Immunomodulatory Activity of Docosahexenoic Acid on RAW264.7

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Immunomodulatory activity of docosahexenoic acid on RAW264.7 cells activa-tion through GPR120-mediated signaling pathway Lirong Han, Jun Yu, Yuanyuan Chen, Dai Cheng, Xu Wang, and Chunling Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05894 • Publication Date (Web): 07 Jan 2018 Downloaded from http://pubs.acs.org on January 7, 2018

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Title: Immunomodulatory activity of docosahexenoic acid on RAW264.7 cells ac-

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tivation through GPR120-mediated signaling pathway

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Authors: Lirong Han, Jun Yu, Yuanyuan Chen, Dai Cheng, Xu Wang and Chunling

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Wang*

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The affiliation and address of the authors: Key Laboratory of Food Nutrition and

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Safety, Ministry of Education, College of food Engineering and Biotechnology, Tian-

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jin University of Science and Technology, No.29, 13th Avenue, Tianjin Economy

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Technological Development Area, Tianjin 300457, People Republic of China.

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*Corresponding author at: Key Laboratory of Food Nutrition and Safety, Ministry of

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Education, College of food Engineering and Biotechnology, Tianjin University of

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Science and Technology, No.29, 13th Avenue, Tianjin Economy Technological De-

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velopment

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+86-022-60912421. E-mail address: [email protected] (Chunling Wang).

Area,

Tianjin

300457,

People

Republic

of

China.

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Conflict of Interest: The authors declare no competing financial interest.

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Abstract

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In this study, we elucidated the immunomodulatory activity of docosahexaenoic acid

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(DHA) on protein expression in RAW264.7 cells and its molecular mechanism. The

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results showed that the proliferation index of RAW264.7 cells at 48 hours was about

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173.03±7.82% after the treatment of 2.4 μM DHA. DHA could activate RAW264.7

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cells by the G-protein coupled cell membrane receptor GPR120- C-Raf- Mito-

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gen-Activated Protein Kinases (MAPKs) - nuclear factor κB (NF-κB) p65 pathway. In

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addition, 2.4 μM of DHA could significantly increase (P99%). 3-(4,

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5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), lipopolysaccharide

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(LPS) and GPR120 antibody were purchased from Sigma (St. Louis, MO, USA).

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Penicillin-streptomycin solution, trypsin, phosphate buffer saline (PBS) and dimethyl

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sulfoxide side (DMSO) were purchased from Thermo (Beijing, China). GW9508 (in-

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hibitor of GPR120) was purchased from Sigma (St. Louis, MO, USA). GW5074 (in-

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hibitor of C-Raf) and GDC-0094 (inhibitor of ERK1/2) were also purchased from

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Selleck Chemicals (Houston, TX, USA). The IκB-α, NF-κB p65, β-actin, phos-

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pho-C-Raf, C-Raf, phospho-ERK1/2, ERK1/2, phospho-p38 MAPK, p38 MAPK,

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phospho-JNK/SAPK, JNK/SAPK and horseradish peroxidase-conjugated secondary

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antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

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DHA were dissolved in dimethyl sulfoxide (DMSO) to 5 mg·mL-1, and then was

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added in the RPMI-1640 medium as the working solutions of DHA. All other chemi-

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cals were of the analytically purest grade available.

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2.2 Cell culture

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RAW 264.7 Abelson murine leukemia virus-induced tumor was obtained from The

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Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai,

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China). The cells were maintained in an RPMI-1640 medium (Gibco Invitrogen Co.,

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San Diego, CA, USA) supplemented with 10% fetal bovine serum (Gibco BRL,

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Grand Island, NY, USA), 100 units·mL-1 penicillin and 0.1 mg·mL-1 streptomycin at

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37 °C in a humidified chamber of 95% air and 5% CO2 atmosphere.

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2.3 Cell proliferation assay

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The effect of DHA on the proliferation of RAW264.7 cells was determined by MTT

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assay (13). The cell suspension (1 × 105 cells·well-1) was planted on coverslips that

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were partitioned previously into a 96-well plate. After 4 hours, the RAW264.7 cells

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were washed twice with pre-warmed Hank’s Balanced Salt Solution and incubated in

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free-serum culture medium with 0.6–3.0 μM DHA for 0–72 hours. The control cells

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were incubated with 0.02% DMSO (equal with the content of DMSO in 3.0 μM DHA

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group) in RPMI-1640 medium. After incubation, the cells were incubated with 20 μL

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MTT solution (5 mg·mL-1) in medium for 4 hours at 37 ℃. Viable cells convert the

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MTT to formazan, which generates a blue-purple color after dissolving in 150 μL

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DMSO. The absorbance at 570 nm was the measured with a microplate reader (Model

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680, Bio-Rad, Hercules, CA, USA). All tests were carried out in 6 independent ex-

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periments.

