Open Access

Anti‑inflammatory effects and enhancing immune response of freshwater hybrid catfish oil in RAW264.7 cells

  • Authors:
    • Bussarin Tongmee
    • Atcharaporn Ontawong
    • Narissara Lailerd
    • Kriangsak Mengamphan
    • Doungporn Amornlerdpisan
  • View Affiliations

  • Published online on: August 26, 2021     https://doi.org/10.3892/etm.2021.10657
  • Article Number: 1223
  • Copyright: © Tongmee et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

The present study assessed the effect of freshwater hybrid catfish oil (FFO) on the inflammatory status of lipopolysaccharide (LPS)‑stimulated RAW264.7 cells and investigated the underlying mechanisms. RAW264.7 cells were supplemented with various concentrations [0.125‑2% in 0.5% propylene glycol (v/v)] of FFO with or without LPS (1 µg/ml) for 24 h. Inflammatory cytokines and mediators were quantified using ELISA and reverse transcription‑quantitative PCR. The results revealed that FFO treatment inhibited the secretion and mRNA expression of the pro‑inflammatory cytokines IL‑6, IL‑1β, TNF‑α. In line with this, FFO suppressed the expression and secretion of the inflammatory mediators cyclooxygenase‑2 and prostaglandin E2. FFO also reduced apoptotic body formation and DNA damage. Correspondingly, FFO enhanced the immune response by modulating the cell cycle regulators p53, cyclin D2 and cyclin E2. Accordingly, FFO may be developed as a nutraceutical product to prevent inflammation.

Introduction

Inflammation is one of the first lines of defense against harmful stimuli, such as pathogens, damaged cells, trauma, bacteria and irritants (1). Macrophages detect and react to certain pathogens and consequently regulate the inflammatory response (2). Lipopolysaccharide (LPS) is an endotoxin derived from the outer membrane of Gram-negative bacteria and also a powerful mediator of systemic inflammation and a driver of septic shock (3). LPS is able to activate macrophages to release several inflammatory cytokines (4). Activation of the inflammatory pathway may be induced by pro-inflammatory mediators and cytokines being secreted, including nitric oxide (NO), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6 and prostaglandin (PG)E2(5). Inflammation is one cause of increased morbidity and mortality in intensive care units, also resulting in elevated hospital-related costs (6,7). Nowadays, several anti-inflammatory drugs are available, such as non-steroidal anti-inflammatory drugs (NSAIDs) (8). However, a previous study suggested that NSAIDs may induce gastrointestinal tract bleeding (9). Safe and effective strategies to prevent and treat inflammation and its associated diseases are thus urgently required.

The freshwater hybrid catfish (Pangasius sp.) belongs to the freshwater catfish family. It has become one of the most popular freshwater fish species and has a high demand, particularly on the European and US markets. Fish contains 2-30% fat and ~50% of its body weight is discarded as waste during the fish processing operation (10). One of the fish processing byproducts is fish oil (FO). FO is a source of long-chain polyunsaturated fatty acid (e.g. omega-3 fatty acids), particularly fish oil extracted from marine fish, which is mainly composed of cis-5,8,11,14,17-eicosapentaenoic acid (EPA) and cis-4,7,10,13,16,19-docosahexaenoic acid (11). As a component in FO, omega-3 fatty acids have several benefits, including protection against atherosclerosis, arrhythmias and chronic obstructive pulmonary diseases (12). They also reduce blood pressure, blood glucose and symptoms of asthma and cystic fibrosis (13-15). However, a previous study by our group demonstrated that fish oil from freshwater hybrid catfish contains a high level of monounsaturated omega-9 fatty acid (MUFA) (16). Furthermore, freshwater hybrid catfish oil (FFO) was indicated to have anti-diabetic effects by improving insulin resistance and adipokine imbalance in a rat model of type 2 diabetes and also suppress pro-inflammatory cytokine protein expressions in the skeletal muscle tissues of those rats (17). The omega-9 fatty acid increased of high-density lipoprotein-cholesterol and decreased low-density lipoprotein-cholesterol (17). However, the effect of FFO on the inflammatory condition and the underlying mechanisms have remained elusive. In the present study, the anti-inflammatory effects of FFO on RAW264.7 macrophages stimulated by LPS were examined and the associated mechanism was investigated.

Materials and methods

Chemicals

Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco; Thermo Fisher Scientific, Inc. β-nicotinamide adenine dinucleotide phosphate and LPS were purchased from Merck KGaA. All other chemicals with high purity were purchased from commercial sources.

Preparation of FFO

FO of freshwater hybrid catfish (Pangasius gigas x Pangasianodon hypophthalmus) was purchased from a private company, Me Natural Co., Ltd., which cooperated and received the adipose tissue from the Center of Excellence in Giant Catfish and Buk Siam Catfish, Faculty of Fisheries Technology and Aquatic Resources, Maejo University (Chiang Mai, Thailand). FO was extracted as previously described, which exhibited a high omega-9 content and biological activity (18). In brief, frozen adipose tissues were purified by cleaning and steaming at 90˚C for 30 min. The liquid oil was subsequently filtered through a filter sack and squeezed using a screw compressor. The squeezed liquid was centrifuged at 2,268 x g for 10 min at 25˚C to separate the solid particles from the oil and the supernatant FFO was separated. Solvent-free extraction was used to obtain FFO. As previously, adipose tissue was extracted and partially purified as aforementioned, resulting in FFO at a yield of 300 ml per 1 kg of adipose tissue.

