Fucoidan A2 from the Brown Seaweed Ascophyllum nodosum Lowers

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Bioactive Constituents, Metabolites, and Functions

The fucoidan A2 from the brown seaweed Ascophyllum nodosum lowers lipid by improving reverse cholesterol transport in C57BL/6J mice fed a high-fat diet Zixun Yang, Guanjun Liu, Jiayu Yin, Yufeng Wang, Jin Wang, Bin Xia, Ting Li, Xiaoqian Yang, Pengbo Hou, Shumei Hu, Weiguo Song, and Shoudong Guo J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 04 May 2019 Downloaded from http://pubs.acs.org on May 6, 2019

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Journal of Agricultural and Food Chemistry

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The fucoidan A2 from the brown seaweed Ascophyllum nodosum lowers lipid by

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improving reverse cholesterol transport in C57BL/6J mice fed a high-fat diet

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Zixun Yanga#, Guanjun Liub#, Yufeng Wangc#, Jiayu Yina, Jin Wanga, Bin Xiaa, Ting

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Lia, Xiaoqian Yanga, Pengbo Houa, Shumei Hua, Weiguo Songa*, Shoudong Guoa*

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aInstitute

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School of Pharmacy, Weifang Medical University, Weifang, 261053, China

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bWeihai

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cNanjing

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#Contribute

of Lipid Metabolism and Atherosclerosis, Innovative Drug Research Centre,

Municipal Hospital, Weihai, 264200, China Well Pharmaceutical Co., Ltd. Nanjing, 210042, China equally to this article.

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*Corresponding author

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Shoudong Guo, Address: 7166# Baotong West Street, Weifang, Shandong Province,

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China. E-mail: [email protected]; Tel: +86 0536 8462014. This article may

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also correspond to Weiguo Song, E-mail: [email protected]; Tel: +86 0536

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8462014.

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ACS Paragon Plus Environment


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ABSTRACT: Reverse cholesterol transport (RCT) is a physiological process in

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which excess peripheral cholesterol is transported to the liver, and further excreted

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into the bile and then feces. Recently, fucoidans are reported to have lipid-lowering

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effect. This study was designed to investigate whether the fucoidan from the brown

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seaweed A. nodosum lowers lipid by modulating RCT in C57 BL/6J mice fed a high-

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fat diet. Our results indicated that fucoidan intervention significantly reduced plasma

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triglyceride, TC and fat pad index, and markedly increased HDL-C in a dose-

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dependent manner. In the liver, the fucoidan significantly increased the expression of

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PPARα and γ, LXRβ, ABCA1, ABCG8, LDLR, SR-B1 and CYP7A1; and decreased

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triglyceride level and expression of PCSK9 and PPARβ; but had no effect on LXRα,

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ABCG1 and ABCG5. In the small intestine, the fucoidan treatment significantly

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reduced the expression of NPC1L1 and improved ABCG5/8. These results

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demonstrated that the fucoidan can improve lipids transfer from plasma to the liver by

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activating SR-B1 and LDLR and inactivating PCSK9; and up-regulate lipid

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metabolism by activating PPARα, LXRβ, ABC transporters and CYP7A1. In the

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small intestine, this fucoidan can decrease cholesterol absorption and increase

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cholesterol excretion by activating NPC1L1 and ABCG5/8, respectively. In

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conclusion, the fucoidan from A. nodosum may lower lipids by modulating RCT-

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related protein expression, and it can be explored as a potential compound for

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prevention or treatment of hyperlipidemia-related diseases.

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Keywords: PPAR agonist; cholesterol metabolism; NPC1L1; ABC transporter

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INTRODUCTION

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Cardiovascular diseases (CVDs) are the major cause of mortality worldwide, and

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lipid-lowering drugs like statins are shown to have side effects.1 Therefore, natural

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products with lower toxicity and powerful hypolipidemic effect have attracted

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researchers’ attentions. Fucoidans are water-soluble, negative charged polysaccharide

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found in great abundance in brown seaweed and sea cucumber.2,3 They possess

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various biological functions as investigated both in vitro and in vivo.4-10

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Based on the previous studies, fucoidan is a potential candidate for lipid-lowering.

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For example, the fucoidan polysaccharide from brown seaweed can significantly

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reduce the serum total cholesterol (TC), triglyceride (TG) and low-density lipoprotein

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cholesterol (LDL-C), increase the high-density lipoprotein cholesterol (HDL-C) and

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the activity of lipoprotein lipase, hepatic lipoprotein and lecithin cholesterol

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acyltransferase in high-fat diet rats, C57 BL/6NtacSam mice and apolipoprotein E-

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deficient mice.11-13 The fucoidan from the sea cucumber can reduce TC, LDL-C and

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fat tissue weight in mice fed a high-fat diet.14,15 What’s more, fucoidan administration

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significantly reduces LDL-C in obese adults.16

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Several studies have partially demonstrated the mechanism of fucoidan on lipid-

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lowering. For instance, the fucoidan from brown seaweed Fucus vesiculosus (family

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Fucaceae) can up-regulate liver low-density lipoprotein receptor (LDLR) and down-

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regulate sterol regulatory element-binding protein (SREBP) 2 in poloxamer-407-

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induced hyperlipidemic mice;12 the fucoidan from Cladosiphon okamuranus can

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activate the mRNA expression of peroxisome proliferator-activated receptor (PPAR)

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α and inactivate the mRNA expression of SREBP1 in apolipoprotein E-deficient

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mice;13 and the fucoidan from sea cucumber Acaudina molpadioides may modulate 3



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the Wnt/β-catenin pathway and down-regulate the SREBP-1c expression in mice fed a

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high-fat diet.14 However, the underlying mechanism of fucoidan on lipid-lowering is

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still far from clarification.

