− mice fed a high-cholesterol diet potentially by inhibiting VCAM-1 expression

Accepted Manuscript −/− Sinigrin attenuates the progression of atherosclerosis in ApoE mice fed a highcholesterol diet potentially by inhibiting VCAM-1 expression Yeon Jeong Jang, Bongkyun Park, Hee-Weon Lee, Hye Jin Park, Hyun Jung Koo, Byung Oh Kim, Eun-Hwa Sohn, Sung Hee Um, Suhkneung Pyo PII:

S0009-2797(17)30214-4

DOI:

10.1016/j.cbi.2017.05.006

Reference:

CBI 7994

To appear in:

Chemico-Biological Interactions

Received Date: 26 February 2017 Accepted Date: 4 May 2017

Please cite this article as: Y.J. Jang, B. Park, H.-W. Lee, H.J. Park, H.J. Koo, B.O. Kim, E.-H. Sohn, −/− S.H. Um, S. Pyo, Sinigrin attenuates the progression of atherosclerosis in ApoE mice fed a highcholesterol diet potentially by inhibiting VCAM-1 expression, Chemico-Biological Interactions (2017), doi: 10.1016/j.cbi.2017.05.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Graphic abstract

HCD-fed ApoE−/− mice LDH, TG, TC, LDL, Ca2+ ↓

TNF-α, IL-6 ↓

VCAM-1, ICAM-1, CCL2, CCL5 ↓ HMG-CoA, LXR, SREBP-2, LDLR ↓

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TNF-α-stimulated MOVAS cells NF-κB ↓

p-p38 MAPK ↓

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p-JNK ↓

Atheroprotective effects

ACCEPTED MANUSCRIPT Sinigrin attenuates the progression of atherosclerosis in ApoE−/− mice fed a high-cholesterol diet potentially by inhibiting VCAM-1 expression

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Yeon Jeong Jang1, Bongkyun Park1, Hee-Weon Lee1, Hye Jin Park1, Hyun Jung Koo2, Byung Oh Kim3, Eun-Hwa Sohn4, Sung Hee Um5*, Suhkneung Pyo1*

1

2

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School of Pharmacy, Sungkyunkwan University, Suwon, 16419, Republic of Korea Department of Medicinal & Industrial Crops, Korea National College of Agriculture

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and Fisheries, Jeonju, 54874, Republic of Korea 3

School of Food Sciences & Biotechnology, College of Agriculture & Life Sciences,

Kyungpook National University, Daegu, 41566, Republic of Korea. 4

Department of Herbal Medicine Resources, Kangwon National University,

5

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Samcheok, 25913, Republic of Korea

Department of Molecular Cell Biology, School of Medicine, Sungkyunkwan

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University, Suwon, 16419, Republic of Korea.

*

Correspondence: S. Pyo: Tel: +82-31-290-7713, E-mail: [email protected] and S. H.

Um: Tel: +82-31-299-6123, E-mail: [email protected] 1

ACCEPTED MANUSCRIPT Abstract Atherosclerosis is a complex inflammatory disease associated with elevated levels of atherogenic molecules for leukocyte recruitment. Sinigrin (2-propenylglucosinolate)

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is found mainly in broccoli, brussels sprouts, and black mustard seeds. Recently, sinigrin has received attention for its role in disease prevention and health promotion. In this study, we examined the effect of sinigrin on development of atherosclerosis in

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ApoE−/− mice and the expression of adhesion molecules in vascular smooth muscle cells (VSMCs). The serum concentrations of lactate dehydrogenase (LDH),

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triglyceride (TG), total cholesterol (TC), low density lipoprotein (LDL), calcium (Ca2+), and pro-inflammatory cytokines were reduced by sinigrin treatment in ApoE−/− mice. In addition, oral administration of sinigrin attenuated the mRNA expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1

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(ICAM-1), C-C motif chemokine ligand 2 (CCL2), and CCL5 on aorta tissues and 3hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), liver X receptor (LXR), sterol regulatory element-binding protein-2 (SREBP-2), and low density lipoprotein

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receptor (LDLR) on liver tissues in ApoE−/− mice. To provide a potential mechanism underlying the action of sinigrin, we evaluated the in vitro effect of sinigrin on the

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expression of the VCAM-1 in TNF-α-induced VSMCs. The increased expression of VCAM-1 by TNF-α stimulation was significantly suppressed by the treatment of sinigrin (1-100 µg/ml) and sinigrin inhibited the nuclear translocation of NF-κB and the phosphorylation of p38 MAPK and JNK pathways, suggesting that sinigrin decreases the TNF-α-stimulated VCAM-1 expression through the suppression of NFκB and MAP kinases signaling pathways. Overall, sinigrin has the potential to be used in reducing the risks of atherosclerosis. 2

ACCEPTED MANUSCRIPT Keywords

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Atherosclerosis; Sinigrin; 2-propenyl glucosinolate; ApoE−/− mice; MOVAS cells.

