Molecular mechanisms and in vivo evaluation of the anti-inflammatory

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Molecular mechanisms and in vivo evaluation of the antiinflammatory effect of seleno-polymannuronate derived from alginate Bi Decheng, Lai Qiuxian, Cai Nan, Li Tong, Zhang Yiyao, Han Qingguo, Peng Yanwen, Hong Xu, Jun Lu, Bao Weiyang, Qiong Liu, and Xu Xu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05719 • Publication Date (Web): 06 Feb 2018 Downloaded from http://pubs.acs.org on February 7, 2018

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Journal of Agricultural and Food Chemistry

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Molecular mechanisms and in vivo evaluation of the anti-inflammatory effect of

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seleno-polymannuronate derived from alginate

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Decheng Bia, Qiuxian Laia, Nan Caia, Tong Lia, Yiyao Zhanga, Qingguo Hana, Yanwen

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Pengb, Hong Xua, Jun Lua, c, Weiyang Baod, Qiong Liua, Xu Xua, *

5

a

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

7

b

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Hospital of Sun Yat-sen University, Guangzhou 510630, PR China.

9

c

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

Cell-gene Therapy Translational Medicine Research Center, The Third Afﬁliated

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

10

and Environmental Sciences, and Institute of Biomedical Technology, Auckland

11

University of Technology, Auckland 1142, New Zealand.

12

d

College of fisheries and life, Dalian Ocean University, Dalian 116023, PR China.

13 14

AUTHOR INFORMATION

15

Corresponding Author

16

*

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[email protected].

Dr Xu Xu: Tel: +86-755-26534977; fax: +86-755-26534274; E-Mails:

18

1

ACS Paragon Plus Environment


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ABSTRACT

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Alginate-derived polymannuronate (PM) is a type of polysaccharide found in edible

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brown seaweeds. Seleno-polymannuronate (Se-PM) was prepared from PM via

22

synthesis

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anti-inflammatory activity of Se-PM and the corresponding molecular mechanisms

24

were investigated. Se-PM significantly attenuated the following in lipopolysaccharide

25

(LPS)-activated murine macrophage RAW264.7 cells: the production of nitric oxide

26

(NO), prostaglandin E2 (PGE2) and reactive oxygen species (ROS); the expression of

27

inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2); and the

28

secretion of pro-inflammatory cytokines. Moreover, Se-PM remarkably suppressed

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LPS-induced activation of the nuclear factor (NF)-κB and mitogen-activated protein

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kinase (MAPK) signalling pathways in RAW264.7 cells. Furthermore, Se-PM also

31

decreased the production of pro-inflammatory mediators in LPS-triggered primary

32

murine macrophages. Additionally, Se-PM inhibited the inflammatory response in the

33

air pouch inflammation model. These results might contribute to the overall

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understanding of the potential health benefits of Se-PM for food and drug

35

applications.

using

a

sulfation

and

selenation

replacement

reaction.

The

36 37

KEYWORDS: seleno-polymannuronate, lipopolysaccharide, anti-inflammation,

38

nuclear factor-κB, mitogen-activated protein kinase

39

2


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INTRODUCTION

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As important components of the innate and adaptive immune system, macrophages

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play crucial roles in host defence against infection through inflammation, in which

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various factors work together.1 Inflammation is a double-edged sword and must be

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precisely regulated. Prolonged and excessive inflammation is involved in the

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pathogenesis of almost all human degenerative diseases, including cancer, arthritis,

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cardiovascular diseases, type 2 diabetes, Alzheimer’s disease and so on.2-4 LPS, the

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molecular basis of the Gram-negative bacterial cell wall, is an important ligand of

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Toll-like receptor 4 (TLR4) and interacts with TLR4 with the assistance of myeloid

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differentiation factor 2 (MD2), eliciting downstream signalling and triggering the

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secretion of inflammatory mediators.5,6 The nuclear factor-κB (NF-κB) and

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mitogen-activated protein kinase (MAPK) signalling pathways have vital functions in

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TLR4 signalling and regulate the immuno-inflammatory response.7 In resting cells,

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NF-κB migrates within the cell matrix in the form of p65:p50 dimers and binds to IκB,

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the inhibitor of NF-κB. Following phosphorylation and proteasomal degradation of

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IκB, NF-κB p65:p50 dimers are released and translocate to the nucleus, where they

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subsequently enhance transcription of target genes.8 MAPKs comprise three protein

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kinase subtypes: p38 kinases, extracellular signal-regulated kinases (ERKs) and c-Jun

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N-terminal kinases (JNKs); these kinases control a number of physiological processes,

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such as the immune response, apoptosis, cell differentiation and proliferation.9

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Inflammatory mediators, including nitric oxide (NO), prostaglandin E2 (PGE2),

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tumour necrosis factor (TNF), the interleukins (ILs) and reactive oxygen species 3



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(ROS) play primary roles in inflammatory responses and are regulated by the NF-κB

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and MAPK signalling pathways.10-12

