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Research Paper

Korean Society for Biotechnology and Bioengineering Journal 2023; 38(2): 121-126

Published online June 30, 2023 https://doi.org/10.7841/ksbbj.2023.38.2.121

Copyright © Korean Society for Biotechnology and Bioengineering.

주름겹홑파래 에탄올 추출물의 항염증 효과

Anti-Inflammatory Effects of Monostroma crassidermum Tokida Ethanol Extract on Lipopolysaccharide-Stimulated RAW 264.7 Cells and Mice Ear Edema

Min-Ji Kim1, Woo-Sin Kang1, So-Mi Jeong2, Ga-Eun Woo1, A-Yeong Park1, Hae-Ji Hwang1, and Dong-Hyun Ahn1*

1Department of Food Science & Technology and Institute of Food Science, Pukyong National University, Busan 48513, Korea
2Institute of Fisheries Sciences, Pukyong National University, Busan 46041, Korea

Correspondence to:Tel: +82-51-629-5831, Fax: +82-51-629-5824, E-mail: dhahn@pknu.ac.kr

Received: January 27, 2023; Revised: May 3, 2023; Accepted: May 4, 2023

In this study, the anti-inflammatory effects of Monostroma crassidermum Tokida ethanol extract (MCEE) in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and croton oil-stimulated ICR mouse models were investigated. MCEE reduced the secretion of pro-inflammatory cytokines, including interleukin-6, IL-1β, tumor necrosis factor-α, and nitric oxide, which are non-toxic to RAW 264.7 cells. In vivo testing showed that the results in the MCEE-treated group (10–250 mg/kg. body weight) were similar to those in the prednisolone-treated group (10 mg/kg. body weight). Photo-micrographs showed that the skin layer was thinned, and mast cell infiltration was reduced in the mouse-ear tissue. Therefore, these results suggested that MCEE exerts an anti-inflammatory effect, indicating the possible applications of MCEE as a natural anti-inflammatory material.

Keywords: anti-inflammatory activity, ear edema, Monostroma crassidermum Tokida

Inflammation is a defense mechanism of the body that helps recovery in damaged areas; however, it causes symptoms, such as pain, redness, fever, edema, dysfunction, and mucosal damage, which can lead to the onset of arthritis and cancer [1].

Macrophages are involved in maintaining homeostasis in several host reactions and play an important anti-inflammatory role by decreasing immune reactions through the release of pro-inflammatory cytokines, including interleukin (IL)-6, IL-1β, and tumor necrosis factor-α (TNF-α). In addition, the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) produces nitric oxide (NO) and prostaglandin E2 (PGE2), which cause inflammatory reactions and promote the movement of immune cells to the inflammatory site [2]. NO is a vital cellular signaling molecule involved in many physiological and pathological processes. It is produced as a metabolic by-product when L-arginine is converted into Lcitrulline via the interference of nitric oxide synthase [3]. NO and pro-inflammatory cytokines are mainly generated through nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPKs). NF-κB synthesizes inflammation-mediated enzymes, including iNOS and COX-2, and inflammatorymediated cytokines as transcription factors in the nucleus. In addition, NF-κB activity is suppressed by binding to IκBα in the cytoplasm [4-6].

MAPKs contain three main inflammatory factors: extracellular signaling-regulated kinase (ERK), c-Jun N-terminal kinases (JNK), and p38. MAPK activity in response to various stimuli, such as lipopolysaccharides (LPS), causes the production of NO and pro-inflammatory cytokines in macrophages [7]. LPS is a component of the cell wall of gram-negative bacteria that cause a strong inflammatory reaction. This stimulates toll like receptor 4 (TLR4, CD284), which is present on the macrophage surface, and induces the activation of MAPK, a downstream of cell signaling pathway. The activated MAPK signaling pathway causes various inflammatory mediators to impair immune system function [8]. Therefore, inhibition of NF-κB and MAPK activities through these signaling pathways is an important factor in controlling the inflammatory response.

Recently, studies on the anti-inflammatory activity of algae have been reported [9-11]. Monostroma crassidermum Tokida is a green alga with a thin fan-shape and a green-yellow blade. It is typically found in the western and intertidal regions of the North Pacific Ocean. The cells in the cross-section of the lower part of the plant are small, thin, and bead-shaped. In addition, this green alga has thick cell walls and is characterized by grain formation on the surface of the blade. Anti-influenza A virus activity [12] of rhamnan sulfate extracted from Monostroma nitidum and anticoagulant properties of sulfated polysaccharides extracted from Monostroma angicava [13] have been documented.

