Sodium butyrate mitigates iE-DAP induced inflammation caused by

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Bioactive Constituents, Metabolites, and Functions

Sodium butyrate mitigates iE-DAP induced inflammation caused by high-concentrate feeding in liver of dairy goats Animesh Chandra Roy, Yan Wang, Huanmin Zhang, Shipra Roy, Hongyu Dai, Guangjun Chang, and Xiangzhen Shen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02732 • Publication Date (Web): 04 Aug 2018 Downloaded from http://pubs.acs.org on August 6, 2018

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Journal of Agricultural and Food Chemistry

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Sodium butyrate mitigates iE-DAP induced inflammation caused by high-concentrate

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feeding in liver of dairy goats

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Animesh Chandra Roy†, Yan Wang†, Huanmin Zhang†, Shipra Roy†, Hongyu Dai†, Guangjun

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Chang†, and Xiangzhen Shen*,†

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†

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*

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College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, P. R. China

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*Phone: +86 25 84395505; Fax: +86 25 84398669; E-mail: [email protected]

College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, P. R. China

Corresponding Author: Xiangzhen Shen

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ABSTRACT

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The aim of this study is to explore the impact of sodium butyrate on iE-DAP-induced liver

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inflammation in dairy goats during subacute ruminal acidosis (SARA) caused by high-

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concentrate feed. To achieve this aim, 12 lactating dairy goats were randomly divided into 2

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groups: a high-concentrate feed group (n = 6, concentrate: forage = 6: 4) as the control group and

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a sodium butyrate with high-concentrate feed group (n = 6, concentrate: forage = 6: 4, with 1%

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SB by wt.) as the treatment group. A rumen pH below 5.6 lasted for at least 4 h/d due to long-

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term HC feeding. The concentration of iE-DAP was significantly lower (11.67 ± 3.85 µg/mL, and

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7.74 ± 1.46 µg/mL, at the 4th h and 6th h of feeding, respectively) in the SB-treated group than that

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in the HC group (51.45 ± 5.71 µg/mL, and 18.31 ± 3.83 µg/mL, at the 4th h and 6th h of feeding,

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respectively). Meanwhile, SB significantly suppressed the mRNA expression of inflammatory

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genes (NOD1, RIPK2, TAK1, NF-κB/p65, ERK, JNK2, p38, IL-1β, TNF-α, CCL5, CCL20,

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CXCL12, FOS, β-defensin/LAP). Moreover, the protein expression of NOD1, p-IκBα, p-NF-

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κB/p-p65, p-ERK1/2, p-JNK, p-p38 and HDAC3 was significantly downregulated in the HC+SB

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group. In conclusion, iE-DAP-induced inflammation and liver disruption generated by the HC

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diet was mitigated by SB treatment.

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KEYWORDS: dairy goat, liver, high-concentrate diet, iE-DAP, inflammation, sodium butyrate,

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histone deacetylation

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INTRODUCTION

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Currently, high levels of grain feed are given to most dairy ruminants reared in intensive

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production systems1 for maximizing energy intake and milk yield.2 The production of short chain

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fatty acids (SCFAs) beyond the rumen’s absorptive, buffering and outflow capacity by excessive

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feeding of non-structural carbohydrates and highly fermentable forage with low physically

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effective fiber in ruminants depresses ruminal pH, resulting in subacute ruminal acidosis

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(SARA),3-5 defined by recurrent events of reduced pH below 5.6 for at least 3 h per day.6, 7 The

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induction of SARA causes papilla destruction8, 9 and, the disruption of rumen epithelial tight

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junctions (TJs),10 leading to increased permeability of the rumen epithelium,4,

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and

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transmigration of toxic and immunogenic compounds into the portal circulation.4,

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The

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induction of SARA leads to changes in ruminal microbial populations, systemic inflammation

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and localized inflammation in papillae tissues of the rumen,2 and liver abscesses,13, 14 as well as a

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decrease in milk fat production.2,

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administration of a highly concentrated diet.15 Very few studies have been done on the detection

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of diaminopimelic acid in the rumen. High-performance liquid chromatography (HPLC) is a

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common technique used for the determination of diaminopimelic acid.16, 17 D-glutamyl-meso-

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diaminopimelic acid (iE-DAP), a common component in the cell wall peptidoglycans (PGNs) of

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most gram-negative and some gram-positive bacteria, is responsible for immunobiological

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activities.18

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translocate into the portal circulation19, 20 and harbor in liver, which is rich in hepatocytes and

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Kupffer cells (liver macrophages).

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Immunity plays a key role in a healthy liver by controlling the resistance strength of the liver,

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modulating cellular reactions to different stresses, and orchestrating cellular repair.21 Liver

6, 12

2, 6-diaminopimelic acid is elevated in the rumen by

DAP, which is released into the rumen as a consequence of bacterial lysis, can



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inflammation can stimulate the production of acute-phase proteins that can be responsible for the

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collapse of gut-derived blood detoxification.22, 23 Aspartate aminotransferase (AST) and alanine

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aminotransferase (ALT) are considered the most important factors for the evaluation of liver

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damage.24 GGT, ALP, LDH, total protein, albumin, globulin, total bilirubin, ammonia (NH3),

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etc., can also be used for the evaluation of liver injury and function.25-29

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Neutrophils have long been considered the first line of defense against infection.30

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Myeloperoxidase (MPO), found mainly in neutrophils, is released during inflammation.31

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Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) is one member of the

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NOD-like receptors (NLRs), which are intracellular pattern recognition receptors that recognize

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bacterial peptidoglycan (PG), containing an adverse range of pathogen-associated molecular

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patterns (PAMPs) such as, iE-DAP after harboring in the liver and activating the innate immune

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system.32-36 NOD1 consists of a tripartite domain structure, with an amino-terminal caspase

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recruitment domain (CARD), a central nucleotide-binding domain (NBD) and a carboxy-

