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Hexane fraction from the ethanolic extract of Sargassum serratifolium suppresses cell adhesion molecules via regulation of NF-κB and Nrf2 pathway in human umbilical vein endothelial cells

Hexane fraction from the ethanolic extract of Sargassum serratifolium suppresses cell adhesion... Sargassum serratifolium ethanolic extract has been known for strong antioxidant and anti-inflammatory properties. We prepared hexane fraction from the ethanolic extract of S. serratifolium (HSS) to improve biological activities. In this study, we investigated the effects of HSS on the inhibition of tumor necrosis factor (TNF)-α-induced monocyte adhesion to human umbilical vein endothelial cells (HUVECs). We found that HSS suppressed the production of cell adhesion molecules such as intracellular adhesion molecule-1 and vascular cell adhesion molecule-1 in TNF-α-induced HUVECs. Moreover, TNF-α-induced production of monocyte chemoattractant protein 1 and keratinocyte chemoattractant was inhibited by HSS treatment. HSS suppressed TNF-α-induced nuclearfactorkappa B(NF-κB) activation via preventing proteolytic degradation of inhibitor κB-α. HSS induced the production of heme oxygenase 1 via translocation of Nrf2 into the nucleus in TNF-α-treated HUVECs. Overall, HSS alleviated vascular inflammation through the downregulation of NF-κB activation and the upregulation of Nrf2 activation in TNF-α-induced HUVECs. These results indicate that HSS may be used as therapeutic agents for vascular inflammatory disorders. Keywords: Sargassum serratifolium,Vascularinflammation,Nuclearfactor-κB, Adhesion molecule, Human umbilical vein endothelial cell Background and Pober 2001; Rao et al. 2007). A vascular endothe- Vascular inflammation has been known to play a key lium is a key target of tumor necrosis factor (TNF)-α-in- role in the progress of atherosclerosis, and enhanced duced inflammation, which promotes the expression of monocyte adhesion to endothelial cells is believed to be ICAM-1 and VCAM-1. Monocyte chemoattractant pro- one of the earliest events in atherogenesis (Packard and tein (MCP)-1 and keratinocyte chemoattractant (KC) Libby 2008). Accumulating evidences have revealed that have pivotal roles in recruiting monocytes to the lesion chronic inflammation plays a crucial role in the of dysfunctional endothelium. Excess chemokines are initiation and progression of atherosclerosis. Monocyte found in human atherosclerotic plaques and regulate the adhesion to endothelial cells is primarily mediated by interaction between monocytes and vascular endothelial several intracellular signaling events that lead to the cells (Catalan et al. 2015). Taking these evidences, inhi- elevated expression of endothelial adhesion molecules, bition of adhesion molecules and chemokines may be a including vascular cell adhesion molecule-1 (VCAM-1) promising approach to prevent atherosclerosis by blocking and intracellular adhesion molecule-1 (ICAM-1) (Madge monocyte invasion to the vascular inflammatory lesion. An accumulative evidence suggests that TNF-α,a * Correspondence: hrkim@pknu.ac.kr pleiotropic pro-inflammatory cytokine in the inflamma- Department of Food Science and Nutrition, Pukyong National University, 45, tory cascade, involves in a critical role in vascular inflam- Yongso-Ro, Nam-Gu, Busan 48513, Republic of Korea mation and the subsequent progress of atherosclerosis Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Gwon et al. Fisheries and Aquatic Sciences (2019) 22:7 Page 2 of 10 (Madge and Pober 2001). TNF-α induces the produc- detection kit was purchased from GE Healthcare tion of reactive oxygen species (ROS) by the activation Bio-Science (Piscataway, NJ, USA). QIAzol lysis reagent of membrane-bound nicotinamide adenine dinucleo- was purchased from Quiagen (Valencia, CA). tide phosphate (NADPH) oxidase in endothelium 2′,7′-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein (Kleinbongard et al. 2010). Excess ROS produced by acetoxy-methyl ester (BCECF-AM), Alexa Fluor® TNF-α stimulate the phosphorylation of the inhibitor kB 488-conjugated secondary antibody, 4′,6-diamidino-2-- kinase (IKKs) complex, leading to the proteasomal phenylindole (DAPI), and dual luciferase assay kits were degradation of phosphorylated IκB-a. Thus, free NF-κB purchased from Invitrogen (Carlsbad, CA, USA). dissociated from IκB is translocated into the nucleus and 2′,7′-dichlorofluorescin diacetate (DCFH-DA), dimethyl binds to the cis-acting element to regulate the expression sulfoxide (DMSO), and phenylmethylsulfonyl fluoride of adhesion molecules in endothelium (Huang et al. 2018). (PMSF) were obtained from Sigma-Aldrich Corporation Thus, approaches to regulate endothelial activation are (St. Louis, MO, USA). The antibodies against ICAM-1, potential strategies to prevent atherosclerosis through the VCAM-1, and HO-1 were obtained from Abcam (Cam- suppression of vascular inflammation. bridge, UK). pIKK-β, IKK-β,pIκB-α, and IκB-α were pur- Sargassaceae family is consumed as food or traditional chased from Cell Signaling (Danvers, MA, USA). NF-κB, medicine in Korea and China and various bioactive com- GAPDH, PARP, β-actin, Nrf2, and secondary antibody pounds have been identified as meroterpenoids, phloro- were purchased from Santa Cruz Biotechnology (Santa tannins, fucoxanthins, and fucosterols (Liu et al. 2012). Cruz, CA, USA). Sargassum serratifolium (C. Agardh), a marine brown alga, is broadly distributed throughout the Korean and Preparation of n-hexane fraction from the ethanolic Japanese coasts. Recently, we reported that ethanolic extract of S. serratifolium extract of S. serratifolium showed antioxidant and anti- S. serratifolium was collected along the coast of Busan, inflammatory activities and active compounds were South Korea, in May 2015. Specimen identity was con- identified as sargahydroquinoic acid (SHQA), sargachro- firmed by an algal taxonomist (C.G. Choi), at the Depart- menol (SCM), and sargaquinoic acid (SQA) (Joung et al. ment of Ecological Engineering, Pukyong National 2017; Lim et al. 2018;Oh etal. 2016). Moreover, University, Republic of Korea, and a voucher specimen SCM and SQA isolated from the ethanolic extract of was deposited in Marine Brown Algae Resources Bank, S. serratifolium showed a potent anti-inflammatory Republic of Korea (TC18835). Collected sample was activity in LPS-stimulated macrophages and anti- air-dried and ground. One and a half kilograms of dried atherogenic activities in TNF-α-treated human umbi- sample was extracted twice with 70% (v/v)ethanol lical vein endothelial cells (HUVECs), respectively (6 L each time) at 70 °C for 3 h. The combined extract was (Gwon et al. 2017;Gwon et al. 2015; Joung et al. 2015). filtered through the ultrafiltration unit (MWCO, 50 kDa) We prepared a meroterpenoid-rich fraction from the etha- and concentrated until the lipophilic fraction was sepa- nolic extract of S. serratifolium (MES) by removing salts rated from salt water. The lipophilic fraction was concen- and water-soluble saccharides, and the combined amount trated by a rotary vacuum evaporator (Eyela N3010, of SHQA, SCM, and SQA was estimated to be 46 g in Tokyo, Japan) at 45 °C after washing three times with 10 100 g of MES (Gwon et al. 2018;Kwonet al. 2018). To times volume of D.W. (meroterpenoid-rich extract further concentrate active compound in MES, we fractio- from S. serratifolium, MES). For further concentrate nated MES with n-hexane, and resulting n-hexane fraction active component in MES, the extract was resuspended from the ethanolic extract of S. serratifolium (HSS) in water/EtOH (1 v/v) and partitioned with n-hexane, contained 64% of three components in HSS. For a better ethyl acetate, n-butanol, and D.W. The n-hexane fraction understanding of pharmacological actions of HSS, we which showed the highest anti-inflammatory activity examined the vascular anti-inflammatory actions of HSS against LPS-induced RAW 264.7 cells was concentrated in TNF-α-stimulated HUVECs. by a rotary vacuum evaporator and kept at − 20 °C for this study. From 1.5 kg of dried sample, 80 g of the HSS was obtained. Isolation and quantification of SHQA, SCM, Methods and SQA were performed according to the method Reagents described previously (Azam et al. 2017; Joung et al. 2017). Endothelial cell growth medium (EGM-2), growth sup- Quantification of SHQA, SCM, and SQA in HSS was per- plements, and primary cultured HUVEC were obtained formed with the method described previously (Joung et al. from Lonza (Walkersville, MD, USA). CellTiter ®AQ 2017), and the contents of SHQA, SCM, and SQA in 100 ue- One Solution Cell Proliferation assay kit and reverse g of HSS were estimated to be 52.4 ± 3.3, 8.26 ± 0.82, and ous transcriptase were purchased from Promega (Madison, 3.0 ± 0.21 g, respectively, as determined by calibration WI, USA). Enhanced chemiluminescence (ECL) curves (Lim et al. 2019). Gwon et al. Fisheries and Aquatic Sciences (2019) 22:7 Page 3 of 10 Cell culture and treatment RNA were used for reverse transcription using oligo- HUVECs were grown in EGM-2 medium with 2% fetal dT-adaptor primer and superscript reverse transcriptase. bovine serum (FBS) and endothelial growth supplement PCR was performed with the gene-specific primers and used between passage 3 and 6. HUVECs were cul- (Table 1). PCR products were observed by electropho- tured in 100-mm dishes at a concentration of 5 × 10 resis in agarose gels and visualized with UV light after cells/dish in EGM-2 medium. Human monocytic THP-1 staining with ethidium bromide. Densitometric analysis