Article pubs.acs.org/JAFC
Protective Effect of Brown Alga Phlorotannins against Hyperinflammatory Responses in Lipopolysaccharide-Induced Sepsis Models Yeong-In Yang,† Jeong-Hwa Woo,† Yun-Ji Seo,† Kyung-Tae Lee,† Yunsook Lim,‡ and Jung-Hye Choi*,† †
Department of Life & Nanopharmaceutical Science and ‡Department of Food and Nutrition, Kyung Hee University, 26 Kyunghee-daero, Dongdaemoon-gu, Seoul 130-701, South Korea S Supporting Information *
ABSTRACT: Brown algae have been recognized as a food ingredient and health food supplement in Japan and Korea, and phlorotannins are unique marine phenol compounds produced exclusively by brown algae. Sepsis is a whole-body inflammatory condition with a mortality rate of 30−40%. Here, we investigated the effects of a phlorotannin-rich extract of the edible brown alga Ecklonia cava against hyper-inflammatory response in LPS-induced septic shock mouse model. E. cava extract significantly increased the survival rate and attenuated liver and kidney damage in the mice. In addition, E. cava attenuated serum levels of NO, PGE2, and HMGB-1. In macrophages, treatment with E. cava extract down-regulated iNOS, COX-2, TNF-α, IL-6, and HMGB-1. In addition, E. cava suppressed the NIK/TAK1/IKK/IκB/NFκB pathway. Moreover, E. cava increased Nrf2 and HO-1 expression. HO-1 knockdown using siRNA restored the extract-suppressed NO and PGE2 production. Dieckol, a major compound in the extract, reduced mortality, tissue toxicity, and serum levels of the inflammatory factors in septic mice. These data suggest that brown algae phlorotannins suppress septic shock through negative regulation of pro-inflammatory factors via the NIK/TAK1/IKK/IκB/NFκB and Nrf2/HO-1 pathways. KEYWORDS: sepsis, Ecklonia cava, phlorotannin, inflammation, Nrf2/HO-1
■
INTRODUCTION Sepsis is a significant health problem, with a mortality rate of 30− 40%.1 The predominant cause of morbidity and mortality is the development of multiple-system organ dysfunctions with subsequent organ failure. Pro-inflammatory cytokines and other mediators have been demonstrated to play a crucial role in the pathogenesis of sepsis.2 Thus, there is an urgent need for effective therapies that target these inflammatory mediators of sepsis. Infection initially stimulates the innate (nonspecific) immune response, mediated mostly via inflammatory cells, such as neutrophils and macrophages. In particular, macrophages play a critical role in regulating the immune response for defensive reactions.3 Macrophages regulate inflammatory processes by secreting pro-inflammatory cytokines, such as IL-6, IL-1β, and TNF-α, and other inflammatory mediators, such as PGE2 and NO, via the inducible isoforms of cyclooxygenase-2 (COX-2) and NO synthase (iNOS), respectively.4 In addition to the wellknown pro-inflammatory factors, high-mobility group box-1 (HMGB-1) has recently been suggested as a key cytokine of local inflammation and lethal systemic inflammation (e.g., sepsis and endotoxaemia).5 Under injurious or inflammatory conditions, HMGB-1 is actively released by innate immune cells such as macrophages and passively released by necrotic cells Brown algae are a health food supplement, popular food ingredient, and an animal feed supplement in East Asia, as they are a rich source of nutrition, such as polysaccharides, dietary fiber, and minerals.6 Ecklonia cava is one of the most abundant brown algae found on the southern coast of Korea and Japan. This seaweed has attracted great interest in recent years because © XXXX American Chemical Society
of its highly bioactive and novel components, which possess a variety of biological activities. E. cava has been shown to contain two major possibly functional components: polyphenol and polysaccharide.6 Unique polyphenolic phlorotannins represent a large class of well-characterized marine secondary metabolites. The phlorotannins eckol, phlorofucofuroeckol, dieckol, and 6,6′bieckol have been isolated from the Ecklonia species and have been revealed to have multiple biological activities, including antibacterial,7 anti-inflammatory,8,9 antioxidant,10,11 antiadipogenic,12 anti-HIV,13 and anticancer14−16 activities. However, the effect of brown alga phlorotannins on sepsis-induced hyperinflammation and its underlying molecular mechanism of action have not been demonstrated. In the present study, we investigated whether a phlorotannin-rich extract of E. cava would prevent lipopolysaccharide (LPS)-induced septic shock in vivo. In addition, the molecular mechanism of phlorotannininduced anti-inflammatory activity was evaluated.