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terminal domain of leucine-rich repeats (LRRs).37 Activation of NF-κB/p65 and MAPK

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pathways produce pro-inflammatory molecules introduced by NOD1.38-40 LRRK2 senses the

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ligand iE-DAP and makes structural changes to unmask the central NBD for oligomerization,

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which leads to the disclosure of the CARD. CARD interacts with the CARD of receptor-

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interacting protein kinase 2 (RIPK2) to activate the serine-threonine kinase RICK/RIPK2.38, 39, 41,

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TAB3 complex, to turn on the IKK complex as well as MAPK, including ERK, Jun N-terminal

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kinase (JNK), and p38.39, 41 The activation of IKK phosphorylates the NF-κB inhibitor IκBα for

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polyubiquitination and gets degraded by the proteasome.39, 41 The degraded IκBα permits the

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translocation of NF-κB to the nucleus and triggers the production of pro-inflammatory cytokines,

Activated RIPK2 engages and stimulates the TAK1 (TGFβ-activating kinase 1)-TAB1-TAB2-


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chemokines and antimicrobial peptides.34, 39, 43, 44 Acute phase proteins (APPs) (SAA3, and HP)

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in the liver are stimulated by activated cytokines (TNF-α, IL-1β, IL-6) and chemokines during

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the acute protein response (APR).23 β-defensins (LAP) can have directly bacteriostatic or

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bactericidal effects.45 C/EBP is a transcription factor involved in inflammation and the immune

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response, and is regulated by cytokines.46 Hepatocyte nuclear factor 3β (HNF3β)/forkhead box

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protein A2 (FOXA2), is a transcription factor that can regulate specific genes in the liver such as

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albumin and transthyretin.47 FOXA2 can aid in cellular signal transduction, metabolism, and

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immunoregulation.48 The MAPK/FOXA2 signaling pathway can interfere in squamous cell

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metaplasia. The inhibition of MAPK increases FOXA2, which can depress the degree of cell

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proliferation.49

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The fermentation of non-digestible carbohydrates by rumen bacteria produces butyric acid (BA),

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a four-carbon short chain fatty acid (SCFA).50 An increased concentration of butyrate increases

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the circulating level of butyrate in the portal vein.50 Butyrate suppresses the expression of pro-

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inflammatory cytokines51-53 through the inhibition of histone deacetylase (HDAC) activity in a

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variety of cells.54-56 Sodium butyrate (Na(C3H7COO)), a sodium salt of butyric acid, is usually

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packaged as a white, water-soluble, crystalline solid with a very strong, unpleasant lingering

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smell.

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Recently, several studies have been done to investigate the relationship between SARA and LPS-

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induced signaling pathways produced by high-concentrate feeding in ruminants. However, very

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few studies have been conducted to characterize the inflammatory damage in the liver by the iE-

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DAP-induced signaling pathway during high-concentrate feeding and evaluate the role of SB in

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suppressing liver alterations. Therefore, the aim of this study was to find the immunological liver

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response to iE-DAP during SARA and SB treatment. We hypothesized that iE-DAP derived



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from the digestive tract reaches the liver via the portal vein during SARA, causes liver injury,

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suppresses it’s function, and stimulates hepatic inflammatory signaling pathways that are

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alleviated by SB application.

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MATERIALS AND METHODS

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Ethics Statement

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The experimental procedures endorsed by the Animal Ethics Committee of Nanjing Agricultural

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University were performed in accordance with the Guidelines for Experimental Animals of the

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Ministry of Science and Technology (2006, Beijing, China). All the surgical procedures such as

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laparotomy and, rumenotomy, as well as making ruminal fistula to collect ruminal fluid were

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practiced under local anesthesia using lidocaine hydrochloride to alleviate pain.

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Reagents and Chemicals

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Sodium butyrate (SB) was purchased from Dongying Degao Biotechnology, Jinan, Shandong,

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China. Chloroform and isopropanol were bought from Shanghai Lingfeng Chemical, Shanghai,

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China. All the reagents were of analytical grade. Ultrapure water was obtained from a Milli-Q

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system (Bedford, MA, United States). H2 (99.999% purity), N2 (99.999% purity), and helium

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(99.999% purity) was purchased from Jiangsu Tianhong Chemical, Nanjing, Jiangsu, China.

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Animals, Diets and Experimental Design

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Twelve healthy multiparous Saanen dairy goats, weighing 40.0 ± 3.22 kg, that were purchased

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from the experimental farm of Nanjing Agricultural University were chosen for this research.

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The goats were randomly divided into two groups: high-concentrate diet (HC, n = 6)

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(concentrate: forage = 6: 4) as the control group, and high-concentrate diet with SB (HC+SB, n =

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6) (concentrate: forage = 6: 4 including 1% SB by weight) as the treatment group. The goats

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were fed for the 24-week experimental period. Tables 1 and 2 show all the items and nutritional


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components of the diets and their formulations. The goats, housed in individual tie stalls, were

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fed their respective diets two times per day at 7: 00 AM, and 5: 00 PM, including free access to

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fresh drinking water ad libitum throughout the entire experimental period. Two weeks prior to

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the start of feeding, trail experimental surgeries were performed to fit rumen fistulae on the flank

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region of the cows for the collection of ruminal fluid, and the goats were then adapted to the

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experimental diet. Catheters were permanently cannulated in the portal and hepatic veins for the

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whole experimental period. Recovery of the goats from surgical stress without showing any

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clinical signs of infection was ensured. The catheters were unblocked regularly by using saline

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(50 IU/mL). The goats were butchered at the termination of the experiment.

