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‡Marine Chemical Research Institute, Qingdao, Shandong 266071, China. 8. 9. *. Corresponding author. 10. Shoudong Guo; Tel: +86-0538-6229517; Fax: +...
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Fucosylated Chondroitin Sulfate from Sea Cucumber Apostichopus japonicus Retards Atherosclerosis in Apolipoprotein E-deficient Mice Ying Zhang, Haihong Sun, Shucun Qin, Yuanyuan Song, Yanhong Si, Pengbo Hou, Nana Yang, and Shoudong Guo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03479 • Publication Date (Web): 05 Oct 2017 Downloaded from http://pubs.acs.org on October 7, 2017

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

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Fucosylated Chondroitin Sulfate from Sea Cucumber Apostichopus japonicus

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Retards Atherosclerosis in Apolipoprotein E-deficient Mice

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Ying Zhang†,*, Haihong Sun‡, Shucun Qin†, Yuanyuan Song†, Yanhong Si†, Pengbo

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Hou†, NaNa Yang†, Shoudong Guo†,*

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Atherosclerosis, Taishan Medical University, Taian, Shandong 271000, China.

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Key Laboratory of Atherosclerosis in Universities of Shandong Province, Institute of

Marine Chemical Research Institute, Qingdao, Shandong 266071, China.

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*

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Shoudong Guo; Tel: +86-0538-6229517; Fax: +86-0538-6225275; E-mail: SD-GUO

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@hotmail.com;

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+86-0538-6118246; Fax: +86-0538- 6225275; E-mail: sdyrx@163. com.

Corresponding author

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Ying

Zhang;

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ABSTRACT: Fucosylated chondroitin sulfate (FucCS) is exclusively found at sea

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cucumbers, and it has been demonstrated to bear lipid-lowering effect in rodent

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models. The aim of this study is to investigate whether FucCS could attenuate

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atherosclerosis. FucCS was successfully obtained from sea cucumber A. japonicas,

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and its effect was investigated in apolipoprotein E-deficient (apoE−/−) mice. Results

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showed that high-dose FucCS (H-FucCS, 150 mg/kg) significantly decreased the

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plasma levels of LDL-C, TG, TNF-α and IL-6; increased the level of HDL-C and

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reduced the lipids accumulation in the aortic sinus and liver compared to the model

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group. Western blotting results showed that H-FucCS could down-regulate the

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proteins expression of MCP-1, VCAM-1, ICAM-1 and the phosphorylation of NF-κB

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p65 compared to the model group. Further study on the proteins expression of MAPK

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subfamilies indicated that FucCS administration had no influence on the

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phosphorylation of JNK1/2 and Erk1/2, but it significantly reduced the

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phosphorylation of p-38. These results suggest that p38/NF-κB pathway may partially

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contribute to the anti-inflammatory activity of FucCS. This study indicated that

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FucCS ameliorates atherosclerosis, at least in part, by its lipid-lowering and

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anti-inflammatory effects in apoE−/− mice. Furthermore, food intake of sea cucumbers

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may benefit the primary prevention of atherosclerosis.

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KEYWORDS: sea cucumber, atherosclerosis, inflammation, lipid-lowering, MAPK

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pathway

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INTRODUCTION

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Atherosclerosis mainly induced by hyperlipideamia and inflammation is the basic

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pathological change of cardiovascular disease (CVD), which is currently the leading

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cause of death worldwide.1 Although there are many therapeutic strategies, the

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mortality of urban and rural residents induced by CVD increases progressively year

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by year. This fact indicates that daily prevention is important for CVD, and the

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traditional food therapy in China and other eastern countries maybe a useful way to

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attenuate the onset and progression of atherosclerosis.