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Alginate is a naturally occurring acidic polysaccharide that comprises alternations

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of β-D-mannuronate (M) and its C-5 epimer α-L-guluronate (G) with 1,4-glycosidic

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linkages;

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polymannuronate (PM) blocks, homopolymeric polyguluronate (PG) blocks and

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heteropolymeric blocks (PMG)13,14 and exist widely in the cell walls of various edible

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brown seaweeds such as Laminaria hyperborean, Macrocystis pyrifera and

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Ascophyllum nodosum.15 Alginate and its derivatives from different sources exert

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various biological and pharmaceutical activities, including neuroprotective,16

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anti-inflammatory,17

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effects.22,23 Due to its stability, biodegradability and lack of toxicity, alginate has been

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widely used in the food, pharmaceutical, cosmetic and textile industries.24

the

residues along

the

chain

immunostimulatory,18-20

are

arranged

anti-tumour21

in

homopolymeric

and

antioxidant

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Selenium is a trace element that is essential for nutrition and is closely related to

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the normal activities of life.25 Se is also an important component of selenoenzymes,

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such as glutathione peroxidase (GPx) and phospholipid hydroperoxide glutathione

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peroxidase (PhGPx), which prevents cells from severe oxidative damage induced by

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ROS.25,26 Organic selenium refers to any organic compound wherein selenium

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replaces sulfur, such as selenoprotein and seleno-polysaccharide (Se-polysaccharide),

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and is normally less toxic and better bioavailable than inorganic selenium although

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different form of organic selenium has different degree of bioavailability.27,28

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Se-polysaccharide is an important organic selenium derivative. Se-polysaccharide 4


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includes natural materials extracted from plants or synthesized compounds with

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selenium and polysaccharides and exerts more efficient antioxidant activities than

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selenium-free polysaccharides in vitro and in vivo.29,30 Selenium-containing

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polysaccharides extracted from Hericium erinaceum present antioxidant activity as

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evidenced by in vitro assays of lipid peroxidation inhibition and scavenging free

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radicals.29 Selenium-polysaccharides from the mycelia of Coprinus comatus inhibit

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the oxidative stress in alloxan-induced diabetic mice.30 Selenylation modification of

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polysaccharide obtained from Radix hedysari inhibits the Aβ25-35-induced oxidative

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damage in SH-SY5Y cells.31

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We recently synthesized a Se-polysaccharide derivative, Se-polymannuronate

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(Se-PM), using alginate-derived PM and sodium selenite (Na2SeO3). Our previous

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study demonstrated that Se-PM could exhibit significant antioxidant activity in H2O2

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induced Neuro-2a (N2a) cells and suppress the apoptosis of N2a-sw-APP695 cells, an

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Alzheimer's disease (AD) cell model.32 In this study, the molecular mechanisms

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underlying the anti-inflammatory activity of Se-PM were investigated in

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LPS-activated RAW264.7 macrophages, LPS-activated primary murine peritoneal

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macrophages and carrageenan-induced air pouch inflammation mouse model.

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MATERIALS AND METHODS

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Materials. Alginate, sulfur trioxide pyridine complex (SO3-Py), LPS, fluorescein

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isothiocyanate (FITC)-conjugated LPS, 4′,6-diamidino-2-phenylindole (DAPI) and

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carrageenan were obtained from Sigma-Aldrich (St. Louis, MO). RPMI-1640 medium,

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penicillin and streptomycin were purchased from HyClone (Logan, UT). Foetal 5



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bovine serum (FBS) was obtained from Biological Industries (Beit-Haemek, Israel).

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Antibodies against Akt, phosphor-Akt (p-Akt), p65, phosphor-p65 (p-p65), IκB-α,

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phosphor-IκB-α (p-IκB-α), p38, phosphor-p38 (p-p38), ERK, phosphor-ERK (p-ERK),

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JNK, phosphor-JNK (p-JNK), iNOS, and COX-2, along with Alexa Fluor

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596-conjugated secondary antibody and horseradish peroxidase (HRP)-conjugated

111

secondary antibody, were purchased from Cell Signaling Technology (Beverly, MA).

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Anti-β-actin and anti-α-tubulin antibodies were purchased from Proteintech (Hubei,

113

China). The enzyme-linked immunosorbent assay (ELISA) kit for PGE2 was

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purchased from Cayman Chemical Co. (Ann Arbor, MI). ELISA kits for TNF-α,

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IL-1β, IL-6 and IL-12 were purchased from Neobioscience Technology Company

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(Guangdong, China). 2′,7′-Dichlorofluorescein diacetate (DCF-DA), Cell Counting

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Kit (CCK)-8 and radioimmunoprecipitation assay (RIPA) buffer were obtained from

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Beyotime Institute of Biotechnology (Jiangsu, China). All other chemicals were

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obtained from Macklin Biochemical Technology (Shanghai, China).

