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Omics Technologies Applied to Agriculture and Food
Proteomic analysis reveals inflammation modulation of #/#carrageenan hexaoses in LPS-Induced RAW264.7 Macrophages Juanjuan Guo, Jinghao Chen, Xu Lu, Zebin Guo, Zhiwei Huang, Shaoxiao Zeng, Yi Zhang, and Baodong Zheng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01144 • Publication Date (Web): 23 Apr 2018 Downloaded from http://pubs.acs.org on April 23, 2018
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Journal of Agricultural and Food Chemistry
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Proteomic analysis reveals inflammation modulation of
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κ/ι-Carrageenan hexaoses in LPS-Induced RAW264.7
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Macrophages
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Juanjuan Guo†,§,#, Jinghao Chen†,§, Xu Lu†,§,#, Zebin Guo†,§,#, Zhiwei Huang†,§,#, Shaoxiao
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Zeng†,§,#, Yi Zhang†,§,#, Baodong Zheng*,†,§,#1
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†
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350002, China
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§
College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian
China-Ireland International Cooperation Centre for Food Material Science and Structure
Design, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China
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#
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Starch, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China
Fujian Provincial Key Laboratory of Quality Science and Processing Technology in Special
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*
Corresponding author : College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, 350002, China. Tel.: +86 59183736738; fax: +86 591 83739118 E-mail address:
[email protected] (B. D. Zheng)
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ABSTRACT
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κ/ι-Carrageenan hexaoses (κ/ι-neocarrahexaoses, KCO-4) are a type of carrageenan
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oligosaccharide that have a broad spectrum of bioactivities due to the presence of sulfate
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groups. However, the anti-inflammatory capacity of purified carrageenan oligosaccharides
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and the underlying mechanism has not been completely elucidated. The present study aimed
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to investigate inflammatory signaling modulation of KCO-4 in LPS-induced macrophages
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using a quantitative proteomic strategy. KCO-4 inhibited the over-secretion of inflammatory
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mediators (i.e. NO, TNF-α, IL-1β, IL-8, iNOS and COX-2). KCO-4 treatment altered
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proteome profile, and metabolic processes in mitochondria were significantly disrupted. The
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IPA
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pathway-dependent anti-inflammation process through the inhibition of CD14/Rel@p50 in
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LPS-induced RAW264.7 macrophages. These data improve our understanding of the
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anti-inflammatory mechanism and contribute to exposure biomarker screening of
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κ-carrageenan oligosaccharides.
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KEYWORDS:
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macrophages
network
analysis
proposed
that
κ/ι-neocarrahexaoses,
KCO-4
triggered
anti-inflammation,
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the
NF-κB
proteomics,
signaling
RAW264.7
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INTRODUCTION
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Inflammation is a natural host-defense response to invading pathogens, and it involves
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innate and adaptive immune systems. During inflammation, a variety of inflammatory cells
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and macrophages are activated to produce inflammatory mediators (e.g., tumor necrosis
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factor-α TNF-α, nitric oxide NO, reactive oxygen species ROS, interleukin-1β IL-1β and
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interleukin-8 IL-8),1 and eliminate invading pathogens. Prolonged increased production of
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these inflammatory mediators cause chronic inflammatory diseases such as autoimmune
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disease, tumorigenesis, cardiovascular disease, osteoporosis, and even neurodegenerative
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disorders.2 Therefore, the suppression of macrophage activation is a potential method to
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ameliorate inflammatory diseases. The endotoxin lipopolysaccharide (LPS)-induced RAW
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264.7 model has been commonly used to represent inflammatory tissues. LPS activates a set
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of
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mitogen-activated protein kinase (MAPK) pathways in RAW264.7 macrophages.3 It also
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induces the overexpression of genes encoding inflammatory mediators including TNF-α, and
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NO, IL-1β.4, 5
inflammatory
signaling
pathways
including
nuclear
factor-κB
(NF-κB)
and
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κ-Carrageenan is a high molecular weight, sulfated D-galactan (an alternating backbone
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of β-1,3-D-galactose-4-sulfate and 3,6-anhydrogro-α-1,4linked-D-galactose), which is
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depolymerized from food grade carrageenan through chemical or enzymatic hydrolysis.6
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Previous
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immunoregulatory activities of carrageenan poly- or oligosaccharides.7,
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bioactivities of purified carrageenan poly- or oligosaccharides and their mechanisms of
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action remain unclear. The comparison between mixed κ-carrageenan oligosaccharides (KOS)
studies
have
demonstrated
the
significant
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anti-tumor,
anti-viral 8
and
However, the
Journal of Agricultural and Food Chemistry
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and desulfated derivatives of KOS showed that the sulfate content was positively associated
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with anti-inflammatory capacity.9, 10 The mixed carrageenan oligosaccharides can inhibit the
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growth of transplantable sarcoma S180 by promoting the immune system in S180-bearing
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mice.11 Furthermore, mixed carrageenan oligosaccharides stimulate IL-10 production in both
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human and mouse blood cells.12 These data demonstrated that carrageenan oligosaccharides
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are promising agents to promote immune responses and to treat inflammatory diseases.
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However, the studies of immunoregulatory or anti-inflammatory activities still rely
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predominantly on the mixed carrageenan oligosaccharides.