Determination of fatty acids, fat-soluble vitamins and heavy metal levels of FFO

The chemical compounds, including the fatty acids and fat-soluble vitamins, were sent for analysis at a certified lab with international standardization in the field of information technology (ISO172025), the Central Laboratory (Thailand) Co. Ltd., Chiang Mai Branch, following the TE-CH 260 in-house method of the Association of Official Analytical Chemists 996.06(19). Heavy metal contamination of FFO was also detected according to this in-house method.

Cell culture

RAW264.7 cells were purchased from the American Type Culture Collection. Cells at passage 2-22 were maintained in DMEM (Thermo Fisher Scientific, Inc) containing 3.7 g/l NaHCO3 supplemented with 10% FBS (Thermo Fisher Scientific, Inc) and 1% penicillin/streptomycin in a humidified atmosphere at 37˚C with 5% CO2 and sub-cultured every 4-5 days using 0.05% trypsin-EDTA in PBS (Thermo Fisher Scientific, Inc.). Cells were seeded at a density of 1x105 cells/well and cultured in 6-, 12- and 96-well plates for 3 days until subsequent experimentation. The medium was replaced every 2 days during culture.

Determination of cell viability

The MTT assay was performed to assess the effect of FFO on cell viability. Cells were incubated with serum-free medium with FFO at 0, 0.125, 0.25, 0.5, 1 or 2% in 0.5% propylene glycol (v/v). Subsequently, serum-free medium containing 0.5 mg/ml of MTT (Thermo Fisher Scientific, Inc.) was added to each well, followed by incubation at 37˚C for 4 h. The MTT solution was then aspirated and cells were washed once with ice-cold PBS. The purple formazan crystals were dissolved in DMSO for 30 min and cell viability was subsequently analyzed by measuring the absorption at a wavelength of 570 nm using an M965 AccuReader microplate reader (Metertech, Inc.). The lysed cells were detected at a wavelength of 680 nm was used as a reference. Cell viability was calculated as follows: Cell viability (%) = [(Absorbance value-reference value) x100]/[mean of (absorbance value-reference value) in untreated cells].

ELISA

RAW264.7 cells were seeded into 12-well plates at a density of 1x105 cells/ml and incubated for 24 h at 37˚C in a humidified atmosphere with 5% CO2. The culture medium was removed and cells were treated with different concentrations of FFO [0.125-2% in 0.5% propylene glycol (v/v)] with or without LPS (1 µg/ml) in fresh medium for 24 h at 37˚C in a humidified atmosphere with 5% CO2. Subsequently, the cells were homogenized and lysed cells were centrifuged at 2,000 x g for 10 min at 4˚C. The supernatant was collected and stored at -80˚C for quantification of IL-6 (cat. no. BIOL-431304), IL-1β (cat. no. BIOL-432604), TNF-α (cat. no. BIOL-430904), NO (cat. no. 780001) and PGE2 (cat. no. ABBK-KTE70765-96T) concentrations using commercial kits (BioLegend, Inc.) according to the manufacturer's protocols.

NO assay

The nitrate/nitrite concentration was determined using a colorimetric assay kit (Cayman Chemical Co.). In brief, cells were treated with different concentrations of FFO [0.125-2% in 0.5% propylene glycol (v/v)] with or without LPS (1 µg/ml) for 24 h at 37˚C in a humidified atmosphere with 5% CO2. Treated cells were centrifuged at 10,000 x g for 20 min at 4˚C. The supernatant was subsequently collected to measure the NO concentration at a wavelength of 540 nm using an M965 AccuReader microplate reader (Metertech, Inc.).

Hoechst 33342 staining

To confirm the effect of FFO on LPS-induced DNA damage, RAW264.7 cells were seeded into 8-well cell culture slides and treated with different concentrations of FFO [0.125-2% in 0.5% propylene glycol (v/v)] with or without LPS (1 µg/ml) for 24 h at 37˚C in a humidified atmosphere with 5% CO2. Treated cells were fixed with 4% paraformaldehyde for 10 min at room temperature and subsequently stained with Hoechst 33342 (5 µg/ml) for 10 min at room temperature. Cells were washed twice with PBS and observed under a Nikon Eclipse Ni-U fluorescent microscope (original magnification, x40; Nikon Corporation).

DNA damage assay

To further determine the protective effect of FFO on LPS-induced DNA damage, the effect of FFO on DNA impairment was investigated via ELISA. RAW264.7 cells were seeded into 12-well plates at a density of 1x105 cells/ml and incubated for 24 h at 37˚C in a humidified atmosphere with 5% CO2. The culture medium was removed and cells were treated with different concentrations of FFO [0.125-2% in 0.5% propylene glycol (v/v)] with or without LPS (1 µg/ml) for 24h at 37˚C in a humidified atmosphere with 5% CO2. Treated cells were centrifuged at 10,000 x g for 20 min at 4˚C. The supernatant was collected and stored at -80˚C for quantification of 8-hydroxy-2'-deoxyguanosine (8-OHdG; cat. no. AB-EIADNAD), a DNA damage marker, using commercial kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.