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Preparation of Se-PM. Se-PM, a seleno-derivative of PM, was prepared from PM

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via synthesis using a seleno-reaction as described in our previous study.32 Briefly, PM

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and SO3-Py (1:6, w/w) were mixed and reacted in dimethyl methanamide at 60°C for

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4 h to obtain the reactant, sulfonated PM (S-PM). The existence of sulphate groups of

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S-PM was confirmed by the barium chloride (BaCl2)-gelatin method. Then, S-PM

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was reacted with Na2SeO3 (1:2, w/w) in H2O using nitric acid (HNO3) and BaCl2 as

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catalysts at 60°C for 8 h to prepare Se-PM. The resulting mixture was then purified

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through dialysis, and Se-PM was obtained after freeze drying and stored prior to 6


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further analyses.

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Se content determination. Ten mg of Se-PM was digested with a mixture of

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perchloric acid and HNO3 (1:4, v/v) overnight in a glass beaker. Then the glass beaker

131

was heated to produce white smoke using an electrothermal furnace. The residue was

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dissolved in 6 M HCl after cooling. The solution was heated again and was diluted

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with 0.6 M HCl to a constant volume. The Se content of Se-PM was determined using

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the atomic fluorescence spectrometer (Titan Instruments Co., Ltd., Beijing, China). Se

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standard solution [GBW(E)080215, 100 pg/ml] was obtained from the National

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Standard Material Research Center (Beijing, China).

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Cell culture. Primary murine peritoneal macrophages were isolated from

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6-week-old female BALB/c mice 4 days after intraperitoneal (i.p.) injections of 4%

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starch medium. Murine RAW264.7 macrophages and primary murine peritoneal

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macrophages were cultured in RPMI-1640 medium, which was supplemented with 10%

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FBS, 100 µg/ml streptomycin and 100 IU/ml penicillin. The cells were grown in an

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RS Biotech incubator with a humidified atmosphere at 37°C with 5% CO2.

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Cell viability assay. Cell viability was tested with the CCK-8 kit according to the

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manufacturer’s instructions. Briefly, RAW264.7 cells (2×105 cells/well) were seeded

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into 96-well plates and incubated with different concentrations of Se-PM (0.2, 0.4 and

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0.8 mg/ml) or 0.8 mg/ml PM for 2 h and treated with 1 µg/ml LPS for 22 h at 37°C.

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After the CCK-8 reagent was added, the absorbance was measured at 540 nm using a

148

Spectra Max microplate reader (Thermo Scientific, Hudson, NH).

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NO assay. RAW264.7 cells (2×105 cells/well) or primary murine peritoneal 7



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macrophages (1×105 cells/well) in 96-well plates were pretreated with different

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concentrations of Se-PM (0.2, 0.4 and 0.8 mg/ml) or 0.8 mg/ml PM for 2 h and then

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co-cultured with 1 µg/ml LPS for an additional 22 h at 37°C. The concentration of

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NO2- in the culture medium was examined with the Griess reagent [1% sulfanilamide,

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0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride and 2.5% phosphoric acid].

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Briefly, 50 µl of cell culture medium was transferred into a new 96-well plate and

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mixed with 100 µl of Griess reagent. After incubation at room temperature (RT) for 5

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min, the absorbance was measured at 540 nm using a Spectra Max microplate reader

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(Thermo Scientific, Hudson, NH).

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PGE2 assay. RAW264.7 cells (2×105 cells/well) in 96-well plates were pretreated

160

with different concentrations of Se-PM (0.2, 0.4 and 0.8 mg/ml) or 0.8 mg/ml PM for

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2 h and then treated with 1 µg/ml LPS for an additional 22 h at 37°C. The

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concentration of PGE2 in the culture medium was examined with ELISA kits

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according to the manufacturer’s instructions.

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Cytokine assay. RAW264.7 cells (2×105 cells/well) or primary murine peritoneal

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macrophages (1×105 cells/well) in 96-well plates were pretreated with different

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concentrations of Se-PM (0.2, 0.4 and 0.8 mg/ml) or 0.8 mg/ml PM for 2 h and then

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treated with 1 µg/ml LPS for an additional 22 h at 37°C. The levels of TNF-α, IL-1β,

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IL-6 and IL-12 in the culture medium were measured with ELISA kits according to

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the manufacturer’s instructions.

170 171

Intracellular ROS measurement. RAW264.7 cells (5×105 cells/well) in 24-well plates were pretreated with different concentrations of Se-PM (0.2, 0.4 and 0.8 mg/ml) 8


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or 0.8 mg/ml PM for 2 h and then treated with 1 µg/ml LPS for an additional 22 h at

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37°C. Then, a fluorescent probe, DCFH-DA (10 µM), was added to the cells and

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incubated at 37°C for 20 min. Then, the cells were suspended in PBS, and

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intracellular ROS was measured by the fluorescence activated cell sorting (FACS)

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system (Becton Deckinson, San Jose, CA) with excitation and emission wavelengths

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of 488 and 525 nm, respectively.

