Immune Activation of RAW264.7 Macrophages by Low Molecular

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Bioactive Constituents, Metabolites, and Functions

Immune activation of RAW264.7 macrophages by low molecular weight fucoidan extracted from New Zealand Undaria pinnatifida Bi Decheng, Boming Yu, Qingguo Han, Jun Lu, William Lindsey White, Qiuxian Lai, Nan Cai, Wenqi Luo, Liang Gu, Sheng Li, Hong Xu, Zhangli Hu, Shao-Ping Nie, and Xu Xu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03698 • Publication Date (Web): 27 Sep 2018 Downloaded from http://pubs.acs.org on September 28, 2018

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Journal of Agricultural and Food Chemistry

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Immune activation of RAW264.7 macrophages by low molecular weight

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fucoidan extracted from New Zealand Undaria pinnatifida

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Decheng Bi a,1, Boming Yu a,1, Qingguo Han a,1, Jun Lu a,b,*, William Lindsey White b,

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Qiuxian Lai a, Nan Cai a, Wenqi Luo a, Liang Gu a, Sheng Li a, Hong Xu a, Zhangli

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Hu a, Shaoping Nie c, Xu Xu a,*

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a

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Bioresources and Ecology, Shenzhen University, Shenzhen 518060, PR China.

8

b

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and Environmental Sciences, Auckland University of Technology, Auckland 1142,

College of Life Sciences and Oceanography, Shenzhen Key Laboratory of Marine

School of Science and School of Interprofessional Health Studies, Faculty of Health

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New Zealand.

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c

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Nanchang 330047, Jiangxi, China.

13

*

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+86-755-26534274, +64-9-9219175

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E-mail addresses: [email protected] (X. Xu) and [email protected] (J. Lu)

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1

State Key Laboratory of Food Science and Technology, Nanchang University,

Corresponding authors. Tel.: +86-755-26534977, +64-9-9219999x7381; fax:

These authors contributed equally to this work.

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ACS Paragon Plus Environment


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Abstract

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Fucoidan, a sulfated polysaccharide extracted from brown seaweeds, has been

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shown to possess various bioactivities. In particular, low molecular weight fucoidan

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(LMWF) has been shown to have better bioactivities. In this study, a LMWF (< 10

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kDa) was extracted from New Zealand Undaria pinnatifida and investigated for its

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immune modulation effects. LMWF at a concentration range from 1 to 50 µg/ml

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exerted an effective immune activation in RAW264.7 macrophages. LMWF treatment

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promoted significant NO release, iNOS expression and TNF-α and IL-6 secretion in a

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concentration-dependent manner. It also significantly stimulated the activation of

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NF-κB and MAPK signalling pathways, and specific inhibitors of NF-κB and MAPK

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pathways diminish the stimulation, confirming the activation pathways. These results

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indicate that LMWF possesses potential health benefits through immune-stimulation,

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which may lead to future pharmaceutical development.

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Keywords: low molecular weight fucoidan; Undaria pinnatifida; immune activation;

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nitric oxide, New Zealand.

34 35


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Introduction

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The innate immune system of mammalian is mediated by phagocytes such as

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macrophages and neutrophils, which is the first line of defence against pathogens.1

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Macrophage, an important component of the innate and adaptive immune system,

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plays a vital role in host defence against infection and tumor through inflammation

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response,2 in which pro-inflammatory mediators are working together.3 The

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inflammatory mediator production in RAW264.7 macrophages have been reported to

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be regulated by NF-κB and MAPK signalling pathways.4 NF-κB (p65:p50 dimer)

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migrates within the cell matrix and binds to the IκB in resting cells. After stimulated

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by various stimulants, the IκB is phosphorylated and releases p65:p50 dimers. Then,

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p65:p50 dimers pass through the nucleopore to enter the nucleus and bind the target

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gene to enhance its transcription.5,6 MAPKs are comprised of three protein kinase

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subtypes: p38, c-Jun JNK and ERK, and are regulated by Ras. These kinases control

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many physiological processes, such as cell differentiation, proliferation and immune

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response.7

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Marine seaweed polysaccharides, including fucoidan, alginate and carrageenan,

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have been studied in diverse research fields including drug and functional food

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development.8,9 Fucoidan mainly exists in the cytoderm and intercellular space of

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edible brown seaweeds where it occupies about 40% dry weight of the alga cell walls

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and approximately 16% dry weight of the whole seaweed.10,11.