cells (KCLB, Seoul, Korea) were maintained in RPMI-1640 was performed using EZ-Capture II (ATTO and Rise containing 10% FBS (GIBCO, Grand Island, NY, USA). Co., Tokyo, Japan) and CS analyzer (ver. 3.00 software, Both cells were maintained at 37 °C in a humidified ATTO). chamber containing 5% CO and 95% air. HSS was dissolved in dimethyl sulfoxide (DMSO), and the final Preparation of cytosolic and nuclear extracts DMSO concentration in all assays was adjusted to 0.1%. HUVECs were seeded in a culture dish at a density of 6×10 cells/dish. Cultured cells were pretreated with Measurement of cell viability 0, 2.5, 5.0, and 10.0 μg/mL of HSS for 1 h and stimulated The cytotoxicity of HSS was determined by MTS assay. with TNF-α for 30 min. Separation of cytosolic and HUVECs were pretreated with a serial dilution of HSS for nucleus fraction was performed as previously described 1 h and then stimulated with TNF-α (10 ng/mL) for an (Gwon et al. 2015). additional 24 h, except for control wells. MTS solution with fresh medium was added to each well. After 1 h, the Western blot analysis absorbance was measured at 490 nm with a microplate HUVECs pretreated with 0, 2.5, 5.0, and 10.0 μg/mL reader (GloMax Multi Detection System, Promega). HSS for 1 h were stimulated with TNF-α (10 ng/mL) at times indicated in the figure legends. Cells were washed Measurement of MCP-1 and KC by ELISA twice with cold PBS and lysed with lysis buffer on ice for Cells were inoculated in 12-well plates at a density of 20 min. After centrifugation at 12,000×g for 20 min, the 1.5 × 10 cells/well and pretreated with 2.5, 5.0, and protein concentrations of the supernatants were deter- 10 μg/mL of HSS for 1 h prior to TNF-α (10 ng/mL) mined and an equal amount of protein was loaded on stimulation for 24 h. Culture medium was collected after sodium dodecyl sulfate-polyacrylamide gel electropho- centrifugation at 2000×g for 10 min and stored at − 80 °C resis (SDS-PAGE) for protein separation. The specific until testing. Levels of MCP-1 and KC in culture media procedure for Western blot is described in our previous were quantitively determined by ELISA kit according to paper (Gwon et al. 2015). The antibodies against the manufacturer’sinstruction. ICAM-1, VCAM-1, HO-1, pIKK-β, IKK-β,pIκB-α, and IκB-α were diluted to 1/3000, and those against NF-κB, Cell-based enzyme-linked immunosorbent assay glyceraldehyde 3-phosphate dehydrogenase (GAPDH), The expression of ICAM-1 and VCAM-1 on the surface poly (ADP-ribose) polymerase (PARP), β-actin, and Nrf2 of HUVECs was assessed using ELISA, as previously were diluted to 1/1000 with Tris-buffered saline. described (Gwon et al. 2015). Briefly, HUVECs were pretreated with HSS (2.5, 5.0, and 10.0 μg/mL) for 1 h Determination of intracellular ROS levels followed by TNF-α treatment (10 ng/mL) for 6 h. The The levels of intracellular ROS in HUVECs were cells were then fixed by 1% paraformaldehyde and then measured using fluorescent probe DCFH-DA. HUVECs blocked with 2% bovine serum albumin (BSA) at room in the 96-well black plate were pretreated with 0, 2.5, temperature. Subsequently, the cells were incubated with 5.0, and 10.0 μg/mL HSS for 1 h and then incubated mouse anti-human ICAM-1 or VCAM-1 antibodies for 2 h and washed with phosphate-buffered saline (PBS). Table 1 Primer sequences for RT-PCR Cells were incubated with the horseradish peroxidase Primer Sequence (HRP)-conjugated secondary antibody and washed, ICAM-1 Forward 5′-GAGATCACCTGGAGCCAAT-3′ followed by peroxidase substrate solution. The absor- Reverse 5′-CCTCTGGCTTCGTCAGAATC-3′ bance at 490 nm was measured using a microplate VCAM-1 Forward 5′-GGAAGCCGATCACAGTCAAG-3′ reader (Glomax Multi Detection System, Promega). Reverse 5′-GCATTTCCAGAAAGGTGCTG-3′ Reverse transcription-polymerase chain reaction HO-1 Forward 5′-AGTCTTCGCCCCTGTCTACT-3′ The mRNA levels of ICAM-1 and VCAM-1 in HUVECs Reverse 5′-GGGGCAGAATCTTGCACTTT-3′ were determined using RT-PCR. Total RNA was GAPDH Forward 5′-ACCACAGTCCATGCCATCAC-3′ extracted from HUVECs by QIAzol reagent according to Reverse 5′-TCCACCACCCTGTTGCTGTA-3′ the manufacturer’s instructions. Five micrograms of total Gwon et al. Fisheries and Aquatic Sciences (2019) 22:7 Page 4 of 10 with 20 μM DCFH-DA for 30 min. The cells were stim- were estimated to be 7.25 ± 0.53 (μg/mL), 4.55 ± 0.52 ulated with TNF-α (10 ng/mL) for 1 h. The fluorescence (μg/mL), and 2.29 ± 0.13 (μg/mL), respectively. IC levels were determined using a fluorescence microplate values of EtOH, HSS, and EtOAc for the inhibition of reader (Em 485 nm and Ex 528 nm). VCAM-1 were also determined to be 6.67 ± 0.31 (μg/mL), 3.61 ± 0.22 (μg/mL), and 4.53 ± 0.28 (μg/mL), respectively. Immunofluorescence analysis With n-Hexane fractionation, HSS showed 1.6-fold and To assess the translocation of NF-κB, HUVECs were 1.8-fold stronger inhibitory activity on the production of grown on 8-well chamber slides (SPL Life Sciences ICAM-1 and VCAM-1, respectively, than MES. However, Co., Gyeonggi-do, Korea) and stimulated with TNF-α the n-butanol and H O fraction were not inhibited (10 ng/mL) for 30 min after HSS pretreatment for 1 h. ICAM-1 and VCAM-1 production. Although EtOAc frac- The cells were fixed with 4% paraformaldehyde in PBS for tion showed selectively higher inhibitory activity for 15 min. After washing with PBS, the cells were perme- ICAM-1 production, active compounds in the EtOAc abilized with 0.5% Triton X-100 in PBS for 10 min. After were not identified. Thus, we investigated molecular blocking with 3% BSA/PBS for 30 min, cells were mechanisms of HSS on the inhibition of vascular inflam- incubated with an anti-NF-κBantibody for 2h followed mation using TNF-α-stimulated HUVECs. by Alexa Fluor® 488-conjugated secondary antibody for 1 h. Cells were stained with 2 μg/mL DAPI and HSS inhibits the production of TNF-α-induced adhesion images were captured using a confocal microscope. molecules To examine the cytotoxicity of HSS, the viability of Statistical analysis HUVECs was measured by the MTS assay. HUVECs Data are expressed as the means ± standard deviations were cultured in 96-well microplates for 24 h and pre- (SDs). Data analysis was performed using ANOVA treated with various concentrations of HSS (2.5, 5.0, and followed by the Bonferroni test. P values < 0.05 were 10.0 μg/mL) followed by TNF-α-stimulation. The via- considered statistically significant. SPSS for Windows, bility was not influenced up to 10.0 μg/mL of HSS in version 10.07 (SPSS Inc., Chicago, IL, USA) was used for HUVECs (Fig. 1a). Since monocyte adhesion to HUVECs all analyses. is critically regulated by ICAM-1 and VCAM-1, we assessed the production of ICAM-1 and VCAM-1 pro- Results tein using Western blot. The expression levels of both Inhibitory activity of EtOH extract and its solvent-soluble adhesion molecules were remarkably elevated in fractions on ICAM-1 and VCAM-1 production TNF-α-treated HUVECs (Fig. 1b). Pretreatment of HSS In order to evaluate the vascular anti-inflammatory at 5.0 μg/mL notably reduced the production of ICAM-1 potential of S. serratifolium, the EtOH extract and its and VCAM-1 by 66.1% and 52.7%, respectively, which solvent-soluble fractions were tested using TNF-α- shows stronger inhibitory activity for adhesion molecules treated HUVECs. Inhibitory effects (IC ) of these frac- than EtOH extract. To further investigate the effect of tions on the adhesion molecules were determined with HSS on ICAM-1 and VCAM-1 mRNA expression, non-toxic concentration by Western blot using TNF- RT-PCR was performed. Our data showed that HSS α-stimulated HUVECs. As shown in Table 2,IC values dose-dependently suppressed mRNA expression of both of EtOH, HSS, and EtOAc for the inhibition of ICAM-1 adhesion genes in TNF-α-treated HUVECs (Fig. 1c). Furthermore, the result of cell-based ELISA assay showed that HSS suppressed the expression of ICAM-1 Table 2 Inhibitory activity of HSS and its solvent-soluble and VCAM-1 in TNF-α-treated HUVECs in a dose- fractions on the expression of ICAM-1 and VCAM-1 in TNF-α- dependent manner (Fig. 1d). These data indicate that the stimulated HUVECs HSS-mediated downregulation of adhesion molecules in Fractions IC50 (μg/mL) Yields b b (%) TNF-α-treated endothelium is primarily attributable to ICAM-1 VCAM-1 their transcriptional regulation. Ethanolic extract 7.25 ± 0.53 6.67 ± 0.31 100 ± 5.2 n-Hexane 4.55 ± 0.52 3.61 ± 0.22 66.1 ± 4.3 HSS suppresses TNF-α-induced chemokine production Ethyl acetate 2.29 ± 0.13 4.53 ± 0.28 9.23 ± 1.2 Recruitment of monocytes to endothelium is largely reg- n-Butanol ≥ 100 ≥ 100 17.5 ± 1.9 ulated by chemokines, including MCP-1 and KC. As shown in Fig. 2, exposure of TNF-α to HUVECs for 6 h Water ≥ 100 ≥ 100 8.12 ± 1.4 enhanced the production of MCP-1 (Fig. 2a) and KC IC50 is calculated to be concentrations with a half-maximal expression of ICAM-1 and VCAM-1 in TNF-α-stimulated HUVECs (Fig. 2b). HSS pretreatment as low as 2.5 μg/mL inhi- The expression of levels of ICAM-1 and VCAM-1 were determined with bited TNF-α-induced production of both chemokines. Western blot Yield is showing the amount of each fraction in 100 g of ethanolic extract Pretreatment with 10 μg/mL HSS decreased the production Gwon et al. Fisheries and Aquatic Sciences (2019) 22:7 Page 5 of 10 A C Fig. 1 Effect of HSS on the production of TNF-α-induced ICAM-1 and VCAM-1 expression in HUVECs. a Cytotoxic effect of HSS was measured by MTS assay. b Cells pretreated with the indicated concentration of HSS for 1 h were stimulated with TNF-α for 6 h. Equal amounts of total protein were separated by 10% SDS-PAGE. The expression