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RNA isolation and RT-PCR. RAW264.7 cells (1×106 cells/well) in 6-well plates

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were pretreated with different concentrations of Se-PM (0.2, 0.4 and 0.8 mg/ml) or

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0.8 mg/ml PM for 2 h and then treated with 1 µg/ml LPS for an additional 10 h at

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37°C. Total RNA was extracted with the RNA Extraction Kit (RNAfast2000, Fastagen,

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shanghai, China), and 1 µg of the RNA was reverse transcribed to cDNA using the

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PrimeScript first strand cDNA Synthesis Kit (TaKaRa Biotechnology Co., Ltd.,

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Liaoning, China). PCR reactions were performed in 50-µl reaction mixtures

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containing 23 µl of Premix Taq (TaKaRa Biotechnology), 1 µl of forward and reverse

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iNOS primers, COX-2 Primers or β-actin primers (20 µM), 5 µl of cDNA, and 20 µl

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of nuclease-free water with the following parameters: 1 cycle for 180 s at 95°C; 26

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cycles for 55 s at 93°C, 45 s at 60°C, and 40 s at 72°C; and 1 cycle for 100 s at 72°C.

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The primer sequences for iNOS, COX-2 and β-actin were as follows: iNOS, Forward

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5′-CAA CCA GTA TTA TGG CTC CT-3′, Reverse 5′-GTG ACA GCC CGG TCT

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TTC CA-3′; COX-2, Forward 5′-CCA CTT CAA GGG AGT CTG GA-3′, Reverse

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5′-AGT CAT CTG CTA CGG GAG GA-3′; and β-actin, Forward 5′-GGA GAA GAT

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CTG GCA CCA CAC C-3′, Reverse 5′-CCT GCT TGC TGA TCC ACA TCT GCT 9



194

GG-3′. Each PCR reaction product was resolved on a 1% agarose gel and observed

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with a G-box imaging system (Syngene, Cambridge, UK).

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Western blot analysis. RAW264.7 cells (1×106 cells/well) in 6-well plates were

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pretreated with different concentrations of Se-PM (0.2, 0.4 and 0.8 mg/ml) or 0.8

198

mg/ml PM for 2 h and then treated with 1 µg/ml LPS for an additional 22 h at 37°C.

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After washed by cold PBS, cells were collected and lysed on ice with RIPA buffer

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containing a protease inhibitor cocktail (Selleck, Shanghai, China). The same amount

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of protein in each sample (30 µg) was resolved by 10% sodium dodecyl

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sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a

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polyvinylidene fluoride (PVDF) membrane. The membranes were blocked with 5%

204

(w/v) skim milk at RT for 2 h and incubated with iNOS and COX-2 primary

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antibodies at 4°C overnight. After 3 washes in Tris-buffered saline Tween-20 (TBST),

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membranes were incubated with HRP-conjugated secondary antibody at RT for 2 h.

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After rinsing, the proteins on the membranes were visualized with the LAS3000

208

luminescent image analyser (Fujifilm Life Science, Tokyo, Japan) using an ECL kit

209

(Thermo Scientific, Hudson, NH), and the band density of each protein was quantified

210

using the Quantity One software (Bio-Rad, Richmond, CA).

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To test the expression levels of Akt, NF-κB and MAPKs, RAW264.7 cells (1×106

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cells/well) in 6-well plates were preincubated with 0.8 mg/ml Se-PM or 0.8 mg/ml

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PM for 2 h and stimulated with 1 µg/ml LPS for 30 min at 37°C. After the proteins

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were obtained, the Western blot analysis was performed as described above using

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phosphorylated and nonphosphorylated Akt, IκB-α, p65, p38, JNK, and ERK primary 10


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antibodies.

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Immunofluorescence analysis. RAW264.7 cells (1×106 cells/well) seeded on

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sterile glass coverslips in 6-well plates were pretreated with 0.8 mg/ml Se-PM or 0.8

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mg/ml PM for 2 h and then treated with 1 µg/ml FITC-LPS for an additional 2 h at

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37°C. The cells on the coverslips were fixed immediately in 4% formaldehyde at RT

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for 30 min. After permeabilized with 0.2% Triton X-100 in PBS for 10 min, cells were

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incubated with DAPI at RT for 2 h. After additional washes, the fluorescence of

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FITC-LPS on RAW264.7 cell surfaces were observed by confocal microscopy (Carl

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Zeiss Jena Gmbh, Jena, Germany) and analysed using the ImageJ software (US

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National Institutes of Health, Bethesda, MD).

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To examine the nuclear translocation of NF-κB/p65, RAW264.7 cells (1×106

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cells/well) seeded on sterile glass coverslips in 6-well plates were pretreated with 0.8

228

mg/ml Se-PM or 0.8 mg/ml PM for 2 h and then treated with 1 µg/ml LPS for an

229

additional 2 h at 37°C. After fixated and permeabilized with the same method as

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above, cells were blocked with 10% (w/v) goat serum in PBS at 37°C for 1 h. Cells

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were then incubated with a NF-κB/p65 primary antibody at 4°C overnight. After

232

washed with cold PBS, cells were incubated with Alexa Fluor 596-conjugated

233

secondary antibody and DAPI at RT for 2 h. The nuclear translocation of the

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NF-κB/p65 subunit was observed by confocal microscopy (Carl Zeiss Jena Gmbh,

235

Jena, Germany) and analysed using the ImageJ software (US National Institutes of

236

Health, Bethesda, MD).

