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. 2022 Aug 15;17(8):e0270794. doi: 10.1371/journal.pone.0270794

Anti-inflammatory effects of neutral lipids, glycolipids, phospholipids from Halocynthia aurantium tunic by suppressing the activation of NF-κB and MAPKs in LPS-stimulated RAW264.7 macrophages

A-yeong Jang 1,#, Weerawan Rod-in 2,3,#, Chaiwat Monmai 2,3, Gyoung Su Choi 1, Woo Jung Park 1,2,3,*
Editor: Charles Michael Greenlief4
PMCID: PMC9377571  PMID: 35969529

Abstract

Halocynthia aurantium is a marine organism that has been considered a promising source for bio-functional materials. Total lipids were extracted from H. aurantium tunic, and then they were separated into neutral lipids, glycolipids, and phospholipids. In the present study, fatty acid profiles of three lipids and their anti-inflammatory effects in RAW264.7 cells were investigated. Among the lipid classes, phospholipids showed the diversity of fatty acid constituents, compared with the glycolipids and neutral lipids. Three lipids contain different contents of fatty acids depending on the kinds of lipids. The most contents were saturated fatty acids (SFAs, 53–69% of the fatty acids) and monounsaturated fatty acids (MUFAs, 15–17% of fatty acids) and polyunsaturated fatty acids (PUFAs, 14–32% of fatty acids) are followed. H. aurantium lipids not only dose-dependently inhibited nitric oxide production but also reduced the expression of inflammatory cytokine genes such as TNF-α, IL-1β, and IL-6 in LPS-stimulated macrophages. It was also demonstrated that the expression of COX-2 was dose-dependently suppressed. Moreover, H. aurantium lipids decreased phosphorylation of NF-κB p-65, p38, ERK1/2, and JNK, suggesting that three lipids from H. aurantium tunic provide anti-inflammatory effects through NF-κB and MAPK signaling. These results indicate that H. aurantium is a potential source for anti-inflammation.

Introduction

Lipids, especially essential fatty acids play an important role in the health and development of humans and play a critical role in the prevention of disease by altering their composition [1]. Lipids are a source of energy and the structure of cell membranes and polyunsaturated fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are essential fatty acids to regulate the inflammatory responses on the macrophage cells, which are considered anti-inflammatory agents [2–5]. Moreover, they can inhibit the activation of the pro-inflammatory transcription factor, such as nuclear factor κB (NF-κB) [3, 5, 6] and suppress the activation of mitogen-activated protein kinases (MAPKs) signaling pathway [6, 7]. These pathways involved inflammatory responses, which produce inflammatory cytokines and inflammatory mediators. [8]. In addition, high levels of EPA and DHA were reported from the lipid extracts including from total lipids, neutral lipids, and polar lipids of various ascidian species [9–12]. The lipid extracts from ascidians have improved beneficial health effects, such as anti-diabetic [13] and antioxidant effects.

Macrophages play important roles in inflammation and they can be activated by endotoxin which cause the production of inflammatory cytokines such as interleukin-1β (IL-1β), IL-6, and tumor necrosis factor (TNF)-α, and releasing cyclooxygenase-2 (COX-2) and nitric oxide synthase (iNOS) catalyze the production of prostaglandin E2 (PGE2) and nitric oxide (NO) as inflammatory mediators [8, 14]. The natural substances such as alkaloids, steroids, polysaccharides, fatty acids, proteins, and other, which were isolated form marine biomaterials have been demonstrated strong anti-inflammatory activities [15]. They were determined by inhibiting the production of NO, TNF-α and suppressing the expression of IL-1β, IL-6, TNF-α, iNOS and COX-2, in RAW264.7 macrophages activated by lipopolysaccharides (LPS) [16–19].

Halocynthia aurantium is a solitary ascidian found in the Southern, Eastern Sea of Korea, and Northern Sea of Japan. The tunic is an essential structure part in the outer protective covering of the body, which contains a cellulose-like substance [20, 21]. Total lipids of H. aurantium tunic composed of the most abundant of palmitic acids (16:0), stearic acids (18:0), α-linolenic acids (ALA, 18:3 n-3), eicosapentaenoic acids (EPA, 20:5 n-3), and docosahexaenoic acids (DHA, 22:6 n-3) [22]. Similar to ascidian species, H. roretzi showed also the same fatty acids in total lipids, neutral lipids, and phospholipids. H. aurantium showed biological effects such as antimicrobial peptides (Dicynthaurin, and Halocidin) [23–25], and antioxidant effects [26]. Vanadium-binding protein from H. roretzi was investigated in macrophage-like RAW264.7 cells stimulated by LPS [27, 28]. However, no previous study has evaluated the fatty acid profiles of individual lipids such as phospholipids, glycolipids, and neutral lipids, which were isolated from H. aurantium tunic. Moreover, few studies have determined how these fractionated lipids exert anti-inflammatory activity on macrophages.