1. Introduction

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Atherosclerosis is a progressive multi-factorial disorder and a chronic inflammatory disease occurring in large and medium-sized arteries. For many years, it was simply

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believed that lipid accumulation in the artery wall was the cause of atherosclerosis. However, the cause and symptoms of atherosclerosis have been proven to be more complex than mere lipid storage, with evidence that inflammation and the immune response cause the ongoing atherosclerosis [1]. Today, atherosclerosis is generally

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identified to be a form of obstinate inflammation caused by interaction of malfunctioned lipoproteins, inflamed vascular smooth muscle cells (VSMCs), immune cells and normal cellular elements of the arterial wall [2, 3]. After an intimal

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injury, inflammatory mediators including growth factors and cytokines released due to endothelial dysfunction induce a phenotype change in the VSMCs, and the cells

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migrate and proliferate from the media to the intima [4]. The recruitment of VSMCs to the intima by intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), is a key event for both the initiation and progression of atherosclerotic plaque, and in fact, VCAM-1 is found on VSMCs of human atherosclerotic plaques [5]. Additionally, the treatment of VSMCs with tumor necrosis factor-alpha (TNF-α) leads to an elevation in both the cell surface expression and the mRNA level of VCAM-1. VCAM-1 expression contributes to the pathophysiology of 3

ACCEPTED MANUSCRIPT inflammatory and immune processes in atherosclerosis [6, 7]. Therefore, the regulation of VCAM-1 expression in VSMCs could be critical to modulate plaque stability as well as the progression of the atherosclerotic lesion.

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There has been an increased interest in fostering the food and nutrition sciences because of the potential health risks and benefits of various foods and food ingredients. It has been suggested that pharmacological and nutritional agents can

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promote optimal health and reduce the risk of various inflammatory diseases like atherosclerosis. Additionally, plant-based dietary supplements have health and

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disease-preventing benefits [8]. However, the pharmacological activity of bioactive materials present in dietary product remains unexplored. Sinigrin

(2-propenyl

glucosinolate)

is

a

phytochemical

discovered

in

the

Brassicaceae family including the seeds of black mustard, brussels sprouts, and

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broccoli. Glucosinolates have diverse pharmacological biological activities that include anti-inflammatory, antioxidant, anti-carcinogenic, antifungal, anti-bacterial, wound healing activities, and biofumigation potential in in vitro/in vivo experimental

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models [9]. It has been also known that sinigrin inhibits hepatocarcinogenesis induced by diethylnitrosamine in male ACI/N rats [10]. Oral administration of sinigrin

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reportedly suppressed the dimethylhydrazine-induced apoptosis and aberrant crypt foci in those cells in the colon of rat [11]. In addition, sinigrin suppresses the nonenzymatic glycation of albumin and lens crystalline. This inhibitory effect was stronger than that exerted by other potent natural inhibitors such as quercetin, apigenin, and curcumin [12]. In preliminary experiments, we revealed that sinigrin has atheroprotective effects in apolipoprotein E-knockout (ApoE−/−) mice [13]. However, other biological activities of sinigrin and its molecular mechanisms have 4

ACCEPTED MANUSCRIPT not yet been completely elucidated. The present study aims to evaluate the effect of sinigrin on the development of atherosclerosis progression. In addition, we further investigated the effect of sinigrin

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underlying the anti-atherogenic effects observed in vivo.

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on adhesion molecule expression to gain insights into the possible mechanisms

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ACCEPTED MANUSCRIPT 2. Materials and methods

2.1. Reagents and Chemicals

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Unless otherwise indicated, all the chemicals were purchased from Sigma-Aldrich Chemical (St Louis, MO). Sinigrin (purity: > 98%) was purchased from Tokyo Chemical Industry Co., LTD (Chuo-ku, Tokyo). Dulbecco’s modified Eagle’s medium

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(DMEM) was obtained from Corning Life Sciences (Tewksbury, MA). Mouse TNF-α and IL-6 ELISA MAX™ Standard were purchased from BioLegend, Inc (San Diego,

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CA). cDNA synthesis kit Super Script II was obtained from Thermo Fisher Scientific (Pittsburgh, PA). TOP real™ qPCR 2X PreMIX (SYBR Green with high ROX) was purchased from Enzynomics (Hanam, Gyeonggi). Antibody against VCAM-1 was purchased from R&D Systems (Minneapolis, MN). Antibodies against p44/42

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extracellular signal-regulated kinase (ERK), phospho-p44/42 ERK, p38 mitogenactivated protein kinase (MAPK), phospho-p38 MAPK, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), phospho-SAPK/JNK, and β-actin were

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purchased from Cell Signaling Technology (Beverly, MA). Antibody against p65 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). DC protein assay

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reagent was obtained from Bio-Rad Laboratories (Hercules, CA). Nitrocellulose membranes was from Merck Millipore (Billerica, MA). Western blotting detection reagent AbSignal was purchased from AbClon (Guro, Seoul). The Trizol Reagent and in-fectTM in vitro Transfection Reagent were purchased from iNtRON Biotechnlogy, Inc (Sungnam, Gyeonggi). pGL3-nuclear factor-kappa B (NF-κB) and the luciferase assay system were obtained from Promega (Fitchburg, WI). pCMV-βgalactosidase (β-gal) was obtained from Lonza (Walkersville, MD). 6

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2.2. Animals and diets Eighteen five-weeks-old male ApoE−/− mice and six five-week-old male C57BL/6J

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mice were purchased from SLC Inc. (Hamamatsu, Shizuoka). Mice were kept in standard plastic cages in a controlled environment at a temperature of 22 ± 2°C, humidity of 50 ± 5%, 10 to 18 circulations per hour and a 12 h light/dark cycle. All

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mice were supplied with a basal diet and sterilized water without any restrictions during the experiment. Food and water were available freely. After one week of

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adaptation, all mice were fed a western type diet (AIN-76A purified rodent diet #101556; Dyets Inc, Bethlehem, PA), which contained 40% fat, 0.44% cholesterol, 20% casein, 15% sucrose, 5% cellulose, and 15% starch, until the end of the study. At six-weeks-of-age, the male C57BL/6J mice and ApoE−/− mice were randomly divided into four groups of six animals: control group (CTR), C57BL/6J mice were fed

