Ecklonia cava ethanolic extracts inhibit lipopolysaccharide-induced cyclooxygenase-2 and inducible nitric oxide synthase expression in BV2 microglia via the MAP kinase and NF-jB pathways Won-Kyo Jung a , Young-Wook Ahn b , Sang-Hoon Lee c , Yung Hyun Choi d , Se-Kwon Kim c , Sung Su Yea e , Inhak Choi f , Sae-Gwang Park f , Su-Kil Seo f , Soo-Woong Lee f , Il-Whan Choi f, * a Department of Marine Life Science, Marine Life Research Center, Chosun University, Gwangju 501-759, Republic of Korea b Department of Microbiology, Pukyong National University, Busan 608-737, Republic of Korea c Marine Bioprocess Research Center, Pukyong National University, Busan 608-737, Republic of Korea d Department of Biochemistry, Dongeui University College of Oriental Medicine, Busan 614-052, Republic of Korea e Department of Biochemistry, Inje University College of Medicine, Busan 633-165, Republic of Korea f Department of Microbiology, College of Medicine and Center for Viral Disease Research, Inje University, Busan 633-165, Republic of Korea article info Article history: Received 27 August 2008 Accepted 25 November 2008 Available online xxxx Keywords: Ecklonia cava Inducible nitric oxide synthase Cyclooxygenase-2 Nuclear factor-jB Mitogen-activated protein kinase abstract Ecklonia cava (EC) is a brown alga that has demonstrated radical scavenging, bactericidal, tyrosinase inhibitory, and protease inhibitory activities. However, the molecular mechanisms underlying its anti- inflammatory action remain unclear. In the current study, we attempted to determine whether pretreat- ment with EC induces a significant inhibition of anti-inflammatory activity in lipopolysaccharide (LPS)- stimulated murine BV2 microglia. Our results indicate that EC inhibits LPS-induced nitric oxide (NO) and prostaglandin E 2 (PGE 2 ) production in a concentration-dependent manner and inhibits inducible nitric oxide (iNOS) and cyclooxygenase (COX)-2 in BV2 microglia without significant cytotoxicity. EC treatment significantly reduced nuclear factor-jB (NF-jB) translocation and DNA-binding in LPS-stimu- lated BV2 microglia. This effect was mediated through the inhibition of the degradation of the inhibitor jB and by inhibition of the mitogen-activated protein kinase (MAPK) phosphorylation, at least in part by inhibiting the generation of reactive oxygen species. Our data also indicate that EC extracts exert anti- inflammatory effects by suppressing proinflammatory cytokines. Collectively, these results suggest that EC suppresses the induction of cytokines by LPS, as well as iNOS and COX-2 expression, by blocking NF- jB and MAPK activation. These findings provide mechanistic insights into the anti-inflammatory and neuroprotective actions of EC in BV2 microglia. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction Ecklonia cava (EC) is a brown alga that grows plentifully in the subtidal regions of Jeju Island, Korea. EC contains a richer supply of total phenolic compounds, phlorotannins, than do other brown seaweeds (Heo et al., 2005). Recently, an increasing amount of evidence has demonstrated that EC exhibits radical scavenging, matrix metalloproteinase inhibitory, bactericidal, protease inhibi- tory, antioxidative, anti-inflammatory, immunomodulatory, and anti-asthmatic activities (Kang et al., 2004; Kim et al., 2006a, 2008; Ahn et al., 2004, 2008; Shin et al., 2006). Nevertheless, no reports have investigated the anti-inflammatory effects and mole- cular mechanisms of EC extracts in LPS-stimulated microglia. Microglia enter the brain early in embryogenesis and develop in parallel with the maturation of the nervous system (Mosley et al., 2006). As such, these cells constitute up to 20% of the cell popula- tion in certain regions of the brain (Dobrenis, 1998; Lawson et al., 0278-6915/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2008.11.041 Abbreviations: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro- mide; AD, Alzheimer’s disease; COX, cyclooxygenase; DCFDA, 2 0 ,7 0 -dichlorofluores- cein diacetate; DMEM, Dulbecco’s modified Eagle medium; EC, Ecklonia cava; ECL, electrochemiluminescence; ELISA, enzyme-linked immunosorbent assay; EMSA, electrophoretic mobility shift assay; ERK, extracellular signal-regulated kinase; FACS, fluorescence-activated cell sorting; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IL, interleu- kin; iNOS, inducible nitric oxide synthase; IjB, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor; JNK, c-Jun N-terminal kinase; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; MS, multiple sclero- sis; NF-jB, nuclear factor-jB; NO, nitric oxide; PD, Parkinson’s disease; PBS, phosphate-buffered saline; PGE 2 , prostaglandin E 2 ; ROS, reactive oxygen species; RT-PCR, reverse transcriptase-polymerase chain reaction; SAPK, stress-activated protein kinase; TNF-a, tumor necrosis factor-a. * Corresponding author. Tel.: +82 51 890 6461; fax: +82 51 891 6004. E-mail address: cihima@inje.ac.kr (I.-W. Choi). Food and Chemical Toxicology xxx (2009) xxx–xxx Contents lists available at ScienceDirect Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox Please cite this article in press as: Jung, W.-K. et al., Ecklonia cava ethanolic extracts inhibit lipopolysaccharide-induced ..., Food Chem. Toxicol. (2009), doi:10.1016/j.fct.2008.11.041 ARTICLE IN PRESS