Therefore, the present study was to identify the fatty acid composition in H. aurantium lipids, containing neutral lipids, glycolipids, and phospholipids, and their anti-inflammatory activities using RAW264.7 cells.

Materials and methods

Preparation of fractionated lipids from H. aurantium tunic

H. aurantium used in this study was obtained in Jumunjin market on the he East Sea near Gangwon Province, South Korea. H. aurantium tunic was dried and homogenized to powder. Total lipids were extracted using a modified method by Bligh and Dyer [29]. A mixture of chloroform/methanol (1:2, v/v) containing 0.01% of butylated hydroxytoluene (BHT) to the solvent as antioxidant [30] was added to 4.5 g of dry weight sample and centrifuged at 3000 rpm for 10 minutes. Subsequently, the organic solvent was collected and filtered. A rotary evaporator (IKA® RV10, EYELA, China) was to remove the solvent, and the residual solvent was removed by nitrogen evaporator (N-EVAP, Organomation Associates Inc., USA). The extracted lipids with a yield of 33.6 mg (w/w) or 0.75% of dry material were then resuspended in hexane for fractionation of lipid extracts.

The total lipids were added into silica gel column filled with silica gel and anhydrous sodium sulfate using chloroform, acetone, and methyl alcohol to produce the neutral lipids, glycolipids, and phospholipids, respectively. After separating, these extracts were evaporated and weighed. The neutral lipids, glycolipids, and phospholipids were shown to have a high lipid yield of 8.63% (2.9 mg), 30.95% (10.4 mg), and 44.94% (15.1 mg) of total lipid, respectively. The H. aurantium lipids at 2 mg/mL (set to 100%) were prepared in dimethyl sulfoxide (DMSO, Sigma-Aldrich, USA, Cat# D8418) and stored at −20 ◦C for further analysis.

Analysis of fatty acid profiles

Fatty acid compositions of neutral lipids, glycolipids, and phospholipids were determined using gas chromatography (GC)-flame ionization detection (FID) (Perkin Elmer, Waltham, MA, USA) as previously described [31]. Quantification of fatty acid peaks were identified by the comparison of their retention times with heptadecanoic acid (C17:0) as internal standard (Sigma-Aldrich, USA, Cat# H3500). Results were presented as quintuplicate (n = 5) independent experiments.

Cell culture and treatment

Mouse macrophages RAW 264.7 cell line was obtained from Koran Cell Line Bank (KCLB, Cat# 40071, RRID: CVCL_0493). The cells were culture in RPMI-1640 medium (Gibco™, Waltham, USA, Cat# 11875–093) supplemented with 10% fetal bovine serum (FBS, Welgene, Korea, Cat# S001-07) and 1% penicillin/streptomycin (Welgene, Korea, Cat# LS202-02), and then incubated at 37°C in a humidified atmosphere of 5% CO2. The lipids were dissolved in RPMI-1640 medium (GibcoTM, Waltham, USA, Cat# 11835–030) supplemented with 1% FBS and 1% penicillin/streptomycin to different concentrations at 0.5%, 1.0%, 2.0% and 4.0%. 100 μL of the lipids were culture into the RAW264.7 cells (at a density of 1 × 105 cell/well) for 1 h. After incubation, the presence or absence of 1 μg/mL lipopolysaccharide (LPS from Escherichia coli O111:B4, Sigma-Aldrich, USA, Cat# L4391-1MG) were added into each well for another 24 h.

Measurement of cell viability and NO production

The cell viability of three lipids from H. aurantium was analyzed using EZ-Cytox Cell Viability Assay Kit (DaeilLab Service, Seoul, Korea, Cat# EZ-3000) as described by Kim et al. [19]. Three independent experiments were performed in triplicate. Griess reagent (Promega, WI, USA, Cat# G2930) was used to evaluate the LPS induced the production of nitric oxide [32] according to the manufacturer’s instructions. Three independent experiments were performed in triplicate.

Analysis of immune gene expression by quantitative real-time PCR

The mRNA expression levels of immune-regulated genes were determined by qRT-PCR. TRI reagent® (Molecular Research Center, Cincinnati, OH, USA, Cat# TR118) was used to extract the total RNA from RAW264.7cells. High capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA, Cat# 4368814) was used to reverse transcribe cDNA. Real-time PCR was performed on QuantStudio™ 7 FlexReal-Time PCR System (Applied Biosystems, Foster City, CA, USA) using TB Green® Premix Ex Taq™ II (Takara Bio Inc., Shiga, Japan, Cat#RR820A). The relative expression levels of IL-1β, IL-6, TNF-α, and COX-2 were normalized using the β-actin (S1 Table). Results were presented as triplicate independent experiments.

