한수지 52(1), 81-86, 2019 Note Korean J Fish Aquat Sci 52(1),81-86,2019 LPS 로유도한마우스의급성신경염증에대한톱니모자반 (Sargassum serratifolium) 추출물의효과 최민우 김형락 이형곤 1 김재일 * 부

한수지 52(1), 81-86, 2019 Note Korean J Fish Aquat Sci 52(1),81-86,2019 LPS 로유도한마우스의급성신경염증에대한톱니모자반 (Sargassum serratifolium) 추출물의효과 최민우 김형락 이형곤 1 김재일 * 부경대학교식품영양학과, 1 산안토니오텍사스주립대학교생물학과 Effect of a Sargassum serratifolium Extract on Neuroinflammation Induced by Lipopolysaccharides in Mice Min-Woo Choi, Hyeung-Rak Kim, Hyoung-Gon Lee 1 and Jae-Il Kim* Department of Food Science and Nutrition, Pukyong National University, Busan 48513, Korea 1 Department of Biology, University of Texas at San Antonio, Texas 78249, U.S.A. The common hallmark of several neurodegenerative disorders, including Alzheimer s disease (AD), is the presence of chronic neuroinflammation, which contributes to the loss of neuronal structure and function. This study investigated the effects of an ethanolic extract of Sargassum serratifolium (SSE) in a lipopolysaccharides (LPS)-induced murine neuroinflammation model. Mice were administered SSE (100 mg/kg body weight) or vehicle for 5 days by oral gavage, and then treated with LPS or saline by intraperitoneal injection. Thereafter, the brain tissues were collected, and the expression of pro-inflammatory cytokines was analyzed by quantitative real-time RT-PCR. There was a marked increase in the spleen weight index in the LPS-treated groups, which indicated the induction of acute systemic inflammation. Based on significant increases in the levels of IL-1 and IL-6 expression, the induction of neuroinflammation was also evident in the cortex and hippocampus of the LPS-treated groups. The overall expression of IL-1 and IL-6 was decreased slightly by SSE administration, compared with the LPS group, and a marked change in IL-1 was observed in the cortex of the SSE-treated (SSE/LPS) group. These results suggest that SSE has potential as an anti-neuroinflammatory nutraceutical. Key words: Neurodegenerative disorders, Neuroinflammation, Brown seaweeds, Sargassum serratifolium, Lipopolysaccharides 서론 (Alzheimer s disease, AD), (prion diseases), (Parkinson s disease), (amyotrophic lateral sclerosis) (neurodegenerative disorders) (neuronal loss) (activated microglia) (Soto, 2003). (macrophages) (neuroinflammation), (McGeer et al., 2016; Molteni and Rossetti, 2017; Schain and Kreisl, 2017). interleukin (IL)-1, IL-6, tumor necrosis factor- (TNF- ) cyokines nitric oxide (free radical) (Molteni and Rossetti, 2017; Schain and Kreisl, 2017). (host)., *Corresponding author: Tel: +82. 51. 629. 5849 Fax: +82. 51. 629. 5842 E-mail address: jikim@pknu.ac.kr This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licens (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. https://doi.org/10.5657/kfas.2019.0081 Korean J Fish Aquat Sci 52(1), 81-86, February 2019 Received 15 February 2019; Revised 21 February 2019; Accepted 21 February 2019 저자직위 : 최민우 ( 대학원생 ), 김형락 ( 교수 ), 이형곤 ( 교수 ), 김재일 ( 교수 ) Copyright 2019 The Korean Society of Fisheries and Aquatic Science 81 pissn:0374-8111, eissn:2287-8815

