An aqueous extract of Nomura’s jellyfish ameliorates inflammatory responses in lipopolysaccharide-stimulated RAW264.7 cells and a zebrafish model of inflammation

Abstract

The recent mass emergence of Nomura’s jellyfish (Nemopilema nomurai) has caused much economic and environmental damage. However, there is no innovative strategy to dispose of or utilize these jellyfish. Some reports suggest that the jellyfish may be bioactive resources and a source of important compounds with antibacterial activity. Here, we examined the effect of an aqueous extract of Nomura’s jellyfish (AENJ) on lipopolysaccharide (LPS)-stimulated Raw 264.7 macrophages and a zebrafish model of inflammation and analyzed the underlying molecular mechanisms.

AENJ downregulated inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA levels in LPS-stimulated Raw 264.7 macrophages, with no apparent cytotoxic effects. However, AENJ had no effect on expression of other inflammation-related genes such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and MCP-1. Furthermore, AENJ reduced expression of nerve injury-induced protein 1 (Ninj1), which is an important adhesion molecule, thereby reducing cell adhesion to the extracellular matrix (ECM) in vitro. The inhibitory effect of AENJ on leukocytes was confirmed in LPS-microinjected zebrafish larvae; AENJ reduced the number of the infiltrate accumulating at the site of inflammation. In addition, AENJ suppressed the expression of matrix metalloproteinase-2 (MMP-2) and MMP-9 in LPS-stimulated Raw 264.7 cells. Finally, AENJ blocked nuclear translocation of nuclear factor kappa B (NF-κB), a key transcription factor for inflammatory responses, in Raw 264.7 cells in a dose-dependent manner.

Collectively, the data suggest that AENJ inhibits expression of COX and iNOS by blocking NF-κB signaling pathways and suppresses the activity of macrophages by downregulating Ninj1 and MMPs. Therefore, AENJ may be a useful preventive neutraceutical, or therapeutic agent against inflammatory disorders.

Introduction

Inflammatory responses represent the body’s defense against diverse stimuli such as microbial invasion and injury [1]. Lipopolysaccharide (LPS) is present in the cell wall of Gram-negative bacteria and is a major cause of sepsis; it is also a powerful activator of the immune system [2,3]. During an inflammatory response, interleukins (ILs) and growth factors trigger migration of leukocytes to the site of tissue injury or infection [4]. If inflammation is sustained, overexpression of inflammatory factors results in capillary leakage and an increase in the amount of leukocytic infiltrate. Therefore, leukocyte accumulation is a characteristic of inflamed tissue. Because infiltrating leukocytes produce excess inflammatory mediators such as cytokines and chemokines, they are main players during chronic inflammatory processes [[5], [6], [7]].

Usually, activated macrophages produce cytokines that protect the host from some viruses or parasites [8,9]. However, overproduction of cytokines may induce an excessive inflammatory response, leading to tissue damage, genetic mutations, and nerve injury [10,11]. Therefore, it is important to normalize the inflammatory responses of macrophages as quickly as possible. NF-κB is a target molecule that can trigger resolution of inflammation because it plays a critical role in expression of genes encoding pro-inflammatory mediators and adhesion molecules [11]. Under normal conditions, the heteromeric NF-κB molecule binds to its inhibitor, IκB, and is localized in the cytosol. When exposed to inflammatory stimuli, IκB undergoes proteasomal degradation to expose the hidden nuclear-localization signal of NF-κB, which allows translocation to the nucleus followed by active transcription of target genes [12,13]. Therefore, NF-κB inhibitors are candidate therapeutic agents for inflammatory disorders [13,14].

The zebrafish (Danio rerio, Hamilton-Buchanan, 1822) is a freshwater teleost belonging to the family Cyprinidae. The fish is a representative model system for human inflammatory diseases and has already contributed to some successful phenotype-based drug discoveries [15,16]. As a vertebrate, Danio rerio is genetically closer to humans than D. melanogaster, C. elegans, or S. cerevisiae. Furthermore, zebrafish embryos and larvae are completely transparent, which makes them ideally suited to real time imaging of leukocytes migration at relatively low cost [17,18]. Here, we used zebrafish larvae to visualize changes in inflammatory infiltrates during inflammatory responses.

Nemopilema nomurai was first described as a distinct species by Kishinouye (1922) and is a rhizostome jellyfish belonging to the phylum Cinidaria and is the largest jellyfish in the world, with a body weight around 200 kg. It blooms in the East Sea of Korea and Japan [19,20]. In the summer, jellyfish numbers soar; the jellyfish can tear fishing nets, thereby causing enormous damage and economic losses to the fishing industry [19]. Besides economic losses, contact with the tentacles of N. nomurai can pose a threat to humans by causing fever, nausea, diarrhea, and respiratory distress [19,21]. Therefore, research into innovative methods of upcycling jellyfish is urgently required. Recent studies show that jellyfish are good sources of green fluorescent protein, collagen, and food [22,23]. Also, the insecticidal compound, JAP-1 is purified from an extract of N. nomurai [21]; this compound also shows potent antimicrobial activity against both Gram-positive and Gram-negative bacteria [24]. Also, a lyophilized powder from N. nomurai has antioxidant activity [20]. However, the anti-inflammatory activity of extracts or compounds derived from Nomura’s jellyfish is largely unknown.

In this study, we sought to investigate the potential effect and underlying molecular mechanisms of aqueous extract of Nomura’s jellyfish (AENJ) on LPS-stimulated RAW 264.7 macrophages and zebrafish model of inflammation. The results suggest that AENJ could suppresses inflammatory responses induced by LPS by inhibiting the NF-κB signaling pathway. Studies in zebrafish revealed that AENJ suppresses the accumulation of leukocytes at site of inflammation. Thus, AENJ is a putative anti-inflammatory agent.