Western blotting analysis

Cell lysates were prepared using RIPA buffer (Tech & Innovation, Hebei, China, Cat# BRI-9001) containing 0.5 mM EDTA solution, and a protease & phosphatase inhibitor cocktail (Thermo Fisher Scientific, USA, Cat# 78440). SDS-PAGE and western blotting were performed. The protein was analyzed by immunoblot using primary antibodies against phospho-nuclear NF-κB-p65 (Cell Signaling Technology, MA, USA, Cat# 3033, RRID: AB_331284), phospho-p38 (Cell Signaling Technology, MA, USA, Cat# 9211, RRID:AB_331641), phospho-ERK1/2 (Cell Signaling Technology, MA, USA, Cat# 9101, RRID: AB_331646), phospho-JNK (Cell Signaling Technology, MA, USA, Cat# 9251, RRID: AB_331659), and α-tubulin (Abcam, Cambridge, UK, Cat# ab15246, RRID:AB_301787), and then was incubated with secondary antibodies as goat anti-rabbit IgG (H+L)-HRP (GenDEPOT, TX, USA, Cat# SA006-500). The protein bands were measured by the ChemiDoc XRS+ imaging system, and ImageLab software (Bio-Rad, Hercules, CA, USA). Results were presented as triplicate independent experiments.

Statistical analysis

All data were subjected to analysis of variance using Statistix 8.1 Statistics Software (Tallahassee, FL, USA). One-way ANOVA followed by the Duncan’s multiple range test was used to evaluate the significance of the differences (p < 0.05). Data are expressed as mean ± standard deviation (SD).

Results

Fatty acid profiles of neutral lipids, glycolipids, and phospholipids, which were isolated from H. aurantium tunic

Fig 1 presents the percentages of fatty acids composition in neutral lipids, glycolipids, and phospholipids, which determined by GC-FID analyses. The results showed that the highest amount of the lipids were SFAs in neutral lipids (63.75%), glycolipids (52.70%), and phospholipids (69.45%). Total amount of MUFAs were 16.92% in neutral lipids, 14.87% in glycolipids, and 16.52% phospholipids. Moreover, total amount of PUFAs are 19.34, 32.44, 14.03% of neutral lipids, glycolipids, and phospholipids, respectively.

Fig 1. Fatty acid composition (%) of H. aurantium tunic lipids.

Fig 1

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(A) Neutral lipids. (B) Glycolipids. (C) Phospholipids. The letters a–h indicate significant differences (p < 0.05) between the amounts of fatty acids, which were obtained from each lipid of H. aurantium tunic. Results represent means ± SD (n = 5). SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid.

At first, myristic acid (14:0), palmitic acid (16:0), and stearic acid (18:0) was mainly contained in SFAs, and in addition the phospholipids also showed arachidic acid (20:0). At second, the major MUFAs are palmitoleic acid (16:1n-7) and oleic acid (18:1n-9) and they were contained with 4.92 and 11.99% in neutral lipids, 8.16 and 2.96% in glycolipids, and 7.25 and 6.82% in phospholipids, respectively. At third, the major PUFAs are linoleic acid (18:2n-6) and eicosapentaenoic acid (20:5n-3) which were 13.87 and 5.46% in neutral lipids, 2.24 and 4.49% in glycolipids, and 5.43 and 2.29% in phospholipids. Especially, the presence of stearidonic acid (18:4n-3) and arachidonic acid (20:4n-6) was also identified in glycolipids (22.20 and 3.51%) and phospholipids (3.64 and 1.27%).

Effect of neutral lipids, glycolipids, and phospholipids from H. aurantium tunic on macrophage cell proliferation

To determine if the fractionated lipids containing neutral lipids, glycolipids, and phospholipids are not toxic to RAW264.7 macrophages, we investigated cytotoxicity using an EZ-Cytox cell viability assay kit. As shown in Fig 2A, our results showed that the neutral lipids, glycolipids, and phospholipids did not give any cytotoxicity at concentrations of 0.5–4.0% of lipids.

Fig 2. The effects of neutral lipids, glycolipids and phospholipids from H. aurantium tunic on macrophage proliferation.

Fig 2

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(A) Cell proliferation. (B) NO production. Significant different at p<0.05 (*) compared with RPMI.

Anti-inflammatory effects of neutral lipids, glycolipids, and phospholipids from H. aurantium tunic on NO production

To investigate the anti-inflammatory activity of fractionated lipids from H. aurantium tunic, we measured lipid-mediated inhibition of NO production in LPS-stimulated RAW264.7 cells using Griess reagent assay. NO production was determined using different of three lipids at 0.5–4.0% concentrations. Neutral lipids, glycolipids, and phospholipids of H. aurantium tunic gradually decreased NO production in LPS-stimulated RAW264.7 cells according to the lipid concentration (Fig 2B).

