Sodium Butyrate Ameliorates High-Fat-Diet-Induced Non-alcoholic

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Bioactive Constituents, Metabolites, and Functions

Sodium butyrate ameliorates high-fat diet-induced NAFLD through PPAR#mediated activation of # oxidation and suppression of inflammation Bo Sun, Yimin Jia, Jian Hong, Qinwei Sun, Shixing Gao, Yun Hu, Nannan Zhao, and Ruqian Zhao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01189 • Publication Date (Web): 01 Jul 2018 Downloaded from http://pubs.acs.org on July 2, 2018

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Journal of Agricultural and Food Chemistry

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Sodium butyrate ameliorates high-fat diet-induced NAFLD through PPARα-mediated

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activation of β oxidation and suppression of inflammation

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Bo Sun1,2, Yimin Jia1,2, Jian Hong2,3, Qinwei Sun1,2, Shixing Gao1,2, Yun Hu1,2, Nannan Zhao1,2,

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Ruqian Zhao1, 2*

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1MOE

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Agricultural University, Nanjing 210095, P. R. China

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2Key

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210095, P. R. China

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* Corresponding author: [email protected]

Joint International Research Laboratory of Animal Health & Food Safety, Nanjing

Laboratory of Animal Physiology & Biochemistry, Nanjing Agricultural University, Nanjing

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Tel. 00862584395047

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Fax: 00862584398669

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ACS Paragon Plus Environment


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Abstract

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PPARα plays a protective role against NAFLD. NaB has been shown to alleviate NAFLD, yet

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whether and how PPARα is involved in the action of NaB remains elusive. In this study, NaB

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administration alleviated high-fat diet-induced NAFLD in adult rats, with a decrease of hepatic

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TG content from 108.18 ± 5.77 µg/mg to 81.34 ± 7.94 µg/mg (P < 0.05), which was associated

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with a significant activation of PPARα. NF-κB-mediated NLRP3 signaling and pro-inflammatory

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cytokines release were diminished by NaB treatment. NaB-induced PPARα up-regulation

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coincided with reduced protein content of HDAC1 and promoted H3K9Ac modification on the

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promoter of PPARα, whereas NaB-induced suppression of inflammation was linked to

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significantly increased PPARα binding with p-p65. Conclusion: NaB acts as a histone deacetylase

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inhibitor to up-regulate PPARα expression with enhanced H3K9Ac modification on it promoter.

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NaB-induced PPARα activation stimulates fatty acid β oxidation and inhibits NF-κB-mediated

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inflammation pathways via protein-protein interaction, thus contributes to amelioration of high-fat

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diet-induced NAFLD in adult rats.

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Key words: fatty liver, NF-κB, NLRP3, PPARα, sodium butyrate.

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1. Introduction

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Non-alcoholic fatty liver disease (NAFLD) has become the most common liver disease in the

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world. The main feature of NAFLD pathogenesis is the accumulation of triglyceride (TG)

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accompanied by metabolic inflammation. It may further progress towards liver cirrhosis and

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hepatocellular carcinoma

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NAFLD has long been a focus of biomedical research.

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Peroxisome proliferator-activated receptors α (PPARα) is a subtype of PPAR superfamily that is

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highly expressed in liver. PPARα acts as a transcription factor to regulate an array of genes

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involved in fatty acid uptake, mitochondrial fatty acid oxidation 4-5, and inflammatory response6 .

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It has been shown that hepatocyte-specific deletion of PPARα impairs fatty acid catabolism,

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resulting in hepatic lipid accumulation and NAFLD 7. Activation of hepatic PPARα signaling

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significantly alleviates NAFLD by promoting mitochondria β oxidation 8. Also, PPARα is reported

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to suppress the expression of pro-inflammatory factors by abolishing p65 binding to NF-κB

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response element (NRE) via protein-protein interaction 6.

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Butyrate is a short-chain fatty acid (SCFA) that naturally exists in butter and cheese 9, and can also

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be produced endogenously by the colonic bacterial anaerobic fermentation of fiber

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polysaccharides

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directly acting in the intestine to inhibit fat absorption 11 or by targeting the liver to increase fatty

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acids oxidation

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pharmacological activities in various tissues

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(TLR4) expression, NF-κB activation and attenuates macrophages infiltration18-19. In adipose

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tissue, NaB alleviates obesity-induced inflammation by inhibiting NLRP3 inflammasome pathway

10.