82 최민우ㆍ김형락ㆍ이형곤ㆍ김재일 (Molteni and Rossetti, 2017; Schain and Kreisl, 2017). (protein aggregates), (Soto, 2003; Schain and Kreisl, 2017). in vitro in vivo, (Soto, 2003; Molteni and Rossetti, 2017; Schain and Kreisl, 2017).. (Sargassum) (Sargassace), meroterpenoids, phlorotannins, fucoxanthins, fucosterols (Liu et al., 2012). (Sargassum serratifolium), (Oak and Lee, 2006). in vivo in vitro, (anti-cancer) (Kang et al., 2015), (hypopigmentation) (Azam et al., 2018), (anti-oxidant) (Lim et al., 2018; 2019), (anti-inflammatory) (Gwon et al., 2015; 2017; 2018; Joung et al., 2015; 2017), (hepatoprotective) (Lim et al., 2018), (neuroprotective) (Oh et al., 2016; Choi et al., 2017; Seong et al., 2017). (Sargachromenol, SCM), (Sargaquinoic acid, SQA), (Sargahydroquinoic acid, SHQA) meroterpenoids (Gwon et al., 2015), (Lim et al., 2018; 2019) (Gwon et al., 2015; 2017; 2018; Joung et al., 2015; 2017). - ( -amyloid) (Choi et al., 2017; Seong et al., 2017),., lipopolysaccharides (LPS), (ethanolic extract of S. serratifolium, SSE). 재료및방법 실험재료 Poly (ethylene glycol)-400 (PEG-400), phosphate-buffered saline (PBS), 2,2,2-tribromoethanol (avertin), LPS Sigma- Aldrich (St Louis, MO, USA), dimethyl sulfoxide (DMSO) Life Sciences (Tewksbury, MA, USA). RNA TRIzol Invitrogen (Carlsbad, CA, USA), tert-amyl alcohol (2-methyl-2-butanol) Fisher Scientific (Pittsburgh, PA, USA), High Capacity cdna Reverse Transcription Kits and RNase Inhibitor PowerUp SYBR Green Master Mix Applied Biosystems (Beverly, MA, USA). 톱니모자반주정추출물 (SSE) 시료의제조 (S. serratifolium) (Gwon et al., 2015; Joung et al., 2015; Choi et al., 2017). 2016 5,., 50 g 10 (95% ) 70 4 2. 40,. (SSE) -20. SSE (sargahydroquinoic acid, SHQA), (sargaquinoic acid, SQA), (sargachromenol, SCM), 45-48% SHQA (Joung et al., 2017; Lim et al., 2018). 동물실험, 경구투여, LPS 에의한급성신경염증의유도 12 C57BL/6J (9 ) Jackson Laboratory (Bar Harbor, ME, USA). 3, Control, LPS, SSE/LPS. SSE DMSO vehicle (25% DMSO/75% PEG- 400) (Gerenu et al., 2015), SSE/LPS kg 100 mg 5 (oral gavage). Control LPS vehicle. LPS Jo et al. (2017) 5 2 LPS (5 mg/kg body weight) LPS SSE/LPS. Control PBS. 4 avertin

급성신경염증에대한톱니모자반추출물의효과 83 (250-500 mg/kg body weight) (cerebral cortex) (hippocampus) -80. (spleen), (spleen weight index). (University of Texas at San Antonio) (Institutional Animal Care and Use Committee, IACUC). Quantitative Real-Time RT-PCR 에의한염증성 cytokine 유전자발현의분석 TRIzol (Invitrogen, Carlsbad, CA, USA) total RNA. Total RNA (400 ng) high capacity cdna reverse transcription kits (Applied Biosystems, Beverly, MA, USA) cdna. cdna (10 ng) PowerUp SYBR green master mix (Applied Biosystems, Beverly, MA, USA), primers real-time PCR. Ct (Choi et al., 2017), real-time PCR primers Table 1. 통계처리 (mean SD), Student s t-test. 결과및고찰 (SSE) LPS. LPS, nutraceutical (Catorce and Gevorkian, 2016). LPS (astrocytes) cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (inos) cytokines (Qin et al., 2007; Erickson and Banks, 2011; Jo et al., 2017). LPS amyloid precursor protein (APP) -amyloid, (hyperphosphorylated) tau (Sheng et al., 2003; Kitazawa et al., 2005). APP, -amyloid hyperphosphorylated tau (McGeer et al., 2016). LPS-. SSE 5 LPS. Table 2. 