Anti-inflammatory effects of neutral lipids, glycolipids and phospholipids from H. aurantium tunic on immune-associated gene expression

Since NO production was significantly inhibited by the fractionated lipids from H. aurantium tunic, we measured the mRNA expression of immune-associated genes such as IL-1β, IL-6, TNF-α, and COX-2 in LPS-stimulated RAW264.7 cells by qRT-PCR. The expression levels of inflammatory cytokines were significantly down-regulated depending on the concentration of H. aurantium lipids, including neutral lipids (Fig 3A), glycolipids (Fig 3B), and phospholipids (Fig 3C). Our results showed that the IL-1β expression was highly reduced by H. aurantium lipids and the expression levels of other cytokine genes such as IL-6 and TNF-α were dose-dependently decreased. Moreover, the expression levels of COX-2, another well-known inflammatory biomarker, were dose-dependently suppressed according to the concentration of neutral lipids, glycolipids, and phospholipids from H. aurantium tunic.

Fig 3. Quantification of immune-associated gene expression (fold) in LPS-stimulated RAW264.7 cells.

Fig 3

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(A) The relative mRNA expression of neutral lipids. (B) The relative mRNA expression of glycolipids. (C) The relative mRNA expression of phospholipids. Significant different at p<0.05 (*) compared with LPS.

Anti-inflammatory effects of neutral lipids, glycolipids and phospholipids from H. aurantium tunic on MAPK and NF-κB signaling pathway

In order to investigate the understanding of the molecular mechanism by which H. aurantium lipids exert their anti-inflammatory effect, immune signaling pathways such as NF-κB and MAPK were analyzed. Our results indicated neutral lipids (Fig 4A), glycolipids (Fig 4B), and phospholipids (Fig 4C), which were isolated from H. aurantium tunic, dose-dependently inhibited the phosphorylation of NF-κB p-65, ERK1/2, JNK, and p38 in a dose-dependent manner. These results showed that fractionated lipids from H. aurantium tunic inhibited inflammation through MAPK and NF-κB signaling pathways in LPS-stimulated RAW246.7 cells.

Fig 4. The effects of fractionated lipids from H. aurantium tunic on the protein expression associated with NF-κB and MAPK pathways in LPS-stimulated RAW264.7 cells.

Fig 4

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(A) Western blot and relative band of neutral lipids. (B) Western blot and relative band blot of glycolipids. (C) Western blot and relative band of phospholipids. Significant different at p<0.05 (*) compared with LPS.

Discussion

Halocynthia aurantium, an edible ascidian species, has not been studied, although tunicates and ascidians are well-known to contain biologically active compounds. This study was undertaken to analyze the fatty acid composition in fractionated lipids including neutral lipids, glycolipids, and phospholipids from H. aurantium tunic, and to investigate their anti-inflammatory effects on LPS-stimulated macrophages.

Recently, the total lipids of H. aurantium tunic were analyzed the fatty acid compositions, consisting of the most abundant of palmitic acids (21.73±2.16), stearic acids (33.13±3.22), oleic acid (6.78±0.28%), LA (2.72±0.23%), dihomo α-linolenic acid (4.09±0.36), EPA (3.88±0.31%), and DHA (3.38±0.34%) [31]. The current results showed the fatty acid profiles of fractionated lipids (neutral lipids, glycolipids, and phospholipids) from the total lipids extracted from H. aurantium tunic (Fig 1).

The lipid extracts including neutral lipids, glycolipids, and phospholipids, which isolated from H. roretzi were found to be similar to the total lipids of H. aurantium [12]. In addition, the lipid extracts of Hippocampus trimaculatus, containing neutral lipids, glycolipids, phospholipids reduced the production of NO, IL-6, IL-1β, and TNF-α in LPS-stimulated RAW264.7 cells [18]. Moreover, many studies investigated the anti-inflammatory activities of various bioactive compounds from ascidian species. The enzymatic hydrolysates derived from Styela clava down-regulated LPS-induced expression of iNOS and COX-2, suppressed the production of pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α and they inhibited LPS-induced phosphorylation of ERK, JNK, and p38 [33]. Thomson et al. reported that polysaccharides isolated from the Ascidiella aspersa exhibited anti-inflammatory effects in vitro and in vivo systems [34]. H. roretzi also exhibited the anti-inflammatory effect of carotenoids and vanadium-binding protein, which down-regulated the expression of pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α as well as iNOS and COX-2 mRNA expression on LPS-stimulated RAW264.7 cells [28]. Similar to previous reports, our results also showed that H. aurantium lipids including neutral lipids, glycolipids, and phospholipids effectively suppressed the expression of inflammatory cytokines, such as IL-1β, IL-6, and TNF-α (Fig 3) which are pro-inflammatory cytokines that activated T cells, maturation of B cells, and activation of NK cells, especially monocytes or macrophages [35]. In addition, the COX-2 expression levels, which is a key enzyme in the production of prostaglandins (PGE2) by LPS-stimulated macrophages in the inflammatory process [36], was down-regulated by H. aurantium lipids depending on the lipid concentration (Fig 3). These results suggested that H. aurantium lipids are biomaterials to contain anti-inflammatory effects on immune systems in a physiological system.

