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ARTICLE Extract from Acanthopanax senticosus prevents LPS-induced monocytic cell adhesion via suppression of LFA-1 and Mac-1 Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by University of Waterloo on 05/30/14 For personal use only.

Hyun Jeong Kim, Danielle McLean, Jaeho Pyee, Jongmin Kim, and Heonyong Park

Abstract: A crude extract from Acanthopanax senticosus (AS) has drawn increased attention because of its potentially beneficial activities, including anti-fatigue, anti-stress, anti-gastric-ulcer, and immunoenhancing effects. We previously reported that AS crude extract exerts anti-inflammatory activity through blockade of monocytic adhesion to endothelial cells. However, the underlying mechanisms remained unknown, and so this study was designed to investigate the pathways involved. It was confirmed that AS extract inhibited lipopolysaccharide (LPS)-induced adhesion of monocytes to endothelial cells, and we found that whole extract was superior to eleutheroside E, a principal functional component of AS. A series of PCR experiments revealed that AS extract inhibited LPS-induced expression of genes encoding lymphocyte function-associated antigen-1 (LFA-1) and macrophage-1 antigen (Mac-1) in THP-1 cells. Consistently, protein levels and cell surface expression of LFA-1 and Mac-1 were noticeably reduced upon treatment with AS extract. This inhibitory effect was mediated by the suppression of LPS-induced degradation of I␬B-␣, a known inhibitor of nuclear factor-␬B (NF-␬B). In conclusion, AS extract exerts anti-inflammatory activity via the suppression of LFA-1 and Mac-1, lending itself as a potential therapeutic galenical for the prevention and treatment of various inflammatory diseases. Key words: Acanthopanax senticosus extract, LFA-1, Mac-1, I-␬B, cell adhesion. Résumé : L'extrait brut d'Acanthopanax senticosus (AS) a été l'objet d'une attention croissante a` cause des ses activités bénéfiques potentielles, incluant des effets antifatigue, anti-stress, contre les ulcères gastriques et immuno-stimulateurs. Nous avions précédemment rapporté que l'extrait brut d'AS exerce une activité anti-inflammatoire par le blocage de l'adhésion des monocytes aux cellules endothéliales. Cependant, les mécanismes sous-jacents demeurent inconnus; conséquemment, l'étude présente a été conçue afin d'examiner les sentiers impliqués. Il a été confirmé que l'extrait d'AS inhibait l'adhésion des monocytes aux cellules endothéliales induite par le LPS, et nous avons trouvé que l'extrait total était supérieur a` l'éleuthéroside E, la principale composante fonctionnelle d'AS. Une série d'expériences par PCR a révélé que l'extrait d'AS inhibait l'expression induite par le LPS de gènes codant LFA-1 (lymphocyte function-associated antigen-1) et Mac-1 (macrophage-1 antigen) chez les cellules THP-1. En conséquence, les niveaux protéiques et l'expression a` la surface cellulaire de LFA-1 et Mac-1 étaient réduits de manière importante a` la suite du traitement a` l'extrait d'AS. L'effet inhibiteur était dépendait de la suppression de la dégradation d'I␬B-␣ induite par le LPS, un inhibiteur connu du facteur nucléaire NF-␬B. En conclusion, l'extrait d'AS exerce une activité antiinflammatoire par la suppression de LFA-1 et de Mac-1, ce qui en fait un médicament galénique potentiel pour la prévention et le traitement de différentes maladies inflammatoires. [Traduit par la Rédaction] Mots-clés : extrait d'Acanthopanax senticosus, LFA-1, Mac-1, I␬B, adhésion cellulaire.

Introduction Acanthopanax senticosus (AS; Siberian ginseng) belongs to the plant family Araliaceae and has been widely used as a traditional medicinal plant in East Asia (Davydov and Krikorian 2000). In recent decades, a number of phytochemical, biological, and clinical studies on AS have been reported. The stem, root, and fruit of AS contain many chemical constituents such as volatile compounds, triterpenoid saponins, lignans, coumarins, flavones, polysaccharides, and eleutheroside E. Among those compounds, eleutheroside E (EE) is functionally the principal constituent of AS, conferring antiinflammatory properties (Davydov and Krikorian 2000; Tokiwa et al. 2006; Huang et al. 2011). AS extracts have also been reported to have anti-ulcer, anti-allergic, anti-oxidant (Fujikawa et al. 1996; Nishibe et al. 1990; Yi et al. 2001, 2002; Yokozawa et al. 2003), and anti-cancer properties (Yoon et al. 2004; Choi et al. 2008; Lin et al. 2008). As a result, AS extracts have been used pharmaceutically to