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1-3.

Therefore, searching for effective strategies to prevent or treat

Previous studies indicate that sodium butyrate (NaB) attenuates NAFLD by

and decrease fatty acid synthesis 13. Moreover, butyrate has anti-inflammatory 14-17.

In liver, butyrate inhibits Toll-like receptor 4

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20.

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Butyrate is reported to restore the hepatic PPARα expression suppressed by high-fat diet feeding,

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which implicates a possible role of PPARα in mediating the NAFLD-alleviating action of butyrate

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21.

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a histone deacetylase inhibitor (HDACi) in the epigenetic gene regulation 22. To date, it remains

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elusive whether PPARα is involved in the action of butyrate in alleviating fat deposition and

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inflammation in liver and whether butyrate regulates hepatic PPARα expression through

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modulating the status of histone acetylation on its promoter.

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Therefore, in this study we fed rats with high-fat diet to establish NAFLD model and

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demonstrated the efficacy of NaB in ameliorating NAFLD. Moreover, we show that

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PPARα-mediated activation of β-oxidation and inhibition of inflammation is associated with NaB

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action and NaB-induced transactivation of hepatic PPARα expression is related to suppressed

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HDAC activity and hyperacetylation of PPARα promoter.

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2. Materials and Methods

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2.1 Chemicals

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Chemicals, including sodium butyrate（98% purity），citric acid trisodium salt dehydrate (99.0%

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purity), glycine, Tris-base and sodium dodecyl sulfate, were obtained from Sigma Chemical

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Company (St Louis, MO, USA). All other reagents were commercial products of the highest

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purity grade available.

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2.2 Animal model and experimental protocol

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A total of 24 male specific-pathogen-free Sprague Dawley rats (aged 6 weeks) purchased from

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Beijing Vital River Laboratory Animal Technology Co., Ltd. were kept in the Animal Core

Butyrate exerts its biological function through different mechanisms, one of which is acting as

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Facility of Nanjing Medical University. The animals were housed under the following typical

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conditions: 22 ± 2°C, 50-60% humidity, 12 h light/12 h dark lighting regime. The rats were

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allowed free access to food and water and assimilated to their environment for one week. The rats

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were randomly divided into two groups, fed a basal diet (CON, n = 8) and a high-fat diet (HF, 45%

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calorie from lard and sesame oil, n = 16), respectively, for 9 weeks to establish obesity and

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NAFLD. The rats in CON group were incessantly kept on the basal diet throughout the experiment,

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whereas the HF rats remained on the HF diet and subsequently assigned into two groups (n = 8).

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One HF group received a vehicle for comparison with HFB, which received 300 mg/kg NaB per

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rat via gavage every other day for 7 weeks. At the end of week 7, the rats were fasted overnight

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and sacrificed with an anesthetic (1% barbital sodium, 500 mg/kg). Blood and liver samples were

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harvested and stored at -80°C. The gavage dosage (300 mg/kg NaB) was determined based on a

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previous study indicating that both 200 and 400 mg/kg could effectively alleviate insulin

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resistance, fat accumulation, and dyslipidemia in rats with type 2 diabetes

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procedure followed the method used in our previous study on HF-induced obese mice 24-25.

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All procedures were approved by the Animal Ethics Committee of Nanjing Agricultural University

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(Project No. 2012CB124703), which complied with the “Guidelines for the Ethical Treatment of

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Experimental Animals” (2006) No. 398 provided by the Ministry of Science and Technology,

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China.

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2.3 Hepatic content of triglyceride and total cholesterol

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Hepatic liver content of triglyceride (TG) and total cholesterol (Tch) was measured using

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respective commercial assay kits (E1013 for TG and E1015 for Tch, Applygen Technologies, Inc.,

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Beijing, China) following the manufacturer’s instruction. 5


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The treatment


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2.4 Hepatic mitochondrial respiratory chain complex Ⅲ and complex V activity

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Mitochondrial respiratory chain complex Ш and complex V was measured using respective

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commercial assay kits (ly3 for complex Ш and ly5 for complex V; Comin Technologies, Co., Ltd.,

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Suzhou, China) according to the manufacturer’s instruction.

