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 1

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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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ACS Paragon Plus Environment

Tel/Fax:


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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


Journal of Agricultural and Food Chemistry

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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

Immunomodulatory Activity of Docosahexenoic Acid on RAW264.7

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