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Cell proliferation rate(%)

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= [(absorbance of cells treated with DHA

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− absorbance of control cells)/absorbance of control cells]

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× 100%

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2.4 Western blot analysis

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RAW 264.7 cells were seeded onto 6-well culture plates at 1×105 cells·mL-1 and al-

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lowed to adhere for 4 h, then the medium was aspirated and new medium with or

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without DHA (0, 1.8, 2.4 and 3.0 μM) was added. After incubation for 48 h, the me-

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dium was aspirated again. After various treatments, RAW264.7 cells were washed in

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cold PBS 3 times and lysed with the Nuclear and Cytoplasmic Protein Extraction Kit

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(Beyotime, Shanghai, China). Protein concentrations were quantified with the BCA

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Protein Assay Kit using bovine serum albumin as a standard. All the primary antibod-

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ies were diluted with PBS for 1000 times (Cell Signaling Technology, Danvers, MA,

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USA). The membranes were washed extensively and incubated with the appropriate

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secondary antibodies bonded to horseradish peroxidase (Amersham Pharmacia Bio-

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tech, Piscataway, NJ, USA). The immune-reactive bands were detected with using an

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enhanced chemiluminescence (ECL) kit (Millipore Co., Billerica, MA, USA). West-

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ern blot analysis was carried out by the method as described previously (14). Each test

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was performed in 6 independent experiments.

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2.5 Immunofluorescence assay for NF-κB p65

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The cell suspension (1 × 105 cells·well-1) was inoculated on coverslips that were par-

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titioned previously into a 6-well plate. After 4 hours, RAW264.7 cells were treated

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with (0 or 2.4 μM) DHA for 48 hours. Cells were fixed with 3% formaldehyde in PBS

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for 20 min and washed with PBS for three times. Washed cells were permeabilized

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using 0.2% Triton X-100, and then blocked in 2% bovine serum albumin in PBS.

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Thereafter, cells were washed three times with PBS and incubated with the antibody

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NF-κB p65 (dilution 1: 200) with 2% BSA in PBS at 37 ℃ for 1 hour. The resulting

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cells were washed with PBS three times and incubated with fluorescein FITC-labeled

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polyclonal goat anti-mouse IgG antibody (dilution 1: 200) at 37 ℃ for 1 hour, after

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which they were stained with propidium iodide (PI) (Sigma, St. Louis, MO, USA).

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The cells were, finally, scanned by laser scanning confocal microscopy (LSCM, Ni-

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kon, D-Eclipse C1, Tokyo, Japan) after being washed again with PBS (15). All images

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were acquired using the same intensity and photodetector gain.

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2.6 Measurement of NO production

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Nitrite accumulated in the culture medium was measured as an indicator of NO pro-

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duction based on the Griess reaction (16). Briefly, 100 μL aliquots of the supernatant

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were distributed in a 96-well plate and then equal volumes of the Griess reaction solu-

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tions (1% sulfanilamide, 0.1% N-(1-naphthyl)-ethylenediamine dihydro-chloride in

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2.5% phosphoric acid) were added (17). The reaction was allowed to proceed for 15

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min at room temperature. The concentration of NO was calculated by extrapolating a

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NaNO2 standard curve.

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2.7 Real-time quantitative polymerase chain reaction (RTQ-PCR) analysis

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RAW 264.7 cells (cell density: 1 × 105 cells·mL-1) were treated with or without lipo-

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polysaccharide (LPS) (5 μg·mL−1) and cultured with the test compound, DHA (0, 1.2,

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1.8, 2.4 and 3.0 μM), for 48 h. The cells were collected, and total RNA was extracted

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by using the TransZol Up reagent (TransGen Biotech Co., Ltd), according to the

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manufacturer’s protocol. RNA (5 ng) was reverse-transcribed using a TransScript

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First-Strand cDNA Synthesis SuperMix kit to produce a cDNA PCR template.