Reverse transcription-quantitative (RT-q)PCR

Total RNA was extracted and purified from RAW264.7 cells using TRIzol® reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol and reverse transcribed into cDNA using the SensiFAST™ cDNA synthesis kit (Bioline). qPCR was subsequently performed using SYBR Real-Time PCR Master Mix (Bioline) on a CFX Touch real-time PCR system (Bio-Rad Laboratories, Inc.). PCR amplifications were performed at a 20-µl volume with the following thermocycling conditions: A polymerase enzyme activation step at 95˚C for 2 min; followed by 40 cycles of denaturation at 95˚C for 5 sec, 10 sec of annealing at 60˚C depending on primers, and 10 sec of elongation at 72˚C. The primer sequences used for qPCR were purchased from Macrogen, Inc. and used at a final concentration of 0.4 µM. The primer sequences for mouse TNF-α, IL-1β, IL-6, COX2, p53, p27, cyclin D2, cyclin E2 and GAPDH are presented in Table I (20-25). Gene expression was calculated using the 2-ΔΔCq method (26) and normalized to GAPDH. Data were reported as the relative fold change. qPCR amplification was performed in duplicate for each synthesized cDNA set.

Table I

Primer sequences and expected amplicon sizes for gene amplification.

Table I

Primer sequences and expected amplicon sizes for gene amplification.

cDNAGenBank accession no.Forward primerReverse primerAmplicon size (bp)
TNF-αNM013693.3 5'-ACCTGGCCTCTCTACCTTGT-3' 5'-CCCGTAGGGCGATTACAGTC-3'161
IL-1βNM008361.4 5'-GCCACCTTTTGACAGTGATGAG-3' 5'-AGTGATACTGCCTGCCTGAAG-3'165
IL-6NM031168.2 5'-CAACGATGATGCACTTGCAGA-3' 5'-TCTCTCTGAAGGACTCTGGCT-3'201
COX-2NM011198.4 5'-CCACTTCAAGGGAGTCTGGA-3' 5'-AGTCATCTGCTACGGGAGGA-3'197
Cyclin D2NM009829.3 5'-ACCTCCCGCAGTGTTCCTATT-3' 5'-CACAGACCTCTAGCATCCAGG-3'93
Cyclin E2NM001037134.2 5'-TCTGTGCATTCTAGCATCGACTC-3' 5'-AAGGCACCATCGTCTACACATTC-3'149
p27NM009875.4 5'-GCGGTGCCTTTAATTGGGTCT-3' 5'-GGCTTCTTGGGCGTCTGCT-3'230
p53NM011640.3 5'-ACCGCCGACCTATCCTTACC-3' 5'-TCTTCTGTACGGCGGTCTCTC-3'118
GAPDHNM001289726.1 5'-TGTGTCCGTCGTGGATCTGA-3' 5'-TTGCTGTTGAAGTCGCAGGAG-3'150

[i] TNF-α, tumor necrosis factor-α; IL, interleukin; COX-2, cyclooxygenase-2.

Statistical analysis

Statistical analysis was performed using SPSS version 23 software (IBM Corp.). Values are expressed as the mean ± standard error of the mean. One-way ANOVA followed by Dunnett's test was used to compare differences between multiple groups. P<0.05 was considered to indicate a statistically significant difference.

Results

Fatty acids and vitamins in FFO

As presented in Table II, FFO contained several fatty acids, including saturated, unsaturated, MUFAs and polyunsaturated fatty acid (PUFAs) at 40.38, 55.80, 46.74 and 12.75 g/100 g of FFO, respectively. Among the detected MUFAs, the predominant fatty acid was omega-9 (42.27±1.76 g/100 g of FFO). In addition, FFO also contained PUFAs and the predominant fatty acids were omega-3 (1.17±0.39 g/100 g of FFO) and omega-6 (10.95±1.46 g/100 g of FFO). In addition, vitamin A was present at 1.80±0.12 µg/100 g of FFO and vitamin E was present at 0.69±0.06 mg/100 g of FFO.

Table II

Fatty acid composition and vitamin content of freshwater hybrid catfish oil.

Table II

Fatty acid composition and vitamin content of freshwater hybrid catfish oil.

Chemical componentAmount
Saturated fatty acids, g/100 g40.38±2.94
Unsaturated fatty acids, g/100 g55.80±0.64
     Monounsaturated fatty acids, g/100 g46.74±2.24
     Oleic acid, g/100 g42.07±1.79
     Omega-942.27±1.76
     Polyunsaturated fatty acids, g/100 g12.75±1.04
     Omega-31.17±0.27
     Omega-610.95±1.03
Vitamins
     Vitamin A (retinol), µg/100 g1.80±0.12
     Vitamin E (α-tocopherol), mg/100 g0.69±0.06

[i] Values are expressed as means ± standard error of the mean (n=3).

Heavy metal content profiles of FFO

The concentrations of arsenic, copper, lead, mercury, tin and zinc in FFO are presented in Table III. The results demonstrated that FFO contained copper and lead at much lower concentrations, while arsenic, mercury, tin and zinc were not detected. Moreover, the USA established a recommended dietary allowance of copper for adults at 0.9 mg/day (27).

