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Anti-Inflammatory Potential of Carpomitra costata Ethanolic Extracts via Inhibition of NF-κB and AP-1 Activation in LPS-Stimulated RAW264.7 Macrophages

Marine algae have valuable health and dietary benefits. The present study aimed to investigate whether an ethanol extract of Carpomitra costata (CCE) could inhibit the inflammatory response to LPS. CCE attenuated the production of proinflammatory mediators, such as prostaglandin E2 (PGE2) and nitric oxide (NO), by inhibiting inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in LPS-induced RAW264.7 macrophages. CCE also inhibited the expression of proinflammatory cytokines such as IL-1β, TNF-α, and IL-6. CCE suppressed the LPS-induced DNA-binding activity of (NF-κB) and activator protein-1 (AP-1). In addition, CCE attenuated the LPS-stimulated phosphorylation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK) and phosphatidylinositol 3′-kinase/Akt (PI3K/Akt). Functional aspects of the JNK and Akt signaling pathways were analyzed using specific inhibitors, which attenuated the LPS-induced production of proinflammatory cytokines, and NO and PGE2 expression by suppressing AP-1 and NF-κB activity. In particular, the AP-1 signaling pathway is not involved in the production of inflammatory cytokines, such as IL-6, TNF-α, and IL-1β. These results suggested that CCE might exert its anti-inflammatory action by downregulating transcriptional factors (NF-κB and AP-1) through JNK and Akt signaling pathways. The current study suggested that CCE might be a valuable candidate for the treatment of inflammatory disorders.


Introduction
Marine algae have long been used as potential medicinal and dietary sources in Asia. Algae are rich in vitamins, minerals, dietary fibers, essential fatty acids, enzymes, polysaccharides, and various functional polyphenols [1]. The brown seaweed Carpomitra costata (Stackhouse) Batters (Sporochnaceae) has been part of the traditional diet in Asia. A previous study reported that C. costata extracts (CCE) have antibacterial, antifungal, and photoprotective effects [2,3].
Inflammation is a defensive reaction in response to different stimuli, such as infection, chemical exposure, tissue damage, or other foreign substances [4]. However, excessive or inappropriate inflammatory responses can cause numerous chronic inflammatory disorders, including inflammatory bowel disease, cancer, atherosclerosis, and diabetes [5,6]. Inflammation is mediated by inflammatory mediators that are generated primarily by macrophages that have been overactivated by various intrinsic or extrinsic stimuli that initiate and maintain immune responses [7]. Thus, the regulation of 2 Evidence-Based Complementary and Alternative Medicine macrophage activation might have therapeutic potential for the treatment of many inflammatory disorders. Lipopolysaccharide (LPS), a major component of Gram-negative bacteria cell walls, induces a number of cellular responses that act as extremely strong stimulators of innate or natural immunity [8]. Therefore, LPS stimulation of macrophages is a powerful model for the study of inflammation that is mediated by inflammatory mediators from activated macrophages.
During the inflammatory process, large amounts of inflammatory mediators are produced. Among them, nitric oxide (NO) and prostaglandin E 2 (PGE 2 ) are products of the inducible isoforms of inducible nitric oxide synthase (iNOS) enzymes and cyclooxygenase-(COX-) 2, respectively [9,10]. NO produced by iNOS has been reported to be a primary biological messenger that is involved in diverse physiological activities, including neural communication, vasodilation, and host defense, and also has beneficial antiparasitic, antiviral, microbiocidal, and antitumoral effects [9]. However, excessive and sustained NO release can damage the host and has been correlated with the progression of many inflammatory diseases. Prostaglandins (PGs) play a critical role in the generation of the inflammatory response and its expression is increased in inflamed tissue. PG, which is synthesized by COX-2, is another important mediator in the pathogenesis of inflammation. COX-2 catalyzes the conversion of arachidonic acid to prostaglandin H 2 , the precursor of a variety of biologically active mediators, such as PGE 2 [11,12]. Additionally, large amounts of inflammatory cytokines [interleukin-1 (IL-1 ), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-)] are released during the response of tissue to injury, resulting in systemic responses to the inflammatory process.
Therefore, controlling inflammatory mediators may have therapeutic applications for the treatment of various inflammatory disorders. In the present study, to evaluate the antiinflammatory potential of CCE, we investigated whether CCE could attenuate the production of NO, PGE 2 , and proinflammatory cytokines that are important targets in the treatment of inflammatory disorders. In addition, we determined the intracellular signaling pathways responsible for the regulatory actions of CCE in mouse RAW264.7 macrophages that are stimulated by LPS.