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a normal diet plus PBS; high cholesterol group (ApoE−/−), ApoE−/− mice were fed with western-type high-cholesterol atherogenic diet (HCD) plus PBS; positive group

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(ApoE−/− + Pravastatin), ApoE−/− mice were fed with HCD plus pravastatin (5 mg/kg) dissolved in PBS; sinigrin group [ApoE−/− + Sinigrin (10 mg/kg) dissolved in PBS],

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ApoE−/− mice were fed with HCD plus sinigrin (10 mg/kg) dissolved in PBS. The mice were received oral gavage of substance 3 times per week for 16 weeks. The dose in this study was selected based on previous studies [9-12] and our preliminary toxicity study which did not show clinical pathology (data not shown). Food intake and body weight were monitored on a regular basis throughout the study. We did not observe any obvious differences between sinigrin-treated mice and sham-operated animals. At the end of the 21 weeks, all animals were fasted overnight and sacrificed. All 7

ACCEPTED MANUSCRIPT animal procedures were performed in accordance with guidelines established by the Institutional Animal Care and Use Committee at Sungkyunkwan University.

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2.3. Serum analysis At the end of the 21 week study, blood samples were collected from the inferior vena cava after completion of treatment, and the sera were separated. Serum

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concentrations of lactate dehydrogenase (LDH), triglyceride (TG), total cholesterol (TC), low density lipoprotein (LDL), high density lipoprotein cholesterol (HDL), and

(RaonBio, Yongin, Gyeonggi).

2.4. Cell proliferation assay

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calcium (Ca2+) were measured using an enzymatic methods with commercial kits

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The mouse vascular aortic smooth muscle cell line, MOVAS, was purchased from the American Type Culture Collection (ATCC, Manassas, VA). MOVAS cells were cultured in DMEM supplemented with 200 µg/ml G418, 1% penicillin/streptomycin

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(P/S), and 10% heat-inactivated fetal bovine serum (FBS). Cultures were maintained in a humidified atmosphere containing 5% CO2 incubator at 37°C. Cell proliferation

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was determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. MOVAS cells were seeded (2 × 104 cells/well) in wells of 96 well culture plate and allowed to adhere for overnight. Cells were treated with various concentrations of sinigrin for 24 h. After incubation for a set period of time, the media were replaced with fresh media containing 10% (v/v) MTT solution (2 mg/ml in phosphate buffered saline (PBS)), and cells were incubated for another 4 h at 37°C. The supernatant was discarded and the formazan blue crystals formed by the 8

ACCEPTED MANUSCRIPT reduction of MTT were dissolved in 150 µl of dimethylsulfoxide (DMSO). The amount of formazan was determined using a microplate reader (Molecular Devices,

2.5. Enzyme-linked immunosorbent assay (ELISA)

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Sunnyvale, CA) at λ = 540 nm.

Cell surface expression of VCAM-1 was quantified by ELISA. MOVAS cells were

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seeded at a concentration of 2 × 104 cells/well in 96 well culture plate, incubated overnight to allow adherence and pretreated with sinigrin (1, 10, and 100 µg/ml) for 2

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h at 37°C. These pretreated cells were then incubated with fresh growth media containing TNF-α (10 ng/ml) for 8 h for measurement of VCAM-1. After incubation, the cells were washed with PBS and fixed with 1% glutaraldehyde for 30 min at 4°C. Bovine serum albumin (BSA) (1% in PBS) was added to the cells to reduce non-

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specific binding. The cells were then incubated with monoclonal antibody against VCAM-1 or isotype matched control antibody overnight at 4°C, washed with PBS, and incubated with goat anti-rat secondary antibody. The cells were then washed

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with PBS and exposed to the peroxidase substrate. The absorbance was measured at 650 nm using the aforementioned microplate reader. The absorbance values of

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the isotype matched control antibody were taken as the blank, and were subtracted from the experimental values.

2.6. Quantitative real time polymerase chain reaction (qRT-PCR) assay Total RNA was extracted from each liver, aorta, and cells using Trizol Reagent, after which 1 µg/µl of the isolated RNA was subjected to reverse transcription using the cDNA synthesis kit. qRT-PCR analysis for VCAM-1 gene was performed using an 9

ACCEPTED MANUSCRIPT ABI Prism 7500 qRT-PCR system (Applied Biosystems, Foster City, CA). Gene expression was detected using SYBR Green and the relative gene expression was determined by normalizing to the reference gene, glutaraldehyde phosphate

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dehydrogenase (GAPDH), with the relative quantitative method. The sequences of the primers that were analyzed in this study are shown in Table 1.

MOVAS

cells

were

collected,

washed in

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2.7. Western blot analysis cold

PBS,

and

suspended

in

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homogenization buffer (50 mM Tris-Cl (pH 6.8), 1% glycerol, 2% sodium dodecyl sulfate (SDS), and protease inhibitor cocktail). To obtain the subcellular fractionation of the cell, MOVAS cells were suspended in ice-cold Buffer A (10 mM HEPES (pH 8.0), 1.5 mM MgCl2, 10 mM KCl, 1 mM dithiothreitol [DTT], 0.1% NP-40, and 1 mM

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phenylmethylsulfonyl fluoride [PMSF]) and incubated on ice for 10 min. The cells were then centrifuged at 13,000 rpm for 10 min at 4°C, and the supernatant was collected as cytoplasmic cell fractions. For nuclear fractionation, the pellet was

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washed with Buffer A’ (10 mM HEPES (pH 8.0), 1.5 mM MgCl2, 10 mM KCl, 1 mM DTT, and 1 mM PMSF), resuspended in Buffer C (20 mM HEPES (pH 8.0), 0.2 M

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NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, and 1 mM PMSF), and then incubated on ice for 40 min with intermittent agitation every 10 min. Nuclear extracts (supernatants) were recovered after centrifugation at 13,000 rpm, 4°C for 10 min. These protein concentration of the lysates was then determined using DC protein assay reagent with BSA as the standard. The proteins in the lysates were separated by 8–15% SDS-polyacrylamide electrophoresis and then transferred to nitrocellulose membranes. The membranes were probed with primary antibody and the 10

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horseradish

peroxidase-conjugated

secondary

antibody.