Table III

Heavy metal content in freshwater hybrid catfish oil.

Table III

Heavy metal content in freshwater hybrid catfish oil.

Element (symbol)Amount (mg/kg)
Arsenic (As)Not detected
Copper (Cu)<0.50
Lead (Pb)<0.050
Mercury (Hg)Not detected
Tin (Sn)Not detected
Zinc (Zn)Not detected
FFO decreases the secretion of pro-inflammatory cytokines in RAW264.7 cells

To determine the anti-inflammatory effect of FFO in RAW264.7 cells, the levels of pro-inflammatory cytokines were detected via ELISA. As presented in Fig. 1A, LPS at 1 µg/ml significantly increased the levels of pro-inflammatory cytokines, while added FFO at 2% significantly decreased IL-6 production compared with LPS-treated cells. Added FFO at 0.25-2% also markedly decreased TNF-α and IL-1β expression compared with LPS-treated cells (Fig. 1B and C). Similarly, celecoxib (CX), an NSAID (28), significantly decreased IL-1β expression. However, in the absence of LPS, there was no significant effect of FFO (0.25-2%) and CX on the viability of RAW264.7 cells compared with the control cells (Fig. 1D). Taken together, these results suggested that FFO exerts an anti-inflammatory effect without a cytotoxic effect.

FFO decreases the mRNA expression levels of pro-inflammatory cytokines in RAW264.7 cells

To confirm the inhibitory effect of FFO on inflammation, RT-qPCR analysis was performed to detect the mRNA expression levels of the pro-inflammatory cytokines IL-6, IL-1β and TNF-α in RAW264.7 cells. A single dose (2%) was selected, as it significantly decreased all inflammatory cytokines (Fig. 1). The results demonstrated that the expression levels of the pro-inflammatory cytokines significantly decreased following additional treatment with FFO and CX compared with LPS-treated cells (Fig. 2). Collectively, these results suggested that FFO inhibits the synthesis of pro-inflammatory cytokines in activated macrophages.

FFO suppresses molecules involved in inflammatory signaling

The present study further investigated the molecular mechanisms by which FFO reduces inflammation and thus, signalling molecules of the inflammatory pathway were assessed. As presented in Fig. 3A, LPS significantly enhanced NO production compared with the control. However, the addition of FFO had no effect on NO production compared with LPS-treated cells. Furthermore, the effects of FFO on the production of PGE2, a principal mediator of inflammation, and COX-2, a prostaglandin-endoperoxide synthase (29), were investigated in the present study. The results demonstrated that FFO significantly decreased COX-2 mRNA expression, which in turn decreased PGE2 production (Fig. 3B and C). Taken together, these results suggested that FFO exerts anti-inflammatory effects on LPS-stimulated RAW264.7 cells.

FFO prevents apoptosis and DNA damage

To validate the inhibitory effect of FFO on cell apoptosis, RAW264.7 cells treated with FFO, with or without LPS for 24 h, were stained with Hoechst 33342. Microscopic observation demonstrated that treatment with LPS increased the rate of cell apoptosis featuring nuclear fragmentation, chromatin condensation and apoptotic body formation compared with the control cells (Fig. 4A and B). However, these features were reduced in FFO- and CX-treated cells (Fig. 4C and D).

In addition, it was investigated whether FFO is able to prevent DNA damage. As presented in Fig. 4E, treatment with LPS markedly increased the production of the 8-OHdG adduct, an oxidative stress-induced DNA damage marker (30), compared with the control cells. It was observed that the production of 8-OHdG induced by LPS significantly decreased in cells co-treated with FFO. Similarly, CX also significantly decreased 8-OHdG levels. Collectively, these results suggested that FFO has a cytoprotective effect.

FFO enhances immune response by modulating cell cycle regulators

A previous study reported that programmed cell death serves an important role in the regulation of inflammation (31). Thus, the current study also investigated the effect of FFO on cell cycle regulators. To identify the effect of FFO that are responsible for enhancing the immune response, the present study also investigated the expression of cell proliferation markers in RAW264.7 cells. As presented in Fig. 5, the gene expression of the cell cycle inhibitors p27 and p53 (32-35) decreased in FFO-treated cells compared with the control. Furthermore, gene expression of the cell cycle inducers cyclin D2 and cyclin E2 increased in RAW264.7 cells treated with FFO. Taken together, these results suggested that FFO improves inflammatory status by modulating cell cycle regulators.

Discussion

To the best of our knowledge, the present study was the first to demonstrate that FFO rich in omega-9 exerts anti-inflammatory effects in vitro by decreasing the expression and secretion of pro-inflammatory cytokines and mediators, preventing DNA damage via reduction of apoptotic body formation and 8-OHdG, and also promotes an immune response. A previous study demonstrated that the activation of tissue macrophages releases various pro-inflammatory cytokines, including TNF-α, IL-1 and IL-6, resulting in autoimmune and inflammatory diseases. In addition, n-3 polyunsaturated fatty acids (PUFAs) serve anti-inflammatory effects by reducing the production of TNF-α, IL-1β, IL-6 and tissue factors by stimulated monocytes (36). Thus, inhibiting the synthesis of these cytokines may prove useful for the treatment of autoimmune and inflammatory diseases. The results of the present study demonstrated that FFO markedly decreased the production of IL-6, IL-1β and TNF-α and mRNA expression levels in RAW264.7 cells, similar to NSAIDs. These results suggested that FFO exerts an anti-inflammatory effect by downregulating pro-inflammatory cytokines at both the transcriptional and translational levels, without any cell toxicity. Similarly, oleic acid, one of the most representative monounsaturated omega-9 fatty acids, was reported to mediate anti-inflammatory effects by inhibiting reactive oxygen species, p38 MAPK and Akt signaling pathways/IKK/NF-κB in BV2 cells (37).