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Carrageenan-induced air pouch inflammation model. Specific pathogen-free 11



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(SPF) BALB/c mice (female, 6-week-old, 21.2 ± 0.7 g) were purchased from the

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Guangdong Laboratory Animal Monitoring Institute (Guangdong, China) and fed

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under the standard laboratory conditions: 12-h light and 12-h dark cycle, temperature

241

at 22 ± 2°C. All experiments were approved by the Regional Ethical Committee for

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Animal Experimentation. The mice received 5 ml of sterile air on an area of the dorsal

243

skin through subcutaneous injection after isoflurane inhalational anesthesia. The air

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pouch was injected with 5 ml of air after 3 days to establish a stable pouch. After 3

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days, the pouches received two injections of 100 µl of PBS, Se-PM (5 mg/mouse) or

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PM (5 mg/mouse) 24 h and 2 h before the injection of 2% (w/v) carrageenan. At 2 h

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following carrageenan administration, the mice were euthanized by inhalational

248

anesthesia with isoflurane and cervical dislocation. The serum samples were collected

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after clotting. Then, the pouch was lavaged with 2 ml of sterile PBS, and the air pouch

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exudate was obtained after the cells were separated by centrifugation at 400 ×g for 10

251

min. The levels of TNF-α and IL-6 in the air pouch exudate and serum were

252

measuring using ELISA kits.

253

Statistical analysis. The data for all experiments shown are the means

254

of triplicate assays in a single experiment and presented as the means ± standard

255

deviation (SD). The results were analysed using the two-tailed Student’s t-test to

256

determine any significant differences by GraphPad prism 5.01 (GraphPad Software,

257

Inc., La Jolla, CA). Differences were considered significant with the following p

258

values: * P < 0.05; ** P < 0.01; *** P < 0.001.

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RESULTS 12


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Se-PM inhibits LPS-triggered NO and PGE2 production in RAW264.7 cells.

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The selenium content of Se-PM was determined to be 197.5 µg/g. RAW264.7 cells

262

were incubated with 0.2, 0.4 and 0.8 mg/ml Se-PM, and 0.8 mg/ml PM respectively in

263

the presence of LPS, and cytotoxicity was evaluated using the CCK-8 kit. As shown

264

in Figure 1A, no cytotoxic effects were observed in response to Se-PM at any tested

265

concentration or PM.

266

NO and PGE2 are critical mediators that are released during the inflammatory

267

response.10,11 LPS-activated macrophages overproduce both NO and PGE2 and serve

268

as a general inflammation model for evaluating the anti-inflammatory activities of

269

agents in vitro. RAW264.7 cells were treated with 0.2, 0.4 and 0.8 mg/ml Se-PM, and

270

0.8 mg/ml PM in the presence of LPS, and the generation of NO and PGE2 was

271

measured using the Griess reagent and ELISA kit, respectively. Compared with the

272

control, NO and PGE2 production were significantly increased by the treatment with

273

LPS. However, pretreatment of RAW264.7 cells with Se-PM dose-dependently

274

decreased NO and PGE2 production compared with the LPS-only treatment, whereas

275

the pretreatment with PM had no effect (Figure 1B and C). In addition, both the

276

precursor S-PM of Se-PM and the inorganic selenium control compound (Na2SeO3)

277

also did not exert any effect on the LPS-induced NO production (data not shown).

278

Se-PM supresses LPS-triggered iNOS and COX-2 expression in RAW264.7

279

cells. The iNOS and COX-2 genes are vital regulators of NO and PGE2 production,

280

respectively, in the macrophage inflammatory response.33, 34 Therefore, the effects of

281

Se-PM and PM on iNOS and COX-2 mRNA and protein expression in RAW264.7 13



282

cells were determined by RT-PCR and Western blot analysis, respectively. As shown

283

in Figure 2A and B, the mRNA expression levels of iNOS and COX-2 were

284

significantly upregulated in the LPS-treated group compared with the control group

285

and notably reduced in a concentration-dependent manner by the pretreatment with

286

Se-PM; PM did not have this effect. The LPS-induced protein expression levels of

287

iNOS and COX-2 were also suppressed by the Se-PM pretreatment dose-dependently

288

but were not suppressed by the PM pretreatment (Figure 2C and D).