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Sample Collection and Preservation

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At the end day of the 21st, 22nd and 23rd weeks, ruminal fluid was collected by cannula placed in

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fistula at 0 h, 1 h, 2 h, 4 h, 6 h, 8 h and 10 h after immediate feeding at 7: 00 AM of every

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collection day to measure the pH value immediately. Simultaneously, the collection of blood

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samples from the hepatic vein was performed, and the blood samples were kept in 5 mL vacuum

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tubes with sodium heparin. Then, 10 mL of ruminal fluids filtered through 2 layers of

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cheesecloth was centrifuged at 10,000 × g for 45 min. The supernatant was drained through a

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disposable 0.22-µm filter to collect the exudates in a sterile glass tube. The tubes were boiled at

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100 °C for 30 min and stored at -20 °C after chilling at room temperature for 15 min for

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subsequent iE-DAP detection. The plasma was separated from the blood samples by

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centrifugation at 3000 × g at 4 °C for 15 min and preserved at -20 °C for the detection of liver

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biomarkers. At the end of the experiment, slaughtering of the goats was performed after

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overnight fasting according to the law of Jiangsu Provincial People’s Government, China. Liver



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tissues were collected from all the slaughtered goats aseptically and were immediately preserved

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in liquid nitrogen at -70 °C within 30 min of slaughtering.

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Detection of Ruminal pH

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The pH value was detected immediately by using a portable pH meter (Sartorius, Basic pH Meter

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PB-10, PB-21, Goettingen, Germany) from rumen fluids collected at 0 h, 1 h, 2 h, 4 h, 6 h, 8 h,

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and 10 h after immediate feeding at 7: 00 AM.

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Rumen iE-DAP Measurements by HPLC with Chiral Derivatization

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The samples were detected by LC-20AT high-performance liquid chromatography (Shimadzu

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Corporation, Nakagyo-ku, Kyoto, Japan) using a Boston Green ODS column (C18; 250 mm×4.6

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mm, 5 µm; Boston Analytics, Boston, MA, USA) with a mobile phase consisting of a mixture of

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acetonitrile: 50 mM ammonium acetate buffer solution (at a ratio of 10: 90, V: V) at a flow rate

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of 0.5 mL/min. The peak detection was performed by a UV detector (345 nm).The iE-DAP chiral

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derivatizations were achieved using o-phthaldehyde (OPA) and N-acetyl-cysteine reagents.

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Measurement of Biochemical Parameters

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Different liver enzymes and protein, biomarkers of liver injury and function were measured in

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both tissue and hepatic plasma by using specific assay kits and machines. Enzymes such as

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ALT/GPT, AST/GOT, AKP/ALP, and γ-GT/GGT were detected in both tissue and plasma by

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using ALT. AST, AKP and γ-GT enzyme activity kits were purchased from Nanjing Jiancheng

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Bioengineering Institute, Nanjing, Jiangsu, China and were performed following the

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manufacturer’s instructions. NH3 was measured in tissue by using an NH3 protein activity kit

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from the same Bioengineering Institute. LDH, total protein, albumin, and total bilirubin were

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measured by using a biochemical analyzer (SPOTCHEMTMEZ SP-4430, Arkray, Kyoto,

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Japan). A spectrophotometer (UV-5600, Metash Instruments Co., Ltd, Shanghai, China) was


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used in the case of γ-GT and NH3 to determine the concentration. The absorbance values of the

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colorimetric reactions were 510 nm, 510 nm, 520 nm, 410 nm, and 630 nm for ALT, AST,

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AKP, γ-GT, and NH3, respectively.

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MPO Activity Assay

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Detection of MPO in the liver tissue and hepatic vein was performed by using an MPO kit

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purchased from Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China following

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the manufacturer’s instructions. A spectrophotometer (UV-5600, Metash Instruments Co., Ltd,

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Shanghai, China) was used to measure the absorbance of the colorimetric reaction at 460 nm.

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MPO activities are shown in U/gm of wet tissue and U/L of plasma.

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RNA Extraction and Real-Time PCR (RT-qPCR)

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Extraction of the total RNA from 100 mg powdered liver tissue (n = 12) was performed using

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RNA iso Plus

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protocol. The purity and concentration of RNA were assessed by both agarose gel (1%)

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electrophoresis and a spectrophotometer (A260/A280) (Nannodrop ND-2000, Thermo Fisher

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Scientific Inc., Waltham, MA, United States). Samples with concentration ratios between 1.8 and

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2.1 were used. The synthesis of cDNA was carried out by reverse transcription using 250 ng/µL

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of RNA with PrimeScript RT Master Mix Perfect Real Time (Takara Co., Otsu, Shiga, Japan).

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The primers of the desirable genes were designed from known sequences using OLIGO 6.44

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(Molecular Biology Insights, Inc, Colorado Springs, Colorado, USA) and Primer Quest Tool

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(Integrated DNA Technologies, Inc, Skokie, Illinois, USA) software. Table 3 represents all the

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primers of the target genes. Quantitative PCR was performed with the ABI 7300 instrument

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(Applied Biosystems, Foster City, CA, USA). Thermal cycling parameters consisted of initial

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

TM

reagent (Takara Co., Otsu, Shiga, Japan) according to the manufacturer’s



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extension at 60 °C for 31 s. The target segment of cDNA was amplified by using SYBR Premix

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EX Taq™ 144 kit (Cat. DRR420A, Takara Co., Otsu, Shiga, Japan), and a single specific PCR

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product for each gene was ensured by melting curve performance. Glyceraldehyde phosphate

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dehydrogenase (GAPDH) served as the housekeeping gene for the normalization of gene

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expression, and the 2-∆∆Ct method was used for relative quantification.

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Western Blotting

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A total of 100 mg liver tissue was homogenized in 1 ml radio immunoprecipitation assay (RIPA)

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lysis buffer (Beyotime Biotechnology, Inc., Shanghai, China) with 0.1 M PMSF by Dounce

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Homogenizer (Polytron PT 1200 E, KINEMATICA AG, Luzern, Switzerland) to extract the total

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protein. After 30 min of incubation on ice, the lysate was centrifuged at 12,000 × g at 4 °C for 20

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min. Bicinchoninic acid (BCA) and bovine serum albumin (BSA) as standards (Thermo Fisher

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Scientific Inc. Waltham, MA, USA) were used to measure the concentration of the supernatant

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and, subsequently, to adjust to 4 µg/µL. Each protein sample was mixed with loading buffer and

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denatured in a water bath at 99 °C for 5 min. The denatured protein was run on 10% sodium

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dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to separate proteins and then

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shifted onto nitrocellulose membranes (Bio Trace, Pall co., NY, USA).