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2.5 Adenosine triphosphate (ATP) content

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Liver ATP levels were measured using a ATP assay kit (Beyotime, China) according to the

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manufacturer’s instructions. Luminance was measured by CLARIOstar microplate reader (BMG

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LABTECH, Ortenberg, GER). The protein concentration was measured by BCA protein assay.

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Data were normalized to the control group and expressed as percentage of control levels.

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2.6 Determination of cytokines by ELISA

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The contents of cytokines including interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor

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necrosis factor α (TNFα) in liver were determined using the ELISA kits (ANG-E11546R for IL-1β,

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ANG-E11755R for IL-6 and ANG-E11546R for TNFα, Angle Gene Bioengineering Co., Ltd.,

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Nanjing, China) according to the manufacturer’s protocol. Detection range: IL-1β from 1.6 ng/L to

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80 ng/L, IL-6 from 8 ng/L to 150 ng/L, TNFα from 15 ng/L to 300 ng/L. The intra- and inter-assay

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coefficients of variation were less than 9% and 15% respectively.

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2.7 Histopathology and immunofluorescence

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The liver tissues were cut and fixed with a 4% paraformaldehyde, and were embedded in paraffin.

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Sections (4 μm) were stained with hematoxylin-eosin (HE) or Oil Red O (ORO) to investigate

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architecture of the liver and hepatic lipid droplets. Stained slides were scanned with the

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Pannoramic SCAN II and images were captured with 3DHISTECH software (3DHISTECH Ltd.

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Budapest, Hungary). Immunofluorescence was used for checking the expression of CD68 (M1 6


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macrophage marker) and CD206 (M2 macrophage marker). Briefly, liver sections (4 μm) were

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deparaffinized with xylene, followed by antigen retrieval by heating in citrate buffer (10 mmol/L)

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at 95°C for 15 min. The following primary antibodies were used: anti-CD68 (ab955, Abcam, USA)

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and CD206 (ab64693, Abcam, USA). The Alexa Fluor® 488 conjugated goat anti-rat IgG for

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CD68 (ab150157, Abcam, USA) and Alexa Fluor® 488 conjugated donkey anti-rabbit IgG for

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CD206 (ab150073, Abcam, USA) were used respectively as the second antibody. DAPI was used

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as a marker for cell nuclei.

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2.8 RNA isolation and quantitative real-time PCR

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Total RNA was isolated from 30 mg liver samples by using 1 mL of TRIzol reagent (Invitrogen,

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USA). Up to 1 µg of the RNA samples was reverse-transcribed following the protocol provided by

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the manufacturer (Vazyme Biotech, Nanjing, China). The cDNA was diluted at 1:25 of which 2 μL

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was used as template in PCR reactions on a real-time PCR system (Mx3000P, Stratagene, USA).

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Moreover, 18S was chosen as a reference gene to normalize the mRNA abundance of target genes.

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All primers were made by Generay Biotech and listed in Table 2. The 2-ΔΔCt method was used to

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analyze real-time PCR data.

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2.9 Protein extraction and Western blot analysis

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Total protein was extracted from 50 mg frozen liver samples as previously described26. Protein

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concentrations were measured using the BCA Protein Assay Kit (No. 23225, Thermo Scientific,

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USA) according to the instructions provided by the manufacturer. Protein (40 µg/lane) was used

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for electrophoresis on a 7.5% or 10% sodium dodecyl sulfate (SDS) polyacrylamide gel

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electrophoresis and then transferred onto a polyvinylidene fluoride membrane. Membranes were

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blocked in 5% milk and then incubated in primary and secondary antibodies. Western blot analysis 7



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for PPARα (BS1689, Bioworld, USA, diluted 1:500) and carnitine palmitoyltransferase 1 (BS7744,

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Bioworld, USA, diluted 1:1000) was performed, using tubulin (BS1699, Bioworld, USA, diluted

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1:5000) as a reference protein for normalization of loading and transfer efficiency. Western blot

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analysis was conducted for cytochrome c oxidase subunit 1 (COX1) (BS70809, Bioworld, USA,

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diluted 1:500), cytochrome c oxidase subunit 4 (COX4) (BS2186, Bioworld, USA, diluted 1:500),