This study was undertaken to confirm the anti-inflammatory activity of Monostroma crassidermum Tokida ethanol extract (MCEE) in the LPS-stimulated RAW 264.7 cells and croton oil-induced mouse model, and to explore the future possibility of a new inflammatory treatment using natural ingredients.

2.1. Preparation of MCEE

M. crassidermum Tokida collected in 2016 from Song-Jeong, Busan, Korea, was used in this study. Powdered M. crassidermum Tokida was extracted with 95% ethanol for 24 h at room temperature using an agitator (H-0820, Dongwon Science Co., Busan, Korea). The extract was then centrifuged at 3,000 rpm for 10 min, and the supernatant was filtered and concentrated using a rotary evaporator (RE200, Yamato Co., Tokyo, Japan).

2.2. Animals

Eight-week-old male ICR mice were purchased from Orient Bio (Seongnam, Korea) and were used for the ear edema test. Mice were preliminarily bred in an animal room under controlled conditions at 20 ± 2°C, relative air humidity 50 ± 10%, and a 12/12-h light–dark cycle, and subsequently maintained for a week for use in tests. Animal experiments were approved by the Animal Ethics Committee of Pukyong National University (2015-04).

2.3. Cell culture

Murine macrophage RAW 264.7 cells were purchased from the Korean Cell Line Bank (KCLB 40071, Seoul, Korea). The cells were cultured in plastic dishes containing Dulbecco’s modified eagle medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin (Logan, UT, USA) in a 5% CO2 incubator (MCO-15AC, Sanyo, Osaka, Japan) at 37°C. Cells were sub-cultured when reaching a density of approxi-mately 80–90%, cells did not exceed 20 passages in the study.

2.4. Cell viability assay

MTT assay was performed to evaluate the celltoxicity of MCEE. RAW 264.7 cells (1 × 106 cells/mL) were cultured in 96-well plates for 20 h. The cells were then cultured with 1 μg/ mL LPS and MCEE (0.1, 1, 10, 50 and 100 μg/mL) for 22 h at 37°C and 5% CO2. MTT reagent (5 mg/mL) was added, and the cells were incubated for 2 h. The medium was then discarded, DMSO was added to each well, and absorbance was measured at 540 nm using a microplate reader (Model 550, Bio-rad, Richmond, VA, USA). Cell proliferation was calculated using the following formula:

Cell viability index (% of control) =

(sample absorbance/control absorbance) × 100

2.5. Nitric oxide determination

Nitrite concentration in the supernatant was measured using the Griess reaction [14]. RAW 264.7 cells (2.5 × 105 cells/mL) were plated in 24-well plates and incubated in a 5% CO2 incubator for 20 h. After pre-incubation, 1 μg/mL LPS and MCEE (0.1, 1, 10, 50 and 100 μg/mL) were added, and the cells were incubated for 24 h. The supernatant (100 μL) was then mixed with the same volume of Griess reagent (1% sulfanilamide + 0.1% naphthalene-diamine dihydrochloride in 5% phosphoric acid, 1:1) and incubated at room temperature for 10 min. Absorbance was measured at 540 nm using a microplate reader, and the nitrite concentration was calculated using standard curves of sodium nitrite (NaNO2).

2.6. Enzyme-linked immunosorbent assay (ELISA)

ELISA was performed to measure the secretion of proinflammatory cytokines. The levels of TNF-α, IL-6 and IL-1β were determined using an ELISA kit (mouse ELISA set). Cultured RAW 264.7 cells (2.5 × 105 cells/mL) in 24-well plates were stimulated by 1 μg/mL of LPS and treated with the previously indicated concentration of MCEE for 24 h. Levels of TNF-α, IL-6, and IL-1β in the culture medium were measured by ELISA using anti-mouse TNFα, IL-6 and IL-1β antibodies and biotinylated secondary antibody according to the manufacturer's instructions. After the reaction, the mixture was washed with PBST, and an OPD solution was added to produce a dark reaction at room temperature for 30 min. To terminate the reaction, 2 M H2SO4 was added, and the absorbance was measured at 490 nm using a microplate reader.