In immune signaling pathways, there are critical two pathways including NF-κB and MAPK pathways, in which NF-κB is a transcription factor, is a critical regulator mediator for iNOS, COX-2 transcription, and the production of cytokines in LPS-induced macrophages [37, 38], and The MAPK pathway is considered one of the main intracellular signaling pathways that regulate inflammatory responses [39]. Our current results showed that three lipids from H. aurantium tunic reduced the activation of the NF-κB pathway by inhibiting the phosphorylation of the NF-κB p-65 subunit (Fig 4). In addition, H. aurantium lipids inhibited the expression of MAPKs (ERK1/2, JNK, and p38) in LPS-induced RAW 264.7 cells in a dose-dependent manner. Similar to ascidian species, vanadium-binding protein from H. roretzi has reported being anti-inflammatory effects. This protein inhibited the LPS-stimulated inflammatory response in RAW264.7 macrophages through NF-κB and MAPK pathways [27]. Skipjack tuna eyeball oil was identified as the main fatty acids of DHA (25%) and EPA (5%), inhibited the production of NO and pro-inflammatory cytokine by suppressing the activation of NF-κB and MAPK signaling pathways in RAW264.7 cells [40]. Taken together, these results indicated that three lipids of H. aurantium tunic induced the suppression of NO, and immune-regulated genes in activated macrophages through the inhibition of NF-κB and MAPK pathways.

Conclusions

The present study demonstrated that the fractionated lipids from H. aurantium tunic, including neutral lipids, glycolipids, and phospholipids composed of the highest contents of 16:0 and 18:0 as SFAs, 16: 1n7 and 18:1n9 as MUFAs, and 18:2n6 and 20:5n3 as PUFAs. Three lipids of H. aurantium tunic significantly inhibited the production of NO and the expression of immune-associated genes such as IL-1β, IL-6, TNF-α, and COX-2. Likewise, the decreased expression levels led to further activation of NF-κB p-65 and MAPK molecules, such as ERK1/2, JNK, and p38, thus alleviating the immune response. These results might be helpful to understand the anti-inflammatory mechanisms of H. aurantium lipids on immune cells and suggested that H. aurantium is a potential source for anti-inflammation.

Supporting information

S1 Raw images. Original western blot gel image data.

(PDF)

Click here for additional data file. (1.8MB, pdf)
S1 Table. Nucleotide primers used in this study.

(PDF)

Click here for additional data file. (55.3KB, pdf)

Data Availability

All relevant data are within the paper and its Supporting information files.

Funding Statement

This study was partially supported by the Basic Science Research Program of the National Research Foundation of Korea (NRF), which is funded by the Ministry of Science, ICT & Future Planning (2019R1A2B5B01070542). This research project is also supported by the University Emphasis Research Institute Support Program (No.2018R1A61A03023584), which is funded by the National Research Foundation of Korea. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Charles Michael Greenlief

20 Apr 2022

PONE-D-22-03751Anti-inflammatory effects of neutral lipids, glycolipids, phospholipids from Halocynthia aurantium tunic by suppressing the activation of NF-κB and MAPKs in LPS-stimulated RAW264.7 macrophagesPLOS ONE

Dear Dr. Park,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Academic Editor

PLOS ONE

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(This study was partially supported by the Basic Science Research Program of the National Research Foundation of Korea (NRF), which is funded by the Ministry of Science, ICT & Future Planning (2019R1A2B5B01070542). This research project is also supported by the University Emphasis Research Institute Support Program (No.2018R1A61A03023584), which is funded by the National Research Foundation of Korea.)

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Please revise your manuscript taking into account the comments of the two reviewers. Point 2 about figure quality from the first reviewer can be disregarded as they did not download the full tiff files.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

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Reviewer #1: The manuscript describes anti-inflammatory effects of lipid extracted from Halocynthia aurantium. Even though results are interesting, need major revision to enhance the quality of the paper sutiable for publication in PLOS ONE.

Major comments –

1. Author described the extraction method of total lipids (lines 86-87), unfortunately no extraction yield was given anywhere. Similarly, lipid extracted from H. aurantium was fractionated into neutral lipid, glycolipid and phospholipids (line 94-96), and neither initial lipid weight nor the fractions yield were given. This is important information to understand the value of the lipid from H. aurantium.