Macrophages are associated with acute and chronic inflammatory responses by stimulating NO generation, resulting in an increment of macrophage activity (38). NO and PGE2 production are critical immune-regulatory biomarkers for chronic inflammatory diseases, such as hepatic dysfunction and pulmonary disease (39). The results of the present study demonstrated that FFO decreased PGE2 and its synthase enzyme COX-2, but not the NO level, similar to the action of NSAIDs. Previous studies have reported that natural products, including coumarin, Indonesian cassia extract and Halocynthia aurantium or docosahexaenoic acid-omega-3, decrease PGE2 and NO expression levels, which suggests that they have potential as anti-inflammatory agents (40-42). Conversely, it has been demonstrated that omega 3 increases the production of PGE2(43). The increment of the PGE2 concentration may be inhibited by the NF-κB signaling pathway and EP4 receptor, resulting in anti-inflammatory effects (44). There are controversial data on the effect of PGE2 in inflammation. The results of the present study demonstrated that FFO contains several fatty acids, including omega-3, -6 and -9. Consistently, previous studies have demonstrated that omega-3 fatty acids decrease PGE2 by decreasing the catalytic monomer of COX-1 dimer by arachidonic acid and inhibiting COX-1 oxygenation (45,46). In addition, omega-9 exerts anti-inflammatory effects in inflammation via a PPAR-γ expression-dependent mechanism (47).

It is well-known that there is a close association between inflammation and DNA damage (48). NO generated by inflammatory cytokine stimulation is sufficient to induce oxidative DNA damage (49). The results of the present study demonstrated that LPS induced DNA damage by nuclear fragmentation, chromatin condensation and apoptotic body formation, the effects of which were reversed following treatment with FFO and NSAIDs. Consistently, n-3 polyunsaturated fatty acids attenuate oxidative stress-induced DNA damage in vascular endothelial cells through upregulation of nuclear factor-mediated antioxidant response and the decrease in intracellular reactive oxygen species (50). In addition, the present study demonstrated that the expression of cell cycle regulators, including cyclin D2 and cyclin E2, increased following treatment with FFO, while p53 expression was inhibited. A previous study reported that cyclin D2 deficiency suppresses immune activity (51). On the other hand, hyperactive cyclin D2 expression promotes autoimmune disease or allograft rejection (52). Other natural products merely promote immune responses by regulating cell cycle regulators. For instance, A. asphodeloides enhances the immune response of RAW264.7 cells by extending the cell cycle S-phase, suppressing p27 and increasing cyclin D2 and cyclin E2 gene expression (53).

In conclusion, the results of the present study demonstrated that FFO improved inflammation by suppressing the mRNA expression and secretion of pro-inflammatory cytokines and their mediators, and inhibiting apoptotic body formation and DNA damage. FFO also enhanced the immune response by modulating cell cycle regulators. Thus, FFO may be used as a natural anti-inflammatory supplement. Moreover, future in vivo studies and clinical trials are required to elucidate whether FFO has an overall anti-inflammatory effect in autoimmune or inflammatory diseases.

Acknowledgements

Not applicable.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

BT performed the experiments, collected and analysed the data, and wrote the first draft of the manuscript. AO designed the experiments, collected and analysed the data and wrote the manuscript. NL and KM provided, analyzed and interpreted the data. DA designed and verified the experiments, analysed the data, and wrote and provided critical feedback for the manuscript. DA and AO confirm the authenticity of all the raw data. All authors read and approved the final version of the study.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References

1 

Pereira DM, Correia-da-Silva G, Valentão P, Teixeira N and Andrade PB: Anti-inflammatory effect of unsaturated fatty acids and Ergosta-7,22-dien-3-ol from Marthasterias glacialis: Prevention of CHOP-mediated ER-stress and NF-κB activation. PLoS One. 9(e88341)2014.PubMed/NCBI View Article : Google Scholar

2 

Lee HJ, Shin JS, Lee KG, Park SC, Jang YP, Nam JH and Lee KT: Ethanol extract of Potentilla supina Linne suppresses LPS-induced inflammatory responses through NF-κB and AP-1 inactivation in macrophages and in endotoxic mice. Phytother Res. 31:475–487. 2017.PubMed/NCBI View Article : Google Scholar

3 

Bennett JE, Dolin R and Blaser MJ: Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases. 8th edition. Bennett JE, Dolin R and Blaser MJ (eds). Elsevier/Saunders, Philadelphia, PA, p27, 2015.