2.7. Ear edema measurement and histopathology analysis

To confirm the anti-inflammatory effect of MCEE in the mouseear edema model, ear edema measurements and histopathological analyses were conducted using the methods of Kim et al. [15] and Saraiva et al. [16]. MCEE (200 μL) was orally administered to ICR mice at 10, 50 and 250 mg/kg body weight. After 1 h, 2.5% croton oil (20 μL/ear) was spread on the inner and outer surfaces of the right ear, and ear thickness was measured after 5 h. An increase in ear thickness after croton oil treatment was regarded as edema. For histopathology analysis, 20 μL of MCEE was spread on the right ear of an ICR mouse at a concentration of 100 mg/mL, and 20 μL of 5% croton oil was spread for 15 min. After 6 h, ear tissue was dissected and fixed in 10% formaldehyde for 72 h. After fixation, the tissue slices were embedded in paraffin. Sections were deparaffinized and stained with hematoxylin–eosin and toluidine blue for the observation of the tissue and mast cells. The rate of edema generation was calculated using the following equation:

Edema formation (% of control) =

(ear thickness of sample/ear thickness of control) × 100.

2.8. Statistical analyses

Statistical analysis was performed using SAS software (Statistical Analytical System V8.2, SAS Institute Inc., Cary, NC, USA). Duncan's multiple range test was used to test for significant differences between control and treatment groups at p < 0.05.

3.1. Effects of MCEE on cell viability

MCEE at concentrations of 0.1, 10, 50 and 100 μg/mL was applied to RAW 264.7 cells to confirm cytotoxicity through MTT assay. There was no significant difference between the PBS treatment and all concentrations of MCEE, confirming that there was no cytotoxicity at the concentrations (Fig. 1(A)).

Figure 1. Effect of Monostroma crassidermum Tokida ethanol extract on proliferation index (% of control, A) of RAW 264.7 cells and the production of NO (B), IL-6 (C), TNF-α (D), and IL-1β (E) in LPS-stimulated RAW 264.7 cells. (a-e)Means with different superscript are significantly different (P < 0.05).

3.2. Inhibitory effect of NO production

NO is an unstable compound that is a major cause of aging and skin damage. NO release is increased by inflammatory stimuli, including LPS, causing inflammation and tissue damage [17]. Therefore, to confirm the inhibitory effect of MCEE treatment on NO production, MCEE was added (0.1, 10, 50 and 100 μg/mL) to LPS-stimulated RAW 264.7 cells, and the production of NO was measured. It was confirmed that NO production was decreased by treatment with MCEE in a dose-dependent manner when compared to LPS treatment (Fig. 1(B)). Following MCEE treatment application at concentrations of 50 μg/mL and 100 μg/mL, the production of NO was reduced by approximately 17% and 40%, respectively, compared to the treatment with LPS alone, confirming the inhibitory effect of MCEE on NO production. The results of this study were similar to the results of an over 50% NO inhibitory effect of Sargassum fulvellum ethanol extract at concentrations of 50 and 100 μg/mL [18]. In addition, Kang et al. [19] reported that approximately 28% of NO production was inhibited following treatment with 100 μg/mL kelp root ethanol extract.

3.3. Inhibitory effect of pro-inflammatory cytokines

Pro-inflammatory cytokines play an important role in cancer development and skin inflammation and are inevitable in inflammatory responses. These are expressed not only in normal tissues but also under pathological conditions [20].

TNF-α and IL-6 are representative pro-inflammatory cytokines [21]. TNF-a is produced by LPS-stimulated RAW 264.7 cells, and it induces local inflammatory reactions leading to NO production and acute inflammatory reactions [22]. IL-1β is also produced by stimulated macrophages as a proprotein that is proteolytically processed to its active form by caspase 1. It is an important mediator of the inflammatory response and involved in a variety of cellular activities, including cell proliferation and apoptosis [2]. Accordingly, Heo et al. [23] reported that there is a high possibility of controlling the inflammatory response through inhibition of pro-inflammatory cytokines, which play an important role in the initial immune response. To investigate the inhibitory effect of MCEE on the secretion of pro-inflammatory cytokines, the levels of TNF-α, IL-6 and IL-1β were assessed after LPS-stimulated RAW 264.7 cells were treated with various concentrations of MCEE (0.1, 1, 10, 50 and 100 μg/mL). The secretion of TNF-α induced by LPS stimulation decreased by up to 26% and 40% at concentrations of 50 and 100 μg/mL of MCEE, respectively (Fig. 1(D)). Similarly, the secretion of IL-6 decreased by approximately 45% at a concentration of 50 μg/mL of MCEE and inhibited by 80% at a concentration of 100 μg/mL of MCEE (Fig. 1(C)). The secretion of IL-1β was also significantly decreased by MCEE at all concentrations and inhibited by approximately 46% and 73% at 50 and 100 mg/mL of MCEE, respectively (Fig. 1(E)). These results suggested that MCEE is involved in anti-inflammatory action by effectively inhibiting proinflammatory cytokines in LPS-stimulated RAW 264.7 cells.