prevent various diseases including diabetes mellitus, chronic bronchitis, tumors, hypertension, and ischemia (Nishibe et al. 1990; Fujikawa et al. 1996; Davydov and Krikorian 2000; Yi et al. 2001). However, despite studies showing pharmaceutical functionality, there are very few reports concerning the underlying mechanisms. Homo- and hetero-typic types of cell adhesion are vital in the generation of an effective immune response, and cell-adhesion molecules in circulating monocytes are key mediators of the inflammatory processes (Bevilacqua et al. 1994). Firm adhesion of monocytes to the endothelium and transmigration of monocytes across vascular endothelial cells are key early events in physiological and pathophysiological processes including inflammation and atherosclerosis (Bevilacqua et al. 1994; Gimbrone et al. 1997). During the initial phases of inflammation, the adhesion of quiescent monocytes to the vascular endothelium is mediated by the

Received 19 October 2013. Accepted 9 January 2014. H.J. Kim, J. Pyee, and H. Park. Department of Molecular Biology & Institute of Nanosensor and Biotechnology, Dankook University, 126, Jukjeon-dong, Suji-gu, Yongin-si, Gyeonggi-do 448-701, Korea. D. McLean. Cardiovascular Research Institute, University of Vermont, 208 South Park Drive, Colchester, VT 05446, USA. J. Kim. Department of Life Systems, Sookmyung Women's University, 52 Hyochangwon-gil, Yongsan-gu, Seoul 140-742, Korea. Corresponding authors: Jongmin Kim (e-mail: [email protected]) and Heonyong Park (e-mail: [email protected]). Can. J. Physiol. Pharmacol. 92: 278–284 (2014) dx.doi.org/10.1139/cjpp-2013-0392

Published at www.nrcresearchpress.com/cjpp on 13 January 2014.

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carbohydrate ligand on monocytes (Phillips et al. 1990; Walz et al. 1990) and endothelial selectin (E-selectin) on vascular endothelium (Bevilacqua et al. 1987; Butcher 1991). Following this, monocytes are activated and the interaction of monocyte integrin beta 2 family members such as activated lymphocyte function-associated antigen-1 (LFA-1) and (or) macrophage-1 antigen (Mac-1) with endothelial adhesion molecules promotes firm cell–cell adhesion (Hmama et al. 1999; Kounalakis and Corbett 2006). Although constitutively expressed at a low level, up-regulation of these adhesion molecules is stimulated in a wide variety of cells by pro-inflammatory cytokines, as well as by activity of mitogen-activated protein kinases (MAPKs) and nuclear factor-␬B (NF-␬B) (Phillips et al. 1990; Aoudjit et al. 1997; Capodici et al. 1998; Hill et al. 2008), key signaling molecules in inflammatory pathways. In this study, we determined the underlying mechanisms by which AS extract plays a critical role in anti-inflammatory processes. These findings clarify the functional properties of AS extract, thereby providing insight into the potential benefits to human health. These mechanistic studies promote the wider utilization of AS extract as a more specific functional food and (or) a pharmaceutical.

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microscope (Zeiss Autoplan 2) and then counted for quantification using Alpha Ease FC version 3.2.1 (Alpha Innotech).