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Subsequently, 7% skim milk (7% BSA in the case of phosphorylated protein) was used for 2 h at

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room temperature to block the membranes. Then, the membranes were incubated with primary

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antibodies anti-NOD1(1: 200, sc-22045, Santa Cruz Biotechnology, Inc., Dallas, Texas, USA),

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anti-IκB alpha (1: 1000, sc-AF1282, Beyotime Biotechnology, Inc., Shanghai, China), anti-p-IκB

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alpha (ser32) (1: 1000, sc-AF1870, Beyotime Biotechnology, Inc., Shanghai, China), anti-p65 (1:

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500, sc-AF1234, Beyotime Biotechnology, Inc., Shanghai, China), anti-p-p65 (ser536) (1: 200,

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sc-33020, Santa Cruz Biotechnology, Inc., Dallas, Texas, USA), anti-ERK1/2 (1: 1000, sc-


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AF1051, Beyotime Biotechnology, Inc., Shanghai, China), anti-p-ERK1 (Thr202/Tyr204)/ anti-

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p-ERK2(Thr 185/Tyr187) (1: 1000, sc-AF1891, Beyotime Biotechnology, Inc., Shanghai,

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China), anti-JNK/SAPK (1: 1000, sc-AJ518, Beyotime Biotechnology, Inc., Shanghai, China),

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anti-p-JNK (1: 1000, sc-AJ516, Beyotime Biotechnology, Inc., Shanghai, China), anti-p38 (1:

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1000, sc-AM063, Beyotime Biotechnology, Inc., Shanghai, China), anti-p-p38 (1: 1000, sc-

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AM063, Beyotime Biotechnology, Inc., Shanghai, China), anti-FOXA2 (1: 1000, sc-8186, Cell

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Signaling Technology, Inc., Danvers, MA, USA), anti-HDAC3 (1: 1000, sc-AH385, Beyotime

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Biotechnology, Inc., Shanghai, China), anti-β-Actin (1: 500, sc-AA128, Beyotime Biotechnology

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Inc., Shanghai, China; 1: 200, sc- sc-130656, Santa Cruz Biotechnology, Inc., Dallas, Texas,

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USA), anti-GAPDH (1: 500, sc-AG019, Beyotime Biotechnology, Inc., Shanghai, China), and

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anti-β-tubulin (1: 2000, sc-SAM1002, Sunshine Biotechnology Co., Ltd, Guangzhou,

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Guangdong, China) overnight at 4 °C. β-Actin, GAPDH and β-tubulin antibodies were used as

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reference proteins for normalization. After washing with Tris-buffered saline containing Tween

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20 (TBST), the membranes were incubated with horseradish peroxidase (HRP)-conjugated

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secondary antibodies, rabbit-anti-goat (1: 5000, sc-E030130-01, EarthOx, LLC., Millbrae, CA,

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USA), goat-anti-rabbit (1: 1000, sc-A0208, Beyotime Biotechnology, Inc., Shanghai, China), and

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goat-anti-mouse (1: 5000, E030110-01, EarthOx, LLC., Millbrae, CA, USA), for 2 h at room

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temperature. After washing with TBST, the results were analyzed using a West Pico ECL

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enhanced chemiluminescent kit (Biosharp life Sciences, Hefei, Anhui, China). BIO-RAD

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Molecular Imager® ChemiDocTm XRS+ Imaging System (BIO-RAD, Life Science Research,

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Hercules, CA, USA) and BIO-RAD Image Lab 5.2.1 Software (BIO-RAD, Life Science

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Research, Hercules, CA, USA) were used to quantify and analyze the results, respectively. The



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relative quantities of proteins were evaluated by a densitometer and expressed as absorbance

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units (AU).

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Milk Yield and Milk Quality Measurement

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The goat milk yields were recorded after milking, twice daily at 8: 00 AM and 16: 00 PM. To

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analyze the milk quality (milk fat, protein, and lactose), 50 mL milk samples were collected at

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the end (last 3 days) of each experimental week and stored at 4 °C adding potassium dichromate

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until analyses were performed with a FossMatic 5000 analyzer (FOSS Electric A/S, Hillerød,

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Denmark).

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Statistical Analysis

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General linear model with repeated measures was used to analyze the pH data by IBM SPSS

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20.0 statistics for Windows (IBM Inc., NY, USA), in which the effects of goats and that of diet

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and time were considered random and fixed effects, respectively. The time within treatments and

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the goats were considered repeated measurements. Means were compared via one-way ANOVA

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to analyze the week-based milk yield, fat, protein and lactose data between two groups in IBM

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SPSS 20.0 statistics for Windows (IBM Inc., NY, USA). All other statistical data analyses

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between two groups were conducted using compare means via independent t-test with IBM SPSS

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20.0 statistics for Windows (IBM Inc., NY, USA). All of the data were expressed as the mean ±

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SEM. Differences were considered statistically significant if P < 0.05 or highly significant if P
0.05).