2. Figures are in poor quality possible due to electronic submission process.

3. Figure 1 described the percentage of individual fatty acids in terms of total fatty acid count. It will not give exact percentage of the fatty acid in the tested fractions i.e., neutral lipid, glycolipids or phospholipids. Lipid fraction such as neutral lipids may contain other components too, better to present the fatty acid data in w/w basis.

4. No positive control was used in any of the bio-assays. Recommended to have positive control for comparison.

5. Author described the final concentration of the lipids 20 mg/ml (line 97), but in Figures 2-4 results are described in percentage lipids, need further clarification how the test sample were prepared from 20 mg/ml to 0.5-4% lipids.

6. At 4.0% lipid concentration neutral lipid and glycolipids fractions showed cytotoxicity effect (Figure 2A, lines 171-174). No explanation was given why these lipids fractions were tested at 4.0% for NO and other assays. NO inhibition and other positive effect at this concentration may due to cell death.

Reviewer #2: This manuscript describes a series of studies looking at the anti-inflammatory effects of lipids extracted form Halocynthia aurantium. Overall the results are interesting, but revisions are needed to help improve the manuscript. These points are listed below.

1. The authors describe an extraction method in lines 86-98. This methodology needs to be more quantitative. How much material was used to perform the extract? What was the mass obtained in each fraction? This will allow one to know the lipid content in the extraction. While each fraction analyzed had the same mass of lipids, it would be more useful to have the number on a weight percent basis.

2. There needs to be a positive control in the bioassays.

3. Why was a 4.0% lipid concentration chosen for the results in Figure 2? No rationale for this choice is given and should be provided.

**********

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Reviewer #2: No

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PLoS One. 2022 Aug 15;17(8):e0270794. doi: 10.1371/journal.pone.0270794.r002

Author response to Decision Letter 0

2 Jun 2022

Reviewer #1

The manuscript describes anti-inflammatory effects of lipid extracted from Halocynthia aurantium. Even though results are interesting, need major revision to enhance the quality of the paper suitable for publication in PLOS ONE.

Major comments

1. Author described the extraction method of total lipids (lines 86-87), unfortunately no extraction yield was given anywhere. Similarly, lipid extracted from H. aurantium was fractionated into neutral lipid, glycolipid and phospholipids (line 94-96), and neither initial lipid weight nor the fractions yield was given. This is important information to understand the value of the lipid from H. aurantium.

- It was corrected in Line 87-88 and 92-95.

- Total lipids produced a yield of 33.6 mg (w/w) or 0.75% of dry material (4.5 g). If 4.5 g of dried material is 100%, yield (33.6 mg) is 0.75% of dried material (4.5 g).

- The neutral lipids, glycolipids, and phospholipids were shown to have a high lipid yield of 8.63 (2.9 mg), 30.95 (10.4 mg), and 44.94 (15.1 mg) % of total lipid (33.6 mg), respectively.

- Thanks.

2. Figures are in poor quality possible due to electronic submission process.

- It was corrected.

- Thanks.

3. Figure 1 described the percentage of individual fatty acids in terms of total fatty acid count. It will not give exact percentage of the fatty acid in the tested fractions i.e., neutral lipid, glycolipids or phospholipids. Lipid fraction such as neutral lipids may contain other components too, better to present the fatty acid data in w/w basis.

- Based on an internal standard, fatty acids were analyzed.

- We are attaching the percentage of fatty acids like the following.

- We calculated all detected lipids by the equation as (each lipid/all fatty acids) * 100.

- Thanks.

4. No positive control was used in any of the bio-assays. Recommended to have positive control for comparison.

- Normally the anti-inflammatory effects used LPS as positive control.

- Thanks.

- References

o Fernando, I. S., Sanjeewa, K. A., Kim, H. S., Kim, S. Y., Lee, S. H., Lee, W. W., & Jeon, Y. J. (2017). Identification of sterols from the soft coral Dendronephthya gigantea and their anti-inflammatory potential. Environmental Toxicology and Pharmacology, 55, 37-43.

o Li, M., Zhang, L., Cai, R. L., Gao, Y., & Qi, Y. (2012). Lipid‐soluble extracts from Salvia miltiorrhiza inhibit production of LPS‐induced inflammatory mediators via NF‐κB modulation in RAW264.7 cells and perform anti-inflammatory effects in vivo. Phytotherapy Research, 26(8), 1195-1204.

o Kim, K. N., Ko, Y. J., Yang, H. M., Ham, Y. M., Roh, S. W., Jeon, Y. J., ... & Oda, T. (2013). Anti-inflammatory effect of essential oil and its constituents from fingered citron (Citrus medica L. var. sarcodactylis) through blocking JNK, ERK and NF-κB signaling pathways in LPS-activated RAW 264.7 cells. Food and Chemical Toxicology, 57, 126-131.

o Wang, T., Wu, F., Jin, Z., Zhai, Z., Wang, Y., Tu, B., ... & Tang, T. (2014). Plumbagin inhibits LPS-induced inflammation through the inactivation of the nuclear factor-kappa B and mitogen activated protein kinase signaling pathways in RAW 264.7 cells. Food and chemical toxicology, 64, 177-183.