Preparation of the Ethanol Extract from C. costata.
The brown seaweed, C. costata, was collected near Ulleung Island, Korea. After collection, C. costata was washed with tap water to remove slats, epiphytes, and sand attached to the surface of the samples and then stored at −20 ∘ C. The frozen samples were lyophilized and homogenized using a grinder before extraction. The dried powder was extracted with 70% EtOH (1 : 10 w/v) for 1 h (five times) by sonication, and then the extract was evaporated in vacuo. The extract was dissolved in DMSO before use in the experiment.

Cell
Culture. RAW264.7 macrophages obtained from the American Type Culture Collection (Manassas, VA, USA) were cultured in DMEM containing 10% fetal bovine serum, 100 U/mL of penicillin, and 100 g/mL of streptomycin at 37 ∘ C in a 5% CO 2 humidified air environment.

Cell Viability Assay.
Cell viability was assessed using the Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan) method. Briefly, 96 wells containing 8 × 10 4 cells/mL were treated with CCE (30-100 g/mL) and the cell culture plate was incubated for 24 h. CCK-8 was then added to each well and the plate was again incubated at 37 ∘ C for 1 h. The absorbance of each well was recorded at 450 nm using a microplate reader (SpectraMax M2e, Molecular Devices, Sunnyvale, CA, USA).

NitricOxide Assay.
After cells (5 × 10 5 cells/ml) were treated with 100 ng/mL LPS alone or with 30, 50, or 100 g/mL CCE in 24-well plates for 24 h, 100 L of each culture medium was mixed with the same volume of Griess reagent. Nitrite levels were determined at 540 nm using an enzyme-linked immunosorbent assay (ELISA) plate reader (SpectraMax M2e).
2.6. Measurement of PGE 2 . RAW264.7 cells were cultured in 6-well plates (5 × 10 5 cells/mL) and incubated with CCE in the presence or absence of LPS (100 ng/mL) for 24 h. The culture medium was collected for determination of the PGE 2 concentration by ELISA (Cayman Chemical, Ann Arbor, MI, USA).

Western Blot
Analysis. The cells (3.2 × 10 5 cells/60 mm culture dish plate) were washed three times with 50 L PBS and lysed with lysis buffer (Mammalian Cell-PE LB6, G-Biosciences, St. Louis, MO, USA). The proteins were separated on polyacrylamide mini-gels and transferred to nitrocellulose membranes (GE Healthcare Life Sciences, Chalfont, UK). Following overnight incubation with the appropriate primary antibody, the membranes were incubated with a secondary antibody conjugated to horseradish peroxidase. The immunoreactive bands were visualized using an ECL detection system (Pierce Biotechnology, Inc., Rockford, IL, USA). ELISA. ELISA kits purchased from BioLegend (San Diego, CA, USA) were used to measure the levels of IL-6 and TNF-, and a kit obtained from R&D Systems (Minneapolis, MN) was used to measure the IL-1 levels. The absorbance was measured at 450 nm using a microplate reader (SpectraMax M2e).