5 1 g Table 1. Primers used for quantitative real-time RT-PCR analysis Gene Sense Antisense GAPDH 5 -AGGTCGGTGTGAACGGATTTG-3 5 -TGTAGACCATGTAGTTGAGGTCA-3 IL-1β 5 -CCAAGCAACGACAAAATACCC-3 5 -GTTGAAGACAAACCGTTTTTCC-3 IL-6 5 -AGTTGCCTTCTTGGGACTGA-3 5 -TCCACGATTTCCCAGAGAAC-3 TNF- α 5 -ATGGCCTCCCTCTCAGTTC-3 5 -TTGGTGGTTTGCTACGACGTG-3 GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IL-1β, interleukin-1β; IL-6, interleukin-6; TNF-α, tumor necrosis factors-α. Table 2. Body weight change and spleen weight index in C57BL/6J mice treated with or without ethanolic extract of Sargassum serratifolium (SSE) followed by lipopolysaccharides (LPS) Groups Body weight (g) Day 1 Day 5 Spleen weight (g) Spleen weight index Control 20.0±1.0 21.1±1. 2 0.067±0.006 3.17±0.17 LPS 19.8±1.6 20.6±1.1 0.084±0.009* 4.11±0.66* SSE/LPS 19.3±1.4 19.5±1.4 0.089±0.006** 4.44±0.26** Data are expressed as mean±sd (n=3). SSE or vehicle only was administered by oral gavage in mice for 5 days. On day 5, the mice were sacrificed after 4 h of intraperitoneal injection of LPS (5 mg/kg body weight) or PBS. The spleens from mice were isolated and weighed, and spleen weight index were calculated as organ weight (mg) per gram (g) of mouse body weight. *P<0.05 and **P<0.01 compared to control group.

84 최민우ㆍ김형락ㆍ이형곤ㆍ김재일 Table 3. Gene expression profile of pro-inflammatory cytokines in the brains of C57BL/6J mice treated with or without ethanolic extract of Sargassum serratifolium (SSE) followed by lipopolysaccharides (LPS) Brain regions Cortex Hippocampus Groups Genes IL-1β IL-6 TNF-α Control 1.04±0.39 1.00±0.12 ND 1 LPS 14.30±2.48** 76.88±26.26* ND SSE/LPS 7.20±3.41* a 47.21±18.74* ND Control 1.02±0.30 1.05±0.39 ND LPS 19.53±7.96* 66.19±9.03** ND SSE/LPS 12.20±4.09* 45.97±25.59* ND Gene expression levels were determined by reverse transcription followed by real-time PCR and were normalized with the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The transcription levels were analyzed after 4 h of intraperitoneal injection of LPS (5 mg/kg body weight) or PBS. Data (mean±sd, n=3) are expressed relative to control group. IL-1β, interleukin-1β; IL-6, interleukin-6; TNF-α, tumor necrosis factor-α. 1 ND, not detected. *P<0.05 and **P<0.01 compared to control group; a P<0.05 compared, vehicle LPS SSE (SSE/LPS). LPS (spleen weight index) (Li et al., 2016; Wang et al., 2017). Table 2 (spleen) LPS., LPS. LPS (Qin et al., 2007; Erickson and Banks, 2011, Jo et al., 2017). (cortex) (hippocampuse) cytokines real-time qunatitative RT-PCR Table 3. LPS IL-1 IL-6 (P<0.05-P<0.01), IL-6. cytokines SSE (SSE/LPS ), IL-1 (P<0.05). SSE. LPS. LPS,, LPS, (Catorce and Gevorkian, 2016). (Jo et al., 2017), LPS cytokines, IL-1 IL-6 2, 6 12. LPS 4. cyokines, SSE.. (Soto C, 2003; Schain and Kreisl, 2017). -amyloid, prion protein, -synuclein,., (Soto, 2003; Schain and Kreisl, 2017). SSE. 사사 (2017 ). References Azam MS, Kwon M, Choi J and Kim HR. 2018. Sargaquinoic acid ameliorates hyperpigmentation through camp and ERK-mediated downregulation of MITF in α-mshstimulated B16F10 cells. Biomed Pharmacother 104, 582-589. https://doi.org/10.1016/j.biopha.2018.05.083.

