Article pubs.acs.org/JAFC
Sargaquinoic Acid Inhibits TNF-α-Induced NF-κB Signaling, Thereby Contributing to Decreased Monocyte Adhesion to Human Umbilical Vein Endothelial Cells (HUVECs) Wi-Gyeong Gwon,†,∥ Bonggi Lee,†,∥ Eun-Ji Joung,† Min-Woo Choi,† Nayoung Yoon,§ Taisun Shin,# Chul-Woong Oh,⊥ and Hyeung-Rak Kim*,† †
Department of Food Science and Nutrition, Pukyong National University, Busan 608-737, South Korea Food and Safety Research Division, National Fisheries Research and Development Institute, Gijang-gun, Busan 619-705, South Korea # Division of Food and Nutrition, Chonnam National University, Buk-gu, Gwangju 500-757, South Korea ⊥ Department of Marine Biology, Pukyong National University, Busan 608-737, South Korea §
S Supporting Information *
ABSTRACT: Sargaquinoic acid (SQA) has been known for its antioxidant and anti-inflammatory properties. This study investigated the effects of SQA isolated from Sargassum serratifolium on the inhibition of tumor necrosis factor (TNF)-α-induced monocyte adhesion to human umbilical vein endothelial cells (HUVECs). SQA decreased the expression of cell adhesion molecules such as intracellular adhesion molecule-1 and vascular cell adhesion molecule-1 as well as chemotactic cytokines such as interleukin-8 and monocyte chemoattractant protein-1 in TNF-α-treated HUVECs. As a result, SQA prevented monocyte adhesion to TNF-α-induced adhesion. SQA also inhibited TNF-α-induced nuclear factor kappa B (NF-κB) translocation into the nucleus by preventing proteolytic degradation of inhibitor κB-α. Overall, SQA protects against TNF-α-induced vascular inflammation through inhibition of the NF-κB pathway in HUVECs. These data suggest that SQA may be used as a therapeutic agent for vascular inflammatory diseases such as atherosclerosis. KEYWORDS: sargaquinoic acid, anti-inflammation, nuclear factor-κB, adhesion molecule, human umbilical vein endothelial cell
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INTRODUCTION Atherosclerosis is an inflammatory disease with the most common pathologicsl process leading to cardiovascular disease. Enhanced monocyte adhesion to endothelial cells is believed to be one of the earliest events in atherogenesis.1 Recent studies have demonstrated that chronic inflammation significantly contributes to the initiation and progression of atherosclerosis.1,2 Monocyte adhesion to endothelial cells is primarily regulated through cellular signaling that stimulates chemokine expression, such as interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1), and endothelial adhesion molecules, including vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM1).2,3 MCP-1 is a chemoattractant of monocytes/macrophages, whereas IL-8 is a chemoattractant of neutrophils. Both chemokines and adhesion molecules are critical to the initiation and development of vascular inflammation in human atheroma.4,5 Growing evidence suggests that tumor necrosis factor alpha (TNF-α), a key pro-inflammatory cytokine in the inflammatory cascade, has a critical role in vascular inflammation and the subsequent progression of atherosclerosis.6 TNF-α is known to exaggerate the adherence of invading monocytes to vascular endothelium that accelerate the development of atherosclerotic activation and translocation of nuclear factor kappa B (NFκB).2 NF-κB is a transcription factor that up-regulates the production of adhesion molecules, such as VCAM-1 and © 2015 American Chemical Society
ICAM-1, and chemotactic cytokines, including IL-8 and MCP1.8,9 TNF-α also elevates the production of superoxide by activating NADPH oxidase in the endothelial cells.10 Superoxide, the primary reactive oxygen species (ROS) produced in endothelium, increases cytoplasmic levels of H2O2 that can activate NF-κB,11 contributing to excess production of proinflammatory cytokines and adhesion molecules that stimulate the development of atherosclerotic lesions.7,11,12 Therefore, regulating TNF-α induced inflammation and oxidative stress is an important strategy for ameliorating vascular inflammatory diseases such as atherosclerosis. Sargassum serratifolium, a marine brown alga belonging to the Sargassaceae family, broadly exists throughout the Korean and Japanese coasts. Recently, sargaquinoic acid (SQA) has been found to exhibit several biological functions, such as neuroprotective,13 anti-inflammatory,14 antiadipogenic,15 and anticarcinogenic16 properties. However, the anti-inflammatory mechanisms of SQA in TNF-α-induced endothelial cells are unclear. To examine roles of SQA in preventing TNF-α-induced endothelial inflammation in human umbilical vein endothelial cells (HUVECs), we assessed the interaction of monocytes and Received: Revised: Accepted: Published: 9053