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Brown seaweeds such as Ascophyllum nodosum, Fucus vesiculosus, Laminaria

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angustata and Undaria pinnatifida are often used for the preparation of fucoidan.8



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Fucoidan is mainly composed of fucose and also contains some galactose, xylose,

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uronic acids and even protein in various quantities.12 It is proposed that the fucose

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units are linked via either (1-3)-glycosidic bonds, or alternating (1-3)- and

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(1-4)-glycosidic bonds. Sulphate groups are mainly at O-2 or O-4 positions of the

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fucose backbone. There are single fucoside or short fuco-oligosaccharide side chains

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at C-2 or C-4 position of the backbone. Acetylation may occur at the O-2 position of

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the fucose unit.13 While fucoidan used to be considered as a polysaccharide consists

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of a fucose-only-backbone, some authors now argue that the term fucoidan should

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include

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(1-2)-β-D-mannose backbones.13 Since fucoidan has a structure similar to heparin, it

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has potent activities of anticoagulation and antithrombosis.14

sulphated

galactofucans

which

have

(1-6)-β-D-galactose

and/or

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It has been reported that fucoidan has numerous other bioactivities such as

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antitumor,15 anti-inflammatory,16 antiviral,13 and immunomodulatory17 activities.

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However, it is difficult for the human body to utilize fucoidan due to its high

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molecular weight (MW) which would affect the absorption.18 Cho et al. have reported

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that low MW fucoidan (LMWF) extracted from Korea U. pinnatifida has more

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effective anticancer activity compared with high MW fucoidan (HMWF).19 As

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reported, fucoidan can induce nitric oxide (NO) production via p38 MAPK and

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NF-κB-dependent signalling pathways.20 Furthermore, enzyme-modified Korea

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Hizikia fusiforme, of which the main component is LMWF, has shown a more

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significant promoting effect of NO and tumor necrosis factor-α (TNF-α) production

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and phagocytotic activity of RAW264.7 cells than normal extracts.21 It is known that


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fucoidan varies significantly between source species, the environment the source

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seaweeds were cultivated in or harvested from, and the time of the harvest during the

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year. No two isolated fucoidans are exactly the same even if they are extracted from

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the same seaweed species; they are all unique in their structure, composition and

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bioactivities.13

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In this research, we assessed the immunomodulatory function of a LMWF

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extracted from New Zealand U. pinnatifida (which has never been studied for its

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effects on the immune system) on murine macrophage RAW264.7 cells in terms of

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NO and cytokine production and the NF-κB and MAPK signalling pathways

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activation.

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Experimental Section

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Materials

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Lipopolysaccharides (LPS), Polymyxin B (PMB), NG-nitro-L-arginine methyl

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ester (L-NAME) and Pyrrolidine dithiocarbamate (PDTC) were purchased from

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Sigma-Aldrich (St. Louis, MO, USA). Inhibitors SB 20358, SP 600125 and PD 98059

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were obtained from Selleck (Shanghai, China). Cell counting kit (CCK)-8 and

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radioimmunoprecipitation assay (RIPA) buffer were obtained from Beyotime (Jiangsu,

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China). Antibodies against inducible nitric oxide synthase (iNOS), IκB-α,

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phosphor-IκB-α (p-IκB-α), p65, p-p65, p38, p-p38, JNK, p-JNK, ERK and p-ERK

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and horseradish peroxidase (HPR)-conjugated secondary antibody were purchased

100

from Cell Signaling Technology (Beverly, MA, USA). Antibodies against α-tubulin

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and GAPDH were obtained from Proteintech (Hubei, China).



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Preparation of the fucoidan and its fractions

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Fucoidan was prepared from New Zealand U. pinnatifida as described previously.22

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In brief, seaweed was soaked in 70ºC hot water overnight. After centrifugation, 2%

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calcium chloride was added to remove alginate. Then, anhydrous ethanol was added

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to precipitate fucoidan. Fucoidan was freeze-dried to obtain powder, packed under

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nitrogen, sealed in foil pack, and stored in dark and room temperature (RT) prior to

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further analyses.