The

immunocomplexes were developed using the Western blotting detection reagent. After measuring the intensity of each band by densitometry using the Image J image

from the corresponding sample.

2.8. Transient transfection and reporter assay

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processing software, relative intensities were calculated by normalization to β-actin

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MOVAS cells were seeded at a concentration of 5 × 105 cells/well in 6 well culture

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plate. The cells were transiently co-transfected with the plasmids, pGL3–NF-κB and pCMV-β-gal, using in vitro transfection reagent. Briefly, a transfection mixture containing 0.5 µg pGL3–NF-κB and 0.2 µg pCMV-β-gal was mixed with the in vitro transfection reagent and added to the cells. After 4 h, the cells were pretreated with

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sinigrin for 2 h followed by the addition of TNF-α for 4 h, and then lysed with 200 µl of lysis buffer (24 mM Tris–HCl (pH 7.8), 2 mM DTT, 2 mM EDTA, 10% glycerol, and 1% Triton X-100) and 10 µl of cell lysates were used for luciferase activity assay. The

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luciferase and β-galactosidase activities were determined. The values shown represent an average of three independent transfections, which were normalized

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with β-galactosidase activity.

2.9. Statistical analysis

All values were expressed as the mean ± S.E.M. Statistical analysis of the data was performed by one-way analysis of variance (ANOVA). Significant values (p < 0.05) are denoted by an asterisk.

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ACCEPTED MANUSCRIPT 3. Results

3.1. Effect of sinigrin on risk factors of atherogenesis in ApoE−/− mice

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To determine the effect of sinigrin administration on atherosclerosis, we used an ApoE−/− mouse model which develop atherosclerosis spontaneously, and the appearance of lesions is accelerated by feeding them a Western-type diet. [14]. The

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total body weight of the mice from each group was measured until the end of the study, and the change of weight between each group was not observed (Table 2). In

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addition, the serum concentrations of triglyceride (TG), total cholesterol (TC), low density lipoprotein (LDL), lactate dehydrogenase (LDH), high density lipoprotein (HDL), and calcium (Ca2+) of the mice from each group were measured (Table 3). The concentrations of clinical atherogenic factors, such as LDH, TC, TG, and LDL,

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and Ca2+ were greater in the ApoE−/− mice than those in the C57BL/6J control mice. However, our data showed that the oral administration of sinigrin suppressed the increase of LDH, TG, TC, LDL, and Ca2+ levels, whereas the HDL level was

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enhanced. It is important to point out that the serum levels of atherogenic factors modulated by sinigrin treatment were similar to those found in groups treated with

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pravastatin which is used to treat dyslipidemia and the prevention of cardiovascular disease. Thus, these data suggest that sinigrin could have an anti-atherosclerotic effect. A plethora of inflammatory cytokines including IL-6 and TNF-α have been reported to be increased in atherosclerotic plaques, and these pro-inflammatory cytokines have a proatherogenic activity altering the endothelial functions [15]. Therefore, we examined the serum levels of pro-inflammatory cytokines (Fig. 1). The level of these cytokines was increased in ApoE−/− mice, whereas treatment with 12

ACCEPTED MANUSCRIPT pravastatin or sinigrin reduced cytokine levels. Collectively, these data suggest that sinigrin prevents atherosclerosis by inhibiting the accumulation of atherogenic risk

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factors and vascular inflammatory cytokines.

3.2. Sinigrin attenuates migration- and cholesterol metabolism-related genes in ApoE−/− mice

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To determine whether sinigrin has an inhibitory effect on migration-related genes, we examined the level of ICAM-1 and VCAM-1 in aorta tissue. As shown in Fig. 2, the

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mRNA levels of VCAM-1 and ICAM-1 in the sinigrin-administrated group were lower than those in untreated ApoE−/− mice. Moreover, we examined the expression of chemokine (C-C motif) ligand 2 (CCL2) and CCL5 in the tissues. In untreated ApoE−/− mice, the mRNA expression of both CCL5 and CCL2 was higher than those

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in other groups. However, the oral administration of sinigrin group notably reduced this rise in the mRNA level of these chemokine ligands (Fig. 2). To further determine whether sinigrin regulates cholesterol metabolism-related genes, we investigated the

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level of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), liver X receptor (LXR), sterol regulatory element-binding protein 2 (SREBP-2), and low density

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lipoprotein receptor (LDLR) expression in the liver tissue (Fig. 3). Sinigrin significantly reduced the mRNA levels of LDLR, LXR, SREBP-2, and HMGR. These data suggest that sinigrin may have an anti-atherosclerotic effect by regulating migration- and cholesterol metabolism-related genes.

