Sargachromenol from Sargassum micracanthum Inhibits the Lipopolysaccharide-Induced Production of Inflammatory Mediators in RAW 264.7 Macrophages

During our ongoing screening program designed to determine the anti-inflammatory potential of natural compounds, we isolated sargachromenol from Sargassum micracanthum. In the present study, we investigated the anti-inflammatory effects of sargachromenol on lipopolysaccharide (LPS)-induced inflammation in murine RAW 264.7 macrophage cells and the underlying mechanisms. Sargachromenol significantly inhibited the LPS-induced production of nitric oxide (NO) and prostaglandin E2 (PGE2) in a dose-dependent manner. It also significantly inhibited the protein expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) in a dose-dependent manner in LPS-stimulated macrophage cells. Further analyses showed that sargachromenol decreased the cytoplasmic loss of inhibitor κBα (IκBα) protein. These results suggest that sargachromenol may exert its anti-inflammatory effects on LPS-stimulated macrophage cells by inhibiting the activation of the NF-κB signaling pathway. In conclusion, to our knowledge, this is the first study to show that sargachromenol isolated from S. micracanthum has an effective anti-inflammatory activity. Therefore, sargachromenol might be useful for cosmetic, food, or medical applications requiring anti-inflammatory properties.


Introduction
Inflammation is the result of the host's immune response to pathogenic challenges or tissue injuries. Once the response ends, tissue structure and function recover their normal states. Moreover, normal inflammatory responses are tightly controlled and self-limiting, with downregulation of proinflammatory proteins and upregulation of anti-inflammatory proteins. Thus, acute inflammation is essentially a beneficial process, particularly that which develops in response to infectious pathogens. Chronic inflammation, however, is an undesirable phenomenon and is involved in the pathogenesis of chronic diseases such as cancer, arthritis, autoimmune disorders, and vascular diseases [1][2][3]. For these reasons, research has focused on the control of inflammatory reactions. One approach has been the investigation of dietary phytochemicals capable of suppressing inflammation. Many attempts have been made to derive new anti-inflammatory agents from natural sources of phytochemicals that have been considered safe, less toxic, and readily available, although their modes of action mostly remain unclear. Thus, elucidating the molecular mechanisms underlying the antiinflammatory actions of naturally occurring phytochemicals might be a good strategy for identifying new therapeutic agents [4,5].
Many studies of seaweed-derived anti-inflammatory compounds have investigated the potential inhibitory effects of natural compounds in an in vitro system, that is, lipopolysaccharide (LPS)-stimulated macrophages [6][7][8][9]. Using this system, LPS from gram-negative bacteria has become one of the best characterized stimuli for induction of the upregulation of proinflammatory proteins such as 2 The Scientific World Journal cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). Inflammation is induced by many factors, including proinflammatory enzymes such as NO and PGE 2 , which are indicators of inflammatory activity. NO is involved in inflammation and autoimmune diseases, and its levels are elevated during inflammatory responses. COX-2 catalyzes the conversion of arachidonic acid to prostaglandins and is induced by proinflammatory cytokines or LPS. In addition, the COX-2 and iNOS expression is regulated by activation of NF-B, a transcription factor. Therefore, iNOS and COX-2 are important anti-inflammatory targets [10][11][12][13].
Numerous studies have focused on natural compounds or extracts for improving human health, owing to their safety and low toxicity. Although many phytochemicals derived from terrestrial plants have shown anti-inflammatory effects, only a few studies have focused on the molecular mechanisms underlying the anti-inflammatory actions of phytochemicals and extracts from marine algae such as Ecklonia cava, Sargassum muticum, and S. micracanthum [5].
S. micracanthum is a brown marine alga distributed worldwide, from temperate to subtropical regions. A number of compounds isolated from S. micracanthum, including sargaquinoic acid, sargachromenol, and sargassumol, have various pharmacological properties, including antioxidant and antiviral activities [14][15][16][17]. During our ongoing screening program designed to identify the anti-inflammatory potential of natural compounds, we isolated fucosterol, sargaquinoic acid, and sargachromenol from S. micracanthum by using activity-directed fractionation and characterized their structures using spectroscopy ( 1 H and 13 NMR, IR, and MS) as described previously [15]. However, the biological activities or modes of action of these compounds have not been reported previously, although another Korean group recently reported that sargaquinoic acid has anti-inflammatory activity [18]. Therefore, the present study investigated whether sargachromenol inhibited LPS-induced production of NO and PGE 2 , or the expression of iNOS and COX-2 proteins, through the inhibition of I B-in macrophages.