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RTQ-PCR was performed with the StepOnePlusTM Real-Time PCR Instrument (Ap-

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plied Biosystems, Scoresby, Vic) (18) using optical grade 96-well plates in a reaction

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volume of 50 μL using the SYBR Green I using the forward and reverse primers and

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UItraSYBR Mixture (with ROX) (Beijing ComWin Biotech Co.,Ltd. China). Samples

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that were detected by RTQ-PCR in 50 μL reactions containing 2 ×UItraSYBR Mix-

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ture (with ROX), 2 μL of the sample DNA and 200 nM each of the specific primers.

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The RTQ-PCR conditions were 95°C for 10 min, followed by 40 cycles of amplifica-

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tion at 95°C for 10 s, 60°C for 30 s and 72°C for 32 s. Reactions were performed in

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triplicate and the mean values calculated. Data analysis was performed using the

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StepOneTM Software version 2.3 supplied by Applied Biosystems. The primers used

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in this experiment including inducible nitric oxide synthase (iNOS)

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were provided by Takara Bio (Japan) Co., Ltd.

and β-actin

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2.8 Cytokine assays

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RAW264.7 cells were pre-incubated at 1 × 106 cells·well-1 in a 12-well plate (NEST

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Biotechnology Co.LTD., Wuxi, China) for 4 hours at 37 °C in a 5% CO2 and then

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were incubated with various concentrations of DHA (0–3.0 μM) at 37 °C for 48 hours.

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Levels of IL-1β, IL-6, IL-10, IL-12, TNF-α, IFN-γ and TGF-β in the culture

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supernatants were evaluated by using commercial ELISA kits (R&D Systems,

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Minneapolis, MN, USA) according to the manufacturer’s instructions. The

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absorbance was measured in an ELISA reader at 450 nm. The concentrations of

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cytokines were calculated according to the standard curve using each of the

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recombinant cytokines in the ELISA kits.

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2.9 Statistical analysis

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The data represented the mean±SD of 6 independent experiments normalized to

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DMSO (0.02%). Statistical analyses were performed using SPSS software (version

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18.0) to determine the significant differences. All values were analyzed by one-way

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analysis of variance (ANOVA) followed by a post hoc Tukey's test for multiple com-

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parisons. Values of P < 0.05 were considered statistically significant.

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3 Results

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3.1 DHA increased the proliferation index of RAW264.7 cells

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To investigate the immunomodulatory effect of DHA, MTT assay was used to evalu-

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ate RAW264.7 cells proliferation. As shown in Fig. 1A, DHA increased the cell pro-

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liferation index at the concentrations of 0–3.0 μM for 48 hours in a dose-dependent

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manner, relative to the control. The cell proliferation index reached the maximum at

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the 2.4 μM of DHA (P< 0.01). Meanwhile, DHA at 2.4 μM also significantly en-

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hanced the proliferation index of RAW264.7 cells in a time-dependent manner (Fig.

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1B). The cell proliferation index reached the maximum at 48 hours after 2.4 μM of

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DHA treatment (P< 0.01). At the concentration of 2.4 μM, the proliferation index at

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48 hours was about 173.03±7.82%. These results suggest that DHA can promote the

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proliferation of RAW264.7 cells.

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3.2 Effect of DHA on GPR120 protein expression

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GPR120 is a G-protein coupled cell membrane receptor expressed on macrophages

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which can bind with long-chain fatty acids. A marked increase in GPR120 protein

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level was detected in RAW264.7 cells after treatment with DHA compared to the con-

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trol, and the protein expression of GPR120 reached peak value at the concentration of

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2.4 μM DHA (Fig. 2A).

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3.3 Measurement of C-Raf and MAPKs

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The stimulation of GPR120 by FFAs may result in activation of the ERK cascade (19).

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Recent studies have shown that the C-Raf protein can mediate diverse biological

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functions such as cell growth, cell survival and cell differentiation (20). As shown in

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Fig.2B, the level of phosphorylated C-Raf in endochylema increased in a

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dose-dependent manner and reached their maximum value at the concentration of 2.4

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μM DHA.

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Furthermore, MAPKs have been shown to modulate the synthesis and release of

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pro-inflammatory cytokines and mediators in LPS stimulated macrophages(21). To

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determine whether the immunological activities of DHA are mediated by the MAPK

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pathway, we examined the effects of DHA on the phosphorylation of p38 MAPK,

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JNK/stress-activated protein kinase and ERK1/2 by western blot analysis. Fig. 2C

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shows that the activation of MAPK (P38 and ERK) and JNK signaling significantly

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occurred in RAW 264.7 cells after the treatment with different concentrations of DHA,

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compared with the control group. The amount of non-phosphorylated p38, ERK 1/2,

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and JNK was apparently influenced by exposure to DHA, and treatment with increas-

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ing concentrations of DHA induced phosphorylation of p38, ERK 1/2, and JNK in a

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concentration-dependent manner.