Electrophoretic Mobility Shift Assay (EMSA).
Nuclear extract was prepared using the NE-PER nuclear extraction reagent (Pierce Biotechnology, Inc.). An oligonucleotide containing the immunoglobulin -chain binding site ( B, 5 -GATCTCAGAGGGGACTTTCCGAGAGA-3 ) and activator protein-1 (AP-1) DNA-binding site (5 -CGCTTGATG-ACTCAGCCGGAA-3 ) was synthesized as a probe for the DNA-protein binding assay. The 3 end of the probe was labeled with biotin using the biotin 3 -end DNA labeling kit (Pierce Biotechnology, Inc.). The binding reactions contained 5 g nuclear extract protein, buffer, 50 ng poly (dI-dC), and 20 fM biotin-labeled DNA. The reaction mixture was incubated for 20 min at room temperature. The competition reactions were performed by the addition of 100-fold excess of unlabeled NF-B p65 and AP-1 to the reaction mixture.
The mixture was then separated using electrophoresis on a 5% polyacrylamide gel in 0.5x Tris-borate buffer and transferred to nylon membranes. The biotin-labeled DNA was detected using a LightShift Chemiluminescent EMSA kit (Pierce Biotechnology, Inc.).

Statistical Analysis.
Data values are presented as the mean ± SEM All statistical analyses were performed with GraphPad Prism software 5.0 (GraphPad Software Inc., La Jolla, CA, USA). Comparisons between groups were performed by Dunnett's multiple range tests. Differences were considered statistically significant at < 0.05.

Effects of CCE on the Viability of RAW264.7 Macrophages.
We first evaluated the cytotoxicity of CCE using CCK-8 assays. CCK-8 assays were performed in cells incubated with CCE for 24 h. At concentrations of ≤100 g/mL, CCE did not affect cell viability (Figure 1(a)). Based on these results, a CCE concentration range of 30-100 g/mL was chosen for subsequent experiments.

CCE Inhibits NO and PGE 2 Production in LPS-Induced RAW264.7 Macrophages.
To evaluate the effects of CCE on NO production in LPS-stimulated RAW264.7 cells, we measured nitrite levels using the Griess reagent. Cells were pretreated with CCE (30, 50, or 100 g/mL) for 1 h before adding LPS (100 ng/mL). Stimulation for 24 h with LPS resulted in a marked increase in NO levels. However, this increase was attenuated by CCE pretreatment in a concentrationdependent manner (Figure 1(b)). Additionally, we investigated the effect of CCE on the production of PGE 2 using an ELISA in LPS-stimulated RAW264.7 cells. Cells were pretreated with CCE for 1 h and then stimulated with LPS (100 ng/mL) for 24 h. Pretreatment with the indicated concentrations of CCE resulted in a significant reduction in PGE 2 production (Figure 1(c)).

CCE Attenuates the Expression of iNOS and COX-2
in LPS-Induced RAW264.7 Macrophages. The levels of NO and PGE 2 are related to the activities of iNOS and COX-2, respectively; therefore, we evaluated the effects of CCE on the production of iNOS or COX-2 in LPS-induced RAW264.7 cells. Cells were treated with CCE (30, 50, or 100 g/mL) for 1 h before adding LPS (100 ng/mL). LPS stimulation for 24 h and 7 h markedly increased the expression of iNOS and COX-2, respectively. However, this high expression was significantly inhibited in cells pretreated with CCE in a concentration-dependent manner (Figure 2). Next, to verify whether the JNK and Akt signaling pathways are involved in the expression of inflammatory mediators, RAW264.7 cells were treated with LPS with or without JNK and Akt inhibitors. The upregulated expression of inflammatory mediators (NO, PGE 2 , IL-6, TNF-, and IL-1 ) was significantly inhibited by SP600125 (an inhibitor of JNK) and LY294002 (an inhibitor of Akt) (Figures 4(b), 4(c), and 4(d)).