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Alterations in Milk Yields and Milk Components of the Dairy Goats

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The average daily milk yield during 24 week experimental period sharply but insignificantly (P >

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0.05) decreased in the HC group (0.88 ± 0.05 kg/day) compared with that in the HC+SB group

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(0.97 ± 0.05 kg/day) (Table 5). The milk yields gradually decreased from 9th week to 23rd week

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except 18th week in both HC group and HC+SB group (Figure 6A). The average percentage of

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daily milk fat and protein was significantly (P < 0.01) down-regulated in the HC group (2.70 ±

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0.08 % and 3.27 ± 0.09 %, respectively) in comparison with that in HC+SB group (3.27 ± 0.09

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% and 3.93 ± 0.09 %, respectively) (Table 5). Milk fat percentage decreased at 2nd week

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followed by slight increase upto 17th week and then, the fat percentage gradually decreased upto

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22nd week (Figure 6B) in both groups. In the HC group, percentage of milk protein sharply

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decreased from 16th week to 21st week, whereas, in the HC+SB group, milk protein percentage

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decreased from 16th week to 24th week (Figure 6C). In addition, the HC group (3.51 ± 0.14 %)

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showed an insignificant (P > 0.05) decrease in average milk lactose percentage in comparison to

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the HC+SB group (3.86 ± 0.16 %) (Table 5). Milk lactose percentage decreased at 2nd week and

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was almost steady upto 16th week except 11th week and then, the lactose percentage suddenly

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increased at 17th week followed by gradual down-regulation upto 23rd week in both groups

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(Figure 6D).

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DISCUSSION

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Our study uncovered different aspects of inflammation including not only the role of a high-

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concentrate diet in the iE-DAP-induced NOD1 inflammatory signaling pathway in hepatocytes



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and Kupffer cells (liver macrophage) by developing SARA in the rumen, but also that of sodium

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butyrate (SB) in combating inflammatory stress in the liver. In our experiments, we observed that

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offering a high-concentrate diet to dairy goats for a prolonged time reduced rumen pH at a level

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that introduced subacute ruminal acidosis (SARA) by the production of volatile fatty acids,

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which is supported by earlier studies.3-5 In our study, pH decreased below 5.6 for more than 240

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minutes per day post-high grain feeding, similar to results of previous studies.2, 6, 7

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According to many scientists, feeding a high-concentrate diet to dairy animals confirmed a

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significant increase in iE-DAP (constituent of peptidoglycans) in the rumen,15 which is released

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from rumenal bacteria (most of the gram-negative bacteria and few gram-positive bacteria)18 by

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lysis and reaches the portal circulation through translocation19, 20 to liver cells. We also noticed a

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significant increase in iE-DAP in the rumen, as detected by high-performance liquid

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chromatography (HPLC) during SARA in the HC group in comparison to the HC+SB group, and

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iE-DAP can ultimately translocate to the liver to initiate innate immunity.

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Biochemical analysis is usually performed to asses liver function.25-27,

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usually express hepatocyte integrity, not liver function. Evaluations of the syntheses of protein

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(albumin, total protein) by the liver are true liver function tests.26, 27 According to a previous

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study, acute or chronic liver injury eventually results in an increase in ALT,25-27, 29 AST,25-27,

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29

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protein29 and albumin.26, 27, 29 Therefore, the concentrations of these enzymes and proteins can be

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considered important markers of a healthy liver. We also observed significant decreases in ALT,

364

AST, GGT, ALP, and LDH as well as significant increases in total protein and, albumin in our

365

HC+SB group, similar to earlier findings.25-29 Sodium butyrate helps to suppress pro-

366

inflammatory cytokines51-53, 55 through the inhibition of histone deacetylase (HDAC) activity,54,

29

Liver enzyme tests

GGT,25-27ALP,25-27 LDH26, ammonia28, and total bilirubin25, 26 as well as a decrease in total


Page 17 of 41


367

56

368

SARA induced by an HC diet and is recovered by SB treatment. There was no significant change

369

in bilirubin.

370

MPO is the key component inside neutrophils. MPO is released from neutrophils during

371

inflammation.31 We monitored the concentration of MPO, which decreased in the HC+SB

372

compared to the HC treatment. This finding reveals that neutrophil infiltration is elevated during

373

inflammation, as reported by former investigators30,

374

treatment.51-53

375

Due to LRRK2 sensing of the iE-DAP ligand, both LRRK2 and CARD (parts of NOD1) have

376

key roles in innate immunity. LRRK2 downregulation interrupts the exposure of the central NBD

377

for oligomerization, which leads to the shutdown of the CARD exposure required for the

378

interaction of RIPK2 with CARD for optimal recruitment and function of RIPK2/RICK, TAK1,

379

NF-κB and MAPKs.34,

380

expression in the HC+SB group due to sodium butyrate51-53 and an increase in the HC group.

381

Thus, upregulated NOD1 during HC treatment was significantly decreased in the HC+SB group.

382

Oligomerization of NOD1 induces the proximity of RIPK2 /RICK molecules, which leads to the

383

autophosphorylation of Ser176.34, 39 RIPK2 is a key factor in triggering both the NF-κB and

384

MAPK signaling pathways. Without binding to RIPK2, the NF-κB essential modulator

385

(NEMO)/inhibitor of NF-κB kinase γ (IKKγ) interrupts ubiquitination and the exposure of IKKα

386

and IKKβ. Inactive IKK cannot phosphorylate the NF-κB inhibitor IκB, resulting in the

387

interruption of its degradation via the proteasome, which is required for the release and

388

translocation of NF-κB to the nucleus.34,

389

phosphorylated IκBα and phosphorylated NF-κB were significantly increased in the HC group,

which is similar to our finding. Therefore, these findings indicate that liver damage during

39, 41, 42

31

and is lessened by sodium butyrate

We also found hindrance in LRRK2 and CARD4 mRNA

38, 39, 41-43, 57

Though the expression of RIPK2,



390

indicating activation of the NF-κB signaling pathway;, the suppression of this pathway in the

391

HC+SB group occurred via the downregulation of molecules in our study, as reported by

392

previous scientists. The TAK1 and RIPK2 interaction is also essential for NOD1-induced MAPK

393

activation. TAK1, a serine-threonine kinase, builds a TAK1-binding protein 1 (TAB1), TAB2,

394

and/or TAB3 complex that phosphorylates IKK and MAPK for maximum activation.34, 39, 41, 42

395

We also observed upregulation of TAK1 and, TAB1, and, the phosphorylation of ERK1/2