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The crude fucoidan’s chemical composition has been analyzed. The total

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carbohydrate content of the fucoidan samples was quantified by the phenol-sulfuric

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acid method using fucose as a standard. The sulphate content was determined by the

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BaCl2-gelatin method with K2SO4 as the standard after hydrolyzing the samples in

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1M HCl at 105˚C for 12 hours. Uronic acid was measured by the carbazole-sulfuric

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acid method using glucuronic acid as the standard. The protein content was estimated

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by a Bicinchoninic acid protein assay kit (Beyotime Biotech., Jiangsu, China). The

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separation and quantification of monosaccharide constituents of the fucoidan

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polysaccharides

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chromatography coupled with a pulsed amperometric detector (HPAEC-PAD)

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(Thermo Fisher Scientific, Waltham, MA, USA ).

were

determined

by

high-performance

anion

exchange

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LMWF was obtained via dialysis. Fucoidan was dissolved in water to make 1%

121

solution. The solution was put into a 10,000 Dalton dialysis bag and kept at 4 oC for

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three days. The solution out of the bag was collected and freeze-dried to obtain

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300

197

kDa), middle molecular weight fucoidan (MMWF, 10-300 kDa) and LMWF (< 10

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kDa) (Fig. 1A). Using dialysis bag to separate fucoidan into different molecular

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fraction recovered 90% of the fucoidan, with 16% 300 kDa

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fractions. Fractions were re-run with HPGPC and the resulting chromatograms were

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overlaid in Figure 1A. The LMWF contains 2.76±0.31% sugar, 1.42±0.03% uronic

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acid, 7.04±0.47% amino acids, and 16.62±1.31% sulfate.

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Endotoxin pollution assay of fucoidan

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Endotoxin LPS, a vital cytoderm constituent of the Gram-negative bacteria, is an

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effective trigger of inflammatory mediators.25,26 We used PMB, an LPS inhibitor, to

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assess whether there was any endotoxin pollution in fucoidan. RAW264.7 cells were

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treated with 100 ng/mL of LPS or 50 µg/mL of fucoidan (Crude, HMWF and LMWF

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fucoidan) with or without PMB (2.5 µg/mL), and NO release in the medium was


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measured. NO secretion in LMWF-treated cells was not impacted by PMB. However,

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NO secretions in Crude and HMWF treatments were inhibited by PMB significantly

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(Fig. 1B). These results suggested that the endotoxin contamination might exist in the

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Crude and HMWF fractions, but not in the LMWF fraction, and the observed

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LMWF-triggered NO release was the result of activation of the macrophage by

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LMWF itself. Therefore, we further studied the macrophage activation induced by

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LMWF instead of by Crude and HMWF.

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LMWF triggers NO release in RAW264.7 macrophages.

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Up to 50 µg/mL of LMWF showed no cytotoxicity on RAW264.7 macrophages

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(Fig. 2A). Next, we investigated NO release triggered by LMWF in RAW264.7

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macrophages. As presented in Fig. 2B and 2C, NO production was induced by

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LMWF in time- and concentration-dependent manners and the NO production in

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macrophage reached a higher level while treated with 50 µg/mL of LMWF for 24 h.

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LMWF enhances iNOS levels in RAW264.7 macrophages.

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NO production in macrophages is from L-arginine and regulated by iNOS.27 LMWF

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treatment significantly up-regulated both the mRNA and protein expression of iNOS

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in RAW264.7 macrophages in a concentration-dependent manner (Fig. 3A and B).

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Furthermore, NO production in LMWF-triggered RAW264.7 macrophages was

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attenuated by NOS inhibitor L-NAME (Fig. 3C).

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LMWF induces TNF-α and IL-6 secretion in RAW264.7 macrophages.

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In the innate immune system, pro-inflammatory mediators secreted by

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macrophages serve vital roles in defence against pathogens.3 TNF-α and IL-6



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secretions in LMWF-treated RAW264.7 macrophages were measured using an

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ELISA kit. As presented in Fig. 4A and B, TNF-α and IL-6 productions were elevated

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dose-dependently with LMWF stimulation. We also determined the mRNA level of

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TNF-α and IL-6 in LMWF-treated RAW264.7 macrophages using RT-PCR analysis.

