The present study was undertaken to examine the anti-inflammatory effect of EMM on LPS-stimulated murine macrophage cells. To further understood the molecular mechanisms of EMM, we investigated the effects of EMM on the secretion of NO, PGE2, TNF-α, IL-1β, and IL-6, the expression of iNOS and COX-2, and the activation of NF-κB. Our results indicated that EMM effectively inhibited the secretion of NO, PGE2, TNF-α, IL-1β, and IL-6 through a blockade of the NF-κB and MAPK pathways in LPS-stimulated macrophages. The inhibitory effect of EMM on the expression of inflammatory mediators suggested one of the mechanisms responsible for its anti-inflammatory action and its potential for use as a therapeutic agent for treating inflammatory diseases.
Under pathological conditions, excessive inflammatory mediators and pro-inflammatory cytokines produced by activated macrophages cause inflammatory process and act synergistically with other inflammatory mediators
[2, 4, 18]. Compounds able to reduce NO or PGE2 production may be attractive as anti-inflammatory agents and, for this reason, the inhibitory effects of natural compounds on NO or PGE2 productions have been rigorously studied to develop therapeutic agent against inflammatory diseases
[17, 19, 20]. Also, excessive production of pro-inflammatory cytokines plays a critical role in acute inflammatory responses as well as chronic inflammatory diseases. Recent studies have shown that in vivo or in vitro treatments of natural compounds are effective in reducing inflammation by the suppression of pro-inflammatory cytokines, which may ameliorate inflammation-related diseases, including atherosclerosis, cancer, and inflammatory arthritis
[21, 22]. Thus, the regulation of those molecules is important to the inhibition of inflammatory response. Our results indicate that the inhibition of NO and PGE2 production by EMM in LPS-stimulated macrophage cells is associated with downregulation of iNOS and COX-2 genes (Figure
2), which seems the first addressing transcriptional inhibition of iNOS and COX-2 by EMM.
NF-κB is a transcription factor actively involved in the transcriptional induction of iNOS and COX-2 gene
[1, 3]. Like NO and PGE2 produced by iNOS and COX-2, respectively, the release of pro-inflammatory cytokines is regulated by NF-κB pathway and plays an important role in the induction of the innate immune response of the acquired immune response
. It has been well studied that the inhibition of NF-κB activation by black tea extract is associated with the phosphorylation, ubiquitination, and subsequent degradation of IκB via ubiquitin-proteosome pathway
. Although biochemical actions of EMM on NF-κB regulations remain unknown, the present study showed that EMM potentially inhibits the proteolytic degradation of IκB-α and the NF-κB promoter-driven luciferase expression induced by LPS in RAW 264.7 cells. Therefore, these results demonstrate the ability of EMM to inhibit NF-κB activation in response to LPS signal in RAW 264.7 cells. Hence it is likely that reductions of iNOS, COX-2, and pro-inflammatory cytokine expression in RAW 264.7 cells are largely associated with activation/deactivation of NF-κB pathway. This report is, to our best knowledge, the novel findings to address the functions of EMM through NF-κB pathway in response to LPS treatment.
NF-κB is also regulated by various signaling kinases including MAPKs (ERK, JNK, and p38) and Akt, which are groups of signaling molecules to play key roles in NF-κB activation
[2, 3]. MAPKs have been suggested to be involved in pro-inflammatory signaling cascades and in the activation of NF-κB in LPS-stimulated immune cells
[2, 11]. Therefore, anti-inflammatory mechanisms are closely related with inhibition of MAPKs in activated RAW 264.7 cells. In this study, we found that phosphorylation of MAPKs in response to LPS was inhibited by EMM treatment (Figure
6A). Interesting finding of this study is that the activation of Akt, a downstream regulator of PI3K, was also inhibited by EMM in response to LPS signal in RAW 264.7 cells. Precise mechanisms by which EMM modulate this PI3K/Akt pathway in the LPS stimulation remains unclear. Considering a recent study with a phlorofucofuroeckol A, which indicated a link between ROS and PI3K/Akt pathway in regulations of inflammatory genes
[7, 11], it is speculated that the antioxidant activity of EMM or unknown components in EMM may be related to inhibition of Akt phosphorylation. Thus, it is likely that inhibition of MAPKs and Akt phosphorylation by EMM may contribute to the EMM-mediated inhibition of NF-κB pathway in LPS-stimulated RAW 264.7 cells.
Topical application of PMA to mouse skin induces inflammatory response and this is a well-established in vivo model for the evaluation of various anti-inflammatory agents. PMA can activate a wide variety of cell types that may contribute to acute inflammation, resulting in an increase in epidermal tissue swelling and infiltration of inflammatory cells
[25, 26]. The results from our in vivo experiments clearly showed that EMM (90 μg/ear) suppressed 66.8% of PMA-induced skin swelling. Furthermore, 6,6’-bieckol (30 μg/ear) isolated firstly from M. myagroides in this study inhibited 64.1% of PMA-induced skin swelling, indicating that it might be a main anti-inflammatory compound in EMM. Although the inhibitory activity of EMM is about one third of 6,6’-bieckol, EMM showed higher anti-edema activity than Indo compared to its concentration. This result demonstrates that EMM can be used as a topical anti-inflammatory agent.
Like other polyphenolic compounds, phlorotannins, a class of compounds polymerized with phloroglucinol units found in brown seaweeds, have strong antioxidant
[18, 27] and anti-inflammatory
[11, 28] activities. The multifunctional antioxidant activity of phlorotannins is highly associated with phenol rings which act as electron traps to scavenge peroxy, superoxide-anions, and hydroxyl radicals
. Specific mechanisms of phlorotannins on the anti-inflammatory actions are not clearly defined. Considering cellular signaling of polyphenols, phlorotannins may not merely exert their effects as free radical scavenger, but may also modulate inflammatory cellular signaling proteins, including NF-κB and AP-1 transcription factors
. Recent studies demonstrated that phlorofucofuroeckol A and 6,6’-bieckol isolated from Ecklonia spp. alleviate inflammatory response to LPS by inhibiting NF-κB pathway with its intrinsic antioxidant activity
[11, 28]. In the present study, the isolated 6,6’-bieckol from EMM remarkably inhibited the production of iNOS and COX-2 proteins, indicating 6,6’-bieckol is one of the principle anti-inflammatory compounds in EMM.