August 18, October 4, October 5, October 5,
2015 2015 2015 2015 DOI: 10.1021/acs.jafc.5b04050 J. Agric. Food Chem. 2015, 63, 9053−9061
Article
Journal of Agricultural and Food Chemistry
Mass Spectrometry Analysis. The molecular weight of SQA was confirmed by mass spectrometry. The mass spectrometry analysis was performed with a Shimadzu GCMS-QP2010 Ultra instrument with a direct sample inlet device (DI-2010, Kyoto, Japan). The initial probe temperature was 40 °C/min; it was ramped to 350 °C and held for 2 min. The ion source temperature was 200 °C, and the injection volume was 0.5 μL. The mass spectrum is shown in Supporting Information Figure S1. Cell Culture and Viability Assay. 3-(4,5-Dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) was used to assess the cytotoxicity of SQA. Briefly, cells were seeded in a 96-well plate at a density of 5 × 103 cells/well. The cells were treated with a serial dilution of SQA indicated in the figure legend. MTS solution was added with fresh medium for 1 h based on the manufacturer’s instruction followed by measurement of the absorbance at 490 nm using a microplate reader (Glomax Multi Detection System, Promega). Cell-Based Enzyme-Linked Immunsorbent Assay (ELISA). The expression of ICAM-1 and VCAM-1 in HUVECs was determined by cell-based ELISA as described by Manduteanu et al.17 Briefly, HUVECs were placed in a 24-well plate at a density of 5 × 104 cells/ well. After 24 h of incubation, cells were exposed to diverse concentrations of SQA for 1 h. After removal of culture medium, the cells were stimulated with TNF-α (10 ng/mL) for 6 h. The cells fixed by 1% paraformaldehyde were blocked with 2% BSA. After exposure to mouse anti-human ICAM-1 or VCAM-1 antibodies for 1 h followed by washing, cells were incubated with the horseradish peroxidase-conjugated secondary antibody for 1 h. Expression levels of ICAM-1 and VCAM-1 were quantified by the addition of peroxidase substrate solution, and the absorbance was measured at 490 nm with a microplate reader (Glomax Multi Detection System, Promega). Monocyte Adhesion Assay. Confluent HUVECs pretreated with SQA for 1 h were stimulated with TNF-α (10 ng/mL) for 6 h. THP-1 monocytes (KCLB, Seoul, Korea) were tagged with the compound BCECF-AM (10 μM). The tagged THP-1 monocytes (2.5 × 105) were gently added to the HUVEC monolayer and incubated for 1 h. Unbound THP-1 cells were discarded by PBS washing. HUVECattached THP-1 cells were observed with an LSM700 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany). In parallel, HUVEC-adherent monocytes were harvested with cell lysis solution (50 mM Tris-HCl, containing 0.1% SDS, pH 8.0), and the fluorescence intensity was determined using a fluorescence microplate reader (Dual Scanning SPECTRAmax, Molecular Devices Co., Sunnyvale, CA, USA) at 485 nm (excitation wavelength) and 535 nm (emission wavelength). The results were shown as relative monocyte adhesion levels compared with the control group. Reverse Transcription-Polymerase Chain Reaction (RT-PCR). SQA-pretreated HUVECs were stimulated with TNF-α for 6 h. RNA was isolated using the QIAzol reagent. cDNA was synthesized with oligo-dT primer and superscript reverse transcriptase using total RNA (5 μg). PCR was conducted with corresponding primers (Supporting Information Table 1). Densitometric analysis was performed using EZCapture II (ATTO & Rise Co., Tokyo, Japan) and CS analyzer (ver. 3.00 software, ATTO). Intracellular ROS Measurement. ROS scavenging activity of SQA was determined with fluorescent probe DCFH-DA. Confluent HUVECs in black 96-well plates were pretreated with 5, 10, and 15 μM SQA for 1 h. After the wells had been washed with PBS, the cells were treated with 20 μM DCFH-DA for 30 min at 37 °C and then stimulated with TNF-α (10 ng/mL) for 1 h. The fluorescence level was determined using a fluorescence microplate reader (Dual Scanning SPECTRAmax) at 485 nm (excitation wavelength) and 528 nm (emission wavelength). Preparation of Cytosolic and Nucleus Extracts. HUVECs were seeded in a culture dishes at a density of 6 × 105 cells/dish and cultured for 24 h. Cultured cells were pretreated with 0, 5, 10, and 15 μM of SQA for 1 h and stimulated with TNF-α for 30 min. Separation of cytosolic and nucleus extracts was performed as previously described.18
HUVECs, the production of adhesion molecules and chemokines, and the activation of NF-κB in HUVECs.