4 

Oh YC, Cho WK, Oh JH, Im GY, Jeong YH, Yang MC and Ma JY: Fermentation by Lactobacillus enhances anti-inflammatory effect of Oyaksungisan on LPS-stimulated RAW 264.7 mouse macrophage cells. BMC Complement Altern Med. 12(17)2012.PubMed/NCBI View Article : Google Scholar

5 

Fard MT, Arulselvan P, Karthivashan G, Adam SK and Fakurazi S: Bioactive extract from Moringa oleifera inhibits the pro-inflammatory mediators in lipopolysaccharide stimulated macrophages. Pharmacogn Mag. 11 (Suppl 4):S556–S563. 2015.PubMed/NCBI View Article : Google Scholar

6 

Adrie C, Alberti C, Chaix-Couturier C, Azoulay E, De Lassence A, Cohen Y, Meshaka P, Cheval C, Thuong M, Troché G, et al: Epidemiology and economic evaluation of severe sepsis in France: Age, severity, infection site, and place of acquisition (community, hospital, or intensive care unit) as determinants of workload and cost. J Crit Care. 20:46–58. 2005.PubMed/NCBI View Article : Google Scholar

7 

O'Brien DJ and Gould IM: Maximizing the impact of antimicrobial stewardship: The role of diagnostics, national and international efforts. Curr Opin Infect Dis. 26:352–358. 2013.PubMed/NCBI View Article : Google Scholar

8 

Parvizi J and Kim GK: High Yield Orthopaedics. Saunders/Elsevier, Philadelphia, PA, pp325-326, 2010.

9 

Dhikav V, Singh S, Pande S, Chawla A and Anand KS: Non-steroidal drug-induced gastrointestinal toxicity: Mechanisms and management. JIACM. 4:315–322. 2003.

10 

Wangcharoen W, Mengumphan K and Amornlerdpison D: Fatty acid composition, physical properties, acute oral toxicity and antioxidant activity of crude lipids from adipose tissue of some commercialized freshwater catfish. Warasan Khana Witthayasat Maha Witthayalai Chiang Mai. 42:626–636. 2015.

11 

Khoddami A, Ariffin A, Bakar J and Mohd Ghazali H: Fatty acid profile of the oil extracted from fish waste (head, intestine and liver) (Sardinella lemuru). World Appl Sci J. 7:127–131. 2009.

12 

Gammone MA, Riccioni G, Parrinello G and D'Orazio N: Omega-3 polyunsaturated fatty acids: Benefits and endpoints in sport. Nutrients. 11(46)2018.PubMed/NCBI View Article : Google Scholar

13 

Kim JS and Park JW: Mince from seafood processing by-product and surimi as food ingredients. In: Maximising the Value of Marine By-Products. Shahidi F (ed). Woodhead Publishing, Sawston, pp196-228, 2007.

14 

Kim SK and Mendis E: Bioactive compounds from marine processing byproducts - A review. Food Res Int. 39:383–393. 2006.

15 

Tawfik M: Proximate composition and fatty acids profiles in most common available fish species in Saudi market. Asian J Clin Nutr. 1:50–57. 2009.

16 

Bussarin T, Kriangsak M, Narissara L and Doungporn A: Comparison of fatty acid profiles of freshwater hybrid catfish. In: Proceedings of MJU Annual Conference. The Office of Agricultural Research and Extension Maejo, Maejo University. pp60-61. 2018.

17 

Keapai W, Apichai S, Amornlerdpison D and Lailerd N: Evaluation of fish oil-rich in MUFAs for anti-diabetic and anti-inflammation potential in experimental type 2 diabetic rats. Korean J Physiol Pharmacol. 20:581–593. 2016.PubMed/NCBI View Article : Google Scholar

18 

Amornlerdpison D, Rattanaphot T, Tongsiri S, Srimaroeng C and Mengumphan K: Effect of omega-9-rich fish oil on antioxidant enzymes and relative immune gene expressions in Nile tilapia (Oreochromis niloticus). J Sci Technol. 41:1287–1293. 2019.

19 

AOAC Official Method of Analysis: Official Method 996.06 Fat (Total, Saturated, and Unsaturated) in Foods. AOAC International Chapter 41. Oils and Fats: 20-24. 2002.

20 

Li Y, Hao N, Zou S, Meng T, Tao H, Ming P, Li M, Ding H, Li J, Feng S, et al: Immune regulation of RAW264.7 cells in vitro by flavonoids from Astragalus complanatus via activating the NF-κB signalling pathway. J Immunol Res. 2018(7948068)2018.PubMed/NCBI View Article : Google Scholar

21 

Yen TL, Chang CC, Chung CL, Ko WC, Yang CH and Hsieh CY: Neuroprotective effects of platonin, a therapeutic immunomodulating medicine, on traumatic brain injury in mice after controlled corticalimpact. Int J Mol Sci. 19(1100)2018.PubMed/NCBI View Article : Google Scholar

22 

Teratake Y, Kuga C, Hasegawa Y, Sato Y, Kitahashi M, Fujimura L, Watanabe-Takano H, Sakamoto A, Arima M, Tokuhisa T, et al: Transcriptional repression of p27 is essential for murine embryonic development. Sci Rep. 6(26244)2016.PubMed/NCBI View Article : Google Scholar

23 

Zhao H, Bauzon F, Fu H, Lu Z, Cui J, Nakayama K, Nakayama KI, Locker J and Zhu L: Skp2 deletion unmasks a p27 safeguard that blocks tumorigenesis in the absence of pRb and p53 tumor suppressors. Cancer Cell. 24:645–659. 2013.PubMed/NCBI View Article : Google Scholar