289

Se-PM decreases LPS-triggered pro-inflammatory cytokine secretion in

290

RAW264.7 cells. Pro-inflammatory cytokines including TNF-α, IL-1β, IL-6 and

291

IL-12 are important markers of the inflammatory response12 and secretion of

292

cytokines in macrophages by stimuli is an important factor in upregulating

293

inflammatory processes.1 An ELISA-based analysis was performed to determine

294

whether Se-PM could inhibit the production of the pro-inflammatory cytokines

295

triggered by LPS in RAW264.7 cells. Compared with the control, TNF-α, IL-1β, IL-6

296

and IL-12 production in the medium were significantly increased by treatment with

297

LPS. When cells were pretreated with 0.2, 0.4 and 0.8 mg/ml Se-PM, production of

298

the abovementioned cytokines stimulated by LPS was significantly decreased in a

299

dose-dependent manner. In contrast, PM did not have any statistically significant

300

effects on the production of pro-inflammatory cytokines in the LPS-activated

301

RAW264.7 cells (Figure 3A).

302

Se-PM attenuates LPS-triggered ROS production in RAW264.7 cells. ROS is

303

closely associated with the macrophage inflammatory response.35 The influence of 14


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Se-PM on ROS production triggered by LPS in RAW264.7 cells was examined by

305

FACS. As demonstrated in Figure 3B, intracellular accumulation of ROS in

306

RAW264.7 cells were markedly increased by LPS stimulation but was gradually

307

decreased by pretreatments with gradually increased doses of Se-PM in the presence

308

of LPS. However, PM also inhibited LPS-triggered intracellular ROS accumulation

309

(Figure 3B). These observations may be the result of polysaccharide involvement in

310

regulation of cellular homeostasis.36

311

Se-PM blocks the binding of LPS to RAW264.7 cells. In the LPS-stimulated

312

macrophage inflammatory response, LPS first interacts with the Toll-like receptor and

313

induces downstream inflammatory signal transduction.6 Therefore, the effect of

314

Se-PM on the binding of LPS to the RAW264.7 cell surface was examined using

315

FITC-LPS and observed by confocal microscopy. Herein, DAPI staining of nucleus is

316

used to test the shape and location of each cell. And the Merge images can display the

317

green fluorescence of FITC-LPS located on the cell surfaces. As shown in Figure 4A,

318

the fluorescence of FITC-labeled LPS on the RAW264.7 cell surfaces was strong

319

when cells were treated with FITC-LPS alone for 2 h but was obviously weakened by

320

pretreatment with Se-PM. Moreover, as expected, the addition of PM did not affect

321

the fluorescence intensity of FITC-LPS on the cell surfaces. The fluorescence

322

intensity of each group was quantified with the ImageJ software and is shown in

323

Figure 4B. The fluorescence intensity of FITC-LPS in the LPS-treated group (16.63 ±

324

1.07) was higher than that of the control group, and the Se-PM pretreatment decreased

325

fluorescence intensity to 14.78 ± 0.65 effectively, while PM pretreatment did not have 15



326

any effect on the fluorescence of FITC-LPS. These results indicate that the

327

anti-inflammatory activity of Se-PM may result from a reduced capacity of LPS

328

binding to macrophages and subsequent block of the initial step of signalling

329

transduction.

330

Se-PM prevents LPS-induced activation of the NF-κB signalling pathway in

331

RAW264.7 cells. The NF-κB signalling pathway is one primary signalling pathway

332

that is activated by LPS in the macrophage inflammatory response, which regulates

333

the production of pro-inflammatory mediators.7 The effect of Se-PM on LPS-induced

334

activation of the NF-κB signalling pathway in RAW264.7 cells was evaluated with

335

Western blot analysis. As expected, the Akt, IκB-α and p65 phosphorylation levels

336

were upregulated after LPS stimulation for 30 min in RAW264.7 cells (Figure 5A

337

and B). Nuclear translocation of NF-κB/p65 is also a critical step for activation of the

338

NF-κB signalling pathway. Therefore, immunofluorescence microscopy was

339

performed to determine whether Se-PM could inhibit nuclear translocation of the

340

NF-κB/p65 subunit. The results indicated that the p65 subunit dramatically

341

translocated to the nucleus after treatment with LPS alone for 2 h compared with the

342

control and that the Se-PM treatment significantly reduced the LPS-induced nuclear

343

accumulation of the p65 subunit (Figure 5C and D). Conversely, PM did not show

344

any significant effect on phosphorylation of Akt, IκB-α and p65 (Figure 5A and B) or

345

nuclear translocation of NF-κB/p65 (Figure 5C and D) compared with the

346

LPS-stimulated group.

347

Se-PM prevents LPS-induced activation of the MAPK signalling pathway in 16


Page 16 of 46

Page 17 of 46


348

RAW264.7 cells. The MAPK signalling pathway also regulates the LPS-induced

349

macrophage inflammatory response through generation of pro-inflammatory

350

mediators.7 A Western blot analysis was performed to determine whether the

351

inhibitory effect of Se-PM on the induction of pro-inflammatory mediator production

352

was associated with the MAPK signalling pathway. As shown in Figure 5E and F,

353

LPS-induced phosphorylation of p38, ERK and JNK was effectively suppressed by

354

the Se-PM treatment. Conversely, PM did not significantly affect LPS-induced

355

phosphorylation of p38, ERK and JNK. These results suggest that LPS-induced

356

activation of the MAPK signalling pathway might be inhibited by Se-PM.