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MATERIALS AND METHODS
Materials. Endothelial cell growth medium (EGM-2) and primary cultured HUVECs were obtained from Lonza (Walkersville, MD, USA). CellTiter96 AQueous One Solution Cell Proliferation assay kit and reverse transcriptase were purchased from Promega (Madison, WI, USA). Primary and secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Abcam (Danvers, MA, USA). An enhanced chemiluminescence (ECL) detection system was purchased from GE Healthcare Bio-Science (Piscataway, NJ, USA). QIAzol lysis reagent was purchased from Quiagen (Valencia, CA, USA). 2′,7′-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxy-methylester (BCECF-AM), Alexa Fluor 488conjugated secondary antibody, and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Invitrogen (Carlsbad, CA, USA). 2′,7′Dichlorofluorescin diacetate (DCFH-DA), dimethyl sulfoxide (DMSO), and phenylmethanesulfonyl fluoride (PMSF) were obtained from Sigma-Aldrich Corp. (St. Louis, MO, USA). Isolation of SQA. S. serratifolium was collected along the coast of Busan, South Korea, in April 2014. Specimen identity was confirmed by an agal biologist (N. G. Kim) at the Department of Marine Biology and Aquaculture, Gyeongsang National University, South Korea. Dried seaweed (1.5 kg) was extracted twice with 95% (v/v) ethanol (6 L/ each) at 70 °C for 3 h. The ethanolic extract (150.7 g) was obtained by concentration under reduced pressure. To obtain the n-hexane-soluble fraction, the extract was suspended in water/ethanol (9:1, v/v) and then partitioned with n-hexane to produce 121 g of n-hexane-soluble fraction. An aliquot of n-hexane fraction was separated by a recycled HPLC system with a Phenomenex Luna RP-18(2) column (250 × 21.2 mm, 15 μm, Phenomenex, Torrence, CA, USA). The elution profile was composed of a linear gradient from methanol (A)/water (B) (90:10) to A/B (94:6) for 35 min, A/B (94:6) to A/B (100:0) for 2 min, and re-equilibrated with A/B (90:10) for 10 min after a 10 min hold with A/B (100:0). Peaks were monitored at 270 nm, and the flow rate was 7 mL/min. The purity of a fraction exhibiting strong antiinflammatory activity based on the inhibition of ICAM-1 production in TNF-α-stimulated HUVECs was improved by the same HPLC system with Luna RP-18 [Luna C18(2), 250 × 10 mm, 5 μm] at a flow rate of 3.0 mL/min. The fraction was eluted with a linear gradient from methanol A/B (93.4:6.6) to A/B (93.8:6.2) for 20 min, A/B (93.8:6.2) to A/B (100:0) for 2 min, and re-equilibrated with A/B (93.8:6.2) for 10 min after a 10 min hold with A/B (100:0). The purity of the separated compound was >98.0%, as evaluated by the same HPLC system with the Luna RP-18 column [Luna C18(2), 3 μm, 150 × 3.0 mm]. The content of the compound was estimated to be 0.62 ± 0.07 g per 100 g of dried S. serratifolium. Spectrometry. A JNM ECP-400 spectrometer (JEOL, Japan) was used to resolve 1 H and 13 C NMR spectra. CD 3 OD with tetramethylsilane was used as an internal standard. Heteronuclear multiple-bond correlation and heteronuclear multiple-quantum correlation spectra were monitored using pulsed field gradients. The structure of SQA is shown in Supporting Information Figure S1. Structural Elucidation of SQA. C27H36O4 (MW = 424); 1H NMR (CD3OD, 400 MHz) δ 3.12 (2H, d, J = 6.9 Hz, H-1), 5.17 (1H, t, J = 7.4 Hz, H-2), 2.08 (2H, m, H-4), 2.09 (2H, m, H-5), 5.10 (1H, m, H-6), 2.09 (2H, m, H-8), 2.59 (2H, dt, J = 7.2 and 