of ICAM-1, VCAM-1, and β-actin was examined by Western blot using corresponding antibodies. c Cells pretreated with the indicated concentration of HSS for 1 h were stimulated with TNF-α for 6 h, and total RNA was prepared for RT-PCR. d Fixed cells with 1% paraformaldehyde were analyzed by ELISA for the cell surface expression of ICAM-1 and VCAM-1. The values are shown as the means ± SDs from three independent experiments. P < 0.05 represents significant differences versus the non-treated group. *P <0.05 represents significant differences versus the TNF-α-only group of both chemokines to control level. This result indicates phosphorylation of IKK-β and led to IκB-α phospho- that HSS suppresses recruitment of monocytes to endothe- rylation, whereas HSS dose-dependently inhibited the lium by inhibition of chemokine production. phosphorylation of both proteins (Fig. 3b). The phos- phorylation level of cytosolic IκB-α at 10 μg/mL HSS HSS inhibits TNF-α-induced NF-κB activation in HUVECs was comparable to the control group possibly due to the To assess whether HSS inhibits the translocation of inhibitory effect of HSS on the phosphorylation IKK-β NF-κB into the nucleus, microscopic analysis was per- (Fig. 3b). In addition, TNF-α-mediated increase in NF-κB formed. Confocal observation indicated that NF-κB/p65 protein level in the nucleus was dose-dependently was predominantly localized in the perinuclear compart- decreased by HSS treatment (Fig. 3b). These results ment under unstimulated condition. With TNF-α treat- suggest that the HSS-mediated inhibition of adhesion ment, most of cytoplasmic NF-κB/p65 translocated to molecules and chemokines are largely regulated by the the nucleus (Fig. 3a). However, HSS pretreatment at NF-κB pathway in the TNF-α-stimulated cells. TNF-α 10 μg/mL markedly inhibited the NF-κB/p65 translo- activates the membrane-bound NADPH oxidase in endo- cation into the nucleus. For further investigating the thelium which stimulates ROS production, a second molecular mechanism underlying HSS-mediated inhi- messenger for NF-κB activation(Kimetal. 2008). We bition of NF-κB/p65 translocation in TNF-α-stimulated examined the inhibitory effects of the HSS on intracellular cells, we examined the phosphorylation of inhibitor κB ROS accumulation in TNF-α-treated HUVECs. TNF-α-in- kinase (IKK)-β and IκB-α that are associated with the duced ROS was markedly reduced by HSS pretreatment NF-κB activation. TNF-α markedly caused the (Fig. 3C, P < 0.05), indicating that intrinsic antioxidant Gwon et al. Fisheries and Aquatic Sciences (2019) 22:7 Page 6 of 10 suppresses vascular inflammation through the induction of HO-1 via Nrf2 activation in TNF-α-treated HUVECs. Discussion Our previous study reported that the ethanolic extract of S. serratifolium suppressed the production of vascular inflammatory proteins in serum and aorta tissue in high cholesterol diet-fed mice. The anti-atherogenic compo- nents in the extract were identified as SHQA, SCM, and SQA which showed strong anti-inflammatory and anti- oxidant activities (Gwon et al. 2018; Lim et al. 2019). The quantification data suggest that the contents of SHQA, SCM, and SQA in 100 g of meroterpenoid-rich extract from S. serratifolium (MES) were estimated to be 37.6 ± 2.1, 6.23 ± 0.36, and 1.89 ± 0.10 g, respectively (Kwon et al. 2018). To obtain a higher concentration of meroterpenoids from MES, we fractionated MES with n-hexane and the content of each component in HSS was comprised to be 52.4 ± 3.3 g for SHQA, 8.26 ± 0.82 g for SCM, and 3.0 ± 0.21 for SQA as determined by cali- bration curves (Lim et al. 2019). In the present study, we found that HSS showed a higher capacity for the sup- pression of ICAM-1, VCAM-1, and MCP-1 expression compared with MES. HSS suppressed the TNF-α-in- duced NF-κB activation by blocking IκB-α degradation. Additionally, HSS alleviated vascular inflammation by the production of HO-1 via Nrf2 activation. To our know- ledge, this is in vitro evidence that HSS is a potent anti-inflammatory agent to alleviate vascular inflammation and to improve endothelial dysfunction. Fig. 2 Effect of HSS on the production of MCP-1 and KC in TNF-α- Adhesion of circulating monocytes to vascular endo- induced HUVECs. Cells pretreated with the indicated concentration of thelium is an initial step in triggering vascular inflamma- HSS for 1 h were stimulated with TNF-α for 24 h. The levels of MCP-1 tion leading to atherosclerosis (Madge and Pober 2001). (a) and KC (b) were examined by ELISA. The values are shown as the means ± SDs from three independent experiments. P <0.05 represents Pro-inflammatory stimuli, such as TNF-α exposure, ini- significant differences versus the control group. *P < 0.05 represents tiate the expression of adhesion molecules that stimulate significant differences versus the TNF-α-only group the attachment of circulating lymphocytes and mono- cytes to endothelium (Packard and Libby 2008). In this activity of HSS is associated with inhibiting NF-κBacti- respect, the adhesion molecules such as VCAM-1 and vation through scavenging ROS induced by TNF-α ICAM-1 are well-known vascular inflammatory markers, treatment. which are related to tight adhesion of monocytes in atherosclerotic lesion of the endothelium (Packard and Libby 2008). Both adhesion molecules are largely pro- HSS induces HO-1 production through Nrf2 activation duced in advanced human coronary atherosclerotic Increased endogenous HO-1 provides cellular protection plaque and in experimental atherosclerosis animals against TNF-α-induced ROS. Therefore, we determined (Catapano et al. 2017; Nallasamy et al. 2014). Our results the effect of HSS on the production of HO-1 protein implied that HSS blocked the TNF-α-induced endothelial and mRNA by Western blot and RT-PCR, respectively. production of VCAM-1 and ICAM-1in HUVECs. These HSS treatment caused an increased production of HO-1 data indicate that the vascular anti-inflammatory action of protein and mRNA in a dose-dependent manner HSS on TNF-α-treated cells is mainly regulated by (Fig. 4a). We further determined the effect of HSS on suppressing the production of adhesion molecules. Speci- the production of Nrf2, a key regulator of the HO-1 fically, the current findings indicate that the HSS-mediated expression. As shown in Fig. 4b, HSS induced the decline of adhesion molecules is closely associated with tran- production of Nrf2 determined by Western blot in scriptional regulation of ICAM-1 and VCAM-1 mRNA ex- TNF-α-induced cells. These results implicate that HSS pression in TNF-α-treated HUVECs. Gwon et al. Fisheries and Aquatic Sciences (2019) 22:7 Page 7 of 10 Fig. 3 Effect of HSS on the NF-κB translocation in TNF-α-treated HUVECs. a Cells were pretreated with or without HSS for 1 h followed by TNF-α treatment for 30 min. The subunits of NF-κB/p65 were detected by using anti-NF-κB polyclonal antibody and Alexa Fluor® 488-conjugated secondary antibody. The nuclei were detected by DAPI staining and confocal microscopy was used to capture the images. b Cells were incubated withanindicated concentrationofHSS for1hand exposedtoTNF-α (10 ng/mL) for 30 min. Cytosolic and nucleus fractions were analyzed by Western blotting. c The cells pretreated with the indicated concentrations of HSS for 1 h were stimulated with TNF-α (10 ng/mL) for 30 min. ROS was determined by DCFH-DA with fluorescent analysis. 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 TNF-α-only group Chemokine produced by activated endothelium play overexpression of MCP-1 in vascular tissues promotes pivotal roles for chemotactic attachment of circulating cooperation of monocytes/macrophages and formation monocytes in the intima of the blood vessel. Chemokine of atherosclerotic lesion (Namiki et al. 2002). Chemo- stimulates sturdy adhesion of infiltrated monocytes to kine KC is identified as the first chemokine respon- vascular endothelium which is a noticeable pathological sible for monocyte arrest on early atherosclerotic mark in the early stage of atherogenesis (Chen et al. lesion (Huo et al. 2001). KC stimulates monocyte 2004). Accumulating evidence demonstrated that local adhesion in carotid arteries in the early atherosclerotic Gwon et al. Fisheries and Aquatic Sciences (2019) 22:7 Page 8 of 10 endothelium is mediated at least partially by inhibition of chemokine production. NF-κB is an essential transcription factor associated with the expression of adhesion molecules and chemo- kines (Huang et al. 2018). As discussed above, adhesion of monocyte to endothelium is stimulated through adhe- sion molecules and chemokines, including ICAM-1, VCAM-1, MCP-1, and KC in endothelial cells (De Filippo et al. 2008). The expression of these molecules is increased by NF-κB activation. In unstimulated condi- tions, NF-κB is primarily distributed in the cytoplasm as an inactive complex bound to IκB-α. Excess ROS pro- duced by TNF-α treatment induce the phosphorylation of IKK complex, which leads to the phosphorylation of IκB-α, thereby leading to the degradation of IκB-α and translocation of NF-κB into the nucleus (Madge and Pober 2001; Zhang et al. 2009). Thus, NF-κBis an important target for designing pharmaceutical agents for interfering with the development and progression of atherosclerosis. Although mechanisms underlying HSS- mediated NF-κB regulation are uncertain, the current study indicates that HSS suppresses NF-κB activation by inhibiting proteolytic degradation of IκB-α induced by TNF-α in HUVECs. The confocal microscopic obser- vation indicates that HSS blocked NF-κB translocation into the nucleus induced