급성신경염증에대한톱니모자반추출물의효과 85 Catorce MN and Gevorkian G. 2016. LPS-induced murine neuroinflammation model: Main features and suitability for pre-clinical assessment of nutraceuticals. Curr Neuropharmacol 14, 155-164. https://doi.org/10.2174/1570 159X14666151204122017. Choi MW, Jung CG, Kim HR and Kim JI. 2017. Effect of Sargassum serratifolium extracts on β-amyloid production. Korean J Fish Aquat Sci 50, 085-091. https://doi.org/10.5657/ KFAS.2017.0085. Erickson MA and Banks WA. 2011. Cytokine and chemokine responses in serum and brain after single and repeated injections of lipopolysaccharide: multiplex quantification with path analysis. Brain Behav Immun 25, 1637-1648. https:// doi.org/10.1016/j.bbi.2011.06.006. Gerenu G, Liu K, Chojnacki JE, Saathoff JM, Martínez-Martín P, Perry G, Zhu X, Lee HG and Zhang S. 2015. Curcumin/ melatonin hybrid 5-(4-hydroxy-phenyl)-3-oxo-pentanoic acid [2-(5-methoxy-1H-indol-3-yl)-ethyl]-amide ameliorates AD-like pathology in the APP/PS1 mouse model. ACS Chem Neurosci 6, 1393-1399. https://doi.org/10.1021/ acschemneuro.5b00082. Gwon WG, Joung EJ, Kwon MS, Lim SJ, Utsuki T and Kim HR. 2017. Sargachromenol protects against vascular inflammation by preventing TNF-α-induced monocyte adhesion to primary endothelial cells via inhibition of NF-κB activation. Int Immunopharmacol 42, 81-89. https://doi.org/10.1016/j. intimp.2016.11.014. Gwon WG, Joung EJ, Shin T, Utsuki T, Wakamatsu N and Kim HR. 2018. Meroterpenoid-rich fraction of the ethanol extract from Sargassum serratifolium suppresses TNF-αinduced monocytes adhesion to vascular endothelium and vascular inflammation in high cholesterol-fed C57BL/6J mice. J Funct Foods 46, 384-393. https://doi.org/10.1016/j. jff.2018.05.013. Gwon WG, Lee B, Joung EJ, Choi MW, Yoon N, Shin T, Oh CW and Kim HR. 2015. Sargaquinoic acid inhibits TNFα-induced NF-κB Signaling, thereby contributing to decreased monocyte adhesion to human umbilical vein endothelial cells. J Agric Food Chem 63, 9053-9061. https://doi. org/10.1021/acs.jafc.5b04050. Jo M, Kim JH, Song GJ, Seo M, Hwang EM and Suk K. 2017. Astrocytic orosomucoid-2 modulates microglial activation and neuroinflammation. J Neurosci 37, 2878-2894. https:// doi.org/10.1523/jneurosci.2534-16.2017. Joung EJ, Gwon YG, Shin T, Jung BM, Choi JS and Kim HR. 2017. Anti-inflammatory action of the ethanolic extract from Sargassum serratifolium on lipopolysaccharide-stimulated mouse peritoneal macrophages and identification of active components. J Appl Phycol 29, 563-573. https://doi. org/10.1007/s10811-016-0954-9. Joung EJ, Lee B, Gwon WG, Shin T, Jung BM, Yoon NY, Choi JS, Oh CW and Kim HR. 2015. Sargaquinoic acid attenuates inflammatory responses by regulating NF-κB and Nrf2 pathways in lipopolysaccharide-stimulated RAW264.7 cells. Int Immunopharmacol 29, 693-700. https://doi.org/10.1016/j. intimp.2015.09.007. Kang CW, Park MS, Kim NH, Lee JH, Oh CW, Kim HR and Kim GD. 2015. Hexane extrdact from Sargassum serratifolium inhibits the cell proliferation and metastatic ability of human glioblastoma U87MG cells. Oncol Rep 34, 2602-2608. https://doi.org/10.3892/or.2015.4222. Kitazawa M, Oddo S, Yamasaki TR, Green KN, LaFerla FM. 2005. Lipopolysaccharide-induced inflammation exacerbates tau pathology by a cyclin-dependent kinase 5-mediated pathway