396

MAPK, JNK MAPK and p38 MAPK in the HC group, which subsequently reached significant

397

negative regulation by SB treatment, similar to previous studies.34, 39, 41, 49, 57 Alleviation of NF-

398

κB and MAPK signaling molecules significantly reduced pro-inflammatory cytokines (IL-1β and

399

TNF-α), chemokines (CCL5, CCL20, and CXCL12), AP1 (FOS) and antimicrobial peptides

400

(LAP/β-defensin) in the HC+SB group. This alleviation also markedly (not significantly)

401

decreased the levels of IL-6, IL-8, IL-10, CXCL10 and JUN in the HC+SB group. These

402

findings matched those of previous reports.32, 34, 37, 40, 41, 45 C/EBPβ showed no significant change

403

between the two groups, which is dissimilar to earlier work.46 SAA3 (APP), which sharply

404

decreased in the HC+SB group via the inactivation of pro-inflammatory cytokines (TNF-α, IL-

405

1β, IL-6) and chemokines, was higher in the HC group.23 NOS2 can be strongly expanded in

406

hepatocytes by cytokines.40 We also noticed the increase in NOS2 expression in the HC group,

407

which was sharply combated by SB treatment. Therefore, sodium butyrate plays a role in

408

alleviating inflammatory factors by suppressing histone deacetylation.

409

NOD1 stimulation with its agonist iE-DAP alone is sufficient to drive a T helper cell 2 (Th2)

410

antigen-specific immune response in vivo in the presence of RIPK2.58 Th2 positively regulates

411

IL-4 and, IL-5 in IL-4Rα-STAT6-mediated inflammation. FOXA2 plays a role in Th2-mediated

412

inflammation. Th2-mediated inflammation downregulates the expression of FOXA2.48, 59 Our


Page 18 of 41

Page 19 of 41


413

findings also showed that FOXA2 expression decreased in the HC group due to inflammation but

414

was markedly upregulated by sodium butyrate treatment. In addition, according to Du et al,

415

MAPK inhibitors upregulate FOXA2 through the MAPK/FOXA2 signaling pathway.49 These

416

findings also support our results.

417

In our study, we observed the co-relation between long-term high-concentrate feeding and milk

418

parameters (milk yield, milk fat, milk protein, and milk energy). According to the previous

419

studies, SARA caused by the high-concentrate feeding decreased the milk production and milk

420

fat, but not protein and energy level.2, 6, 12 We also observed that long-term high-grain diets

421

reduced not only the milk yield, and milk fat but also milk protein, and milk energy. The

422

alteration in the rumen fermentation might be responsible for the reduction in milk yield and

423

milk quality.2 Though the milk yield, milk fat, milk protein, and milk energy were higher in the

424

HC+SB group than that in the HC group in our week-based study, sodium butyrate was not

425

enough to normalize the milk parameters.

426

In conclusion, iE-DAP, derived from the rumen and circulated to the liver via the portal vein

427

during SARA induced by a high-concentrate diet, triggers the NOD1/RIPK2-mediated

428

inflammatory pathway and inhibits liver function via liver injury. Sodium butyrate lessened the

429

effects on the iE-DAP-induced NOD1/RIPK2-mediated inflammatory pathway and recovered

430

liver function.

431 432 433 434 435



436

LIST OF ABBREVIATIONS SCFA SARA HPLC

Short chain fatty acid Subacute ruminal acidosis High-performance liquid chromatography

iE-DAP

D-glutamyl-meso-diaminopimelic acid

PGNs

Peptidoglycans

ALT/GPT

Alanine aminotransferase/ Glutamate-pyruvate transaminase

AST/GOT Aspartate aminotransferase/Glutamic-oxaloacetic transaminase GGT

Gamma-glutamyltransferase

ALP

Alkaline phosphatase

LDH

Lactate dehydrogenase

TP

Total protein

AL

Albumin

MPO

Myeloperoxidase

NOD1

Nucleotide-binding oligomerization domain-containing protein 1

NLR

NOD-like receptors

PAMP

Pathogen-associated molecular patterns

CARD

Caspase recruitment domain

NBP

Nucleotide-binding domain

LRRK2

Leucine-rich repeat kinase 2

RIPK2

Receptor-interacting protein kinase 2

TAK1

TGFβ-activating kinase1

MAPK

Mitogen activated protein kinase

APP

Acute phase protein

SAA

Serum amyloid A

HP

Haptaglobin

LAP

Lingual antimicrobial peptide

IL

Interleukin

TNF

Tumor necrosis factor

CCL

Chemokine (C-C motif) ligand

CXCL

C-X-C motif chemokine ligand

C/EBP

CCAAT enhancer binding protein

FOXA2

Forkhead box protein A2

HDAC

Histone deacetylase 20 ACS Paragon Plus Environment

Page 20 of 41

Page 21 of 41


437

ACKNOWLEDGMENTS

438

The authors would like to thank Qingqi Yuan, and Yaqiong Wang for assistance with animal

439

feeding and specimen sampling

440

Funding

441

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

442

China (Grants 31672618 and 31172371), and the Priority Academic Program Development of

443

Jiangsu Higher Education Institutions (PAPD).

444

Notes

445

The authors declare no competing financial interest.

446 447 448 449 450 451 452 453 454 455 456 457 458 459



460

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625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 29 ACS Paragon Plus Environment


Page 30 of 41

Table 1. The Ingredients in the Diets and Nutritional Composition Items Concentrate

Ingredients Corn (g/100 g) Bran (g/100 g )

Coarse material

Nutritional index

Quantity 23.07 28.03

Soyabean meal (g/100 g)

2

Rape seed meal (g/100 g)

3.7

Lime stone meal (g/100 g)

1.43

Dicalcium phosphate (g/100 g)

0.6

*

0.4

Premix (g/100 g)

Salt ( g/100 g)

0.5

Oat grass (g/100 g)

32

Alfa alfa (g/100 g)

8

Net energy ( MJ/kg)

17.56

Water content

1.89

Ash (%)

8.22

Fat (%)

3.6

Crude Protein (%)

16.76

Neutral detergent fiber NDF (%)

42.64

Acid detergent fiber ADF (%)

7.22

Ca (%)

1.55

P (%)

0.76

*The premix formulation consists of following ingredients per kg of diet: 6.00 × 103 U of vitamin A, 2.5 × 103 U of vitamin D, 80.0 mg of vitamin E, 6.25 mg of Cu, 62.5 mg of Fe, 62.5 mg of Zn, 50.0 mg of Mn, 0.125 mg of I, 0.125 mg of Co, and 0.125 mg of Mo.