by TNF-α in HUVECs. There- fore, these results showed the ability of HSS to suppress TNF-α-mediated NF-κB activation in HUVECs. In the current study, we found that HSS treatment remarkably reduced TNF-α-induced ROS production in HUVECs, suggesting that HSS retains strong antioxidant capacity. Hence, it is likely that the reductions of ICAM-1, VCAM-1, MCP-1, and KC expression in HUVECs are mainly related to the regulation of NF-κB pathway through the removal of ROS by HSS’s intrinsic anti- oxidant activity. Nrf2 is the primary transcription factor to regulate anti- Fig. 4 Effect of HSS on HO-1 production and Nrf2 activation in TNF- oxidant proteins including HO-1 which is an enzyme that α-induced HUVECs. a Cells pretreated with the indicated concentration of HSS for 1 h were stimulated with TNF-α for 16 h (RT-PCR) or 24 h catalyzes the degradation of heme to ferric iron, carbon (Western blot). b Cells pretreated with the indicated concentration of monoxide, and biliverdin as reaction products. Resulting HSS for 1 h were stimulated with TNF-α for 30 min. The proteins were biliverdin is enzymatically converted to bilirubin by analyzed by Western blotting using corresponding antibodies. The biliverdin reductase (Ayer et al. 2016). HO-1 metabolites values are shown as the means ± SDs from three independent are known to have antioxidant, anti-inflammatory, and experiments. P < 0.05 represents significant differences versus the control group. *P < 0.05 represents significant differences versus the anti-atherogenic effects (Ishikawa et al. 2001). In vitro TNF-α-only group studies show that overexpression of HO-1 attenuates endothelial dysfunction via suppressing the production of VCAM-1, MCP-1, and macrophage stimulating factor in lesion in mice (Huo et al. 2001). Both MCP-1 and KC are TNF-α-treated vascular endothelial cells (Kawamura et al. crucial chemokines to the progression of atherosclerosis, 2005). Recent studies demonstrate that HO-1 enhances due to their abilities to recruit monocytes to the intima of vascular protective and anti-atherogenic actions, and its the blood vessel (Chen et al. 2004). In this study, HSS expression in endothelial cells attenuates atherosclerosis inhibited the TNF-α-induced production of MCP-1 and (Choi et al. 2018;Kato et al. 2001). In this study, we found KC in TNF-α-treated HUVECs. These results suggest that that HSS pretreatment markedly increased the production the anti-inflammatory action of HSS on TNF-α-induced of HO-1 protein and mRNA which are caused by Gwon et al. Fisheries and Aquatic Sciences (2019) 22:7 Page 9 of 10 translocation of Nrf2 into the nucleus. Thus, enhanced Choi ES, Yoon JJ, Han BH, Jeong DH, Lee YJ, Kang DG, Lee HS. Ligustilide attenuates vascular inflammation and activates Nrf2/HO-1 induction and, NO HO-1 expression by HSS attributes to the removal of synthesis in HUVECs. Phytomedicine. 2018;38:12–23. ROS induced by TNF-α treatment. This study appears De Filippo K, Henderson RB, Laschinger M, Hogg N. Neutrophil chemokines KC to be the first finding to address characteristics of HSS in and macrophage-inflammatory protein-2 are newly synthesized by tissue macrophages using distinct TLR signaling pathways. J Immunol. 2008;180:4308–15. the inhibition of vascular inflammation in TNF-α-induced Gwon WG, Joung EJ, Kwon MS, Lim SJ, Utsuki T, Kim HR. Sargachromenol HUVECs. protects against vascular inflammation by preventing TNF-α-induced monocyte adhesion to primary endothelial cells via inhibition of NF-κB activation. Int Immunopharmacol. 2017;42:81–9. Conclusions Gwon WG, Joung EJ, Shin T, Utsuki T, Wakamatsu N, Kim HR. Meroterpinoid-rich In summary, we have shown that HSS inhibits the fraction of the ethanol extract from Sargassum serratifolium suppresses mRNA and protein expression of adhesion molecules TNF-α-induced monocytes adhesion to vascular endothelium and vascular inflammation in high cholesterol-fed C57BL/6J mice. J Func including ICAM-1 and VCAM-1 in TNF-α-stimulated Foods. 2018;46:384–93. HUVECs. Furthermore, the vascular anti-inflammatory Gwon WG, Lee B, Joung EJ, Choi MW, Yoon N, Shin T, Oh CW, Kim HR. roles of HSS in TNF-α-treated cells appear to be closely Sargaquinoic acid inhibits TNF-α-induced NF-κB signaling, thereby contributing to decreased monocyte adhesion to human umbilical vein related to the inactivation of the NF-κB as well as the endothelial cells (HUVECs). J Agric Food Chem. 2015;63:9053–61. activation of Nrf2 pathways. Further confirmation of Huang W, Huang M, Ouyang H, Peng J, Liang J. Oridonin inhibits vascular vascular anti-inflammatory effects of HSS and under- inflammation by blocking NF-κB and MAPK activation. Eur J Pharmacol. 2018;826:133–9. lying mechanisms in animal models will be important Huo Y, Weber C, Forlow SB, Sperandio M, Thatte J, Mack M, Jung S, Littman DR, for the therapeutic application of HSS for treating Ley K. The chemokine KC, but not monocyte chemoattractant protein-1, vascular inflammation-associated diseases. triggers monocyte arrest on early atherosclerotic endothelium. J Clin Invest. 2001;108:1307–14. Acknowledgements Ishikawa K, Sugawara D, Wang X, Suzuki K, Itabe H, Maruyama Y, Lusis AJ. Heme This work was supported by a Research Grant of Pukyong National University oxygenase-1 inhibits atherosclerotic lesion formation in ldl-receptor knockout (2017). mice. Circ Res. 2001;88:506–12. Joung EJ, Gwon WG, Shin T, Jung BM, Choi J, Kim HR. Anti-inflammatory action Authors’ contributions of the ethanolic extract from Sargassum serratifolium on lipopolysaccharide- WGG carried out the experiments and participated to write the manuscript. stimulated mouse peritoneal macrophages and identification of active SGL and JIK corrected the manuscript. YMK and SBK analyzed the content of components. J Appl Phycol. 2017;29:563–73. meroterpenoids in HSS. HRK designed the study. All authors read and Joung EJ, Lee B, Gwon WG, Shin T, Jung BM, Yoon NY, Choi JS, Oh CW, Kim HR. approved the final manuscript. Sargaquinoic acid attenuates inflammatory responses by regulating NF-κB and Nrf2 pathways in lipopolysaccharide-stimulated RAW 264.7 cells. Int Competing interests Immunopharmacol. 2015;29:693–700. The authors declare that they have no competing interests. Kato H, Amersi F, Buelow R, Melinek J, Coito AJ, Ke BBRW, Kupiec-Weglinski JW. Heme oxygenase-1 overexpression protects rat livers from ischemia/ reperfusion injury with extended cold preservation. Am J Transplantation. Publisher’sNote 2001;1:121–8. Springer Nature remains neutral with regard to jurisdictional claims in Kawamura K, Ishikawa K, Wada Y, Kimura S, Matsumoto H, Kohro T, Itabe H, published maps and institutional affiliations. Kodama T, Maruyama Y. Bilirubin from heme oxygenase-1 attenuates vascular endothelial activation and dysfunction. Arterioscler Thromb Vasc Author details 1 Biol. 2005;25:155–60. Department of Food Science and Nutrition, Pukyong National University, 45, Kim JH, Na HJ, Kim CK, Kim JY, Ha KS, Lee H, Chung HT, Kwon HJ, Kwon YG, Kim Yongso-Ro, Nam-Gu, Busan 48513, Republic of Korea. Department of Food YM. The non-provitamin A carotenoid, lutein, inhibits NF-κB-dependent gene Technology, Pukyong National University, 45, Yongso-Ro, Nam-Gu, Busan expression through redox-based regulation of the phosphatidylinositol 3- 48513, Republic of Korea. kinase/PTEN/Akt and NF-κB-inducing kinase pathways: role of H O in NF-κB 2 2 activation. Free Rad Biol Med. 2008;45:885–96. Received: 22 November 2018 Accepted: 18 February 2019 Kleinbongard P, Heusch G, Schulz R. TNFα in atherosclerosis, myocardial ischemia/reperfusion and heart failure. Pharmacol Ther. 2010;127:295–314. Kwon M, Lim SJ, Joung EJ, Lee B, Oh CW, Kim HR. Meroterpenoid-rich fraction of References an ethanolic extract from Sargassum serratifolium alleviates obesity and non- Ayer A, Zarjou A, Agarwal A, Stocker R. Heme oxygenases in cardiovascular alcoholic fatty liver disease in high fat-fed C57BL/6J mice. J Func Foods. health and disease. Physiol Rev. 2016;96:1449–508. 2018;47:288–98. Azam MS, Joung EJ, Choi J, Kim HR. Ethanolic extract from Sargassum Lim S, Choi AH, Kwon M, Joung EJ, Shin T, Lee SG, Kim NG, Kim HR. Evaluation of serratifolium attenuates hyperpigmentation through CREB/ERK signaling antioxidant activities of various solvent extract from Sargassum serratifolium pathways in α-MSH-stimulated B16F10 melanoma cells. J Appl Phycol. and its major antioxidant components. Food Chem. 2019;278:178–84. 2017;29:2089–96. Lim S, Kwon M, Joung EJ, Shin T, Oh CW, Choi JS, Kim HR. Meroterpenoid-rich Catalan U, Hazas ML, Rubio L, Fernandez-Castillejo S, Pedret A, de la Torre R, fraction of the ethanolic extract from Sargassum serratifolium suppressed Motilva MJ, Sola R. Protective effect of hydroxytyrosol and its predominant oxidative stress induced by tert-butyl hydroperoxide in HepG2 cells. Mar plasmatic human metabolites against endothelial dysfunction in human Drugs. 2018;16:374. aortic endothelial cells. Mol Nutri Food Res. 2015;59:2523–36. Liu L, Heinrich M, Myers S, Dworjanyn SA. Towards a better understanding of Catapano AL, Pirillo A, Norata GD. Vascular inflammation and low-density medicinal uses of the brown seaweed Sargassum in traditional Chinese lipoproteins: is cholesterol the link? A lesson from the clinical trials. Br J medicine: a phytochemical and pharmacological review. J Ethnopharmacol. Pharmacol. 2017;174:3973–85. 