CCE Inhibits LPS-Stimulated NF-B and AP-1 Activation in RAW264.7 Macrophages. The production of inflammatory mediators is regulated by nuclear factor-kappa B (NF-B)
and activator protein-1 (AP-1) transcription factors in the inflammatory process [13][14][15]. Therefore, to assess the mechanism by which CCE affects the expression of inflammatory mediators, we examined the effects of CCE on the DNAbinding activity of NF-B and AP-1 using EMSA. We found that LPS stimulation significantly increased the DNA-binding activity of AP-1 and NF-B, whereas pretreatment with CCE significantly suppressed the LPS-induced DNA-binding activity of NF-B and AP-1 ( Figure 5(a)). Next, to confirm whether the transcription factor signaling pathways for AP-1 and NF-B are involved in the expression of inflammatory mediators, RAW264.7 cells were treated with LPS with or without NF-B inhibitors (BAY 11-7082 and parthenolide) and an AP-1 inhibitor (SR 11032). Based on the results shown in Figures 5(b) and 5(c), we found that NF-B inhibitors and the AP-1 inhibitor suppressed the LPS-stimulated expression of NO and PGE 2 . However, the upregulated production of proinflammatory cytokines (TNF-, IL-6, and IL-1 ) by LPS stimulation was not inhibited by the AP-1 inhibitor ( Figure 5(d)). To elucidate what upstream pathways control AP-1 and NF-B activation, we treated the cells with JNK and Akt inhibitors and used EMSA to assess AP-1 and NF-B activation. As shown in Figure 5(e), treatment with SP600125 or LY294002 markedly reduced the LPS-induced DNA-binding activities of AP-1 and NF-B.