24 

Tokumoto M, Fujiwara Y, Shimada A, Hasegawa T, Seko Y, Nagase H and Satoh M: Tokumoto1 M, Fujiwara Y, Shimada A, Hasegawa T, Seko Y, Nagase H, Satoh M: Cadmium toxicity is caused by accumulation of p53 through the down-regulation of Ube2d family genes in vitro and in vivo. J Toxicol Sci. 36:191–200. 2011.PubMed/NCBI View Article : Google Scholar

25 

Chen YG, Zhang Y, Deng LQ, Chen H, Zhang YJ, Zhou NJ, Yuan K, Yu LZ, Xiong ZH, Gui XM, et al: Control of methicillin-resistant Staphylococcus aureus pneumonia utilizing TLR2 agonist Pam3CSK4. PLoS One. 11(e0149233)2016.PubMed/NCBI View Article : Google Scholar

26 

Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001.PubMed/NCBI View Article : Google Scholar

27 

Institute of Medicine (US) Panel on Micronutrients: Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc. National Academies Press, Washington, DC, 2001.

28 

Shin S: Safety of celecoxib versus traditional nonsteroidal anti-inflammatory drugs in older patients with arthritis. J Pain Res. 11:3211–3219. 2018.PubMed/NCBI View Article : Google Scholar

29 

Park JY, Pillinger MH and Abramson SB: Prostaglandin E2 synthesis and secretion: The role of PGE2 synthases. Clin Immunol. 119:229–240. 2006.PubMed/NCBI View Article : Google Scholar

30 

Valavanidis A, Vlachogianni T and Fiotakis C: 8-Hydroxy-2'-deoxyguanosine (8-OHdG): A critical biomarker of oxidative stress and carcinogenesis. J Environ Sci Health Part C Environ Carcinog Ecotoxicol Rev. 27:120–139. 2009.PubMed/NCBI View Article : Google Scholar

31 

Yuan L, Zhang Y, Xia J, Liu B, Zhang Q, Liu J, Luo L, Peng Z, Song Z and Zhu R: Resveratrol induces cell cycle arrest via a p53-independent pathway in A549 cells. Mol Med Rep. 11:2459–2464. 2015.PubMed/NCBI View Article : Google Scholar

32 

Yuan L, Zhang Y, Xia J, Liu B, Zhang Q, Liu J, Luo L, Peng Z, Song Z, Zhu R, Zhu R, et al: Resveratrol induces cell cycle arrest via a p53-independent pathway in A549 cells. Mol Med Rep. 11:2459–2464. 2015.PubMed/NCBI View Article : Google Scholar

33 

Shaw PH: The role of p53 in cell cycle regulation. Pathol Res Pract. 192:669–675. 1996.PubMed/NCBI View Article : Google Scholar

34 

Donehower LA: Phosphatases reverse p53-mediated cell cycle checkpoints. Proc Natl Acad Sci USA. 111:7172–7173. 2014.PubMed/NCBI View Article : Google Scholar

35 

Møller MB: P27 in cell cycle control and cancer. Leuk Lymphoma. 39:19–27. 2000.PubMed/NCBI View Article : Google Scholar

36 

Priante G, Bordin L, Musacchio E, Clari G and Baggio B: Fatty acids and cytokine mRNA expression in human osteoblastic cells: A specific effect of arachidonic acid. Clin Sci (Lond). 102:403–409. 2002.PubMed/NCBI

37 

Oh YT, Lee JY, Lee J, Kim H, Yoon KS, Choe W and Kang I: Oleic acid reduces lipopolysaccharide-induced expression of iNOS and COX-2 in BV2 murine microglial cells: Possible involvement of reactive oxygen species, p38 MAPK, and IKK/NF-kappaB signaling pathways. Neurosci Lett. 464:93–97. 2009.PubMed/NCBI View Article : Google Scholar

38 

Olefsky JM and Glass CK: Macrophages, inflammation, and insulin resistance. Annu Rev Physiol. 72:219–246. 2010.PubMed/NCBI View Article : Google Scholar

39 

Ansar W and Ghosh S: Inflammation and inflammatory diseases, markers, and mediators: Role of CRP in some inflammatory diseases. In: Biology of C Reactive Protein in Health and Disease. pp67-107, 2016.

40 

Kondreddy VK and Kamatham AN: Celecoxib, a COX-2 inhibitor, synergistically potentiates the anti-inflammatory activity of docosahexaenoic acid in macrophage cell line. Immunopharmacol Immunotoxicol. 38:153–161. 2016.PubMed/NCBI View Article : Google Scholar

41 

Monmai C, Go SH, Shin IS, You SG, Lee H, Kang SB and Park WJ: Immune-enhancement and anti-inflammatory activities of fatty acids extracted from Halocynthia aurantium tunic in RAW264.7 cells. Mar Drugs. 16(16)2018.PubMed/NCBI View Article : Google Scholar

42 

Sandhiutami NM, Moordiani M, Laksmitawati DR, Fauziah N, Maesaroh M and Widowati W: In vitro assesment of anti-inflammatory activities of coumarin and Indonesian cassia extract in RAW264.7 murine macrophage cell line. Iran J Basic Med Sci. 20:99–106. 2017.PubMed/NCBI View Article : Google Scholar