2012;142:591–619. Chen YM, Chiang WC, Lin SL, Wu KD, Tsai TJ, Hsieh BS. Dual regulation of tumor Madge LA, Pober JS. TNF signaling in vascular endothelial cells. Exp Mol Pathol. necrosis factor-α-induced CCL2/monocyte chemoattractant protein-1 2001;70:317–25. expression in vascular smooth muscle cells by nuclear factor-κB and activator protein-1: modulation by type III phosphodiesterase inhibition. J Pharmacol Nallasamy P, Si H, Babu PV, Pan D, Fu Y, Brooke EA, Shah H, Zhen W, Zhu H, Liu Exp Ther. 2004;309:978–86. D, Li Y, Jia Z. Sulforaphane reduces vascular inflammation in mice and Gwon et al. Fisheries and Aquatic Sciences (2019) 22:7 Page 10 of 10 prevents TNF-α-induced monocyte adhesion to primary endothelial cells through interfering with the NF-κB pathway. J Nutr Biochem. 2014;25:824–33. Namiki M, Kawashima S, Yamashita T, Ozaki M, Hirase T, Ishida T, Inoue N, Hirata K, Matsukawa A, Morishita R, Kaneda Y, Yokoyama M. Local overexpression of monocyte chemoattractant protein-1 at vessel wall induces infiltration of macrophages and formation of atherosclerotic lesion: synergism with hypercholesterolemia. Arterioscler Thromb Vas Biol. 2002;22:115–20. Oh SJ, Joung EJ, Kwon MS, Lee B, Utsuki T, Oh CW, Kim HR. Anti-inflammatory effect of ethanolic extract of Sargassum serratifolium in lipopolysaccharide- stimulated BV2 microglial cells. J Med Food. 2016;19:1023–31. Packard RR, Libby P. Inflammation in atherosclerosis: from vascular biology to biomarker discovery and risk prediction. Clin Chem. 2008;54:24–38. Rao RM, Yang L, Garcia-Cardena G, Luscinskas FW. Endothelial-dependent mechanisms of leukocyte recruitment to the vascular wall. Circ Res. 2007;101:234–47. Zhang H, Park Y, Wu J, Chen X, Lee S, Yang J, Dellsperger KC, Zhang C. Role of TNF-α in vascular dysfunction. Clin Sci. 2009;116:219–30. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Fisheries and Aquatic Sciences Springer Journals

Hexane fraction from the ethanolic extract of Sargassum serratifolium suppresses cell adhesion molecules via regulation of NF-κB and Nrf2 pathway in human umbilical vein endothelial cells

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Life Sciences; Fish & Wildlife Biology & Management; Marine & Freshwater Sciences; Zoology; Animal Ecology
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10.1186/s41240-019-0122-7
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Abstract

Sargassum serratifolium ethanolic extract has been known for strong antioxidant and anti-inflammatory properties. We prepared hexane fraction from the ethanolic extract of S. serratifolium (HSS) to improve biological activities. In this study, we investigated the effects of HSS on the inhibition of tumor necrosis factor (TNF)-α-induced monocyte adhesion to human umbilical vein endothelial cells (HUVECs). We found that HSS suppressed the production of cell adhesion molecules such as intracellular adhesion molecule-1 and vascular cell adhesion molecule-1 in TNF-α-induced HUVECs. Moreover, TNF-α-induced production of monocyte chemoattractant protein 1 and keratinocyte chemoattractant was inhibited by HSS treatment. HSS suppressed TNF-α-induced nuclearfactorkappa B(NF-κB) activation via preventing proteolytic degradation of inhibitor κB-α. HSS induced the production of heme oxygenase 1 via translocation of Nrf2 into the nucleus in TNF-α-treated HUVECs. Overall, HSS alleviated vascular inflammation through the downregulation of NF-κB activation and the upregulation of Nrf2 activation in TNF-α-induced HUVECs. These results indicate that HSS may be used as therapeutic agents for vascular inflammatory disorders. Keywords: Sargassum serratifolium,Vascularinflammation,Nuclearfactor-κB, Adhesion molecule, Human umbilical vein endothelial cell Background and Pober 2001; Rao et al. 2007). A vascular endothe- Vascular inflammation has been known to play a key lium is a key target of tumor necrosis factor (TNF)-α-in- role in the progress of atherosclerosis, and enhanced duced inflammation, which promotes the expression of monocyte adhesion to endothelial cells is believed to be ICAM-1 and VCAM-1. Monocyte chemoattractant pro- one of the earliest events in atherogenesis (Packard and tein (MCP)-1 and keratinocyte chemoattractant (KC) Libby 2008). Accumulating evidences have revealed that have pivotal roles in recruiting monocytes to the lesion chronic inflammation plays a crucial role in the of dysfunctional endothelium. Excess chemokines are initiation and progression of atherosclerosis. Monocyte found in human atherosclerotic plaques and regulate the adhesion to endothelial cells is primarily mediated by interaction between monocytes and vascular endothelial several intracellular signaling events that lead to the cells (Catalan et al. 2015). Taking these evidences, inhi- elevated expression of endothelial adhesion molecules, bition of adhesion molecules and chemokines may be a including vascular cell adhesion molecule-1 (VCAM-1) promising approach to prevent atherosclerosis by blocking and intracellular adhesion molecule-1 (ICAM-1) (Madge monocyte invasion to the vascular inflammatory lesion. An accumulative evidence suggests that TNF-α,a * Correspondence: hrkim@pknu.ac.kr pleiotropic pro-inflammatory cytokine in the inflamma- Department of Food Science and Nutrition, Pukyong National University, 45, tory cascade, involves in a critical role in vascular inflam- Yongso-Ro, Nam-Gu, Busan 48513, Republic of Korea mation and the subsequent progress of atherosclerosis Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Gwon et al. Fisheries and Aquatic Sciences (2019) 22:7 Page 2 of 10 (Madge and Pober 2001). TNF-α induces the produc- detection kit was purchased from GE Healthcare tion of reactive oxygen species (ROS) by the activation Bio-Science (Piscataway, NJ, USA). QIAzol lysis reagent of membrane-bound nicotinamide adenine dinucleo- was purchased from Quiagen (Valencia, CA). tide phosphate (NADPH) oxidase in endothelium 2′,7′-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein (Kleinbongard et al. 2010). Excess ROS produced by acetoxy-methyl ester (BCECF-AM), Alexa Fluor® TNF-α stimulate the phosphorylation of the inhibitor kB 488-conjugated secondary antibody, 4′,6-diamidino-2-- kinase (IKKs) complex, leading to the proteasomal phenylindole (DAPI), and dual luciferase assay kits were degradation of phosphorylated IκB-a. Thus, free NF-κB purchased from Invitrogen (Carlsbad, CA, USA). dissociated from IκB is translocated into the nucleus and 2′,7′-dichlorofluorescin diacetate (DCFH-DA), dimethyl binds to the cis-acting element to regulate the expression sulfoxide (DMSO), and phenylmethylsulfonyl fluoride of adhesion molecules in endothelium (Huang et al. 2018). (PMSF) were obtained from Sigma-Aldrich Corporation Thus, approaches to regulate endothelial activation are (St. Louis, MO, USA). The antibodies against ICAM-1, potential strategies to prevent atherosclerosis through the VCAM-1, and HO-1 were obtained from Abcam (Cam- suppression of vascular inflammation. bridge, UK). pIKK-β, IKK-β,pIκB-α, and IκB-α were pur- Sargassaceae family is consumed as food or traditional chased from Cell Signaling (Danvers, MA, USA). NF-κB, medicine in Korea and China and various bioactive com- GAPDH, PARP, β-actin, Nrf2, and secondary antibody pounds have been identified as meroterpenoids, phloro- were purchased from Santa Cruz Biotechnology (Santa tannins, fucoxanthins, and fucosterols (Liu et al. 2012). Cruz, CA, USA). Sargassum serratifolium (C. Agardh), a marine brown alga, is broadly distributed throughout the Korean and Preparation of n-hexane fraction from the ethanolic Japanese coasts. Recently, we reported that ethanolic extract of S. serratifolium extract of S. serratifolium showed antioxidant and anti- S. serratifolium was collected along the coast of Busan, inflammatory activities and active compounds were South Korea, in May 2015. Specimen identity was con- identified as sargahydroquinoic acid (SHQA), sargachro- firmed by an algal taxonomist (C.G. Choi), at the Depart- menol (SCM), and sargaquinoic acid (SQA) (Joung et al. ment of Ecological Engineering, Pukyong National 2017; Lim et al. 2018;Oh etal. 2016). Moreover, University, Republic of Korea, and a voucher specimen SCM and SQA isolated from the ethanolic extract of was deposited in Marine Brown Algae Resources Bank, S. serratifolium showed a potent anti-inflammatory Republic of Korea (TC18835). Collected sample was activity in LPS-stimulated macrophages and anti- air-dried and ground. One and a half kilograms of dried atherogenic activities in TNF-α-treated human umbi- sample was extracted twice with 70% (v/v)ethanol lical vein endothelial cells (HUVECs), respectively (6 L each time) at 70 °C for 3 h. The combined extract was (Gwon et al. 2017;Gwon et al. 2015; Joung et al. 2015). filtered through the ultrafiltration unit (MWCO, 50 kDa) We prepared a meroterpenoid-rich fraction from the etha- and concentrated until the lipophilic fraction was sepa- nolic extract of S. serratifolium (MES) by removing salts rated from salt water. The lipophilic fraction was concen- and water-soluble saccharides, and the combined amount trated by a rotary vacuum evaporator (Eyela N3010, of SHQA, SCM, and SQA was estimated to be 46 g in Tokyo, Japan) at 45 °C after washing three times with 10 100 g of MES (Gwon et al. 2018;Kwonet al. 2018). To times volume of D.W. (meroterpenoid-rich extract further concentrate active compound in MES, we fractio- from S. serratifolium, MES). For further concentrate nated MES with n-hexane, and resulting n-hexane fraction active component in MES, the extract was resuspended from the ethanolic extract of S. serratifolium (HSS) in water/EtOH (1 v/v) and partitioned with n-hexane, contained 64% of three components in HSS. For a better ethyl