Materials and methods

RT–PCR THP-1 cells were treated with LPS (1 ␮g/mL) alone, or in the presence of AS extract (100 ␮g/mL), or EE (100 ng/mL). Then, total RNA was extracted from THP-1 using QIAzol Lysis Reagent (QIAZEN). Following reverse transcription of RNA with M-MLV Reverse Transcriptase (Promega), cDNA was amplified by PCR reaction with Taq DNA Polymerase (Biolabs) under the following conditions: denaturation at 98 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 60 s (35 cycles). Primers employed for amplification were as follows; LFA-1 gene: sense, 5=-CATCCACGACCACAACAT-3=; antisense, 5=-CTCCTGCCTG AAGACAAC-3=. Mac-1 gene: sense, 5=-AGAACAACATGCCCAGAA CC-3=; antisense, 5=-GCGGTCCCATATGACAGTCT-3=. Integrin ␤2 gene: sense, 5=-CAAGCTGGCTGAAAACAACA-3=; antisense, 5=ATTGCTGCAGAAGGAGTCGT-3=. PAF receptor gene: sense, 5=ATCAACACCTACTGCTCTG-3=; antisense, 5=-GCTGAACACG ATGAAGATG-3=. L-selectin gene: sense, 5=-AAACCCATGAACTG GCAAAG-3=; antisense, 5=-CGCAGTCCTCCTTGTTCTTC-3=. GAPDH gene: sense, 5=-CCAACGTGTCTGTTGTGGATCTGA-3=; antisense, 5=CAACCTGGTCCTCAGTGTAGCCTA-3=.

Preparation of Acanthopanax senticosus extract AS extract was obtained from Lifetree Biotech. Briefly, AS extract was produced by a series of processes, such as mixing and shaking 6% (w/v) AS in distilled water at 60 °C, separating the supernatant by centrifugation (6200g, 15 min), and filtering the supernatant. Dried AS extract was found to contain about 0.9 mg/g of eleutheroside E.

Preparation of cell lysate THP-1 cells were washed in ice-cold PBS and then lysed by a 1 h rocking incubation in RIPA buffer (50 mmol/L Tris, pH 7.5, 150 mmol/L NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 1 mmol/L phenylmethylsulfonyl fluoride). Soluble lysates were fractionated by centrifugation. Total protein content of soluble cell lysate was measured using the BCA assay (Intron).

Cell culture THP-1 cells isolated from human monocytic leukemia were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (Welgene) and antibiotics (penicillin/streptomycin). The culture was maintained at 37 °C in humidified air containing 5% CO2. Bovine aortic endothelial cells (BAECs) obtained from the bovine descending thoracic aorta were cultured in DMEM (glucose 1 g/L; Welgene) containing 20% fetal bovine serum (Welgene) and antibiotics (penicillin/streptomycin) at 37 °C with 5% CO2 (Jo et al. 1991). For all experiments, BAECs were used at passages 7 to 10.

Western blotting Proteins (10⬃25 ␮g) in the soluble lysates were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride membrane (Millipore), and blotted with antibodies specific to LFA-1 (CD11a, abcam), Mac-1 (CD11b, abcam), Tubulin, I␬B-␣, p-ERK, ERK, p-p38, and p38 (Cell Signaling Technology). Subsequently, the membranes were incubated with HRP-conjugated secondary antibodies and developed using an enhanced chemiluminescence detection method (Amersham).

Cell viability assay Cell cytotoxicity was measured using a commercial cell viability assay kit (Daeil LabService, Seoul, Korea). According to the manufacturer's protocol, THP-1 cells (1 × 105/mL) were treated with various concentrations of the AS extract or EE for 24 h, followed by 1 h incubation with WST-1. In viable cells, WST-1 was reduced by mitochondrial dehydrogenases, thus producing formazan, a water-soluble orange dye. Consequently, the amount of formazan represents the viable cell number, and so the relative cell viability was calculated after measuring absorbance at 450 nm with an ELISA plate reader (Model 550; Bio-Rad).

Flow cytometry THP-1 cells were treated with LPS (1 ␮g/mL) alone or in the presence of AS extract (100 ␮g/mL) for 24 h. Then, cells were fixed using 0.5% paraformaldehyde (10 min) and permeabilized with 0.1% Tween 20–PBS for 20 min. The cells were then incubated in 10% bovine serum albumin–PBS to block nonspecific binding. Subsequently, cells were immunostained by incubation with antiLFA-1 or anti-Mac-1 antibodies (1/200 dilution; Abcam) for 30 min at room temperature followed by Alexa-Fluor 555 antibody (1/500 dilution; Invitrogen) for 30 min at room temperature. Fluorescently stained cells were detected by flow cytometry (Guava easyCyte; Millipore). Data were analyzed using Guavasoft version 2.5.