Cell Culture and Cell Viability
Assay. RAW 264.7 murine macrophages obtained from the Korean Cell Bank (Seoul, Korea) were cultured in DMEM containing 10% FBS, 100 U/mL penicillin, and 100 g/mL streptomycin at 37 ∘ C in 5% CO 2 . Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RAW 264.7 cells were cultured in 96-well plates for 18 h, followed by treatment with LPS (1 g/mL) in the presence of various concentrations of sargachromenol for 24 h. MTT was then added to the medium for 4 h. Finally, the supernatant was removed, and the formazan crystals were dissolved in DMSO. Absorbance was measured at 540 nm. The percentage of cells showing cytotoxicity relative to the control group was determined.

Measurement of Nitrite and
Prostaglandin E 2 . RAW 264.7 cells were plated at 1.8 × 10 5 cells/well in 24-well plates and then incubated with or without LPS (1 g/mL) in the absence or presence of sargachromenol (12.5, 25, 50, and 100 M) for 24 h. The NO determination was carried out as described previously [14] by using the Griess reaction. Briefly, conditioned cell culture media (100 L) were mixed with 100 L of the Griess reagent (1% sulphanilamide and 0.1% N- [1-naphthyl]-ethylenediamine dihydrochloride in 5% phosphoric acid) for 10 min. Then, absorbance was measured at 540 nm using a spectrophotometer. Fresh culture media were used as blanks in all experiments. NO levels in the samples were calculated from a standard curve constructed using sodium nitrite. Sandwich enzyme-linked immunosorbent assay (ELISA) was used to determine the production of cytokines and prostaglandin E 2 (PGE 2 ) in the LPS-treated RAW 264.7 cells in the presence of sargachromenol (12.5, 25, 50, and 100 M). The RAW 264.7 cells were stimulated by LPS for 24 h before the supernatant was harvested and assayed using the relevant ELISA kit in accordance with the manufacturer's instructions (R&D Systems Inc., Minneapolis, MN, USA). Results from 3 independent experiments were used for statistical analysis.

Statistical Analysis.
Results are presented as the mean ± standard deviation of at least 3 replicates. Student's -test was used for statistical analyses of the differences noted. values of 0.05 or less were considered statistically significant.