357

Se-PM inhibits LPS-stimulated pro-inflammatory mediator production in

358

primary murine peritoneal macrophages. The effect of Se-PM on the production of

359

LPS-stimulated

360

macrophages was evaluated. As shown in Figure 6, compared with the control, the

361

production levels of NO, TNF-α, IL-1β and IL-6 in the medium were significantly

362

increased after the LPS treatment, while the production levels of the LPS-stimulated

363

pro-inflammatory mediators were significantly decreased in a dose-dependent manner

364

by the Se-PM pretreatment. In PM pretreatment group, PM did not show any obvious

365

effect on LPS stimulated production of pro-inflammatory mediators in primary

366

murine peritoneal macrophages.

367

Se-PM

pro-inflammatory

inhibits

mediators

pro-inflammatory

in

primary

cytokine

murine

production

peritoneal

in

the

368

carrageenan-induced air pouch inflammation model. The air pouch inflammation

369

model induced by subcutaneous injection of carrageenan (2%) was also utilized to 17



370

evaluate the anti-inflammatory activity of Se-PM in vivo. Se-PM (5 mg/mouse) or PM

371

(5 mg/mouse) was administered by subcutaneous injection 24 and 2 h before the

372

carrageenan injection. As shown in Figure 7, the injection of carrageenan into the air

373

pouch induced an increase in the TNF-α and IL-6 levels in both the air pouch exudate

374

and serum, whereas carrageenan-induced secretion of TNF-α and IL-6 were

375

significantly decreased in both the air pouch exudate and serum from the mice that

376

were injected with Se-PM; PM had no effect.

377

DISCUSSION

378

PM is a type of acidic polysaccharide that is derived from alginate and has

379

antioxidant and anti-coagulant activities.37,38 Selenium, as an essential trace element,

380

is closely associated with the normal activities of life because of its role in an

381

antioxidant enzyme.25 Although selenosis toxicity in humans is very rare, endemic

382

selenium toxicity in some parts of China is still existed,28 and selenosis from

383

industrial accidents also happened occasionally due to inhalation. Organic selenium is

384

regarded to be far less toxic and mutagenicity, and have a better bioavailability than

385

inorganic selenium.27,28 Seleno-polysaccharide is an organic selenium resource and

386

exhibits low toxicity and has antioxidant activity29,30 and neuroprotective effects in

387

vitro.31,32

388

In this study, we synthesized a seleno-polysaccharide, Se-PM. We found that

389

Se-PM, rather than its mother compound PM or precursor S-PM (data not shown),

390

statistically inhibited the inflammatory response in LPS-stimulated RAW264.7

391

macrophages, in LPS-activated primary murine peritoneal macrophages and in 18


Page 18 of 46

Page 19 of 46


392

carrageenan-induced air pouch inflammation mouse model. To our knowledge, this is

393

the first study to investigate the anti-inflammatory activities of a Se-derivative of PM

394

in an inflammation model.

395

In the presence of a stimulus such as LPS, activated macrophages are a widely used

396

model for evaluating and exploring the potent mechanisms of anti-inflammatory drugs

397

and natural compounds in vitro.39 The NO and PGE2 levels closely correlate with the

398

degree of inflammation, and excessive NO and PGE2 production via changes in iNOS

399

and

400

inflammation.40,41 In this study, we found that the Se-PM treatment decreased the

401

generation of NO and PGE2 and the mRNA and protein expression of iNOS and

402

COX-2, in a dose-dependent manner in LPS-induced RAW264.7 cells (Figure 1B, 1C

403

and 2). Furthermore, Se-PM also reduced the production of NO in LPS-induced

404

primary murine peritoneal macrophages (Figure 6A). Meanwhile, our results showed

405

that the production levels of pro-inflammatory cytokines, including TNF-α, IL-1β,

406

IL-6 and IL-12, and ROS were significantly suppressed by the Se-PM treatment in

407

LPS-activated RAW264.7 cells and primary murine peritoneal macrophages (Figure

408

3A and 6B).

COX-2 expression,

respectively,

occurs

in

both acute and

chronic

409

Several intracellular signalling pathways, including the NF-κB and MAPK

410

signalling pathways, can be activated by LPS, which is the key activator of the

411

inflammatory response in macrophages.7 In resting cells, NF-κB migrates in the cell

412

matrix in the form of p65:p50 dimers and binds to IκB. Upon exposure to

413

pro-inflammatory stimuli, such as LPS, TNF-α and IL-1, IκB is rapidly 19



414

phosphorylated and undergoes proteasomal degradation. The NF-κB p65:p50 dimers

415

are then released and translocate into the nucleus, where they regulate transcription of

416

target genes, such as iNOS, TNF-α and IL-1β.8 Our results demonstrated that the

417

Se-PM treatment could block LPS-induced phosphorylation of IκB-α and Akt, its

418

upstream kinase, leading to reduced phosphorylation and nuclear translocation of p65

419

(Figure 5A-D). The p38, ERK and JNK MAPKs are the major components in the

420

MAPK signalling pathway.9 We found that the Se-PM treatment significantly

421

decreased phosphorylation of p38, ERK and JNK (Figure 5E and F). Meanwhile, we

422

also found that Se-PM might block the binding of LPS to cell surface (Figure 4).

