Brown seaweeds and their extracts are a well-known sources of antioxidation or anti-inflammation due to their appreciable amount of polyphenols and pigments. Although significant differences were observed in both total polyphenolic contents and antioxidant activities of extracts from various species, high correlation was found between total polyphenolic contents and their antioxidant capacity to scavenge ROS
. Polyphenolic compounds from marine algae may prevent inflammatory disorders, cancer, and diabetes which are associated with the regulation of free radicals generated in the cells
[30, 31]. In this regard, we analyzed antioxidant and anti-inflammatory activities of five representative brown algae along the southeastern coast of Korea. Among them, M. myagroides showed the highest phenolic contents and ROS scavenging activity as well as anti-inflammatory activity. High phenolic content in M. myagroides may participate in the inhibition of NO production in LPS-treated BV-2 cells. As anti-inflammatory activities from M. myagroides, fucoxanthin
, fatty acid
, and phlorofucofuroeckol B
 have shown potent activities in macrophage or microglial cells. However, anti-inflammatory activity of MME in this study may not be due to fucoxanthin, fatty acid or phlorotannins, since the peaks of those compounds were not detected in the chromatogram (Figure
[20, 21]. Thus, we hypothesized strong anti-inflammatory compounds are contained in MME and we separated the components from MME using C18 column and isolated sargachromenol having strong anti-inflammatory activity (Table
1). Base on the inhibitory activity of NO production in LPS-stimulated BV-2 cells, sargachromenol might be a main anti-inflammatory compound in MME.
NO and PGE2 are crucial inflammatory and neurotoxic mediators. These inflammatory mediators are responsible for the harmful effects on brain diseases, including ischemia, Alzheimer’s disease, and neuronal death
. In vitro and in vivo studies have revealed that overproductions of NO and PGE2 by enhanced iNOS and COX-2 protein levels, are associated with central nervous injuries and diseases
. iNOS and COX-2 proteins have been over-expressed in microglial cells from the rodent brain treated with LPS
. In addition, iNOS and COX-2 inhibitors provide neuroprotective effects against LPS-induced neurotoxicity, suggesting NO and PGE2 have important roles in neurotoxicity
[6, 35]. In this regard, inhibition of inflammatory mediator production is considered as a key step in the control of neuroinflammatory diseases. In the present study, we demonstrated that MME inhibited productions of both NO and PGE2 in LPS-stimulated BV-2 cells (Figure
1). Moreover, we provide evidence that MME-mediated inhibition of NO and PGE2 production was the consequence of the suppression of both mRNA and protein levels of iNOS and COX-2 in LPS-stimulated BV-2 cells (Figure
2). Furthermore, we found that the suppression of COX-2 mRNA expression by MME was more marked than that of COX-2 protein in BV-2 cells, indicating that inhibition of PGE2 by MME is associated with downregulation of COX-2 at both transcriptional and translational levels in LPS-stimulated BV-2 cells. Thus, the present findings may address that MME has protective effects on neurodegenerative diseases induced by neuroinflammation.
Pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6 are small secreted proteins that regulate immunity and inflammation. Their production is increased in inflammatory states and they function by regulating the intensity and duration of the immune response
. TNF-α plays a central role in initiating and regulating the cytokine signaling cascade during an inflammatory response in neuronal cells. In inflammatory disease states, TNF-α along with other pro-inflammatory mediators and neurotoxic substances is predominantly produced by activated microglia
. IL-1β is an important initiator of the immune response, playing a key role in the onset and development of a complex hormonal and cellular inflammatory cascade. IL-6 is a multifunctional cytokine that plays an important role in host defense, with major regulatory effects upon the inflammatory response
. Excessive productions of these pro-inflammatory cytokines activate microglia and lead to neural cell deaths, resulting in the pathogenesis of several neurological and neurodegenerative disorders
[4, 6, 37]. As an alternative chemoprevention of inflammatory diseases, natural compounds able to inhibit the production of pro-inflammatory cytokines may be attractive as anti-inflammatory agents and, for this reason, the inhibitory effects of phytochemicals on the production of pro-inflammatory cytokines have been intensively studied to develop anti-inflammatory agents for preventing inflammatory diseases
. In the present study, we have demonstrated that MME remarkably suppressed the secretions of TNF-α, IL-1β, and IL-6 in LPS-stimulated BV-2 cells (Figure
3). Thus, the present findings may further support the potential of MME as a neuroprotective by reducing inflammation.
NF-κB plays an important role in the regulation of cell survival and coordinates the expression of pro-inflammatory proteins and cytokines, including iNOS, COX-2, TNF-α, IL-1β, and IL-6
. NF-κB is present as an inactive complex associated with an inhibitory subunit, IκB-α, in cytoplasm. Activation of NF-κB caused by LPS or pro-inflammatory cytokines leads to degradation of IκB-α and inducing translocation of NF-κB into nucleus
. Recently, we demonstrated that extract of brown algae including Saccharina japonica
[19, 20], M. myagroides
[21, 22], Ecklonia stolonifera
, and Sargassum fulvellum
 inhibited the activation of NF-κB signaling pathway through the blockade of proteolytic degradation of IκB-α. In this study, we observed that enhanced phosphorylation of IκB-α by LPS was reduced by MME treatment, suggesting that MME protected the proteolytic degradation of IκB-α (Figure
4B). Degradation of IκB-α involves its dissociation from the inactive complex, leading to activation of NF-κB in response to LPS, which is demonstrated by NF-κB promoter activity (Figure
4C). Moreover, the nuclear translocation of NF-κB was significantly inhibited by MME, supporting the inhibition of NF-κB activation by MME (Figure
4A). From these data, the MME-mediated down-expression of LPS-induced inflammatory mediators and cytokines in BV-2 cells is partially associated with the ability of MME to inhibit the IκB/NF-κB signaling pathway.
NF-κB activation is alternatively regulated by various cellular kinases including MAPKs and Akt, which are the groups of protein kinases to play key roles in inflammatory reactions
[38, 40]. MAPKs are involved in inflammatory signaling cascades and regulation of iNOS and COX-2 through the activation of NF-κB in LPS-stimulated immune cells
[9, 12, 38]. Therefore, anti-inflammatory mechanisms are closely related to inhibition of MAPKs in stimulated BV-2 cells. In this study, we have specifically shown that MME inhibits the activation of ERKs and JNKs, but not Akt and little p38 MAPK, in response to LPS in BV-2 cells, suggesting that ERKs and JNKs are additional targets of MME. Although, hexane fraction of MME down-regulated the phosphorylation of MAPKs and Akt in LPS-stimulated RAW 264.7 cells
, it is hard to conclude why MME did not inhibit the phosphorylation of p38 MAPK and Akt in LPS-stimulated BV-2 cells. To further confirm the involvement of ERK1/2 and JNK1/2 on the activation of NF-κB, NO production and iNOS and COX-2 expression were determined. The inhibitory level of NO production showed 45% in the PD98059 treated cells and 65% in the SP600125 treated cells. MME treatment showed higher inhibitory effect of NO production than both inhibitors, which indicates that MME inhibits both phosphorylation of ERK1/2 and JNK1/2 (Figure
5B). In addition, treatment of both inhibitors resulted in suppression of iNOS and COX-2 expressions. Considering roles of MAPKs in inflammatory gene expression, MME inhibited, at least in part, LPS-induced NF-κB activation in the microglial cells by inhibiting the JNK and ERK pathways.