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Reverse cholesterol transport (RCT) is a physiological process in which excess

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peripheral cholesterol is transported by high-density lipoprotein (HDL) or non-HDL

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to the liver, and further excreted into the bile and then feces.17,18 In the plasma, ATP

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binding cassette (ABC) A1 and G1 mediate the transfer of peripheral cholesterol and

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cholesterol ester to apolipoprotein (apo) A1 and HDL, respectively. The plasma lipids

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carried by HDL and non-HDL can be transported to the liver by scavenger receptor B

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type 1 (SR-B1) and LDLR, respectively. Furthermore, proprotein convertase

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subtilisin/kexin type 9 (PCSK9) binds to the epidermal growth factor-like repeat A

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domain of the LDLR and induces LDLR degradation. In the liver, PPARs regulate a

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network of genes involved in lipid metabolism, and the activation of PPARα and β

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can accelerate lipid utilization and the activation of PPARγ can induce fat storage. On

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the one hand, lipids in the liver can be excreted by ABC transporters such as, ABCG5

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and ABCG8, which are regulated by Liver X receptor (LXR)s; on the other hand,

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cholesterol can be transformed to bile acid by enzymes such as, cholesterol 7 alpha-

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hydroxylase A1 (CYP7A1) which is also a target gene of LXRs. In the small intestine,

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Niemann-Pick C1-like 1 (NPC1L1) is responsible for the uptake of cholesterol

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molecules from the gut lumen and bile, whereas ABCG5 and ABCG8 are responsible

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for

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hyperlipidemia.17,18 To our best knowledge, we report for the first time that fucoidan

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from A. nodosum lowers lipid by improving the RCT-related protein expression, and

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especially via modulating the cholesterol absorption and excretion in the small

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intestine of the C57 BL/6J mice fed high-fat diet.

the

cholesterol

excretion.

Therefore,

RCT

4


is

believed

to

facilitate

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MATERIALS AND METHODS

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Materials

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The brown seaweed Ascophyllum nodosum was collected at Saint Lawrence River in

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Quebec, Canada. The croud fucoidan was prepared and given as a gift by Weihai

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Rensheng Pharmaceutical Group Co.,Ltd. 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 (L-rhamnose, L-fucose, D-xylose, D-mannose, D-galactose, D-glucose, D-

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glucuronic acid and D-glucosamine), dextran standards (Mw: 11.6, 23.8, 48.6, 147.6,

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409.8, 1100.0 and 2000 kDa) and 1-phenyl-3-methyl-5-pyrazolone (PMP) were from

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Sigma-Aldrich (St. Louis, MO, USA). Fenofibrate (S1794) was the product of Selleck

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(Shanghai, China). RIPA lysis buffer was a product of Merck (3108491, Darmstadt,

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Germany). Rabbit polyclonal antibody against LDLR (ab30532, 1:500, Mw 95 kDa),

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LXRα (ab3585, 1:200, Mw 50 kDa) and LXRβ (ab28479, 1:500, Mw 51 kDa); rabbit

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monoclonal antibody against SR-B1 (ab217318, 1:1000, Mw 80 kDa), ABCG1

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(ab52617, 1:1000, Mw 110 kDa) and mouse monoclonal antibody against ABCA1

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(ab18180, 1:200, Mw 254 kDa) were from Abcam (Cambridge, MA, USA). Mouse

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monoclonal antibody against NPC1L1 (sc-166802, 1:200, Mw 142-148 kDa), and

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peroxisome proliferator-activated receptor (PPAR) α (sc-398394, 1:100, Mw 55 kDa),

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PPARβ (sc-74517, 1:100, Mw 55 kDa) and PPARγ (sc-7273, 1:100, Mw 55 kDa)

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were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse

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monoclonal antibody against β-actin (66009-1-Ig, 1:5000, Mw 43 kDa), rabbit

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monoclonal antibody against PCSK9 (55206-1-AP, 1:500, Mw 71 kDa) and rabbit

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polyclonal antibody against ABCG5 (27722-1-AP, 1:1000, Mw 72 kDa) were the

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products of Proteintech (Chicago, IL, USA). Mouse monoclonal antibody against 5



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ABCG8 (1B10A5, 1:1000, Mw 76 kDa) and enhanced chemiluminescence (ECL) kits

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were purchased from Thermo Scientific Pierce (Rockford, IL, USA). Rabbit

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polyclonal antibody against CYP7A1 (TA351400, 1:1000, Mw 58 kDa) was the

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product of OriGene (Shanghai, China). Complete protease inhibitor, the secondary

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antibodies and the BCA protein assay kits (CW0014S) were from CWBIO (Beijing

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China). TC and TG assay kits were the products of Biosino Bio-technology and

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Science Inc. (Beijing, China). All reagents used in this study were of analytical grade.