43 

Denkins Y, Kempf D, Ferniz M, Nileshwar S and Marchetti D: Role of omega-3 polyunsaturated fatty acids on cyclooxygenase-2 metabolism in brain-metastatic melanoma. J Lipid Res. 46:1278–1284. 2005.PubMed/NCBI View Article : Google Scholar

44 

Liu Y, Chen LY, Sokolowska M, Eberlein M, Alsaaty S, Martinez-Anton A, Logun C, Qi HY and Shelhamer JH: The fish oil ingredient, docosahexaenoic acid, activates cytosolic phospholipase A2 via GPR120 receptor to produce prostaglandin E2 and plays an anti-inflammatory role in macrophages. Immunology. 143:81–95. 2014.PubMed/NCBI View Article : Google Scholar

45 

Wada M, DeLong CJ, Hong YH, Rieke CJ, Song I, Sidhu RS, Yuan C, Warnock M, Schmaier AH, Yokoyama C, et al: Enzymes and receptors of prostaglandin pathways with arachidonic acid-derived versus eicosapentaenoic acid-derived substrates and products. J Biol Chem. 282:22254–22266. 2007.PubMed/NCBI View Article : Google Scholar

46 

Yuan C, Sidhu RS, Kuklev DV, Kado Y, Wada M, Song I and Smith WL: Cyclooxygenase allosterism, fatty acid-mediated cross-talk between monomers of cyclooxygenase homodimers. J Biol Chem. 284:10046–10055. 2009.PubMed/NCBI View Article : Google Scholar

47 

Medeiros-de-Moraes IM, Gonçalves-de-Albuquerque CF, Kurz AR, Oliveira FM, de Abreu VH, Torres RC, Carvalho VF, Estato V, Bozza PT, Sperandio M, et al: Omega-9 oleic acid, the main compound of olive oil, mitigates inflammation during experimental sepsis. Oxid Med Cell Longev. 2018(6053492)2018.PubMed/NCBI View Article : Google Scholar

48 

Kawanishi S, Ohnishi S, Ma N, Hiraku Y and Murata M: Crosstalk between DNA damage and inflammation in the multiple steps of carcinogenesis. Int J Mol Sci. 18(18)2017.PubMed/NCBI View Article : Google Scholar

49 

Jaiswal M, LaRusso NF, Burgart LJ and Gores GJ: Inflammatory cytokines induce DNA damage and inhibit DNA repair in cholangiocarcinoma cells by a nitric oxide-dependent mechanism. Cancer Res. 60:184–190. 2000.PubMed/NCBI

50 

Sakai C, Ishida M, Ohba H, Yamashita H, Uchida H, Yoshizumi M and Ishida T: Fish oil omega-3 polyunsaturated fatty acids attenuate oxidative stress-induced DNA damage in vascular endothelial cells. PLoS One. 12(e0187934)2017.PubMed/NCBI View Article : Google Scholar

51 

Chunder N, Wang L, Chen C, Hancock WW and Wells AD: Cyclin-dependent kinase 2 controls peripheral immune tolerance. J Immunol. 189:5659–5666. 2012.PubMed/NCBI View Article : Google Scholar

52 

Laphanuwat P and Jirawatnotai S: Immunomodulatory roles of cell cycle regulators. Front Cell Dev Biol. 7(23)2019.PubMed/NCBI View Article : Google Scholar

53 

Ji KY, Kim KM, Kim YH, Im AR, Lee JY, Park B, Na M and Chae S: The enhancing immune response and anti-inflammatory effects of Anemarrhena asphodeloides extract in RAW 264.7 cells. Phytomedicine. 59(152789)2019.PubMed/NCBI View Article : Google Scholar

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November-2021
Volume 22 Issue 5

Print ISSN: 1792-0981
Online ISSN:1792-1015

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Spandidos Publications style
Tongmee B, Ontawong A, Lailerd N, Mengamphan K and Amornlerdpisan D: Anti‑inflammatory effects and enhancing immune response of freshwater hybrid catfish oil in RAW264.7 cells. Exp Ther Med 22: 1223, 2021
APA
Tongmee, B., Ontawong, A., Lailerd, N., Mengamphan, K., & Amornlerdpisan, D. (2021). Anti‑inflammatory effects and enhancing immune response of freshwater hybrid catfish oil in RAW264.7 cells. Experimental and Therapeutic Medicine, 22, 1223. https://doi.org/10.3892/etm.2021.10657
MLA
Tongmee, B., Ontawong, A., Lailerd, N., Mengamphan, K., Amornlerdpisan, D."Anti‑inflammatory effects and enhancing immune response of freshwater hybrid catfish oil in RAW264.7 cells". Experimental and Therapeutic Medicine 22.5 (2021): 1223.
Chicago
Tongmee, B., Ontawong, A., Lailerd, N., Mengamphan, K., Amornlerdpisan, D."Anti‑inflammatory effects and enhancing immune response of freshwater hybrid catfish oil in RAW264.7 cells". Experimental and Therapeutic Medicine 22, no. 5 (2021): 1223. https://doi.org/10.3892/etm.2021.10657