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3.4 Effect of DHA on the expression of NF-κB p65

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NF-κB is an important transcription factor in the regulation of pro-inflammatory me-

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diators in activated macrophages. IκB is a protein bound to dimers of NF-κB, which

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retains these transcription factors in the cytoplasm. An activation of IκB kinase (IKK)

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leads to the phosphorylation of IκB and its degradation which allow nuclear localiza-

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tion of NF-κB (22, 23). To investigate whether DHA activates the NF-κB signaling

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pathway, the nuclear level of NF-κB p65 and IκB-α were analyzed by Western blot.

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As shown in Fig. 3A, IκB-α proteolytic degraded and free NF-κB p65 translocated

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into the nucleus. Meanwhile, under the LSCM, the intensity of NF-κB p65 fluores-

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cence in the nucleus treated by DHA (2.4 μM) was significantly stronger than that of

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the control (Fig. 3B). The results above indicate that DHA can induce NF-κB activa-

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tion in RAW264.7 cells.

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3.5 Relationship between GPR120 and C-Raf

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In order to investigate whether DHA can activate GPR120 and the relationship be-

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tween GPR120 and C-Raf, the cells were pre-incubated with GW9508 (inhibitor of

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GPR120, at 5 μM) prior to adding DHA at a concentration of 2.4 μM. The expressions

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of GPR120 and C-Raf were then analyzed by western blot. The level of GPR120 was

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reduced by GW9508 (Fig. 4A), which indicated that GPR120 expression is dependent

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on DHA treatment. Furthermore, the protein expression of C-Raf was decreased by

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GW9508 (Fig. 4A), which indicated that C-Raf phosphorylation was dependent on

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GPR120.

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3.6 Relationship between C-Raf and ERK1/2

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The Raf proteins are located upstream of MEK and activated Raf phosphorylates

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MAPK kinase 1/2 (MEK 1/2), which in turn phosphorylates and activates extracellu-

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lar signal-regulated kinase 1/2 (ERK 1/2) (24). In this study, the results above demon-

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strate that phosphorylated C-Raf and phosphorylated ERK1/2 in endochylema are in-

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volved in DHA-induced RAW264.7 cells activation. In order to further assess whether

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the phosphorylated ERK1/2 is located downstream of C-Raf phosphorylation, cells

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were pre-incubated with GW5074 (GW, an inhibitor of C-Raf, at 5 μM) prior to add-

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ing DHA at a concentration of 2.4 μM. The expression of phosphorylated ERK1/2

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was then analyzed by western blot. The level of ERK1/2 phosphorylation was reduced

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by GW5074 (an inhibitor of C-Raf) (Fig. 4B), which indicates that ERK1/2 phos-

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phorylation is dependent on C-Raf phosphorylation.

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3.7 Relationship between ERK1/2 and NF-κB p65

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ERK activation leads to phosphorylation of a variety of transcription factors and re-

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sults in induction of gene expression and proliferation (25). The above results demon-

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strate that phosphorylated ERK1/2 and phosphorylated NF-κB p65 in endochylema

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are involved in DHA-induced RAW264.7 cells activation. In order to further assess

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the relationship between them, cells were pre-incubated with GDC0094 (GDC, an in-

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hibitor of ERK1/2, at 5 μM) prior to adding DHA at a concentration of 2.4 μM. The

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expression of and phosphorylated NF-κB p65 was then analyzed by western blot. The

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degradation level of IκB-α was effectively blocked by GDC0094 (an inhibitor of

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ERK1/2) in RAW264.7 cells. Simultaneously, the expression of NF-κB p65 phos-

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phorylation was significantly decreased by GDC0094 (Fig. 4C). These results sug-

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gested that the expression of phosphorylated NF-κB p65 was modulated by ERK1/2

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in DHA-induced RAW264.7 cells activation.

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3.8 DHA induced NO production and expression of iNOS in RAW264.7 cells

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NO has been demonstrated to play an important role in a variety of physiological

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process, host defenses and immunity responses (26). To investigate whether or not

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DHA was capable of inducing NO production in macrophages, RAW264.7 cells were

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treated with DHA (0–3.0 μM) for 48 hours. As shown in Fig. 5A, a minimum amount

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of NO was released in the control, however cells treated with DHA showed signifi-

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cantly higher (P