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Preparation of the Fucoidan

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The crude fucoidan was purified by an anion exchange column Q-SepharoseTM Fast

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Flow (2.6 × 30 cm). This column was connected to an ÄKTA FPLC system and

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eluted with a linear gradient of 0-2 mol·L-1 NaCl. In this study, the fraction containing

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the highest content of fucose was concentrated, dialyzed against distilled water, and

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then further purified on a Sephacryl S200HR column (2.6 × 90 cm) with 0.2 mol·L-1

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NH4HCO3 as the eluent. Sugar-containing fractions were concentrated and freeze-

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dried for further investigation.19,20

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Component Analysis of the Fucoidan

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Carbohydrate content was detected by the phenol-sulfuric acid with fucose as the

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standard.21 Sulfate ester content was determined according to the method previously

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reported.22 Protein content was quantified by a BCA assay kit from CWBIO (Beijing

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China).

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Monosaccharide Component Analysis

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The monosaccharides obtained after complete acid hydrolysis by 2.0 mol/L

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trifluoroacetic acid were pre-column derivatized by PMP. The derivative products 6


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were determined by an Agilent Eclipse XDB-C18 column (5 μm, 4.6×250 mm)

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connected to a high performance liquid chromatography (HPLC) system.23-26 The

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flow phase was freshly prepared phosphate buffer saline (PBS, 0.1 M, pH=6.7) and

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acetonitrile in a ratio of 83: 17 (v/v), and the flow rate was 1.0 mL/min.

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Monosaccharide composition was determined by comparison with reference sugars

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(L-rhamnose, L-fucose, D-xylose, D-mannose, D-galactose, D-glucose, D-glucuronic

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acid and D-glucosamine). Calculation of the molar ratio of the monosaccharide was

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performed based on the peak area of the reference sugars.

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Purity and Molecular Weight Determination

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Purity and molecular weight were determined by high performance gel permeation

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chromatography (HPGPC) with a RID-10A refractive index detector (Shimadzu,

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Japan) and a TOSOH TSKgel G4000PWXL column (7.8 × 300 mm) eluting with 0.1

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M Na2SO4. The flow rate of the mobile phase was 1.0 mL/min, and the column was

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maintained at 40 °C by a temperature-controlled cabinet. The molecular weight was

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calculated according to the standard curve made by dextran standards combined with

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GPC software.26-28

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FTIR Spectroscopy Analysis

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The FTIR spectrum of the polysaccharide was recorded on a Thermo Scientific

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Nicolet iS10 spectrometer. The preparation process was the same as we previously

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reported.20,28

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Animals

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Twenty-five C57BL/6J mice (male, 22 ± 2 g body weight) were purchased from

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Beijing HFK Bioscience Co., Ltd. (license number: SCXK2014-0004). All7



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experiments were approved by the Laboratory Animal Ethical Committee of Weifang

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Medical University and followed the NIH Guidelines for the Care and Use of Animals.

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After acclimatization for 1week, the mice were randomly divided into four groups:

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the chow diet group (n = 5, water by gavage), the model group (n = 5, water by

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gavage), the fenofibrate group (n = 5, 50 mg/kg/d by gavage), the low-dose fucoidan

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A2 group (n = 5, An 50, 50 mg/kg/d by gavage) and the high-dose fucoidan A2 group

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(n = 5, An200, 200 mg/kg/d by gavage).29,30 Mice were fed a high-fat diet (21% fat

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and 0.15% cholesterol) for 6 weeks except the chow diet group.

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Plasma Analysis

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TC, TG and HDL-C levels in the plasma were measured using commercially available

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assay kits of Biosino Bio-technology and Science Inc. (Beijing, China) according to

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the instructions.

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Protein Isolation, Electrophoresis, and Western Blotting

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Total proteins from the mice liver or small intestine were extracted using RIPA lysis

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buffer with complete protease inhibitor according to the manufacturer’s instructions.

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Equal amounts of protein were subjected to 6 % or 12 % SDS-PAGE and transferred

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onto Polyvinylidene fluoride membranes by electroblotting as we previously

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reported.28-31 Images were captured by Clinx ChemiScope 6000 pro (Shanghai, China),

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and densitometry analysis was conducted using Clinx Image Analysis Software

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(Shanghai, China). The expression of the proteins was normalized by housekeeping

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protein β-actin.

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Data Analysis

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

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least three independent experiments. Statistical analysis was performed using one-way

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analysis of variance (ANOVA) followed by Tukey’s test. Differences were

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considered to be significant at a P

Fucoidan A2 from the Brown Seaweed Ascophyllum nodosum Lowers

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