3.3. Sinigrin inhibited VCAM-1 expression in TNF-α-treated MOVAS cells To explore the potential mechanism underlying the in vivo effect which shows that 13

ACCEPTED MANUSCRIPT sinigrin treatment protect against atherosclerosis in ApoE−/− mice, the effect of sinigrin on VCAM-1 expression which is critical in atherosclerosis was examined in mouse vascular smooth muscle cells. MOVAS cells were treated with various

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concentrations of sinigrin for 24 h. As shown Fig. 4A, the concentrations of sinigrin used in our experiments did not affect cell proliferation and viability. Next, to determine whether sinigrin affects VCAM-1 expression, MOVAS cells were treated

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with sinigrin for 2 h and then stimulated with TNF-α for 8 h. Expression of cell surface VCAM-1 upregulated by TNF-α was reduced concentration-dependently by

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pretreatment with sinigrin in MOVAS cells (Fig. 4B). Furthermore, increased protein and mRNA expression of VCAM-1 by the TNF-α treatment were decreased concentration-dependently by sinigrin (Fig. 4C and D). Together, these data suggest that sinigrin is effectively capable of suppressing the expression of VCAM-1 induced

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by TNF-α in MOVAS cells. To further validate these results, we examined the effect of allyl isothiocyanate (AITC), a sinigrin metabolite, on VCAM-1 expression level in TNF-α-treated MOVAS cells (Supplemental Fig. 1). The treatment with AITC resulted

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in the reduced expression of VCAM-1, suggesting that sinigrin and its metabolite AITC appear to have the same anti-atherogenic effect and that these results further

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support the observations made in vivo.

3.4. Sinigrin inhibits the expression of the adhesion molecule via NF-κB and MAP kinase pathways

Transcription factors are important mediators of atherosclerosic responses by regulating subclinical vascular inflammation. The ubiquitous transcription factor NFκB is known to be an important mediator of VCAM-1 and ICAM-1 expression [16, 17]. 14

ACCEPTED MANUSCRIPT Additionally, binding sites of NF-κB transcription factor are located in the VCAM-1 gene promoter [18, 19]. Therefore, we determined whether sinigrin affects the level of NF-κB transcriptional activation using the luciferase reporter assay. The data

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showed that TNF-α treatment caused an increase in NF-κB activity compared to the untreated cells, and this increase in NF-κB activity was concentration-dependently suppressed by sinigrin treatment (Fig. 5A). Further, we examined the p65 of NF-κB

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protein levels in cytosol and nuclear extracts. As shown in Fig. 5B, treatment of sinigrin significantly reduced the p65 translocation to the nucleus, indicating that anti-

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translocation activity of sinigrin was associated with decreased NF-κB activity. It is reported that upregulation of MAP kinase activity by TNF-α stimulation influences the expression of inflammatory mediators in atherosclerosis [20, 21]. Therefore, to evaluate whether sinigrin regulates the MAP kinase pathways, we

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examined the phosphorylation of MAP kinases in TNF-α-induced MOVAS cells. Fig. 5C shows that sinigrin significantly reduced the upregulated levels of JNK and p38 by TNF-α. Next, to confirm whether the MAP kinase pathways were involved in the

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expression of VCAM-1 in the TNF-α-induced MOVAS cells, we evaluated the protein expression of VCAM-1 in TNF-α-stimulated MOVAS cells after pretreatment of the

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ERK inhibitor, PD98059, the p38 MAPK inhibitor, SB203580, and the JNK inhibitor, SP600125, respectively. The results showed that treatment of MAP kinase inhibitors attenuated the protein expression of VCAM-1 elevated by TNF-α, indicating that MAP kinase pathways are involved in the adhesion molecule expression (Fig. 5D). Taken together, our data demonstrate that sinigrin suppressed the TNF-α-stimulated expression of VCAM-1 via the regulation of JNK and p38 MAPK pathways in VSMCs.

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ACCEPTED MANUSCRIPT 4. Discussion

It is well investigated that foods and their components offer health benefits related to

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prevention and treatment of various diseases including atherosclerotic coronary arterial disease. The accumulation of intracellular cholesterol which leads to atherosclerotic coronary arterial disease is a target of anti-atherosclerotic therapy.

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Consumption of natural food product can prevent intracellular cholesterol retention [22]. In this study, we demonstrated the inhibitory effects of sinigrin on the ongoing

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atherosclerosis in ApoE−/− mice and the VCAM-1 expression in MOVAS cells. Atherosclerosis is initiated by the accumulation of LDL in the intima of the artery, which then builds up on the walls of arteries and leads to an increase in incidence of heart disease [23]. The atherogenic factors involved in the formation of

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atheromatous plaques in the arteries are considered to be therapeutic targets for atherosclerosis. In our study, we measured the serum levels of atherosclerosisrelated factors such as LDH, TG, TC, LDL, HDL, and Ca2+ in ApoE−/− mouse model.