7.2 Hz, H-9), 5.84 (1H, t, J = 7.3 Hz, H-10), 2.19 (2H, t, J = 7.2 Hz, H-12), 2.12 (2H, m, H-13), 5.06 (1H, tt, J = 7.3 and 1.4 Hz, H-14), 1.65 (3H, s, CH3-16), 1.58 (3H, s, CH3-17), 1.60 (3H, s, CH3-19), 1.62 (3H, s, H20), 6.42 (1H, m, H-3′), 6.56 (1H, quin, J = 1.4 Hz, H-5′), 2.12 (3H, d, J = 1.5 Hz, aromatic-CH3); 13C NMR (CD3OD, 100 MHz) δ 28.5 (C-1), 120.1 (C-2), 140.4 (C-3), 40.7 (C-4), 27.3 (C-5), 125.7 (C-6), 135.8 (C-7), 40.3 (C-8), 29.0 (C-9), 142.6 (C-10), 132.9 (C-11), 36.1 (C-12), 28.9 (C-13), 124.8 (C-14), 133.4 (C-15), 25.9 (C-16), 17.8 (C-17), 171.6 (C-18), 15.97 (C-19), 16.0 (C-20), 189.4 (C-1′), 150.0 (C-2′), 133.0 (C-3′), 188.9 (C-4′), 133.96 (C-5′), 147.6 (C-6′), 16.1 (aromatic-CH3). . 9054
DOI: 10.1021/acs.jafc.5b04050 J. Agric. Food Chem. 2015, 63, 9053−9061
Article
Journal of Agricultural and Food Chemistry
Figure 1. Effect of SQA on the adhesion of THP-1 monocytes to TNF-α-stimulated HUVECs. Cells were treated with the indicated concentrations of SQA for 1 h and then stimulated with TNF-α for 6 h. Fluorescence-labeled THP-1 was added to the HUVECs and allowed to adhere for 1 h, and adhesion was determined using a fluorescence microplate reader at excitation and emission wavelengths of 485 and 535 nm (A). Pictures are representative fields captured by fluorescence microscope (B): (a) control; (b) TNF-α; (c) cotreated with TNF-α and SQA (5 μM); (d) cotreated with TNF-α and SQA (10 μM); (e) cotreated with TNF-α and SQA (15 μM). The values are shown as the means ± SDs from three independent experiments. (#) P < 0.05 represents significant differences versus the control group; (∗) P < 0.05 represents significant differences versus the TNFα-only group.
pretreated with various final concentrations of SQA (5, 10, and 15 μM) in TNF-α-stimulated HUVECs. The viability was not influenced up to 15 μM SQA in HUVECs (Supporting Information Figure S2). Thus, the HUVECs were treated with SQA in the concentration range of 5, 10, and 15 μM for further experiments. Because inflammation-derived monocyte adhesion to HUVECs is an essential step in atherosclerosis development, we investigated whether SQA inhibits the adhesion of THP-1 monocytes to TNF-α-treated HUVECs. Stimulation of the HUVECs with TNF-α for 6 h markedly increased cell adhesion. However, SQA dose-dependently inhibited TNF-α-induced binding of monocytes to HUVECs (Figure 1A). To verify the inhibition of SQA on the monocyte recruitment to the TNF-αstimulated vascular endothelium, microscopic observation was performed using BCECF-AM staining assay. As shown in Figure 1B, heavy fluorescent staining was shown on the HUV treated with TNF-α alone for 6 h, indicating marked adhesion of THP-1 to the stimulated HUVECs. However, SQA
Western Blotting Analysis. HUVECs pretreated with 0, 5, 10, and 15 μM SQA for 1 h were stimulated with TNF-α (10 ng/mL) at times indicated in the figure legends. The specific procedure for Western blotting is described in our previous paper.18 Immunofluorescence Analysis. HUVECs were cultured on glass coverslips (SPL Life Sciences Co., Gyeonggi-do, Korea) for 24 h. After treatment of cells with SQA for 1 h, cells were stimulated with TNF-α (10 ng/mL) for 30 min. The specific procedure for immunofluorescence analysis of NF-κB is described in our previous paper.19 Statistical Analysis. Data are expressed as the means ± standard deviations (SDs). Data analysis was performed using ANOVA followed by Bonferroni test. P values