5. Author described the final concentration of the lipids 20 mg/ml (line 97), but in Figures 2-4 results are described in percentage lipids, need further clarification how the test sample were prepared from 20 mg/ml to 0.5-4% lipids.

- It was corrected in Line 99.

- The stock of lipids has some mistakes in that the final concentration of lipids is 2 mg/mL.

- Stock of lipids has concentration at 2 mg/mL (w/v) as 100%, and then lipids were diluted to 0.5, 1.0, 1.5 and 2.0% (v/v) with medium.

- Thanks.

6. At 4.0% lipid concentration neutral lipid and glycolipids fractions showed cytotoxicity effect (Figure 2A, lines 171-174). No explanation was given why these lipids fractions were tested at 4.0% for NO and other assays. NO inhibition and other positive effect at this concentration may due to cell death.

- Figure 2A showed cell proliferation in RAW264.7 cells (in the absence of LPS) that are toxic at high concentrations while cell proliferation in LPS-stimulated RAW264.7 cells showed non-toxicity.

- The purpose of our study is to test the anti-inflammatory effects. Therefore, we tested the three lipids with the addition of LPS. It showed the highest NO production at a concentration of 4%, especially phospholipids, which reduced NO production by 9.52 % while causing no toxic in the cells.

- We changed the previous one to new figure 2A including the addition of LPS.

- In our study, the three lipids showed the highest NO production at a concentration of 4%, especially phospholipids, which reduced NO production by 9.52 % while causing no toxic in the cells.

- All experiments with three lipids should use the same concentration.

- Following references showed the cell viability of a sample to be lower than control (≤ 80%), and that they can also be evaluated for NO production, gene expression, and protein expression.

- References

o Qiao, J., Xu, L. H., He, J., Ouyang, D. Y., & He, X. H. (2013). Cucurbitacin E exhibits anti-inflammatory effect in RAW 264.7 cells via suppression of NF-κB nuclear translocation. Inflammation Research, 62(5), 461-469.

o Konishi, I., Hosokawa, M., Sashima, T., Maoka, T., & Miyashita, K. (2008). Suppressive effects of alloxanthin and diatoxanthin from Halocynthia roretzi on LPS-induced expression of pro-inflammatory genes in RAW264. 7 cells. Journal of Oleo Science, 57(3), 181-189.

o Orecchini, E., Mondanelli, G., Orabona, C., Volpi, C., Adorisio, S., Calvitti, M., ... & Belladonna, M. L. (2021). Artocarpus tonkinensis extract inhibits LPS-triggered inflammation markers and suppresses RANKL-induced osteoclastogenesis in RAW264. 7. Frontiers in Pharmacology, 2417.

o Li, C. Y., Meng, Y. H., Ying, Z. M., Xu, N., Hao, D., Gao, M. Z., ... & Ying, X. X. (2016). Three novel alkaloids from Portulaca oleracea L. and their anti-inflammatory effects. Journal of Agricultural and Food chemistry, 64(29), 5837-5844.

o Yang, H., Xue, Y., Kuang, S., Zhang, M., Chen, J., Liu, L., ... & Deng, C. (2019). Involvement of Orai1 in tunicamycin-induced endothelial dysfunction. The Korean Journal of Physiology & Pharmacology, 23(2), 95-102.

Reviewer #2

This manuscript describes a series of studies looking at the anti-inflammatory effects of lipids extracted form Halocynthia aurantium. Overall the results are interesting, but revisions are needed to help improve the manuscript. These points are listed below.

1. The authors describe an extraction method in lines 86-98. This methodology needs to be more quantitative. How much material was used to perform the extract? What was the mass obtained in each fraction? This will allow one to know the lipid content in the extraction. While each fraction analyzed had the same mass of lipids, it would be more useful to have the number on a weight percent basis.

- It was corrected in Line 87-88 and 92-95.

- Total lipids produced a yield of 33.6 mg (w/w) or 0.75% of dry material (4.5 g). If 4.5 g of dried material is 100%, yield (33.6 mg) is 0.75% of dried material (4.5 g).

- The neutral lipids, glycolipids, and phospholipids were shown to have a high lipid yield of 8.63 (2.9 mg), 30.95 (10.4 mg), and 44.94 (15.1 mg) % of total lipid (33.6 mg), respectively.

- Thanks.

2. There needs to be a positive control in the bioassays.

- Normally the anti-inflammatory effects used LPS as positive control.

- Thanks.