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Normally, HDL, the “good cholesterol,” acts as a cholesterol scavenger, mopping up excess cholesterol in the blood, and transporting it back to the liver where the

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complex is broken down with the cholesterol being excreted or recycled. On the other hand, LDL is a fundamental contributor to the ongoing atherosclerosis [24, 25]. ApoE−/− mice treated with sinigrin displayed low serum LDL levels and high serum HDL levels. Sinigrin also inhibited the increase of TG, TC and Ca2+ levels. Taken together, the present data indicate that sinigrin could attenuate the development of atherosclerosis in ApoE−/− mice. Atherosclerosis induced by hypercholesterolaemia is related to inflammation and the 16

ACCEPTED MANUSCRIPT excessive intake of fats and cholesterol could lead the vascular inflammation [1]. In addition, it is reported that pro-inflammatory mediators induce the atherosclerotic inflammation in the aorta tissue [15, 26]. Among the chemokines that are the

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important mediators for atherogenesis, CCL2 and CCL5 have been most strongly implicated in atherogenesis [27, 28]. CCL2/monocyte chemotactic protein-1 (MCP-1) which is elevated in atherosclerosis is not ordinarily found in the wall of blood vessel

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but found in not only macrophage-abundant areas but also smooth and endothelial muscle cells in ongoing atherosclerosis of mouse and human [29]. In addition,

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CCL5/regulated on activation, normal T cell expressed and secreted (RANTES) is found in lots of amounts by stimulated T cells and foam cells in the atheromas of mouse and human [30]. The level of IL-6 and TNF-α have been known to be increased in atherosclerotic plaques and have a proatherogenic activity [15]. In our

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experiments, sinigrin treatment resulted in reduced expression of TNF-α, IL-6, adhesion molecules, CCL2 and CCL5. As a key regulator of cholesterol, SREBP-2 controls the cholesterol-related genes, such as a rate-limiting enzyme related to the

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cholesterol synthesis, HMGR, and LDLR gene [31]. Our data showed that oral administration of sinigrin significantly reduced the mRNA levels of cholesterol and

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lipid metabolism-related genes compared when compared to the high cholesterol group. Interestingly, although sinigrin treatment shows less inhibitory effects on the expression of other genes compared to those of pravastatin, sinigrin reduced the SREBP-2 mRNA levels similar to pravastatin. However, our data totally rule out the possibility that sinigrin attenuates atherosclerosis by modulating the level of some microRNA (miRNA) in vivo. Recently, it has been shown that miRNA controls endothelial cell (EC), VSMC and macrophage functions, and thereby regulate the 17

ACCEPTED MANUSCRIPT progression of atherosclerosis [32, 33]. MiR-10a inhibits various pro-inflammatory genes and miR-146a is assumed to be protective against atherosclerosis. Nevertheless, the simplest interpretation of our data is that sinigrin has anti-

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atherogenic activities like pravastatin by modulating cholesterol-related genes. To gain an insight into the possible mechanisms mediating the in vivo effects of sinigrin on ongoing atherosclerosis, we evaluated the inhibitory effect of sinigrin on

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VCAM-1 expression in MOVAS cells. This is because cell adhesion molecule VCAM1 is involved in the inflammatory response in the atherosclerotic arterial wall and may

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be actively related to the lesions of ongoing atherosclerosis and in intimal neovasculature [34]. This suggests that the discovery of therapeutic new agents specially targeting adhesion molecules may be proper to reduce the development of atherosclerotic lesions. The in vitro data showing that the expression of VCAM-1 was notably inhibited by sinigrin treatment in MOVAS cells are in keeping with the

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atherogenic factors-reducing effects observed in vivo. Signal transduction cascades by TNF-α stimulation regulate gene expressions via

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phosphorylation of various protein kinases [35]. Accumulating evidences have shown that MAP kinases are involved in the mechanisms of intracellular signaling regulating

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the function of TNF-α-induced cell adhesion molecules expressed on VSMCs [20, 21]. Here we investigated whether sinigrin alters the MAPK cascades comprised of JNK, p38 MAPK, and ERK pathways in TNF-α-treated MOVAS cells. Activation of JNK and p38 MAPK, but not ERK, was significantly blocked by pretreatment of sinigrin in TNF-α-induced MOVAS cells. Therefore, it is possible that TNF-αupregulated expression levels of VCAM-1 were reduced by treatment of sinigrin in MOVAS cells through the blocking JNK and p38 MAPK signaling pathways. MAP 18

ACCEPTED MANUSCRIPT kinases activation is crucial for the action of several transcription factors such as SP1, AP-1, Nrf2, and NF-κB which are involved in regulation of multiple genes related to modulation of inflammatory reactions [36-38]. Especially, VCAM-1 promoter has

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binding sites of transcription factor NF-κB located at 65 to 75 bp upstream from the translation start site (TSS), indicating that NF-κB is particularly important factor involved in the gene expression and transcriptional regulation of cell adhesion

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molecule [39]. In the present study, sinigrin inhibited TNF-α-stimulated activation of NF-κB by inhibiting the p65 translocation into the nucleus. Collectively, these results

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suggest that the inhibitory effect of sinigrin is mediated by blocking the phosphorylation of p38 MAPK and JNK as well as NF-κB activation.

5. Conclusions

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Treatment with sinigrin effectively prevented the ongoing atherosclerosis in ApoE−/− mice. The preventing effect of sinigrin may be due to the ability to suppress the

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production of inflammatory cytokines and the expression of pro-atherogenic factors in serum, aorta, and liver tissue. In addition, our data suggests that sinigrin inhibits

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the VCAM-1 expression via preventing NF-κB and p38 MAPK/JNK pathways in MOVAS cells. Taken together, the present data provide the possible mechanisms of the inhibitory effects of sinigrin and implicate that sinigrin has the potential to exhibit health-benefiting effect for preventing atherosclerotic cardiovascular disease.

Conflict of interest 19

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The authors declare that there are no conflicts of interest.

20

ACCEPTED MANUSCRIPT References [1] P. Libby, Inflammation in atherosclerosis, Nature. 420 (2002) 868-874. [2] M. Navab, J.A. Berliner, A.D. Watson, S.Y. Hama, M.C. Territo, A.J. Lusis, D.M.