Results and Discussion
In the course of investigations on the biologically active metabolites from S. micracanthum [14], three known compounds, sargaquinoic acid, sargachromenol, and fucosterol, were isolated as major constituents in our previous study ( Figure 1). Also, we evaluated their radical scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl radicals using an electron spin trapping technique [15]. Therefore, the present study was undertaken to elucidate the pharmacological and biological effects of sargachromenol on the production of inflammatory mediators in macrophages.
Overproduction of NO, an inflammatory mediator involved in host defense mechanisms, is involved in the pathogenesis of several diseases, including periodontitis, bacterial sepsis, atherosclerosis, bowel disease, rheumatoid arthritis, and septic shock. Pharmacological manipulation of NO production has therefore been speculated to be useful in the alleviation of numerous disease states mediated by increased and/or protracted activation of macrophages [12,[19][20][21]. PGE 2 , which is produced at inflammatory sites by COX-2, has also been implicated as an important inflammatory mediator. Interestingly, the induction of COX-2 activity and the subsequent generation of PGE 2 are closely related to NO production. Thus, inhibition of PGE 2 production is an important therapeutic target in the development of antiinflammatory agents [22,23].
In order to determine the potential anti-inflammatory properties of sargachromenol on LPS-induced NO/PGE 2 production, RAW 264.7 cells were treated with sargachromenol (12.5, 25, 50, and 100 M) or left untreated for 1 h, followed by treatment with LPS (1 g/mL) for 24 h. NO and PGE 2 concentrations were measured in the cell culture media via the Griess reaction and ELISA assays, respectively. LPS treatment significantly increased the concentrations of NO and PGE 2 in the conditioned media. As shown in Figure 2(a), sargachromenol inhibited LPS-induced NO production in a concentration-dependent manner (by 7.7%, 16.8%, 31.0%, and 61.6% in the presence of 12.5, 25, 50, and 100 M sargachromenol, resp.). Moreover, sargachromenol markedly suppressed LPS-induced PGE 2 production, with an IC 50 of 30.2 M (Figure 2(b)). The cytotoxic effects of sargachromenol were assessed in the presence or absence of LPS by using an MTT assay. As shown in Figure 2(a), sargachromenol did not affect the viability of RAW 264.7 cells at the concentrations employed in this study. Thus, the inhibitory effects on NO and PGE 2 production were not attributable to cytotoxic effects.
In an effort to characterize the anti-inflammatory activities of sargachromenol, we assessed the effects of sargachromenol on LPS-induced iNOS and COX-2 protein upregulation in RAW 264.7 cells by using Western blotting. The levels of iNOS and COX-2 proteins were greatly increased by LPS treatment. However, sargachromenol was found to inhibit the induction of iNOS protein expression in a dosedependent manner. A densitometric analysis of 3 different experiments demonstrated that LPS-induced iNOS protein expression was inhibited by 94.9% in the presence of 50 M sargachromenol (Figure 3(a)). In parallel, the LPS-induced expression of COX-2 protein was reduced in the presence of sargachromenol in a concentration-dependent manner (Figure 3(b)). These results led us to evaluate the effects of sargachromenol on the expression of the iNOS and COX-2 enzymes in greater detail.
NF-B is one of the most important transcription factors involved in the transactivation of a variety of genes associated with the regulation of immune and inflammatory responses, including iNOS and COX-2. Activation of NF-B is correlated with a variety of inflammatory diseases, and many anti-inflammatory drugs inhibit the production of proinflammatory mediators via suppression of the activation of NF-B suppressors [4][5][6][7][8][9]. NF-B is normally bound to an inhibitory subunit, I B, which is present in the cytoplasm in an inactive form. Foreign stimuli (e.g., LPS or pathogens) induce activation of NF-B by the phosphorylation, ubiquitination, and subsequent proteolytic degradation of I B via activated I B kinase (IKK). The liberated NF-B transcription factor then translocates to the nucleus and binds to B motifs in the promoters of target genes, such as those encoding iNOS, COX-2, and cytokines, to promote their transcription [24,25]. Yoon et al. [26] showed that Artemisia fukudo essential oil prevented the degradation of I B induced by diverse stimuli, and thus, interfered with a step in the signaling cascade leading to the activation of NF-B. Hyun et al. [27] demonstrated that the anti-inflammatory effects of Acanthopanax koreanum fruit waste in RAW 264.7 cells involved prevention of I B degradation. Therefore, we studied whether sargachromenol inhibited the degradation of I B . As a control, we also exposed cells to pyrrolidine dithiocarbamate (PDTC), a specific NF-B inhibitor. As shown in 4 The Scientific World Journal  In conclusion, the results of the present study provide the first evidence that sargachromenol isolated from S. micracanthum inhibits inflammatory mediators, including NO and PGE 2 , in LPS-stimulated RAW 264.7 cells. These inhibitory effects were attributable to the prevention of I B-degradation, thereby suppressing NF-B activation. Although the exact mechanisms regulating the anti-inflammatory activity of sargachromenol are not yet fully known, our findings suggest that sargachromenol may have the potential to prevent inflammatory diseases and may act as a modulator of macrophage activation. Future studies are expected to confirm the anti-inflammatory effects of sargachromenol in a representative anti-inflammatory animal model.

Conflict of Interests
The authors declare that there is no conflict of interests regarding the publication of this paper.