423

Thus, we deduce that the anti-inflammatory capacity of Se-PM is primarily originated

424

from this blocking effect and inhibition of the NF-κB and MAPK signalling pathways

425

activation.

426

Additionally, the mouse air pouch model of carrageenan-induced inflammation was

427

used to investigate the potential anti-inflammatory effect of Se-PM in vivo. Our

428

results further confirmed that Se-PM could significantly ameliorate excessive

429

production of pro-inflammatory mediators in the carrageenan-induced air pouch

430

inflammation mouse model (Figure 7).

431

Although the relationships between the structures and functions of the alginate

432

derivatives are not completely characterized to date, due to the structural diversity and

433

heterogeneity of the derivatives, at least some bioactivities of the alginate derivatives

434

have distinct structural specificities. Our previous reports have shown that the

435

pharmacological activities of alginate are strongly influenced by structural details, 20


Page 20 of 46

Page 21 of 46


436

such as molecular size, M/G composition, and the entire molecular conformation.42-44

437

In this study, Se-PM, rather than PM, exhibited a distinguished anti-inflammatory

438

effect. The differences in between the biological activities of Se-PM and PM might be

439

partly caused by the existence of selenium.

440

To our knowledge, this study is the first to investigate the mechanisms that underlie

441

how the seleno-derivative of PM regulates the inflammatory response of macrophages.

442

We will further study the utility and application of Se-PM in anti-inflammatory food

443

additive in our future research work.

444

ABBREVIATIONS USED

445

Se-PM,

seleno-polymannuronate;

FBS,

foetal

bovine

serum;

LPS,

446

lipopolysaccharide; FITC, fluorescein isothiocyanate; Akt, protein kinase B; NF-κB,

447

nuclear factor-κB; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal

448

kinase; MAPK, mitogen-activated protein kinase; ROS, reactive oxygen species;

449

PGE2, prostaglandin E2; TNF, tumour necrosis factor; IL, interleukin.

450

ACKNOWLEDGEMENTS

451

This work was supported financially by the Science and Technology Innovation

452

Commission

of

Shenzhen

(JCYJ20170302144535707

453

JSGG20160229120821300), the National Natural Science Foundation of China

454

(31000770, 31470804 and 31540012), Science and Technology Planning Project of

455

Guangdong Province (2014A020212488), Yangzhou University Innovation Fund

456

(2014CXJ032) and Natural Science Foundation of Jiangsu Province (BK20141276).

457

NOTES 21


and


458

All authors declare that there are no conflicts of interest.

459

22


Page 22 of 46

Page 23 of 46


460

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585 586

28


Page 28 of 46

Page 29 of 46


587

FIGURE LEGENDS

588

Figure 1. Se-PM reduces the production of NO and PGE2 in LPS-activated

589

RAW264.7 cells. (A) Cell viability was measured using the CCK-8 kit. (B) NO

590

production in the culture supernatant was measured with the Griess reagent. (C)

591

Production of PGE2 in the culture supernatant was detected with an ELISA kit.

592

Representative results from three independent experiments are shown. * P < 0.05; **

593

P < 0.01; *** P < 0.001.

594 595

Figure 2. Se-PM suppresses the expression of iNOS and COX-2 in LPS-activated

596

RAW264.7 cells. (A-B) The mRNA expression levels of iNOS and COX-2 were

597

determined by RT-PCR (A) and normalized to β-actin mRNA (B). (C-D) The protein

598

expression levels of iNOS and COX-2 were detected by Western blot analysis (C) and

599

normalized to β-actin (D). Representative results from three independent experiments

600

are shown. * P < 0.05; ** P < 0.01; *** P < 0.001.

601 602

Figure 3. Se-PM decreases the secretion of pro-inflammatory cytokines and the

603

accumulation of intracellular ROS in LPS-treated RAW264.7 cells. (A) Production of

604

TNF-α, IL-1β, IL-6 and IL-12 in the culture supernatant was detected using an ELISA

605

kit. (B) Accumulation of intracellular ROS was measured by FACS using the

606

DCFH-DA fluorescent probe. Representative results from three independent

607

experiments are shown. * P < 0.05; ** P < 0.01; *** P < 0.001.

608 29



609

Figure 4. Se-PM blocks the binding of LPS to RAW264.7 cells. (A-B) LPS binding to

610

RAW264.7 cells were observed by confocal microscopy (A) and analysed with the

611

ImageJ software (B). DAPI was used to label the nuclei. Representative images and

612

the results from three independent experiments are shown. Scale bar = 20 µm. ** P

Molecular mechanisms and in vivo evaluation of the anti-inflammatory

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