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F4/80 (70076s, Cell Signaling Technology, USA, diluted 1:1000), and CD206 (ab64693, Abcam,

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USA, diluted 1:1000) by using GAPDH (AP0063, Bioworld, USA, dilute 1:5000) as a reference

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protein. The same GAPDH image was used for the normalizations of six target proteins, including

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the phospho–inhibitor of nuclear factor kappa-B kinase alpha/beta (p-IKKα/β) (2697s, Cell

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Signaling Technology, USA, diluted 1:1000), inhibitor of nuclear factor kappa-B kinase γ (IKKγ)

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(sc-166700, Santa Cruz, USA, diluted 1:200), p-p65 (ab6503, Abcam, USA, diluted 1:1000),

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NLRP3 (ab214185, Abcam, USA, diluted 1:1000), cleaved caspase-1 (ab179515, Abcam, USA,

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diluted 1:1000), and histone deacetylases 1 (HDAC1) (BS6485, Bioworld, USA, diluted 1:500) as

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they were detected in the same blot. Proteins of distinct sizes, such as cleaved caspase-1 (20 kDa),

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NLRP3 (110 kDa), and p-IKKα/β (85 kDa), were considerably well separated and could be

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detected using their respective membrane slices probed with their respective antibodies. Stripping

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and re-probing were applied for proteins of the similar size. Images were captured using the

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VersaDoc 4000MP system (Bio-Rad, USA), and band density was analyzed using the software

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Quantity One (Bio-Rad, USA).

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2.10 Chromatin immunoprecipitation assay

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Chromatin immunoprecipitation (ChIP) was analyzed as described in a previous study, with

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several modifications27. Frozen liver samples (300 mg) in liquid nitrogen were homogenized and 8


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then resuspended in PBS containing Protease Inhibitor Cocktail (No. 11697498001; Roche).

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Protein was cross-linked to DNA by adding formaldehyde to a final concentration of 1%. The

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reaction was stopped subsequently with 2.5 mol/L glycine at room temperature. The mixture

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resulting from the aforementioned procedure was centrifuged (5000 rpm for 5 min at 4°C). Pellets

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were rinsed with PBS and homogenized in an SDS lysis buffer containing protease inhibitors.

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Crude chromatin preparations were sonicated on ice to yield DNA fragments of 150-500 bp in

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length and pre-cleared with salmon sperm DNA-treated protein G agarose beads (40 μL, 50%

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slurry, sc-2003, Santa Cruz, USA). The mixture of pre-cleared chromatin preparations and 2 μg of

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primary antibody (H3 lysine 9 acetylation, ab4441, Abcam, USA) were incubated overnight at

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4°C. A negative control was included with normal IgG. Protein G agarose beads (40 μL, 50%

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slurry, sc-2003; Santa Cruz) were added to capture the immunoprecipitated chromatin complexes.

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Finally, DNA fragments were released from the immunoprecipitated complexes by reverse

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cross-linking at 65°C for 1 h, and quantitative real-time PCR was used to quantify the fragments

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of target gene promoters with specific primers (Table 2) using purified immunoprecipitated DNA

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as the template.

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2.11 Co-immunoprecipitation

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Co-immunoprecipitation was conducted as previously described, with slight modifications

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Total protein amounting to 500 µg was incubated with 20 μL of 50% protein A/G plus beads for 2

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h at 4°C and then centrifuged at 7,500 g for 1 min. The supernatants were incubated with 3 µg

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p-p65 antibodies and rotated overnight at 4°C. Subsequently, 20 μL of agarose beads was

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incubated with protein-antibody complexes for 10 h at 4°C. The agarose beads were washed with

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cold PBS after centrifugation, and the immunoprecipitated proteins were run on 10% 9


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SDS-polyacrylamide gel for Western blot analysis.

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2.12 Statistical analysis

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All data are presented as mean ± SEM. One-way ANOVA with a Bonferroni post hoc test was

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applied to estimate the differences among the three groups. P < 0.05 was considered statistically

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significant.

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3. Results

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3.1 NaB reduces high-fat diet-induced body weight gain and hepatic lipid deposition

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Compared to the rats fed control diet, the rats fed high-fat diet for 9 weeks had significantly (P

Sodium Butyrate Ameliorates High-Fat-Diet-Induced Non-alcoholic

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