Page 31 of 41


Table 2. Formulation of Diets for the High-concentrate Group (HC) and the High-concentrate with Sodium Butyrate Group (HC+SB) Concentrate powdered diet (gm)

Alfalfa

Oat grass

(gm) Sodium butyrate

HC group

600

133

267

-

HC+SB group

600

133

267

10

Animals

Roughage (gm)

Buffer agent (gm)

Table 3. List of Primer Sequences of Target Genes for Real-Time PCR Target Genes LRRK2 NOD1 CARD4 RIPK2 IκBα NF-κB TAK1 TAB1 ERK JNK1 JNK2 p38 IL-6 IL-1β TNF-α IL-8 IL-10 CXCL10 CXCL12 CCL5 CCL20 JUN FOS SAA3 HP LAP NOS2 C/EBPβ FOXA2 GAPDH

Forward Primer

Reverse Primer

CCACAGGCCTGTAATAGAGC CAACACTGACCCAGTGAGCA TGCCTAACTGTGCAAGTGCT ATCATCGGGCAGCGTTCTG GGTGAAGGAGCTGCGAGAG CTTCCATCCTGGAACCACTAAA CTGCGCACTGAGTACAATTC CACCACAGAGAATGAGGATGAG GGCCGTCTTTGTCTCATAGA TCCACTGCGCTCACA CCATCCTGCCAAGCCTACAAG GAAAGCAGGGACCTCCTTATAG CGAAGCTCTCATTAAGCACATC CCGTGATGATGACCTGAGGAG CAACAGGCCTCTGGTTCAGAC CTGAGAGTTATTGAGAGTGGGC GTGATGCCACAGGCTGAGAAC ACGGAAAGAGGCATAATCAC TCTCACTGGGCGCTTATCAC CTACACCAGCAGCAAGTGCT GAAGCAGCAAGCAGCTTTGAC ACGACCTTCTACGACGATGC TGAGTCGGCGCATTACAGAG GACATTCCTCAGGGAAGCTG GGAGTACTCGGTTCGCTATCA CTGCTGGGTCAGGATTTACTC GTGGCAAGCACCACATTGAG GTTCATGCAACGCCTGGTG CTCAGACACCTCCTACTACCA GGGTCATCATCTCTGCACCT

ATGCCTTGTCGCCAGTA CAGCAGCTCCTCCTTCTGAG CTGTGGTCTTGTCCCACGAA CTCTGGACAGGAGGGCGTCTA GCTCACAGGCAAGGTGTAGG ACCTCTCTGTCGTCACTCTT TCTGAAGGGCTGACGG ACCCGTACTTGACCTTGTAATC TTCGGGAGAGTTTACCAACC AAGAATGGCATCATAAGCTG TCGCAAAGAGTTGGACACGAC CAGACACTCAAGACTCCATCTC AGTGGTTATTAGACCTGCGATC CAAGACAGGTATAGATTCTTGTC GGACCTGCGAGTAGATGAGG CAGTACTCAAGGCACTGAAGTAG GAAGATGTCAAACTCACTCATGG CAGGGCAGAGTCACTACTGA CCGGTGGTCTCCTTAGGG CAAGCTGCTTAGGACAAGAGG GTTCCATTCCAGGGAGCATC GCCAGATTCAGGGTCATGCT CACAGCCTGGTGTGTTTCAC CTCTCTGGCGTTACTGATCAC CCATCGTTCATTGATGAGTGTG GTTTCTGACTCCGCATCAGTC AGCCTCATGGTGAACACAGG AAGCAGTCCGCCTCGTAGTA ACTTCCTGTCTCTCCCATCT GGTCATAAGTCCCTCCACGA

Product length (bp) 189 100 81 198 326 108 166 134 187 162 180 109 334 303 209 259 213 219 160 190 244 110 99 178 280 192 151 111 111 176

Gen bank accession XM_018047920.1 XM_005679255.1 XM_005686398.2 XM_018058185 XM_018066509 XM_018049264.1 XM_005684675.3 XM_018048785.1 XM_005694690.3 XM_018042431.1 XM_018051623.1 XM_018038792.1 NM_001285640.1 XM_013967700.2 XM_005696606.3 XM_005681749.3 XM_005690416.3 NM_001285721.1 XM_018042300.1 XM_005693201.3 XM_005676644.3 XM_018044742.1 XM_005686096.3 XM_018043001.1 XM_005692202.3 XM_018042143.1 XM_013971952.2 XM_018058020.1 XM_018057273.1 XM_005680968.3

Table 4. iE-DAP Concentration in Rumen Fluid of Dairy Goats at Two Time Points during SARA Item iE-DAP

Time(h) post-feeding 4 6

HC±SEM (µg/mL) 51.45 ± 5.71 18.31 ± 3.83

(HC+SB)±SEM (µg/mL) 11.67 ± 3.85 7.74 ± 1.46


p-value 0.0012 0.0418


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Table 5. Milk Yields and Milk Components of the Dairy Goats Items Milk yield (Kg/day) Fat (%) Protein (%) Lactose (%)

HC±SEM 0.88 ± 0.05 2.70 ± 0.08 3.27 ± 0.09 3.51 ± 0.14

(HC+SB)±SEM 0.97 ± 0.05 3.27 ± 0.09 3.93 ± 0.09 3.86 ± 0.16

p-value 0.1847 0.0001 0.0001 0.1010

Figure 1. pH of rumen fluid at different time points in between high-concentrate (HC) and high-concentrate with sodium butyrate (HC+SB) groups post-feeding (n = 6). HC group showed a clear decrease in pH in comparison to the HC+SB group (P < 0.01). All of the results were expressed as mean±SEM). P ≤ 0.05 between two groups was considered a significant change.