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Sea cucumbers belong to Holothuroidea in biological classification. They are

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nutritional foods and medicinal resources extensively consumed in China, Japan,

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Korea, and other Asian countries.2 Sea cucumbers are rich in sulfated polysaccharides,

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such as fucosylated chondroitin sulfate (FucCS) and fucoidan.2-4 FucCS is a unique

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glycosaminoglycan (GAG) found exclusively at sea cucumbers,5-8 and its structural

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characteristics have been clarified by depolymerization.9-11 FucCS from A. japonicas

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is generally composed of alternating 3-linked N-acetyl β-D-galactosamine (GalNAc)

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and 4-linked β-D-glucuronic acid (GlcUA) units with sulfated fucose branches either

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at the O-3 position of GlcUA or O-6 and O-4 positions of the GalNAc.2,12 Its

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conformational and physicochemical properties have been elucidated by high

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performance size exclusion chromatography combined with multi-angle laser

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scattering, viscometry, and differential refractive index detection and atomic force

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microscopy.7,13

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Previous publications suggested that FucCS from the sea cucumbers have

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antithrombotic14-15

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anti-adipogenic activity by regulating the Wnt/β-catenin pathway.18 Furthermore, a

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effects.11,13,16-17

hypolipidemic

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FucCS

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previous publication demonstrated that chondroitin sulfate (CS) can retard the

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progression of atherosclerosis in apolipoprotein E-deficient (apoE-/-) mice via its

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lipid-lowering and anti-inflammatory effect.19 More importantly, the presence of

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fucose (Fuc) at the O-3 position of the GlcUA is reported to benefit the medical

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properties of CS.20 However, the effect of the FucCS on atherosclerosis is rarely

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known. The aim of this study is to investigate whether FucCS can attenuate

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atherosclerosis and the underlying mechanisms.

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MATERIALS AND METHODS

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Materials

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Dry body wall of sea cucumber Apostichopus japonicus was purchased from a local

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market (Qingdao, Shandong, China). Q-sepharoseTM Fast Flow and Sephacryl

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S200HR were the products of GE healthcare (Piscataway, NJ, USA). Monosaccharide

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standards, dextran standards (Mw: 5.0, 11.6, 23.8, 48.6, 147.6, 273.0, 409.8, 667.8,

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1100.0 kDa) and 1-phenyl-3-methyl-5-pyrazolone (PMP) were bought from

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Sigma-Aldrich (St. Louis, MO, USA). Dialysis membrane (molecular weight cut off

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3500) was purchased from Lvniao (Yantai, Shandong, China). Simvastatin was the

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product of Sine Wanxiang Pharmaceutical CO. LTD (Shanghai, China). Rat

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monoclonal antibody against intercellular adhesion molecule-1 (ICAM-1), rabbit

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monoclonal antibodies against vascular cell adhesion molecule-1 (VCAM-1), Jun

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N-terminal protein kinase (JNK), phospho-JNK, nuclear factor of kappa protein 65

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(NF-κB p65) and phosphor-NF-κB p65, and rabbit polyclonal antibodies against p38

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mitogen-activated protein kinase (MAPK), monocyte chemoattractant protein-1

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(MCP-1), extracellular signal regulated kinase 1 and 2 (Erk1/2), and phospho-Erk1/2

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were obtained from Abcam (Cambridge, MA, USA). Mouse monoclonal antibody 4

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against β-actin and rabbit monoclonal antibody against phospho-p38 MAPK were

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obtained from Cell Signaling Technology Inc. (Danvers, MA, USA). Secondary

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antibodies were from Beijing ComWin Biotech Co., Ltd. (Beijing, China). Assay kits

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for triglyceride (TG), total cholesterol (TC), high density lipoprotein cholesterol

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(HDL-C) and low density lipoprotein cholesterol (LDL-C) were the products of

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Biosino Bio-technology and Science Incorporation (Beijing, China). Enzyme-linked

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immunosorbent assay (ELISA) kits for TNF-α and IL-6 were BlueGene products

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(Shanghai, China). Bicinchoninic acid (BCA) protein quantitative kit was the product

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of Solarbio (Beijing, China). High-fat diet (15 % fat and 1.25 % cholesterol) was

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provided by Keaoxieli Feed Co., Ltd. (Beijing, China). All other chemicals and

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reagents used were analytical purity grade.

