Sea cucumber, Stichopus japonicus ethyl acetate fraction modulates the lipopolysaccharide induced iNOS and COX-2 via MAPK signaling pathway in murine macrophages

Abstract

The sea cucumber Stichopus japonicus is an important food and traditional medicine in Asian countries. However, ethyl acetate solvent fraction of S. japonicus (SCEA-F) is largely unknown for its anti-inflammatory activity and related molecular mechanisms. In this study, effect of SCEA-F on inflammation was investigated in LPS stimulated RAW264.7 cells. SCEA-F significantly inhibited the productions of NO and PGE₂ by inhibiting iNOS and COX-2 at their protein and gene levels. The production and the gene transcription of pro-inflammatory cytokines are also inhibited. The responsible molecular signaling for these inhibitory actions was found to be through suppression of the phosphorylation of MAPK molecules; ERK and p38 MAPK. These results indicate that SCEA-F inhibits LPS-induced inflammatory response via blocking of MAPK signaling pathway in murine macrophages, thus demonstrated its in vitro anti-inflammatory potential. Therefore it could be suggested that SCEA-F could be effectively used in functional food preparations.

Introduction

Inflammation represents a highly coordinated set of events that allows tissues to respond to injury or infection, which requires the participation of various cell types expressing and reacting to diverse mediators in a sequential manner (Sebban and Courtois, 2006). The inflammatory response consists of sequential release of mediators and recruitment of circulating leukocytes, which become activated at the inflammatory site and release further mediators (Lawrence et al., 2002). In most cases, the inflammatory response is resolved by the release of endogenous anti-inflammatory mediators (anti-inflammatory cytokines) as well as the accumulation of intracellular negative regulatory factors. Thus, the inflammatory cells are cleared at an appropriate time. However, the persistent accumulation and activation of leukocytes are a hallmark of chronic inflammation, leading to a dysfunction of these negative regulatory mechanisms (Hanada and Yoshimura, 2002, Lawrence et al., 2002). Therefore, it is crucial to identify molecular mechanisms that are involved in these self-limiting, self-resolving inflammatory lesions. Identification of the responsible molecular mechanisms will be beneficial to be used as therapeutic targets to control the transition from acute to chronic inflammation, by making these molecular signals are absent or deregulated.

Macrophages are major immune cells in the innate immune system. The activation of macrophages plays a key role in inflammatory responses when infected with pathogens. Macrophages can kill pathogens directly by phagocytosis and indirectly through secretion of various pro-inflammatory mediators such as bioactive lipids (prostanoids and other arachidonic acid-derived metabolites), reactive oxygen and nitrogen species, metalloproteinases, cycloxygenase-2 (COX-2), and pro-inflammatory cytokines, tumor necrosis factor-α, interleukine-1β and interleukine-1 (TNF-α, IL-1β, and IL-6) (Moncada, 1999, Nathan, 1992). Overproduction of the inflammatory mediators by activated macrophages has been implicated in the patho-physiology of many inflammatory diseases, including rheumatoid arthritis, atherosclerosis, chronic hepatitis, pulmonary fibrosis, and inflammatory brain diseases (Bosća et al., 2005). Lipopolysaccharide (LPS)-induced macrophage activation increased the production of pro-inflammatory cytokines, nitric oxide (NO) by inducible nitric oxide synthase (iNOS) and prostaglandin E₂ (PGE₂) by cyclooxygenase (COX-2), which are the main cytotoxic and pro-apoptotic mechanisms participating in the innate response in many mammals (Nathan, 1992, Moncada, 1999, Bosća et al., 2005). Therefore, LPS stimulated macrophages can be effectively used as a model to study inflammation and potential anti-inflammatory mediators with their action mechanisms.

Sea cucumbers are soft-bodied worm-like echinoderms which belong to the class Holothuroidae (De Moncerrat Iiguez-Martinez et al., 2005). They are considered as an important food in Asian countries including Philippines, Malaysia, Japan, Korea, and China (Zhong et al., 2007). In addition to their food use, sea cucumbers are also used in traditional medicinal applications in East Asia, for the treatment of asthma, hypertension, rheumatism, anemia, and sinus congestion (Fredalina et al., 1999, Zhong et al., 2007). Moreover, the high eicosapentaenoic acid (EPA) content of the sea cucumber has been characterized to induce tissue repair and wound healing (Fredalina et al., 1999, Hankenson et al., 2000). Solvent extracts and the compounds isolated from sea cucumbers have shown variety of biological activities such as antifungal, anticancer, hemolytic, cytostatic, antioxidant and immuno-modulatory effects (Dang et al., 2007, Han et al., 2008, Mamelona et al., 2007, Zhang et al., 2006, Zhong et al., 2007). Among more than 900 species of sea cucumbers (Whitehouse and Fairlie, 1994), Stichopus japonicus belong to the phylum Echinodermata and is a famous traditional delicacy consumed by Chinese and Japanese (Cui et al., 2007). And also this species is used in Chinese oriental medicine. However, there are few studies conducted on exploring the anti-inflammatory activities of S. japonicus. Therefore, present study is focused on evaluating the biological effects of solvent fractions derived from sea cucumber S. japonicus on the production of inflammatory mediators including NO, PGE₂, and pro-inflammatory cytokines (TNF-α and IL-1β) in murine macrophages (RAW264.7) stimulated with LPS. Furthermore, efforts have taken to identify the molecular mechanisms through mitogen-activated protein kinase (MAPK) signaling pathway.