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[39] S.R. Kim, Y.H. Bae, S.K. Bae, K.S. Choi, K.H. Yoon, T.H. Koo, H.O. Jang, I. Yun, K.W. Kim, Y.G. Kwon, M.A. Yoo, M.K. Bae, Visfatin enhances ICAM-1 and VCAM-1 expression through ROS-dependent NF-kappaB activation in endothelial cells,

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ACCEPTED MANUSCRIPT Figure legends

Fig. 1. Effect of sinigrin on inflammatory cytokines in the experimental mice

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models Serum levels of IL-6 and TNF-α were determined by ELISA. The results illustrated are from a single experiment, and are representative of three separate experiments performed in triplicate. Values have been expressed as the mean ± S.E.M. of 6 mice

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per group. *Significantly different (p < 0.05) from ApoE−/− group not treated with

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pravastatin or sinigrin.

Fig. 2. Effect of sinigrin on migration-related genes in the experimental mice models

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mRNA expression of VCAM-1, ICAM-1, CCL2, and CCL5 on aorta tissue were determined by qRT-PCR. Data were normalized to the mRNA levels of GAPDH. The data are expressed as a fold of free control without any stimulation. The results

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illustrated are from a single experiment, and are representative of three separate experiments performed in triplicate. Values have been expressed as the mean

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± S.E.M. of 6 mice per group. *Significantly different (p < 0.05) from ApoE−/− group not treated with pravastatin or sinigrin.

Fig. 3. Effect of sinigrin on liver tissue in the experimental mice models mRNA expression of HMGR, SREBP-2, LXR, and LDLR on liver tissue were determined by qRT-PCR. Data were normalized to the mRNA levels of GAPDH. The data are expressed as a fold of free control without any stimulation. The results 26

ACCEPTED MANUSCRIPT illustrated are from a single experiment, and are representative of three separate experiments performed in triplicate. Values have been expressed as the mean ± S.E.M. of 6 mice per group. *Significantly different (p < 0.05) from ApoE−/− group

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not treated with pravastatin or sinigrin.

Fig. 4. Effect of sinigrin on adhesion molecule in TNF-α-stimulated MOVAS

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cells.

(A) MOVAS cells were treated with the various concentrations of sinigrin for 24 h.

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Cell proliferation was then determined by MTT assay. The data are expressed as a percentage of sinigrin-free control. (B, C) MOVAS cells were pretreated with sinigrin for 2 h followed by treatment 10 ng/ml TNF-α for 8 h. (B) Expression of VCAM-1 was measured by ELISA. The data are expressed as a percentage of sinigrin-free control without inducer (100%). (C) Protein expression of VCAM-1 was determined by

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western blot assay. β-actin was considered as an internal control. (D) MOVAS cells were pretreated with sinigrin for 2 h followed by treatment 10 ng/ml TNF-α for 6 h.

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mRNA expression of VCAM-1 were determined by qRT-PCR. Data were normalized to the mRNA levels of GAPDH. The data are expressed as a fold of sinigrin-free

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control without inducer. The results illustrated are from a single experiment, and are representative of three separate experiments performed in triplicate. Data are expressed as the mean ± S.E.M. of 3 experiments. *Significantly different (p < 0.05) from TNF-α-stimulated cells not treated with sinigrin.

Fig. 5. Effect of sinigrin on the activation of NF-κB and MAP kinases in TNF-αstimulated MOVAS cells. 27

ACCEPTED MANUSCRIPT (A) MOVAS cells were transfected with a pGL3–NF-κB–Luc reporter plasmid and pCMV-β-gal, pretreated sinigrin for 2 h, and stimulated with TNF-α for 4 h. The data are expressed as a percentage of sinigrin-free control without inducer (100%). The

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results illustrated are from a single experiment, and are representative of three separate experiments performed in triplicate. Data are expressed as the mean ± S.E.M. of 3 experiments. *Significantly different from TNF-α-stimulated cells not

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treated with sinigrin (p < 0.05). (B) MOVAS cells were pretreated with sinigrin for 2 h, then stimulated with 10 ng/ml TNF-α for 4 h. The level of p65 was detected by

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western blot assay to analyze the translocation of NF-κB. Lamin A and α-tubulin were used as loading controls for nuclear and cytosolic protein fractions, respectively. NE, nuclear extracts; CE, cytoplasmic extracts. (C) MOVAS cells were preincubated with or without various concentrations of sinigrin for 2 h, then treated with 10 ng/ml TNF-α for 30 min. The levels of MAP kinases were detected by western blot assay. (D)

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MOVAS cells were pretreated with 10 µM inhibitors of MAP kinases (PD98059, SB203580, and SP600125) and sinigrin for 2 h, then stimulated with 10 ng/ml TNF-α

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for 8 h. Protein expression of VCAM-1 were determined by western blot assay. β-

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actin was considered as an internal control.