acetate, n-butanol, and D.W. The n-hexane fraction understanding of pharmacological actions of HSS, we which showed the highest anti-inflammatory activity examined the vascular anti-inflammatory actions of HSS against LPS-induced RAW 264.7 cells was concentrated in TNF-α-stimulated HUVECs. by a rotary vacuum evaporator and kept at − 20 °C for this study. From 1.5 kg of dried sample, 80 g of the HSS was obtained. Isolation and quantification of SHQA, SCM, Methods and SQA were performed according to the method Reagents described previously (Azam et al. 2017; Joung et al. 2017). Endothelial cell growth medium (EGM-2), growth sup- Quantification of SHQA, SCM, and SQA in HSS was per- plements, and primary cultured HUVEC were obtained formed with the method described previously (Joung et al. from Lonza (Walkersville, MD, USA). CellTiter ®AQ 2017), and the contents of SHQA, SCM, and SQA in 100 ue- One Solution Cell Proliferation assay kit and reverse g of HSS were estimated to be 52.4 ± 3.3, 8.26 ± 0.82, and ous transcriptase were purchased from Promega (Madison, 3.0 ± 0.21 g, respectively, as determined by calibration WI, USA). Enhanced chemiluminescence (ECL) curves (Lim et al. 2019). Gwon et al. Fisheries and Aquatic Sciences (2019) 22:7 Page 3 of 10 Cell culture and treatment RNA were used for reverse transcription using oligo- HUVECs were grown in EGM-2 medium with 2% fetal dT-adaptor primer and superscript reverse transcriptase. bovine serum (FBS) and endothelial growth supplement PCR was performed with the gene-specific primers and used between passage 3 and 6. HUVECs were cul- (Table 1). PCR products were observed by electropho- tured in 100-mm dishes at a concentration of 5 × 10 resis in agarose gels and visualized with UV light after cells/dish in EGM-2 medium. Human monocytic THP-1 staining with ethidium bromide. Densitometric analysis cells (KCLB, Seoul, Korea) were maintained in RPMI-1640 was performed using EZ-Capture II (ATTO and Rise containing 10% FBS (GIBCO, Grand Island, NY, USA). Co., Tokyo, Japan) and CS analyzer (ver. 3.00 software, Both cells were maintained at 37 °C in a humidified ATTO). chamber containing 5% CO and 95% air. HSS was dissolved in dimethyl sulfoxide (DMSO), and the final Preparation of cytosolic and nuclear extracts DMSO concentration in all assays was adjusted to 0.1%. HUVECs were seeded in a culture dish at a density of 6×10 cells/dish. Cultured cells were pretreated with Measurement of cell viability 0, 2.5, 5.0, and 10.0 μg/mL of HSS for 1 h and stimulated The cytotoxicity of HSS was determined by MTS assay. with TNF-α for 30 min. Separation of cytosolic and HUVECs were pretreated with a serial dilution of HSS for nucleus fraction was performed as previously described 1 h and then stimulated with TNF-α (10 ng/mL) for an (Gwon et al. 2015). additional 24 h, except for control wells. MTS solution with fresh medium was added to each well. After 1 h, the Western blot analysis absorbance was measured at 490 nm with a microplate HUVECs pretreated with 0, 2.5, 5.0, and 10.0 μg/mL reader (GloMax Multi Detection System, Promega). HSS for 1 h were stimulated with TNF-α (10 ng/mL) at times indicated in the figure legends. Cells were washed Measurement of MCP-1 and KC by ELISA twice with cold PBS and lysed with lysis buffer on ice for Cells were inoculated in 12-well plates at a density of 20 min. After centrifugation at 12,000×g for 20 min, the 1.5 × 10 cells/well and pretreated with 2.5, 5.0, and protein concentrations of the supernatants were deter- 10 μg/mL of HSS for 1 h prior to TNF-α (10 ng/mL) mined and an equal amount of protein was loaded on stimulation for 24 h. Culture medium was collected after sodium dodecyl sulfate-polyacrylamide gel electropho- centrifugation at 2000×g for 10 min and stored at − 80 °C resis (SDS-PAGE) for protein separation. The specific until testing. Levels of MCP-1 and KC in culture media procedure for Western blot is described in our previous were quantitively determined by ELISA kit according to paper (Gwon et al. 2015). The antibodies against the manufacturer’sinstruction. ICAM-1, VCAM-1, HO-1, pIKK-β, IKK-β,pIκB-α, and IκB-α were diluted to 1/3000, and those against NF-κB, Cell-based enzyme-linked immunosorbent assay glyceraldehyde 3-phosphate dehydrogenase (GAPDH), The expression of ICAM-1 and VCAM-1 on the surface poly (ADP-ribose) polymerase (PARP), β-actin, and Nrf2 of HUVECs was assessed using ELISA, as previously were diluted to 1/1000 with Tris-buffered saline. described (Gwon et al. 2015). Briefly, HUVECs were pretreated with HSS (2.5, 5.0, and 10.0 μg/mL) for 1 h Determination of intracellular ROS levels followed by TNF-α treatment (10 ng/mL) for 6 h. The The levels of intracellular ROS in HUVECs were cells were then fixed by 1% paraformaldehyde and then measured using fluorescent probe DCFH-DA. HUVECs blocked with 2% bovine serum albumin (BSA) at room in the 96-well black plate were pretreated with 0, 2.5, temperature. Subsequently, the cells were incubated with 5.0, and 10.0 μg/mL HSS for 1 h and then incubated mouse anti-human ICAM-1 or VCAM-1 antibodies for 2 h and washed with phosphate-buffered saline (PBS). Table 1 Primer sequences for RT-PCR Cells were incubated with the horseradish peroxidase Primer Sequence (HRP)-conjugated secondary antibody and washed, ICAM-1 Forward 5′-GAGATCACCTGGAGCCAAT-3′ followed by peroxidase substrate solution. The absor- Reverse 5′-CCTCTGGCTTCGTCAGAATC-3′ bance at 490 nm was measured using a microplate VCAM-1 Forward 5′-GGAAGCCGATCACAGTCAAG-3′ reader (Glomax Multi Detection System, Promega). Reverse 5′-GCATTTCCAGAAAGGTGCTG-3′ Reverse transcription-polymerase chain reaction HO-1 Forward 5′-AGTCTTCGCCCCTGTCTACT-3′ The mRNA levels of ICAM-1 and VCAM-1 in HUVECs Reverse 5′-GGGGCAGAATCTTGCACTTT-3′ were determined using RT-PCR. Total RNA was GAPDH Forward 5′-ACCACAGTCCATGCCATCAC-3′ extracted from HUVECs by QIAzol reagent according to Reverse 5′-TCCACCACCCTGTTGCTGTA-3′ the manufacturer’s instructions. Five micrograms of total Gwon et al. Fisheries and Aquatic Sciences (2019) 22:7 Page 4 of 10 with 20 μM DCFH-DA for 30 min. The cells were stim- were estimated to be 7.25 ± 0.53 (μg/mL), 4.55 ± 0.52 ulated with TNF-α (10 ng/mL) for 1 h. The fluorescence (μg/mL), and 2.29 ± 0.13 (μg/mL), respectively. IC levels were determined using a fluorescence microplate values of EtOH, HSS, and EtOAc for the inhibition of reader (Em 485 nm and Ex 528 nm). VCAM-1 were also determined to be 6.67 ± 0.31 (μg/mL), 3.61 ± 0.22 (μg/mL), and 4.53 ± 0.28 (μg/mL), respectively. Immunofluorescence analysis With n-Hexane fractionation, HSS showed 1.6-fold and To assess the translocation of NF-κB, HUVECs were 1.8-fold stronger inhibitory activity on the production of grown on 8-well chamber slides (SPL Life Sciences ICAM-1 and VCAM-1, respectively, than MES. However, Co., Gyeonggi-do, Korea) and stimulated with TNF-α the n-butanol and H O fraction were not inhibited (10 ng/mL) for 30 min after HSS pretreatment for 1 h. ICAM-1 and VCAM-1 production. Although EtOAc frac- The cells were fixed with 4% paraformaldehyde in PBS for tion showed selectively higher inhibitory activity for 15 min. After washing with PBS, the cells were perme- ICAM-1 production, active compounds in the EtOAc abilized with 0.5% Triton X-100 in PBS for 10 min. After were not identified. Thus, we investigated molecular blocking with 3% BSA/PBS for 30 min, cells were mechanisms of HSS on the inhibition of vascular inflam- incubated with an anti-NF-κBantibody for 2h followed mation using TNF-α-stimulated HUVECs. by Alexa Fluor® 488-conjugated secondary antibody for 1 h. Cells were stained with 2 μg/mL DAPI and HSS inhibits the production of TNF-α-induced adhesion images were captured using a confocal microscope. molecules To examine the cytotoxicity of HSS, the viability of Statistical analysis HUVECs was measured by the MTS assay. HUVECs Data are expressed as the means ± standard deviations were cultured in 96-well microplates for 24 h and pre- (SDs). Data analysis was performed using ANOVA treated with various concentrations of HSS (2.5, 5.0, and followed by the Bonferroni test. P values < 0.05 were 10.0 μg/mL) followed by TNF-α-stimulation. The via- considered statistically significant. SPSS for Windows, bility was not influenced up to 10.0 μg/mL of HSS in version 10.07 (SPSS Inc., Chicago, IL, USA) was used for HUVECs (Fig. 1a). Since monocyte adhesion to HUVECs all analyses. is critically regulated by ICAM-1 and VCAM-1, we assessed the production of ICAM-1 and VCAM-1 pro- Results tein using Western blot. The expression levels of both Inhibitory activity of EtOH extract and its solvent-soluble adhesion molecules were remarkably elevated in fractions on ICAM-1 and VCAM-1 production TNF-α-treated HUVECs (Fig. 1b). Pretreatment of HSS In order to evaluate the vascular anti-inflammatory at 5.0 μg/mL notably reduced the production of ICAM-1 potential of S. serratifolium, the EtOH extract and its and VCAM-1 by 66.1% and 52.7%, respectively, which solvent-soluble fractions were tested using TNF-α- shows stronger inhibitory activity for adhesion molecules treated HUVECs. Inhibitory effects (IC ) of these frac- than EtOH extract. To further investigate the effect of tions on the adhesion molecules were determined with HSS on ICAM-1 and VCAM-1 mRNA expression, non-toxic concentration by Western blot using TNF- RT-PCR was performed. Our data showed that HSS α-stimulated HUVECs. As shown in Table 2,IC values dose-dependently suppressed mRNA expression of both of EtOH, HSS, and EtOAc for the inhibition of ICAM-1 adhesion genes