Discussion
Marine algae represent a resource that may provide a large number of therapeutic agents with the potential to attenuate the production of inflammatory mediators in various inflammatory diseases [16]. Therefore, the present study was undertaken to evaluate the pharmacological activity of CCE on the production of proinflammatory mediators such as NO, PGE 2 , IL-1 , IL-6, and TNF-by investigating inhibitory effects in LPS-stimulated murine macrophage RAW264.7 cells. To further understand the molecular mechanisms of CCE activity, we examined the effects of CCE on the phosphorylation of MAPKs and Akt and the activation of the transcription factor NF-B and AP-1. The results of this study show that CCE effectively attenuates LPS-induced production of inflammatory mediators through blockade of the JNK, Akt, NF-B, and AP-1 pathways in RAW264.7 macrophages. The inhibitory activity of CCE suggests one of the regulatory mechanisms responsible for its anti-inflammatory action and its potential utility to treat LPS-stimulated inflammatory diseases. The overactivation of macrophages by inflammatory stimulants leads to the overproduction of proinflammatory mediators, including PGE 2 and NO, and inflammatory cytokines, resulting in various chronic inflammatory diseases [17]. Thus, the elimination of the excessive expression of proinflammatory mediators is an effective therapeutic strategy to control chronic inflammation. In this study, we observed that CCE significantly attenuated the LPS-induced increase in proinflammatory mediators (NO, PGE 2 , TNF-, IL-6, and IL-1 ) without cytotoxicity in RAW264.7 macrophages (Figures  2 and 3). Thus, the results suggest that CCE is a promising agent for inhibiting inflammatory disorders.
The MAPKs (ERK-1/2, p38, and JNK) are members of proinflammatory signal transduction pathways that appear to play important roles in the inflammatory processes of LPSactivated macrophages [17]. Phosphatidylinositol 3-kinase/ Akt (PI3K/Akt) also contributes to the induction of proinflammatory factors [18]. Therefore, application of antiinflammatory agents that effectively modulate MAPK and Akt pathways are good approaches to treat various inflammatory disorders. To understand the signaling mechanisms associated with CCE-mediated regulation of proinflammatory mediator expression, the effect of CCE on the phosphorylation of MAPKs and Akt was evaluated ( Figure 4). As expected, when macrophages were stimulated with LPS, MAPKs and Akt were phosphorylated; however, pretreatment with CCE significantly attenuated LPS-induced phosphorylation of JNK and Akt, but not p38 and ERK. CCE showed a preference for p-JNK, without affecting p-ERK or p-p38 in the MAPK signaling pathway. In other words, CCE targeted only p-JNK. To confirm the above results, we investigated the effects of JNK and Akt inhibitors (SP600125 and LY294002, resp.) on the LPS-induced production of proinflammatory mediators by macrophages. Results showed that CCE inhibits the production of NO, PGE 2 , TNF-, IL-6, and IL-1 in LPS-stimulated macrophages by suppressing the activation of JNK and Akt, consequently attenuating the expression of proinflammatory mediators. These results suggested that CCE could inhibit LPS-induced inflammatory responses that are mediated by the JNK and Akt signaling pathways.
Previous studies reported that the transcription factors NF-B and AP-1 regulate the production of many inflammatory mediators [13,14]. Therefore, in this study, we evaluated the effects of CCE on the LPS-stimulated activation of NF-B and AP-1 in macrophages. Activated transcription factors enter the nucleus and induce inflammatory mediator production; therefore, we examined the DNA-binding activity of NF-B and AP-1. Studies using EMSA revealed that stimulation with LPS increased the DNA-binding activities of NF-B and AP-1 in the nuclear compartment. However, pretreatment with CCE attenuated the induced DNA-binding activities of the transcription factors in a concentration-dependent manner ( Figure 5(a)). To confirm the anti-inflammatory activity of CCE via regulation of the binding activities of transcription factors, we examined the effects of BAY 11-7082, parthenolide (NF-B inhibitors) and SR11302 (an AP-1 inhibitor) on the production of inflammatory mediators in LPS-stimulated macrophages. As shown in Figures 5(b)-5(d), we found that the NF-B inhibitors attenuated LPS-induced production of NO, PGE 2 , IL-6, TNF-, and IL-1 in the macrophages. Additionally, the AP-1 inhibitor blocked LPSinduced NO and PGE 2 production. However, interestingly, we found that proinflammatory cytokines (IL-6, TNF-, and IL-1 ) were not inhibited by treatment with the AP-1 inhibitor ( Figure 5(e)). This result showed that the production of proinflammatory cytokines, such as IL-6, TNF-, and IL-1 , is not directly related to the AP-1 signaling pathway. Previous reports showed that the expression of inflammatory cytokines is regulated by activation of the AP-1 transcription factor [19][20][21]. However, since these previous studies did not confirm this finding using an AP-1 inhibitor, the results that we obtained would not have been observed in the previous studies. To the best of our knowledge, our AP-1 inhibitor results demonstrated, for the first time, that LPS directly stimulates the production of inflammatory mediators in RAW264.7 macrophages. In addition, using inhibitors of JNK and Akt, we showed that LPS stimulation attenuates the activation of NF-B and AP-1. It has been shown that the JNK and Akt signaling pathways can be upstream activators of the NF-B and AP-1 signaling cascade. Based on the above results, we demonstrated that LPS induced NF-B and AP-1 activation in RAW264.7 macrophages via upstream signaling molecules that are involved in the JNK and Akt pathways.
The present study demonstrated that CCE exerts inhibitory effects on NO and PGE 2 production in the LPS-induced RAW264.7 macrophages. Furthermore, CCE markedly attenuated the expression of proinflammatory cytokines, including IL-1 , IL-6, and TNF-. These effects of CCE are associated with the inactivation of LPS-induced NF-B and AP-1 via inhibition of JNK and Akt phosphorylation. In particular, we have found, for the first time, that the production of proinflammatory cytokines, such as IL-6, TNF-, and IL-1 , is not directly related to the AP-1 signaling pathway. Thus, we concluded that CCE possesses potential anti-inflammatory activities and may have therapeutic benefits in the treatment of various inflammatory disorders. Further studies are needed to investigate the components of CCE that may have beneficial and physiologically active effects.

Disclosure
Part of this study was presented in the Korean Society of Fisheries and Aquatic Science (KOSFAS) in 2017.