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As shown in Fig. 4C, LMWF treatment significantly up-regulated the mRNA

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expression of TNF-α and IL-6 in a concentration-dependent manner.

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LMWF activates the NF-κB and MAPK signalling pathways in RAW264.7

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macrophages.

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NF-κB

and

MAPK

signalling

pathways

are

the

major

regulators

of

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pro-inflammatory mediator production in activated macrophages.28,29 The NF-κB and

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MAPK signalling pathways activation were analyzed using Western blot analysis

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after RAW264.7 macrophages had been stimulated by LMWF or LPS for 15, 30 and

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60 min. As shown in Fig. 5A, the levels of phosphorylated IκB-α and p65 induced by

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LMWF in RAW264.7 macrophages were in a dose-dependent manner. However, the

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p-IκB-α and p-p65 levels in macrophage treated with LMWF for 60 min were lower

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than that for 30 min (Fig. 5A). As for the MAPK signalling pathway, LMWF rapidly

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induced the phosphorylation of p38 in time- and concentration-dependent manners

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(Fig. 5B). Interestingly, LMWF induced phosphorylation of JNK showed a significant

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change after 30 min stimulation, however, the p-ERK levels show a weak change

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until 60 min stimulation (Fig. 5B).

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In order to confirm whether the activation of NF-κB and MAPK signalling

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pathways was associated with NO, TNF-α and IL-6 production in RAW264.7


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macrophages stimulated by LMWF, inhibitors of NF-κB, p38, JNK and ERK were

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used. It was demonstrated that NO, TNF-α and IL-6 secretion in LMWF-induced

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RAW264.7 macrophages was attenuated by NF-κB inhibitor PDTC, p38 inhibitor SB

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20358 or JNK inhibitor SP 600125 (Fig. 5C, D and E); while there was no effect of

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ERK inhibitor PD 98095 on NO and TNF-α production (Fig. 5C and D). However,

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the PD 98095 could attenuate the IL-6 production in LMWF-triggered RAW264.7

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cells, it suggests that the weak activation of ERK MAPK induced by LMWF play an

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important role in the IL-6 secretion. Above results suggest that LMWF from New

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Zealand U. pinnatifida activated macrophages mainly via NF-κB and MAPK

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signalling pathways.

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Discussion

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This is the first study to investigate the immune activation and its mechanism of

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LMWF from New Zealand U. pinnatifida. As reported, fucoidan has various

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bioactivities, and these bioactivities are associated with the molecular weight,24

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monosaccharide composition, the degree of sulphation,30 glycosidic linkages, the

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degree of branching and substitution, and chain conformation.31 It has been reported

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that fucoidan has the ability to activate immunity.20,32 However, structure and

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bioactivity of fucoidan vary significantly owing to the source species, cultivated

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environment, time of harvesting and method of extraction.33 Recent studies have

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discovered that LMWFs from Korea Hizikia fusiforme and Iran Sargassum

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angustifolium have a much stronger ability on promoting NO secretion in RAW264.7

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macrophages.21,32 Another study finds that fucoidan purchased from Sigma-Aldrich



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can promote NO release in RAW264.7 macrophages through p38 MAPK and NF-κB

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signalling pathways.20 However, the species from which this fucoidan was extracted

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has not been mentioned. There are three types of fucoidan available from

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Sigma-Aldrich, extracted from Fucus vesiculosus, U. pinnatifida and Macrocystis

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pyrifera, respectively. One report shows that fucoidan provided by Sigma-Aldrich

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(Fucus vesiculosus) does not have a strong immune activation ability.8 Another study

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suggests

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immunomodulatory function than that derived from U. pinnatifida, both from

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Sigma-Aldrich.34 As mentioned before, various fucoidans extracted from different

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species have different bioactivities.33 Therefore, it is important to study the

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immunomodulatory effect of fucoidan extracted from different species and areas. In

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this study, we prepared an LMWF (

Immune Activation of RAW264.7 Macrophages by Low Molecular

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