in TNF-α-treated HUVECs (Fig. 1c). Furthermore, the result of cell-based ELISA assay showed that HSS suppressed the expression of ICAM-1 Table 2 Inhibitory activity of HSS and its solvent-soluble and VCAM-1 in TNF-α-treated HUVECs in a dose- fractions on the expression of ICAM-1 and VCAM-1 in TNF-α- dependent manner (Fig. 1d). These data indicate that the stimulated HUVECs HSS-mediated downregulation of adhesion molecules in Fractions IC50 (μg/mL) Yields b b (%) TNF-α-treated endothelium is primarily attributable to ICAM-1 VCAM-1 their transcriptional regulation. Ethanolic extract 7.25 ± 0.53 6.67 ± 0.31 100 ± 5.2 n-Hexane 4.55 ± 0.52 3.61 ± 0.22 66.1 ± 4.3 HSS suppresses TNF-α-induced chemokine production Ethyl acetate 2.29 ± 0.13 4.53 ± 0.28 9.23 ± 1.2 Recruitment of monocytes to endothelium is largely reg- n-Butanol ≥ 100 ≥ 100 17.5 ± 1.9 ulated by chemokines, including MCP-1 and KC. As shown in Fig. 2, exposure of TNF-α to HUVECs for 6 h Water ≥ 100 ≥ 100 8.12 ± 1.4 enhanced the production of MCP-1 (Fig. 2a) and KC IC50 is calculated to be concentrations with a half-maximal expression of ICAM-1 and VCAM-1 in TNF-α-stimulated HUVECs (Fig. 2b). HSS pretreatment as low as 2.5 μg/mL inhi- The expression of levels of ICAM-1 and VCAM-1 were determined with bited TNF-α-induced production of both chemokines. Western blot Yield is showing the amount of each fraction in 100 g of ethanolic extract Pretreatment with 10 μg/mL HSS decreased the production Gwon et al. Fisheries and Aquatic Sciences (2019) 22:7 Page 5 of 10 A C Fig. 1 Effect of HSS on the production of TNF-α-induced ICAM-1 and VCAM-1 expression in HUVECs. a Cytotoxic effect of HSS was measured by MTS assay. b Cells pretreated with the indicated concentration of HSS for 1 h were stimulated with TNF-α for 6 h. Equal amounts of total protein were separated by 10% SDS-PAGE. The expression of ICAM-1, VCAM-1, and β-actin was examined by Western blot using corresponding antibodies. c Cells pretreated with the indicated concentration of HSS for 1 h were stimulated with TNF-α for 6 h, and total RNA was prepared for RT-PCR. d Fixed cells with 1% paraformaldehyde were analyzed by ELISA for the cell surface expression of ICAM-1 and VCAM-1. The values are shown as the means ± SDs from three independent experiments. P < 0.05 represents significant differences versus the non-treated group. *P <0.05 represents significant differences versus the TNF-α-only group of both chemokines to control level. This result indicates phosphorylation of IKK-β and led to IκB-α phospho- that HSS suppresses recruitment of monocytes to endothe- rylation, whereas HSS dose-dependently inhibited the lium by inhibition of chemokine production. phosphorylation of both proteins (Fig. 3b). The phos- phorylation level of cytosolic IκB-α at 10 μg/mL HSS HSS inhibits TNF-α-induced NF-κB activation in HUVECs was comparable to the control group possibly due to the To assess whether HSS inhibits the translocation of inhibitory effect of HSS on the phosphorylation IKK-β NF-κB into the nucleus, microscopic analysis was per- (Fig. 3b). In addition, TNF-α-mediated increase in NF-κB formed. Confocal observation indicated that NF-κB/p65 protein level in the nucleus was dose-dependently was predominantly localized in the perinuclear compart- decreased by HSS treatment (Fig. 3b). These results ment under unstimulated condition. With TNF-α treat- suggest that the HSS-mediated inhibition of adhesion ment, most of cytoplasmic NF-κB/p65 translocated to molecules and chemokines are largely regulated by the the nucleus (Fig. 3a). However, HSS pretreatment at NF-κB pathway in the TNF-α-stimulated cells. TNF-α 10 μg/mL markedly inhibited the NF-κB/p65 translo- activates the membrane-bound NADPH oxidase in endo- cation into the nucleus. For further investigating the thelium which stimulates ROS production, a second molecular mechanism underlying HSS-mediated inhi- messenger for NF-κB activation(Kimetal. 2008). We bition of NF-κB/p65 translocation in TNF-α-stimulated examined the inhibitory effects of the HSS on intracellular cells, we examined the phosphorylation of inhibitor κB ROS accumulation in TNF-α-treated HUVECs. TNF-α-in- kinase (IKK)-β and IκB-α that are associated with the duced ROS was markedly reduced by HSS pretreatment NF-κB activation. TNF-α markedly caused the (Fig. 3C, P < 0.05), indicating that intrinsic antioxidant Gwon et al. Fisheries and Aquatic Sciences (2019) 22:7 Page 6 of 10 suppresses vascular inflammation through the induction of HO-1 via Nrf2 activation in TNF-α-treated HUVECs. Discussion Our previous study reported that the ethanolic extract of S. serratifolium suppressed the production of vascular inflammatory proteins in serum and aorta tissue in high cholesterol diet-fed mice. The anti-atherogenic compo- nents in the extract were identified as SHQA, SCM, and SQA which showed strong anti-inflammatory and anti- oxidant activities (Gwon et al. 2018; Lim et al. 2019). The quantification data suggest that the contents of SHQA, SCM, and SQA in 100 g of meroterpenoid-rich extract from S. serratifolium (MES) were estimated to be 37.6 ± 2.1, 6.23 ± 0.36, and 1.89 ± 0.10 g, respectively (Kwon et al. 2018). To obtain a higher concentration of meroterpenoids from MES, we fractionated MES with n-hexane and the content of each component in HSS was comprised to be 52.4 ± 3.3 g for SHQA, 8.26 ± 0.82 g for SCM, and 3.0 ± 0.21 for SQA as determined by cali- bration curves (Lim et al. 2019). In the present study, we found that HSS showed a higher capacity for the sup- pression of ICAM-1, VCAM-1, and MCP-1 expression compared with MES. HSS suppressed the TNF-α-in- duced NF-κB activation by blocking IκB-α degradation. Additionally, HSS alleviated vascular inflammation by the production of HO-1 via Nrf2 activation. To our know- ledge, this is in vitro evidence that HSS is a potent anti-inflammatory agent to alleviate vascular inflammation and to improve endothelial dysfunction. Fig. 2 Effect of HSS on the production of MCP-1 and KC in TNF-α- Adhesion of circulating monocytes to vascular endo- induced HUVECs. Cells pretreated with the indicated concentration of thelium is an initial step in triggering vascular inflamma- HSS for 1 h were stimulated with TNF-α for 24 h. The levels of MCP-1 tion leading to atherosclerosis (Madge and Pober 2001). (a) and KC (b) were examined by ELISA. The values are shown as the means ± SDs from three independent experiments. P <0.05 represents Pro-inflammatory stimuli, such as TNF-α exposure, ini- significant differences versus the control group. *P < 0.05 represents tiate the expression of adhesion molecules that stimulate significant differences versus the TNF-α-only group the attachment of circulating lymphocytes and mono- cytes to endothelium (Packard and Libby 2008). In this activity of HSS is associated with inhibiting NF-κBacti- respect, the adhesion molecules such as VCAM-1 and vation through scavenging ROS induced by TNF-α ICAM-1 are well-known vascular inflammatory markers, treatment. which are related to tight adhesion of monocytes in atherosclerotic lesion of the endothelium (Packard and Libby 2008). Both adhesion molecules are largely pro- HSS induces HO-1 production through Nrf2 activation duced in advanced human coronary atherosclerotic Increased endogenous HO-1 provides cellular protection plaque and in experimental atherosclerosis animals against TNF-α-induced ROS. Therefore, we determined (Catapano et al. 2017; Nallasamy et al. 2014). Our results the effect of HSS on the production of HO-1 protein implied that HSS blocked the TNF-α-induced endothelial and mRNA by Western blot and RT-PCR, respectively. production of VCAM-1 and ICAM-1in HUVECs. These HSS treatment caused an increased production of HO-1 data indicate that the vascular anti-inflammatory action of protein and mRNA in a dose-dependent manner HSS on TNF-α-treated cells is mainly regulated by (Fig. 4a). We further determined the effect of HSS on suppressing the production of adhesion molecules. Speci- the production of Nrf2, a key regulator of the HO-1 fically, the current findings indicate that the HSS-mediated expression. As shown in Fig. 4b, HSS induced the decline of adhesion molecules is closely associated with tran- production of Nrf2 determined by Western blot in scriptional regulation of ICAM-1 and VCAM-1 mRNA ex- TNF-α-induced cells. These results implicate that HSS pression in TNF-α-treated HUVECs. Gwon et al. Fisheries and Aquatic Sciences (2019) 22:7 Page 7 of 10 Fig. 3 Effect of HSS on the NF-κB translocation in TNF-α-treated HUVECs. a Cells were pretreated with or without HSS for 1 h followed by TNF-α treatment for 30 min. The subunits of NF-κB/p65 were detected by using anti-NF-κB polyclonal antibody and Alexa Fluor® 488-conjugated secondary antibody. The nuclei were detected by DAPI staining and confocal microscopy was used to capture the images. b Cells were incubated withanindicated concentrationofHSS for1hand exposedtoTNF-α (10 ng/mL) for 30 min. Cytosolic and nucleus fractions were analyzed by Western blotting. c The cells pretreated with the indicated concentrations of HSS for 1 h were stimulated with TNF-α (10 ng/mL) for 30 min. ROS was determined by DCFH-DA with fluorescent analysis. 