3.4. In vivo anti-inflammatory activity of MCEE

Increased cell membrane fluidity and physiological phenomena, including vasodilation and edema, which occur during inflammatory reactions, are a series of biological processes that repair areas damaged by the external environment, for example, as typical reactions to skin inflammation [24]. Croton oil, an inflammatory stimulant extracted from Croton tyrium L., is used to stimulate the skin and cause edema [25]. When Croton oil is applied to the skin, it causes edema due to vascular expansion of tissue and polymorphonuclear leukocyte infiltration by 12-Ο-tetradecanoylphorbol-13-acetate, a component of phorbol esters, leading to an acute inflammatory reaction. This reaction is induced by the activity of protein kinase C (PKC), which promotes phospholipase A2 activity and increases the levels of metabolites, including prostaglandin, arachidonic acid, and leukotriene. PKC also promotes inflammatory responses by increasing the secretion of inflammatory mediators, including chemokines and cytokines [26].

The inhibitory effects of MCEE on ear edema and mast cell infiltration, an inflammatory reaction, were confirmed in a croton oil mouse model. Prednisolone, a synthetic steroid currently used as a common treatment for inflammation, was orally administered once to the ICR mice at concentrations of 10 and 50 mg/kg · body weight, and MCEE was administered once to the ICR mice at concentrations of 10, 50 and 250mg/kg· body weight. Changes in ear thickness via croton oil induced ear edema were measured. Ear thickness was decreased significantly at all concentrations of MCEE, compared to the control mice data (Fig. (2)). A similar level of ear edema inhibitory effect was observed between the positive control prednisolone treatment (10 mg/kg) and treatment with 250 mg/kg MCEE. In addition, the thickness of the dermis increased when only croton oil was used, but the dermis thickness decreased when MCEE was used at a concentration of 100 mg/mL (Fig. 3(A)). Observations of mast cell infiltration in toluidine blue-stained tissues (Fig. 3(B)) demonstrated that mast cell infiltration in the dermis was suppressed to a degree like that of prednisolone treatment. These results suggest that MCEE is effective in alleviating edema, an inflammatory reaction as a natural anti-inflammatory material.

Figure 2. Inhibition of Monostroma crassidermum Tokida ethanol extract against croton oil-induced mouse-ear edema. Means with different superscript (a–d) are significantly different (P < 0.05).

Figure 3. Photomicrograph of transverse sections of mice ears sensitized with topical application of 5% croton oil (v/v) in acetone (a–c) or acetone alone (d), stained with (A) hematoxylin-eosin and (B) toluidine-blue examined under light microscopy (magnification, ×200). Treatments: vehicle 2% Tween 80 (a), prednisolone 0.08 mg/ear (b), and Monostroma crassidermum Tokida ethanol extract 20 μL/ear (c). The numbers 1 and 2 in (A) indicate dermis and epidermis, respectively, and the arrow in (B) indicates infiltration of mast cells.

Many researchers have discovered various types of antioxidants in plants, and vitamin C, tocopherols, flavonoids, polyphenol compounds, and carotenoids are known to be excellent antioxidants present in land plants such as fruits and vegetables. Total polyphenols have been reported to have excellent anticancer and antioxidant effects [27]. In a study on the anti-inflammatory effect of Ulva lactuca Linnaeus extract involved in the green algae plant group, it was reported that high polyphenol content showed antioxidant and antiinflammatory effects [28]. Therefore, it is expected that MCEE used in this study also contains a large amount of polyphenols, and it is considered to have a high anti-inflammatory effect.

In this study, the anti-inflammatory effects of MCEE were demonstrated by testing the inhibitory effect on inflammatory-related substances in vivo and in vitro. This can be used as basic data in various fields that can utilize marine natural products. In particular, it is judged to be of high value as a functional food resource for which safety must be secured.

This research was a part of the project titled 'Bioactive material for algae-based bio-health care substantiation', funded by the Ministry of Oceans and Fisheries, Korea.

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