in a transgenic model of Alzheimer's disease. J Neurosci 25, 8843-8853. Li QH, Jin G, Wang JY, Li HN, Liu H, Chang XY, Wang FX and Liu SL. 2016. Live attenuated Salmonella displaying HIV-1 10E8 epitope on fimbriae: systemic and mucosalimmune responses in BALB/c mice by mucosal administration. Sci Rep 6, 29556. https://doi.org/10.1038/srep29556. Lim S, Choi AH, Kwon M, Joung EJ, Shin T, Lee SG, Kim NG and Kim HR. 2019. Evaluation of antioxidant activities of various solvent extract from Sargassum serratifolium and its major antioxidant components. Food Chem 278, 178-184. https://doi.org/10.1016/j.foodchem.2018.11.058. Lim S, Kwon M, Joung EJ, Shin T, Oh CW, Choi JS and Kim HR. 2018. Meroterpenoid-rich fraction of the ethanolic extract from Sargassum serratifolium suppressed oxidative stress induced by tert-butyl hydroperoxide in HepG2 cells. Mar Drugs 16, pii: E374. https://doi.org/10.3390/ md16100374. Liu L, Heinrich M, Myers S and Dworjanyn SA. 2012. Towards a better understanding of medicinal uses of the brown seaweed Sargassum in traditional Chinese medicine: A phytochemical and pharmacological review. J Ethnopharmacol 142, 591-619. https://doi.org/10.1016/j.jep.2012.05.046. McGeer PL, Rogers J and McGeer EG. 2016. Inflammation, antiinflammatory agents, and Alzheimer's disease: The last 22 years. J Alzheimers Dis 54, 853-857. https://doi. org/10.3233/jad-160488. Molteni M and Rossetti C. 2017. Neurodegenerative diseases: The immunological perspective. J Neuroimmunol 313, 109-115. https://doi.org/10.1016/j.jneuroim.2017.11.002. Oak JH and Lee IK. 2006. Taxonomy of the Genus Sargassum (Fucales, Phaeophyceae) from Korea II. Subgenus Bactrophycus section Halochloa and Repentia. Algae 21, 393-405. Oh SJ, Joung EJ, Kwon MS, Lee B, Utsuki T, Oh CW and Kim HR. 2016. Anti-inflammatory effect of ethanolic extract of Sargassum serratifolium in lipopolysaccharide-stimulated BV2 microglial Cells. J Med Food 19, 1023-1031. https:// doi.org/10.1089/jmf.2016.3732. Qin L, Wu X, Block ML, Liu Y, Breese GR. Hong JS, Knapp DJ and Crews FT. 2007. Systemic LPS causes chronic neuro-

86 최민우ㆍ김형락ㆍ이형곤ㆍ김재일 inflammation and progressive neurodegeneration. Glia 55, 453-462. https://doi.org/10.1002/glia.20467. Schain M and Kreisl WC. 2017. Neuroinflammation in neurodegenerative disorders-a Review. Curr Neurol Neurosci Rep 17, 25. https://doi.org/10.1007/s11910-017-0733-2. Seong SH, Ali MY, Kim HR, Jung HA and Choi JS. 2017. BACE1 inhibitory activity and molecular docking analysis of meroterpenoids from Sargassum serratifolium. Bioorg Med Chem 25, 3964-3970. https://doi.org/10.1016/j. bmc.2017.05.033. Sheng JG, Bora SH, Xu G, Borchelt DR, Price DL, Koliatsos VE. 2003. Lipopolysaccharide-induced-neuroinflammation increases intracellular accumulation of amyloid precursor protein and amyloid beta peptide in APPswe transgenic mice. Neurobiol Dis 14, 133-145. Soto C. 2003. Unfolding the role of protein misfolding neurodegenerative diseases. Nat Rev Neurosci 4, 49-60. https://doi. org/10.1038/nm1007. Wang Z, Xie J, Yang Y, Zhang F, Wang S, Wu T, Shen M and Xie M. 2017. Sulfated Cyclocarya paliurus polysaccharides markedly attenuates inflammation and oxidative damage in lipopolysaccharide-treated macrophage cells and mice. Sci Rep 7, 40402. https://doi.org/10.1038/srep40402.