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 TNF-α-only group Chemokine produced by activated endothelium play overexpression of MCP-1 in vascular tissues promotes pivotal roles for chemotactic attachment of circulating cooperation of monocytes/macrophages and formation monocytes in the intima of the blood vessel. Chemokine of atherosclerotic lesion (Namiki et al. 2002). Chemo- stimulates sturdy adhesion of infiltrated monocytes to kine KC is identified as the first chemokine respon- vascular endothelium which is a noticeable pathological sible for monocyte arrest on early atherosclerotic mark in the early stage of atherogenesis (Chen et al. lesion (Huo et al. 2001). KC stimulates monocyte 2004). Accumulating evidence demonstrated that local adhesion in carotid arteries in the early atherosclerotic Gwon et al. Fisheries and Aquatic Sciences (2019) 22:7 Page 8 of 10 endothelium is mediated at least partially by inhibition of chemokine production. NF-κB is an essential transcription factor associated with the expression of adhesion molecules and chemo- kines (Huang et al. 2018). As discussed above, adhesion of monocyte to endothelium is stimulated through adhe- sion molecules and chemokines, including ICAM-1, VCAM-1, MCP-1, and KC in endothelial cells (De Filippo et al. 2008). The expression of these molecules is increased by NF-κB activation. In unstimulated condi- tions, NF-κB is primarily distributed in the cytoplasm as an inactive complex bound to IκB-α. Excess ROS pro- duced by TNF-α treatment induce the phosphorylation of IKK complex, which leads to the phosphorylation of IκB-α, thereby leading to the degradation of IκB-α and translocation of NF-κB into the nucleus (Madge and Pober 2001; Zhang et al. 2009). Thus, NF-κBis an important target for designing pharmaceutical agents for interfering with the development and progression of atherosclerosis. Although mechanisms underlying HSS- mediated NF-κB regulation are uncertain, the current study indicates that HSS suppresses NF-κB activation by inhibiting proteolytic degradation of IκB-α induced by TNF-α in HUVECs. The confocal microscopic obser- vation indicates that HSS blocked NF-κB translocation into the nucleus induced by TNF-α in HUVECs. There- fore, these results showed the ability of HSS to suppress TNF-α-mediated NF-κB activation in HUVECs. In the current study, we found that HSS treatment remarkably reduced TNF-α-induced ROS production in HUVECs, suggesting that HSS retains strong antioxidant capacity. Hence, it is likely that the reductions of ICAM-1, VCAM-1, MCP-1, and KC expression in HUVECs are mainly related to the regulation of NF-κB pathway through the removal of ROS by HSS’s intrinsic anti- oxidant activity. Nrf2 is the primary transcription factor to regulate anti- Fig. 4 Effect of HSS on HO-1 production and Nrf2 activation in TNF- oxidant proteins including HO-1 which is an enzyme that α-induced HUVECs. a Cells pretreated with the indicated concentration of HSS for 1 h were stimulated with TNF-α for 16 h (RT-PCR) or 24 h catalyzes the degradation of heme to ferric iron, carbon (Western blot). b Cells pretreated with the indicated concentration of monoxide, and biliverdin as reaction products. Resulting HSS for 1 h were stimulated with TNF-α for 30 min. The proteins were biliverdin is enzymatically converted to bilirubin by analyzed by Western blotting using corresponding antibodies. The biliverdin reductase (Ayer et al. 2016). HO-1 metabolites values are shown as the means ± SDs from three independent are known to have antioxidant, anti-inflammatory, and experiments. P < 0.05 represents significant differences versus the control group. *P < 0.05 represents significant differences versus the anti-atherogenic effects (Ishikawa et al. 2001). In vitro TNF-α-only group studies show that overexpression of HO-1 attenuates endothelial dysfunction via suppressing the production of VCAM-1, MCP-1, and macrophage stimulating factor in lesion in mice (Huo et al. 2001). Both MCP-1 and KC are TNF-α-treated vascular endothelial cells (Kawamura et al. crucial chemokines to the progression of atherosclerosis, 2005). Recent studies demonstrate that HO-1 enhances due to their abilities to recruit monocytes to the intima of vascular protective and anti-atherogenic actions, and its the blood vessel (Chen et al. 2004). In this study, HSS expression in endothelial cells attenuates atherosclerosis inhibited the TNF-α-induced production of MCP-1 and (Choi et al. 2018;Kato et al. 2001). In this study, we found KC in TNF-α-treated HUVECs. These results suggest that that HSS pretreatment markedly increased the production the anti-inflammatory action of HSS on TNF-α-induced of HO-1 protein and mRNA which are caused by Gwon et al. Fisheries and Aquatic Sciences (2019) 22:7 Page 9 of 10 translocation of Nrf2 into the nucleus. Thus, enhanced Choi ES, Yoon JJ, Han BH, Jeong DH, Lee YJ, Kang DG, Lee HS. Ligustilide attenuates vascular inflammation and activates Nrf2/HO-1 induction and, NO HO-1 expression by HSS attributes to the removal of synthesis in HUVECs. Phytomedicine. 2018;38:12–23. ROS induced by TNF-α treatment. This study appears De Filippo K, Henderson RB, Laschinger M, Hogg N. Neutrophil chemokines KC to be the first finding to address characteristics of HSS in and macrophage-inflammatory protein-2 are newly synthesized by tissue macrophages using distinct TLR signaling pathways. J Immunol. 2008;180:4308–15. the inhibition of vascular inflammation in TNF-α-induced Gwon WG, Joung EJ, Kwon MS, Lim SJ, Utsuki T, Kim HR. Sargachromenol HUVECs. protects against vascular inflammation by preventing TNF-α-induced monocyte adhesion to primary endothelial cells via inhibition of NF-κB activation. Int Immunopharmacol. 2017;42:81–9. Conclusions Gwon WG, Joung EJ, Shin T, Utsuki T, Wakamatsu N, Kim HR. Meroterpinoid-rich In summary, we have shown that HSS inhibits the fraction of the ethanol extract from Sargassum serratifolium suppresses mRNA and protein expression of adhesion molecules TNF-α-induced monocytes adhesion to vascular endothelium and vascular inflammation in high cholesterol-fed C57BL/6J mice. J Func including ICAM-1 and VCAM-1 in TNF-α-stimulated Foods. 2018;46:384–93. HUVECs. Furthermore, the vascular anti-inflammatory Gwon WG, Lee B, Joung EJ, Choi MW, Yoon N, Shin T, Oh CW, Kim HR. roles of HSS in TNF-α-treated cells appear to be closely Sargaquinoic acid inhibits TNF-α-induced NF-κB signaling, thereby contributing to decreased monocyte adhesion to human umbilical vein related to the inactivation of the NF-κB as well as the endothelial cells (HUVECs). J Agric Food Chem. 2015;63:9053–61. activation of Nrf2 pathways. Further confirmation of Huang W, Huang M, Ouyang H, Peng J, Liang J. Oridonin inhibits vascular vascular anti-inflammatory effects of HSS and under- inflammation by blocking NF-κB and MAPK activation. Eur J Pharmacol. 2018;826:133–9. lying mechanisms in animal models will be important Huo Y, Weber C, Forlow SB, Sperandio M, Thatte J, Mack M, Jung S, Littman DR, for the therapeutic application of HSS for treating Ley K. The chemokine KC, but not monocyte chemoattractant protein-1, vascular inflammation-associated diseases. triggers monocyte arrest on early atherosclerotic endothelium. J Clin Invest. 2001;108:1307–14. Acknowledgements Ishikawa K, Sugawara D, Wang X, Suzuki K, Itabe H, Maruyama Y, Lusis AJ. Heme This work was supported by a Research Grant of Pukyong National University oxygenase-1 inhibits atherosclerotic lesion formation in ldl-receptor knockout (2017). mice. Circ Res. 2001;88:506–12. Joung EJ, Gwon WG, Shin T, Jung BM, Choi J, Kim HR. Anti-inflammatory action Authors’ contributions of the ethanolic extract from Sargassum serratifolium on lipopolysaccharide- WGG carried out the experiments and participated to write the manuscript. stimulated mouse peritoneal macrophages and identification of active SGL and JIK corrected the manuscript. YMK and SBK analyzed the content of components. J Appl Phycol. 2017;29:563–73. meroterpenoids in HSS. HRK designed the study. All authors read and Joung EJ, Lee B, Gwon WG, Shin T, Jung BM, Yoon NY, Choi JS, Oh CW, Kim HR. approved the final manuscript. Sargaquinoic acid attenuates inflammatory responses by regulating NF-κB and Nrf2 pathways in lipopolysaccharide-stimulated RAW 264.7 cells. Int Competing interests Immunopharmacol. 2015;29:693–700. The authors declare that they have no competing interests. Kato H, Amersi F, Buelow R, Melinek J, Coito AJ, Ke BBRW, Kupiec-Weglinski JW. Heme oxygenase-1 overexpression protects rat livers from ischemia/ reperfusion injury with extended cold preservation. Am J Transplantation. Publisher’sNote 2001;1:121–8. Springer Nature remains neutral with regard to jurisdictional claims in Kawamura K, Ishikawa K, Wada Y, Kimura S, Matsumoto H, Kohro T, Itabe H, published maps and institutional affiliations. Kodama T, Maruyama Y. Bilirubin from heme oxygenase-1 attenuates vascular endothelial activation and dysfunction. Arterioscler Thromb Vasc Author details 1 Biol. 2005;25:155–60. Department of Food Science and Nutrition, Pukyong National University, 45, Kim JH, Na HJ, Kim CK, Kim JY, Ha KS, Lee H, Chung HT, Kwon HJ, Kwon YG, Kim Yongso-Ro, Nam-Gu, Busan 48513, Republic of Korea. Department of Food YM. The non-provitamin A carotenoid, lutein, inhibits NF-κB-dependent gene Technology, Pukyong National University, 45, Yongso-Ro, Nam-Gu, Busan expression through redox-based regulation of the phosphatidylinositol 3- 48513, Republic of Korea. kinase/PTEN/Akt and NF-κB-inducing kinase pathways: role of H O in NF-κB 2 2 activation. Free Rad Biol Med. 2008;45:885–96. Received: 22 November 2018 Accepted: 18 February 2019 Kleinbongard P, Heusch G, Schulz R. TNFα in atherosclerosis, myocardial ischemia/reperfusion and heart failure. Pharmacol Ther. 2010;127:295–314. Kwon M, Lim SJ, Joung EJ, Lee B, Oh CW, Kim HR. 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