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KCO-4 (κ/ι-neocarrahexaoses) are purified, heterogeneous carrageenan oligosaccharides,
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which have two types of sulfated groups on their galactose unit: DA2S and G4S, with a
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molecular weight of 1,273 Da. KCO-4 cause no cytotoxicityat concentrations up to and
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including 75 µM in RAW264.7 cells.6 However, the bioactivities of purified carrageenan
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poly- or oligosaccharides and their mechanisms of action remain unclear. In this, we
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investigated the anti-inflammatory effect of KCO-4 in LPS-induced RAW264.7, and
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measured proteomic responses using a high-throughput, label-free proteomics approach, and
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further analyzed the signaling pathways involved in the anti-inflammatory mechanism. The
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results may be useful in providing comprehensive insights into the anti-inflammatory
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mechanism of KCO-4, and developing potential biomarkers for chronic inflammatory
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diseases.
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MATERIALS AND METHODS
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Purification of KCO-4. Heterogeneous κ/ι-neocarrahexaoses (KCO-4) were prepared
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from κ-carrageenase in Thalassospira sp. Fjfst-332,13, 14 and purified using an MPLC-ELSD
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system. The structural sequence of KCO-4 is α-DA/DA2S-1,3-β-G4S-1,4- α-DA-1,3-β-G4S
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-1,4-α-DA-1,3-G4Srα/β.6
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Cell culture and treatment. RAW264.7 macrophage cell lines were obtained from
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the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in
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Dulbecco’s Modified Eagle’s Medium (DMEM, GIBCO, USA) supplemented with 10% fetal
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bovine serum (ExCell Biology, USA) at 37oC in a 5% CO2 incubator. Purified KCO-4 were
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dissolved in deionized water at 10, 25, and 50 µM concentrations. The RAW 264.7 cells were
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pretreated with various concentrations of KCO-4 or 50 µM dexamethasone (DXM, an
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representative anti-inflammatory agent15, Sigma, St. Louis, MO) for 4 h and stimulated with
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10 µM LPS for additional 24 h. The control group was the cells cultured in DMEM for 24 h
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in the absence of any stimulants.
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Determination of cytokine, NO levels in LPS-activated RAW264.7 cells.
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RAW 264.7 cells were seeded onto 96-well plates (100 µL/well, 5×104 cells/mL) and
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incubated as described above. The levels of TNF-α, IL-1β, and IL-8 in the supernatant were
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quantified using Biotrak™ ELISA kits (GE Healthcare, Beijing, China) according to the
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manufacturer’s instructions. Nitrite concentration was measured as a proxy of NO levels
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using the Griess method (Supplemental material).
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Quantitative RT-PCR. Total RNA was isolated using TRIzol reagent (Invitrogen Co.,
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Carlsbad, CA, USA). The cDNA was amplified and quantitative real-time PCR was
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conducted as previously described.16 The first-strand cDNA was amplified using the
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following primers (Eurofins Biolab S.L.U., Barcelona, Spain): forward strand TNF-α, 5'-ATG
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GCC TCC CTC TCA TCA GT-3'; reverse strand TNF-α, 5'-TTT GCT ACG ACG TGG GCT
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AC-3'; forward strand IL-1β, 5'-GCC ACC TTT TGA CAG TGA TGA G-3'; reverse strand
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IL-1β, 5'-GAC AGC CCA GGT CAA AGG TT-3'; forward strand IL-8, 5'-GGC TTT CCA
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CAT TTG AGG ACG-3'; reverse strand IL-8, 5'-CGT GGC GGT ATC TCT GTC TC-3';
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forward strand GAPDH, 5'-TAT GTC GTG GAG TCT ACT GGT-3'; reverse strand GAPDH,
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5'-GAG TTG TCA TAT TTC TCG TGG-3'.
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Western blot analysis. After treatment, RAW 264.7 cells were washed with ice-cold
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PBS three times and lysed in RIPA buffer (St. Louis, MO, USA). A Bradford protein assay
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was used to calculate the concentration of total protein in each sample. A routine western blot
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operation was conducted to detect iNOS and COX-2 expression (Supplemental material).
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Label-free quantitative proteome analysis. The total proteins were extracted from
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RAW264.7 cells. The extracted proteins were reduced with dithiotreitol, alkylated with
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iodoacetamidek, and then digested with trypsin overnight. Tryptic peptides were desalted,
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lyophilized and reconstituted (Supplemental material). . Proteome analysis were conducted
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using EASY-nLC coupled with the Orbitrap Fusion Lumos mass spectrometer system
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(Thermo Fisher Scientific, MA, USA). The samples were separated using a water/acetonitrile
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solvent system containing 0.1% formic acid and a linear gradient (75 min) from 5% to 95%
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acetonitrile. The column flow rate was maintained at 600 nL/min and the column temperature
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was kept at 35oC. An electrospray voltage of 2 kV for mass spectrometry was used. The MS -6-
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spectra (300–1400 m/z) were collected with a resolution of 70,000. The 20 most abundant
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isotope patterns with a charge of 2 were subjected to collisional dissociation with normalized
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collision energy of 35. The raw data were processed using Maxquant software
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((http://maxquant.org/, version 1.5.7.0). The parameters of Maxquant were set as follows:
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variable modifications, acetyl (protein N-term) and oxidation (M); fixed modification,
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Carbamidomethyl (C). The first search peptide tolerance was set to 20 ppm and main
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tolerance was 4.5 ppm. The maximum charge of peptide modifications was set as 7. The
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identified proteins were filtered using a 1% false discovery rate (FDR). Protein quantification
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was conducted using label-free quantification algorithms (LFQ). Protein fold changes were
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calculated by normalizing the LFQ intensity of treatment groups to that of the LPS stimulated
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group (p