■
MATERIALS AND METHODS
Materials. The phlorotannin-rich extract of E. cava and phlorotannins used for this study were kindly provided by Livechem, Inc. (Daejeon, South Korea). The brown alga E. cava, collected between August and October from the coasts of Jeju Island in Koream was taxonomically identified by professor Bong Ho Lee (Hanbat National University). The method to prepare the E. cava extract has been previously described.8 The total polyphenol content of E. cava extract as Received: September 18, 2015 Revised: January 2, 2016 Accepted: January 3, 2016
A
DOI: 10.1021/acs.jafc.5b04482 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry phloroglucinol equivalent was about 98%, and major phlorotannins in the extract were as follows: eckol, phlorotannin A, and fucofuroeckol A, 2-phloroeckol, dieckol, phlorofurofucoeckol A, 8,8′-bieckol, 7-phloroeckol, 6,6′-bieckol, and 2-O-(2,4,6-trihydroxyphenyl)-6,6′-bieckol, as analyzed by HPLC [Waters Spherisorb S10ODS2 column (20 × 250 mm2); eluent, 30% aqueous EtOH; flow rate, 3.5 mL/min]. The purity of phlorotannins was >99%, according to the peak area of components absorbed at each specific wavelength in HPLC analysis. Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s minimum essential medium (DMEM), streptomycin, and penicillin were purchased from Life Technologies Inc. (Grand Island, NY, USA). Escherichia coli LPS, L-N6-(1-iminoethyl)lysine (L-NIL), 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and all other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA). NS398 was obtained from Calbiochem (La Jolla, CA, USA). The enzyme immunoassay (EIA) kits for PGE2 were purchased from R&D Systems (Minneapolis, MN, USA). Luciferase assay kit was obtained from Promega (Madison, WI, USA). iNOS, COX-2, p50, p65, PARP, p-IκBα, IκBα, p-NIK, NIK, and β-actin monoclonal antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The antibodies anti-IKK, anti-p-IKK, anti-TAK1, and anti-pTAK1 were obtained from Cell Signaling Technologies (Beverly, MA, USA). Animal Model of LPS-Induced Septic Shock. Septic shock was induced in C57BL/6 mice (male, 6 weeks old) by injection of bacterial endotoxin LPS (Escherichia coli). To evaluate the effect of E. cava phlorotannins on survival rate in septic mice, mice were divided into six groups of eight mice each. Mice were orally administered either vehicle (200 μL of PBS) alone or vehicle with E. cava extract and dieckol (10, 50, and 100 mg/kg/d) for 7 days. Mice received an intraperitoneal (ip) injection of LPS (25 mg/kg) 1 day after the last administration of E. cava extract and dieckol, and their survival was monitored every 12 h after the LPS injection up to 100 h. To further evaluate the levels of sepsis markers in blood, mice were randomly divided into six groups of three mice each, and the mice were orally administered either vehicle (200 μL of PBS) alone or vehicle with E. cava extract and dieckol (10, 50, and 100 mg/kg/day) for 7 days. Mice received an ip injection of LPS (25 mg/kg) 1 day after the last administration of E. cava extract and dieckol. Twelve hours after LPS treatment, mice were anesthetized, and blood was collected from the inferior vena cava. Serum was collected by centrifuging the blood at 15000g for 15 min at 4 °C and analyzed for organ damage markers and pro-inflammatory mediators (NO, PGE2, and HMGB-1), as described below. The serum levels of organ damage markers and pro-inflammatory mediators were measured in the same blood samples. Plasma glutamic oxaloacetic transaminase (GOT/AST), glutamic pyruvic transaminase (GPT/ALT), and blood urea nitrogen (BUN) levels were determined using a colorimeter testing kit (Asan Pharmaceutical, Seoul, South Korea) according to the manufacturer’s instruction. Cell Culture and Isolation of Peritoneal Macrophage Cells. The murine macrophage cell line RAW 264.7 was purchased from the Korean Cell Line Bank (Seoul, South Korea). Cells were cultured in DMEM containing 10% heat-inactivated FBS, streptomycin sulfate (100 μg/mL), and penicillin (100 units/mL) in an atmosphere of 5% CO2. Five percent thioglycollate (TG)-elicited macrophages were collected 3−4 days after ip injection of 2.5 mL of TG to C57BL6/J mice and adherence-purified, as previously described.17−19 Briefly, peritoneal lavages were carried out using 8 mL of Hank’s balanced salt solution (HBSS) containing 10 U/mL heparin, and the cells were distributed in 24-well plates (2.5 × 105 cells/well) with DMEM containing 10% heatinactivated FBS. Then, to isolate the macrophage using adherence purification method, the cells were further incubated for 3 h at 37 °C in an atmosphere of 5% CO2. Then, nonadherent cells were washed out with HBSS, and only adherent cells were equilibrated with DMEM. Cell Viability Analysis. Cell viability was estimated using the MTT assay. Cells were treated with E. cava extract for 1 h before LPS (1 μg/ mL) treatment for 24 h. After incubation with MTT solution at 37 °C for 4 h, the MTT solution was removed, and then the purple formazan crystals produced were solubilized in DMSO (100 μL/well). The optical
density at 540 nm was measured using a microplate spectrophotometer (SpectraMax; Molecular Devices, Sunnyvale, CA, USA). Nitrite Determination. NO production was determined by measuring the nitrite in culture medium using the Griess reagent. The culture supernatant (100 μL) was mixed with 100 μL of Griess reagent [equal volumes of 1% (w/v) sulfanilamide in 0.1% (w/v) naphthylethylenediamine−HCl and 5% (v/v) phosphoric acid] for 10 min. The optical density at 540 nm was measured using a microplate spectrophotometer. PGE2 Assay. PGE2 levels in culture medium were determined by EIA kits (R&D Systems) according to the manufacturer’s protocol. Western Blot Analysis. The cells were washed once with ice-cold PBS and extracted in protein lysis buffer (50 mM HEPES, pH 7.0, 5 mM EDTA, 250 mM NaCl, 0.1% Nonidet P-40, 0.5 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride, 0.5 mM sodium orthovanadate, and 5 mM sodium fluoride) containing 5 mg/mL each of aprotinin and leupeptin. The Bradford assay was performed to determine the protein concentration. After mixing with 5× sodium dodecyl sulfate (SDS) sample buffer, 40 μg of cellular protein was boiled for 3 min, separated on 10−12% SDS−polyacrylamide gel electrophoresis, and then electroblotted onto polyvinylidene fluoride (PVDF) membranes. The membrane was immunoblotted using primary antibodies at 4 °C overnight, followed by incubation for 1 h with a horseradish peroxidaseconjugated secondary antibody (Santa Cruz Biotechnology, Inc.). The signals were visualized using an enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech, Canada) and Image Quant LAS4000 (GE Healthcare Life Science, Milwaukee, WI, USA). RNA Isolation and Real-Time RT-PCR. Easy Blue kits (Intron Biotechnology, Seoul, South Korea) were used to isolate total cellular RNA. Total RNA (1 μg) was reverse-transcribed (RT) using MLV reverse transcriptase, 1 mM dNTP, and 0.5 μg/μL oligo (dT12−18). Oligonucleotide primers (COX-2, iNOS, IL-6, TNF-α, HMGB-1, and β-actin) were obtained from Bioneer (Seoul, South