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Figure 2. Comparison of the concentrations of ALT, AST, GGT, and AKP in both hepatic plasma (A1, B1, C1, D1 respectively) and liver tissue (A2, B2, C2, D2 respectively), and that of LDH, total protein, albumin, NH3, and total bilirubin in hepatic plasma (E, F, G, H respectively) between the high concentrate group (HC) and high concentrate with sodium butyrate (HC+SB) group of dairy goats (n = 6). Plasma samples were collected from hepatic veins 4 h after feeding.



Significant changes were observed in all of the parameters except GGT in tissue and total bilirubin in plasma. All of the results were expressed as the mean ± SEM. * indicates P ≤ 0.05, ** indicates P ≤ 0.01, and *** indicates P ≤ 0.001. P ≤ 0.05 was considered a significant change. ALT/GPT= Alanine aminotransferase/ Glutamate-pyruvate transaminase, AST/GOT = Aspartate aminotransferase/Glutamic-oxaloacetic transaminase, γ-GT/GGT = Gamma-glutamyltransferase, AKP/ALT = Alkaline phosphatase, LDH = Lactate dehydrogenase

Figure 3. Concentration of MPO in liver tissue (A) and hepatic plasma (B) of goats in the HC, and HC+SB groups (n = 6). Plasma samples were collected from hepatic veins 4 h after feeding. The concentrations of MPO in both tissue (P < 0.05) and plasma (P < 0.01) samples significantly decreased in the HC+SB group compared with that of the HC group. All of the results were expressed as the mean ± SEM. * indicates P ≤ 0.05 and ** indicates P ≤ 0.01. P ≤ 0.05 between two groups was considered a significant change. MPO = Myeloperoxidase


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Figure 4. The relative mRNA expression of immune genes in the liver of dairy goats between high-concentrate (HC) and high-concentrate with sodium butyrate (HC+SB) groups measured by real-time qPCR. (A). Immune genes involved in iE-DAP-induced inflammatory signaling 35 ACS Paragon Plus Environment


pathway. The expression levels of NOD1, and RIPK2 in the liver were significantly downregulated in the HC+SB group compared to those of the HC group. (B). Immune genes in the signaling pathway were TAK1, TAB1, and NF-κB and their expressions were also significantly lower in the HC+SB group compared to the HC group. (C). MAPK genes in signal pathway. HC+SB group significantly represented decreased expression of ERK, JNK2 and p38. (D). Genes responsible for acute phase proteins and antimicrobial peptides were SAA3, LAP, NOS2, and C/EBPβ, and expressions declined in the HC+SB group in comparison with the HC group. (E). Transcription factor gene, FOXA2 showed positive regulation with the HC+SB group. (F). Pro and anti-inflammatory cytokines. The expression of IL-1β, TNF-α, CCL5, CCL20, CXCL12, and FOS was significantly lower in the HC+SB group than in the HC group. The data are expressed as the mean ± SEM and asterisks indicate the differences between two groups (* indicates P ≤ 0.05, ** indicates P ≤ 0.01, n = 6).


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642

643 644

Figure 5. Protein expression of NOD1 (A), FOXA2 (B), IκBα and p-IκBα (C), p65 and p-p65

645

(D), ERK1/2 and p-ERK1/2 (E), JNK and p-JNK (F), p38 and p-p38 (G), and HDAC3 (H) in

646

liver tissue of goats between HC and HC+SB groups (n = 6). NOD1, FOXA2, and all of the



647

phosphorylated proteins including IκBα, p65, ERK1/2, JNK and p38 significantly decreased in

648

the HC+SB group compared to those in the HC group. The results were expressed as the relative

649

intensity compared to housekeeping proteins (β-actin, β-tubulin, and GAPDH). All of the results

650

were expressed as the mean ± SEM. Asterisks indicate the differences between two groups (*

651

indicates P ≤ 0.05, ** indicates P ≤ 0.01, n = 6).


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652

Figure 6. Milk yields (kg/day), percentage of milk fat, protein and lactose in dairy goats fed

653

high-concentrate diets and high-concentrate diets with SB. (A). Although the milk yields were

654

consistent from 1st week to 8th week, the milk yields gradually decreased from 9th week to 17th

655

week in both HC group and HC+SB group. At the 18th week, there was slight increase in milk

656

yields, but subsequently the milk yields gradually decreased upto 23rd week in both HC group

657

and HC+SB group. HC+SB group showed an increased milk yield than HC group. (B). Milk fat

658

percentage decrease at 2nd week followed by slight increased trend upto 17th week and then the

659

fat percentage gradually decreased upto 22nd week. Fat percentage in the HC group was lower

660

than that in the HC+SB group. (C). In the HC group, percentage of milk protein fluctuated upto

661

15th week and then, it showed a decreased trend from 16th week to 21st week. In the HC+SB

662

group, milk protein percentage increased upto 15th week except 11th week and then decreased

663

upto 24th week. Protein percentage in the HC+SB group was higher than that in the HC group.

664

(D). Milk lactose percentage decreased at 2nd week and was almost steady upto 16th week except

665

11th week and then the lactose percentage suddenly increased at 17th week followed by gradual

666

down-regulation upto 23rd week. Lactose percentage in the HC group was lower than that in the

667

HC+SB group.


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Journal of Agricultural and Food Chemistry jf-2018-02732c

The following graphic will be used for the TOC:


Sodium butyrate mitigates iE-DAP induced inflammation caused by

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