28

ACCEPTED MANUSCRIPT Tables

Table 1. The sequences of the forward and reverse primers Primers for quantitative real time PCR

VCAM-1

Forward: 5’-CTC AGG TGG CTG CAC AAG TT-3’ Reverse: 5’-AGA GCT CAA CAC AAG CGT GG-3’

ICAM-1

Forward: 5’-GTG GGT CGA AGG TGG TTC TT-3’ Reverse: 5’-GCA GTT CCA GGG TCT GGT TT-3’

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Genes

Forward: 5’-AGG TCC CTG TCA TGC TTC TGG-3’ Reverse: 5’-CTG CTG CTG GTG ATC CTC TTG-3

CCL5

Forward: 5’-AGA TCT CTG CAG CTG CCC TCA-3’ Reverse: 5’-GGA GCA CTT GCT GCT GGT GTA G-3’

HMGR

Forward: 5’-GTG GCA GAA AGA GGG AAA GG -3’ Reverse: 5’-CGC CTT TGT TTT CTG GTT GA -3’

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CCL2

Forward: 5’-GGC CTC TCC TTT AAC CCC TT-3’ Reverse: 5’-CAC CAT TTA CCA GCC ACA GG-3’

LXR

Forward: 5’-TCC TAC ACG AGG ATC AAG CG -3’ Reverse: 5’-AGT CGC AAT GCA AAG ACC TG -3’

LDLR

Forward: 5’-GCG TAT CTG TGG CTG ACA CC-3’ Reverse: 5’-TGT CCA CAC CAT TCA AAC CC -3’

GAPDH

Forward: 5′-TGC ATC CTG CAC CAC CAA-3′ Reverse: 5′-TCC ACG ATG CCA AAG TTG TC-3′

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SREBP-2

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ACCEPTED MANUSCRIPT Table 2. Body weights (g) at starting week and final week, respectively Final week (21)

CTR

18.23 ± 0.66 ㅇ

26.75 ± 1.75 ㅇ

ApoE−/−

17.33 ± 0.75 ㅇ

24.13 ± 1.50 ㅇ

ApoE−/− + Pravastatin

16.63 ± 0.85 ㅇ

24.43 ± 0.75 ㅇ

ApoE−/− + Sinigrin

18.83 ± 0.90 ㅇ

23.35 ± 0.76 ㅇ −/−

−/−

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Start week (6)

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CTR: C57BL/6J mice were fed a normal diet; ApoE : ApoE mice were fed with western-type high−/− −/− cholesterol atherogenic diet (HCD); ApoE + Pravastatin: ApoE mice were fed with HCD plus −/− −/− pravastatin (5 mg/kg); ApoE + Sinigrin: ApoE mice were fed with HCD plus sinigrin (10 mg/kg). # Values have been expressed as the mean ± S.E.M. of 6 mice per group. p < 0.05, compared with * −/− CTR group; p < 0.05, compared with ApoE group not treated with pravastatin or sinigrin.

Table 3. Effect of sinigrin on the levels of lipid profiles (LDH, TG, TC, LDL, HDL) and

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calcium (Ca2+) in the experimental mice models TG (mg/dl)

CTR

635 ± 36.09#

ApoE−/−

935 ± 41.88#

ApoE−/− + pravastatin

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LDH (U/L)

LDL (mg/dl)

HDL (mg/dl) Ca2+ (mg/dl)

250 ± 05.00#

143 ± 07.02#

544 ± 33.05#

10 ± 0.25#

108 ± 05.57# 1328 ± 11.55#

211 ± 06.66#

160 ± 18.93#

17 ± 0.58#

73 ± 10.02

635 ± 32.23*

76 ± 18.82# 1024 ± 02.65*

127 ± 19.66*

455 ± 04.93*

11 ± 0.56*

750 ± 71.01*

94 ± 14.73# 1086 ± 14.15*

139 ± 45.31*

468 ± 38.79*

11 ± 0.78*

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ApoE−/− + sinigrin

TC (mg/dl)

−/−

−/−

CTR: C57BL/6J mice were fed a normal diet; ApoE : ApoE mice were fed with western-type high−/− −/− cholesterol atherogenic diet (HCD); ApoE + Pravastatin: ApoE mice were fed with HCD plus −/− −/− pravastatin (5 mg/kg); ApoE + Sinigrin: ApoE mice were fed with HCD plus sinigrin (10 mg/kg). # Values have been expressed as the mean ± S.E.M. of 6 mice per group. p < 0.05, compared with * −/− CTR group; p < 0.05, compared with ApoE group not treated with pravastatin or sinigrin.

.

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60 40

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α (ρ ρ g/ml) TNF-α

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CCL2 mRNA expression (Relative Quantitation)

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ICAM-1 mRNA expression (Relative Quantitation)

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*

2.0

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VCAM-1 mRNA expression (Relative Quantitation)

Figure 2.

0.5 0.0

ApoE−/− Pravastatin Sinigrin

+ + -

+ +

ApoE−/− Pravastatin Sinigrin

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0.0

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LXR mRNA expression (Relative Quantitation)

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+ +

-

+ -

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3 LOX-1 LDLR mRNA expression (Relative Quantitation)

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SREBP-2 mRNA expression (Relative Quantitation)

1.0

*

*

1.5

1.5

*

2.0

2.0

*

2.5

*

HMGR mRNA expression (Relative Quantitation)

Figure 3.

+ + -

+ +

ApoE−/− Pravastatin Sinigrin

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Figure 4. B

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VCAM-1 expression (% of control)

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Sinigrin (µ µg/ml)

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Cell Proliferation (% of control)

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*

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Figure 5. B

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200 180 160 140 120 100 80 60 40 20 0

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C + -

+ 1

+ + TNF-α 10 100 Sinigrin p-ERK

+ 100

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ERK

p-p38 p38

p-JNK JNK

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p65 Lamin A

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Luciferase Activity (% of control)

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ACCEPTED MANUSCRIPT Highlights

Sinigrin reduced the clinical atherogenic factors in ApoE−/− mice.

Sinigrin could inhibit the expression of VCAM-1.

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Sinigrin attenuated the level of migration- and cholesterol metabolism-related genes.

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Sinigrin suppressed TNF-α-stimulated NF-κB and MAP kinases signaling pathways.