Korea). The primers used for SYBR Green real-time RT-PCR were as follows: for iNOS, 5′CATGCTACTGGAGGTGGGTG-3′ and 5′-CATTGATCTCCGTGACAGCC-3′; for COX-2, 5′-TGCTGTACAAGCAGTGGCAA-3′ and 5′-GCAGCCATTTCCTTCTCTCC-3′; for TNF-α, 5′-AGCACAGAAAGCATGATCCG-3′ and 5′-CTGATGAGAGGGAGGCCATT3′; for IL-6, 5′-GAGGATACCACTCCCAACAGACC-3′ and 5′AAGTGCATCATCGTTGTTCATACA-3′; for HMGB-1, 5′-GCGGACAAGGCCCGTTA-3′ and 5′-AGAGGAAGAAGGCCGAAGGA3′; for β-actin, 5′-ATCACTATTGGCAACGAGCG-3′ and 5′-TCAGCAATGCCTGGGTACAT-3′. Semiquantitative real-time PCR was carried out using a Thermal Cycler Dice System (Takara, Otsu, Japan). Mean Ct of each gene was determined from triplicate measurements and normalized with the mean Ct of β-actin, a control gene. Nuclear Extraction. The cell were washed with ice-cold PBS, resuspended in hypotonic buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT, 0.2 mM PMSF, 10 μg/mL aprotinin), and incubated on ice for 15 min. The cells were lysed by adding 0.1% Nonidet P-40 and centrifuged at 12000g for 1 min at 4 °C. The nuclear pellet was resuspended in high-salt buffer (20 mM HEPES, pH 7.9, 400 mM KCl, 25% glycerol, 1.5 mM MgCl2, 0.5 mM DTT, 0.2 mM EDTA, 1 mM sodium orthovanadate, 1 mM NaF). After determination of protein concentration, the nuclear protein extract was used for Western blot analysis. Luciferase Assay. The cells were transfected with the NFκB-Luc reporter vector (Clontech, Shiga, Japan) using Lipofectamine LTX (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Following incubation with the transfection reagent for 24 h, cells were pretreated with E. cava extract for 1 h and then treated with LPS (1 μg/mL) for 18 h. At the end of the treatment period, the luciferase activity was measured using the dual luciferase assay system (Promega). HO-1 Knockdown Using siRNA. The cells were transfected with control siRNA or HO-1 siRNA (Bioneer) using Lipofectamine LTX according to the manufacturer’s instructions. After incubation in serumfree media for 24 h, the transfected cells were pretreated with E. cava extract for 1 h before stimulation with LPS (1 μg/mL). After 4 h of B
DOI: 10.1021/acs.jafc.5b04482 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Figure 1. Effect of E. cava extract on LPS-induced septic shock in mice. Mice were treated with E. cava extract (10, 50, or 100 mg/kg/day, po) for 1 week and then administered LPS (25 mg/kg, ip). (A) Survival rates of these mice were observed over the next 100 h. (B, C) Plasma AST, ALT, and BUN levels were measured using a colorimeter testing kit. Statistical analysis was carried out using one-way ANOVA. (#) p < 0.05 versus the control group; (∗) p < 0.05 versus the LPS-stimulated group.
Figure 2. Effect of E. cava extract on serum NO, PGE2, and HMGB-1 levels in septic mice. Mice were treated with E. cava extract (10, 50, or 100 mg/kg/ day, po) for 1 week and then administered LPS (25 mg/kg, ip). (A) The amounts of NO were measured by a Griess reaction assay. (B, C) The amounts of PGE2 (B) and HMGB-1 (C) in serum were measured by an EIA kit. Statistical analysis was carried out using one-way ANOVA. (#) p < 0.05 versus the control group; (∗) p < 0.05 versus the LPS-stimulated group. stimulation, the levels of NO and PGE2 in macrophage culture medium were measured using Griess reagent and EIA kits. Statistical Analysis. Statistical data are presented as the mean ± SD of three individual experiments performed in triplicate. Student’s t test or one-way ANOVA followed by Tukey’s test was performed using GraphPad Prism version 5 (GraphPad, SanDiego, CA, USA). p values
