gelman Laboratory, Ann Arbor, MI, USA). Subsequently, the

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membranes were blocked in 5% skim milk (5% BSA for phosphorylated p65) for 2 h

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at room temperature (25°C), followed by incubation with the primary antibodies anti-

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p65 (1:300, sc-109, Santa Cruz Biotechnology, Inc., Dallas, Texas, USA), anti-p

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p65(Ser 536) (1:300, sc-33020, Santa Cruz Biotechnology, Inc.), anti-IL-1β (1:300,

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sc-7884, Santa Cruz Biotechnology, Inc.), anti-TNF-α (1:300, sc-8301, Santa Cruz

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Biotechnology, Inc.), anti-IL-6 (1:300, sc-1265, Santa Cruz Biotechnology, Inc.) and

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anti-β-actin (1:10000, BS6007M, Bioworld Technology, Inc., Louis Park, USA)



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overnight at 4°C. The membranes were subsequently washed in Tris-buffered saline

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containing Tween 20 (TBST) 6 times (10 min/wash), followed by incubation with

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horseradish peroxidase (HRP)-conjugated secondary antibodies, goat-anti-rabbit

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(1:5000, sc-2004, Santa Cruz Biotechnology, Inc.) and goat anti-mouse (1:10000,

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SN-133, Sunshine Biotechnology (Nanjing) Co., Ltd, China), for 2 h at room

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temperature. The membranes were washed 6 times with TBST (10 min/wash), and the

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results were visualized using an ECL Plus kit (Vazyme, Nanjing, China). The signals

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were recorded on a LAS4000 imaging system (GE Healthcare Bio-Sciences AB,

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Uppsala, Sweden) and were analyzed using Quantity One software (Bio-Rad

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laboratories, Inc., Hercules, CA, USA).

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Histological analysis

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Sections were prepared as previously described, with some modifications 29. Briefly,

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the fixed tissues were embedded in paraffin after dehydration, and 5-µm sections were

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cut on a microtome (Leica Biosystems Nussloch GmbH, Nussloch, Germany),

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mounted on adhesive-coated slides and stained with hematoxylin and eosin (H & E).

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The slides were visualized under a light microscope (Nikon ECLIPSE 50i), and the

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images were captured using a high-resolution digital camera (Nikon Digital Sight

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DS-Fi1; Nikon Corporation, Minato-ku, Tokyo, Japan) and NIS Elements F 3.0

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(Nikon Corporation, Minato-ku, Tokyo, Japan) image acquisition software.

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Macroscopic damage was assessed blindly using a score system described previously

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30

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score for the severity of epithelial injury (graded 0–3, from absent to mild including

. Briefly, the damage of the tissues was assigned a numerical score that includes a


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superficial epithelial injury, moderate including focal erosions, and severe including

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multifocal erosions) and the extent of inflammatory cell infiltrate (graded 0–3, from

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absent to transmural). Three tissue sections from each animal were coded and

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examined by two blinded observers to avoid observer bias.

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Radioimmunoassay

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The concentrations of IL-1β, IL-6 and TNF-α in peripheral plasma collected 4h after

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feeding were determined by a Gamma Radioimmunoassay Counter (HesuoRihuan

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Photoelectric Instrument Co., Ltd, Shanghai, China) using radioimmunoassay kits

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(IL-1β, C09DJB; IL-6, C12DJB; TNF-α, C06PJB) purchased from Beijing North

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Institute of Biological Technology and following the manufacturer’s instructions. The

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detected range of the radioimmunoassay kits for IL-1β, IL-6 and TNF-α were

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0.1–8.1 ng/mL, 50–4000 pg/mL and 9–590 fmol/mL, respectively.

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MPO activity assay

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The MPO activity was measured using an MPO kit (Nanjing Jiancheng

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Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions.

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The

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spectrophotometer, and the MPO activity was expressed in units per gram of wet

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tissue.

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Gelatin zymography analysis

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Gelatin zymography was used to assess the activities of two MMPs: MMP-2 and

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MMP-9. Total protein was isolated from a sample of approximately 100 mg of ground

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rumen epithelium, and gelatin zymography analysis was performed as previously

absorbance

of

the

colorimetric

reaction

was


determined

using

a


233

described 31, 32, with some modifications. Briefly, 60 µg of total protein was loaded on

234

a 12% polyacrylamide gel (with 1% gelatin), and electrophoresis was conducted at a

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constant 100 V for 90 min. Next, the gel was incubated in renaturing buffer at room

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temperature for 30 min. After incubating in developing buffer at 37°C for 36 h, the gel

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was stained with 0.5% Coomassie blue R-250 for 3 h and washed 3 times in different

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de-staining buffers. The bands on the zymogram were indicative of gelatinase activity.

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The relative intensity of the zymography was analyzed using Quantity One software

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(Bio-Rad Laboratories, Inc., Hercules, CA, USA). The experiments were duplicated,

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and the values obtained for the relative intensity were subjected to statistical analysis.

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Statistical analysis

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The results were expressed as the means ± SEM. All of the data, except pH, were

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analyzed using one-way ANOVA with Dunnett's posttest by IBM SPSS 20.0 statistics

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for Windows (IBM Inc., New York, NY, USA). The pH of the rumen fluid was

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analyzed using General Linear Model Repeated Measures by IBM SPSS 20.0

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Statistics for Windows (IBM Inc.). The data were considered statistically significant

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at P

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