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Preparation and Chemical Analysis of FucCS

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FucCS from the sea cucumber A. japonicus was prepared according to a previous

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publication.21 Briefly, the dried body walls (100 g) were cut into small pieces,

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suspended in 2 L of 0.1 mol/L sodium acetate buffer (pH=6) containing 10 g of

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papain, 5 mM EDTA, and 5 mM cysteine, and then incubated at 60 °C for 24 h. The

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mixture was centrifugated at 2000 × g for 10 min at 4 °C to obtain the supernatant,

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which was then precipitated with 2 volumes of 95 % ethanol. The precipitate was

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collected by centrifugation at 2000 × g for 20 min at 4 °C, dialyzed against distilled

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water, condensed and lyophilized. The crude extract was used for anion exchange

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separation with ÄKTA-FPLC. The Q-sepharoseTM Fast Flow column was eluted with

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a linear gradient of NaCl (0~2.0 mol/L) according to the previous publications.21-22

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The flow rate was 2.0 mL/min, and the eluent was collected at 5.0 mL/tub. The

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FucCS containing fractions were collected, dialyzed and lyophilized. The dried

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FucCS was further purified by a Sephacryl S200 HR column (2.6 × 90 cm) taking 0.2 5

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mol/L ammonium bicarbonate as eluent. The fractions of the major peak were

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collected and used for the following studies.

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The purity and molecular weight of the FucCS was determined by size-exclusion

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chromatographic analysis (TOSOH TSKgel G4000PWXL) according to the

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publication.22 Sulfate ester content was detected according to the method reported by

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Therho and Hartiala.23 The monosaccharide composition was determined by

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pre-column PMP derivatization.24

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Anti-atherosclerosis Experiment in ApoE-/- Mice

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Male apoE–/– mice (20.0 ± 2.0 g) were purchased from Beijing HFK Bioscience Co.,

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LTD (Beijing, China; license number: SCXK2009-0004). All care and handling of

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animals were approved by the Animal Care and Use Committee of Taishan Medical

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University. After 10 days of adaptation to the facilities, the mice were randomly

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allocated to four groups (n = 10 each): High-fat diet group (model group), simvastatin

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group (Simv. 20 mg/kg/d), low-dose FucCS group (L-FucCS, 50 mg/kg/d) and

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high-dose FucCS group (H-FucCS, 150 mg/kg/d). Mice in each group were fed a

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high-fat diet (15 % fat and 1.25 % cholesterol) for 8 weeks. Then, the mice were

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anaesthetized with 10 % chloral hydrate, and blood was sampled. Plasma was

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obtained by centrifugation at 1100 × g for 5 min at 4 °C. The mice were perfused with

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5 mL of PBS through left ventricle, and then heart and aortas were removed. 5 hearts

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and aortas in each group were perfusion-fixed with 4 % paraformaldehyde for

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histological and morphological staining. The rest of the aortas were used for Western

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blotting analysis.

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Plasma Analysis

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Plasma TG, TC, HDL-C and LDL-C were measured by assay kits from Biosino 6

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Bio-technology and Science Incorporation (Beijing, China) according to the

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manufacturers’ instructions. Plasma levels of TNF-α and IL-6 were determined by

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ELISA kits from BlueGene (Shanghai, China) according to the manufacturers’

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instructions.

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Histopathologic Analysis

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Heart samples were embedded in optimal cutting temperature compound, and

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5.5-µm-thick cryosections were cut from the aorta to left ventricle. Cryosections were

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stained with hematoxylin and eosin (H & E). The plaques were identified by Oil-Red

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O staining. All images were captured with an Olympus BX51 microscope equipped

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with a video camera. The extent of atherosclerosis was quantified using an Image

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Pro-Plus software.25

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Western Blot Analysis

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This experiment was performed according to our previous publications.25-26

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Data Analysis

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All of the bioassay results were expressed as the mean ± standard deviation (SD) for

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at least three independent experiments. Statistical analysis was performed using

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one-way analysis of variance followed by Student-Newmann-Keuls multiple

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comparison tests with SPSS 13.0 software. Differences were considered to be

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significant at a P