- References

o Li, M., Zhang, L., Cai, R. L., Gao, Y., & Qi, Y. (2012). Lipid‐soluble extracts from Salvia miltiorrhiza inhibit production of LPS‐induced inflammatory mediators via NF‐κB modulation in RAW264.7 cells and perform anti-inflammatory effects in vivo. Phytotherapy Research, 26(8), 1195-1204.

o Kim, K. N., Heo, S. J., Yoon, W. J., Kang, S. M., Ahn, G., Yi, T. H., & Jeon, Y. J. (2010). Fucoxanthin inhibits the inflammatory response by suppressing the activation of NF-κB and MAPKs in lipopolysaccharide-induced RAW 264.7 macrophages. European journal of pharmacology, 649(1-3), 369-375.

o Zhang, W., Yan, J., Wu, L., Yu, Y., Richard, D. Y., Zhang, Y., & Liang, X. (2019). In vitro immunomodulatory effects of human milk oligosaccharides on murine macrophage RAW264. 7 cells. Carbohydrate polymers, 207, 230-238.

3. Why was a 4.0% lipid concentration chosen for the results in Figure 2? No rationale for this choice is given and should be provided.

- Figure 2A showed cell proliferation in RAW264.7 cells (in the absence of LPS) that are toxic at high concentrations while cell proliferation in LPS-stimulated RAW264.7 cells showed non-toxicity.

- The purpose of our study is to test the anti-inflammatory effects. Therefore, we tested the three lipids with the addition of LPS. It showed the highest NO production at a concentration of 4%, especially phospholipids, which reduced NO production by 9.52 % while causing no toxic in the cells.

- We changed the previous one to new figure 2A including the addition of LPS.

- All experiments with three lipids should use the same concentration.

- Following references showed the cell viability of a sample to be lower than control (≤ 80%), and that they can also be evaluated for NO production, gene expression, and protein expression.

- Thanks.

- References

o Qiao, J., Xu, L. H., He, J., Ouyang, D. Y., & He, X. H. (2013). Cucurbitacin E exhibits anti-inflammatory effect in RAW 264.7 cells via suppression of NF-κB nuclear translocation. Inflammation Research, 62(5), 461-469.

o Konishi, I., Hosokawa, M., Sashima, T., Maoka, T., & Miyashita, K. (2008). Suppressive effects of alloxanthin and diatoxanthin from Halocynthia roretzi on LPS-induced expression of pro-inflammatory genes in RAW264. 7 cells. Journal of Oleo Science, 57(3), 181-189.

o Orecchini, E., Mondanelli, G., Orabona, C., Volpi, C., Adorisio, S., Calvitti, M., ... & Belladonna, M. L. (2021). Artocarpus tonkinensis extract inhibits LPS-triggered inflammation markers and suppresses RANKL-induced osteoclastogenesis in RAW264. 7. Frontiers in Pharmacology, 2417.

o Li, C. Y., Meng, Y. H., Ying, Z. M., Xu, N., Hao, D., Gao, M. Z., ... & Ying, X. X. (2016). Three novel alkaloids from Portulaca oleracea L. and their anti-inflammatory effects. Journal of Agricultural and Food chemistry, 64(29), 5837-5844.

o Yang, H., Xue, Y., Kuang, S., Zhang, M., Chen, J., Liu, L., ... & Deng, C. (2019). Involvement of Orai1 in tunicamycin-induced endothelial dysfunction. The Korean Journal of Physiology & Pharmacology, 23(2), 95-102.

Attachment

Submitted filename: Response to Reviewers 1.docx

Click here for additional data file. (831.3KB, docx)

Decision Letter 1

Charles Michael Greenlief

20 Jun 2022

Anti-inflammatory effects of neutral lipids, glycolipids, phospholipids from Halocynthia aurantium tunic by suppressing the activation of NF-κB and MAPKs in LPS-stimulated RAW264.7 macrophages

PONE-D-22-03751R1

Dear Dr. Park,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

C. Michael Greenlief, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: The authors have addressed all previous concerns. The authors have added some additional experimental work that helps to strengthen the manuscript.

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Reviewer #1: Yes: Arjun H. Banskota

Reviewer #2: No

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Acceptance letter

Charles Michael Greenlief

5 Aug 2022

PONE-D-22-03751R1

Anti-inflammatory effects of neutral lipids, glycolipids, phospholipids from Halocynthia aurantium tunic by suppressing the activation of NF-κB and MAPKs in LPS-stimulated RAW264.7 macrophages

Dear Dr. Park:

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Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Raw images. Original western blot gel image data.

    (PDF)

    Click here for additional data file. (1.8MB, pdf)
    S1 Table. Nucleotide primers used in this study.

    (PDF)

    Click here for additional data file. (55.3KB, pdf)
    Attachment

    Submitted filename: Response to Reviewers 1.docx

    Click here for additional data file. (831.3KB, docx)

    Data Availability Statement

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