Adhesion assay THP-1 cells were treated with 1 ␮g/mL lipopolysaccharide (LPS; Sigma–Aldrich) for 24 h alone, or in the presence of AS extract, or eleutheroside E (EE; Sigma–Aldrich). For the signaling study, the cells were pretreated with SB202190 (50 ␮mol/L; ENZO life science) or MG-132 (50 ␮mol/L; Sigma–Aldrich) for 30 min before LPS treatment. The LPS-treated THP-1 cells were stained with 2.5⬃10 ␮mol/L calcein AM (Sigma–Aldrich) for 45 min at 37 °C and then thoroughly washed with phosphate buffered saline (PBS) to remove free forms of calcein AM. Stained THP-1 cells (5 × 105/mL) were then incubated with confluent BAEC cultures for 1 h and washed twice with PBS to remove non-adherent THP-1 cells. Finally, the adherent THP-1 cells were observed under a fluorescent

Results Acanthopanax senticosus extract and eleutheroside E did not exhibit in vitro cytotoxicity against THP-1 cells We first examined whether the extract of Acanthopanax senticosus (AS) and eleutheroside E (EE), an active lignan component of AS, have a cytotoxic effect on THP-1 cells. THP-1 cells were treated with various concentrations of the AS extract and EE for 24 h and cell viability was monitored by conversion of WST-1 to formazan. As shown in Fig. 1, the cells remained viable when exposed to up to 100 ␮g/mL AS extract and 100 ng/mL EE, indicating that neither agent exhibited cytotoxicity. Published by NRC Research Press

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Fig. 1. Effect of Acanthopanax senticosus (AS) extract and eleutheroside E on THP-1 cell viability. THP-1 cells were treated with various doses of AS extract and eleutheroside E (EE) for 24 h. Cell viability was determined using the MTT assay. Values are the mean ± SE, n = 3.

Inhibition of LPS-induced THP-1 cell adhesion to BAECs by AS extract and eleutheroside E Monocytic adhesion to the endothelium plays a central role in the early inflammatory response. Accordingly, we investigated the effects of AS extract and EE on adhesion of LPS-induced THP-1 cells to BAECs. LPS-induced THP-1 adhesion was markedly inhibited (by 60%) by AS extract, within the 10 to 100 ␮g/mL treatment range, whereas EE only elicited a 10% inhibition (Figs. 2A and 2B). We also confirmed these data in a second monocytic cell line, U937 cells (Data not shown). These data reveal that the inhibitory activity of AS extract is much greater than that of EE, suggesting that the use of whole AS extract may confer more potent antiinflammatory activity. AS extract markedly inhibits mRNA expression of LPS-induced LFA-1 and Mac-1 in THP-1 cells Given that monocyte adhesion to endothelial cells is largely controlled by the expression of cell adhesion molecules (CAMs), it is plausible that AS extract and EE regulate this expression, crucially modifying monocytic adhesion to endothelial cells. Previously it was reported that T cell binding to endothelial cells was mediated by 5 important molecules, including PAF receptor, LFA-1, Mac-1, integrin ␤2, and L-selectin (Hynes and Lander 1992). Therefore, we examined the effects of AS extract and EE on the expression of these 5 CAMs by RT–PCR analysis. As shown in Fig. 3, the mRNA levels of the 5 CAMs were increased by 1 ␮g/mL LPS. Inhibitory experiments with AS extract and EE showed that LFA-1 and Mac-1 mRNAs were down-regulated by treatment with AS extract, whereas EE only had a minimal effect on the mRNA expression of Mac-1 (Figs. 3A and 3B). These expressional data are consistent with the monocytic adhesion data, where EE only produced a minimal inhibitory effect on THP-1 cell adhesion to BAECs (see Fig. 2). These data suggest that the anti-adhesion properties of AS extract stem from the additional constituents contained in the whole extract, rather than from the EE component alone. Reduction of LFA-1 and Mac-1 protein and their surface expression by AS extract in LPS-activated THP-1 cells We next assessed whether AS extract regulates the protein levels of LFA-1 and Mac-1 in LPS-activated THP-1 cells. Treatment of THP-1 with LPS caused a significant increase in the protein expression levels of LFA-1 and Mac-1. However, LPS-enhanced protein levels were shown to be significantly diminished by treatment with AS extract (Figs. 4A and 4B). These Western blotting data are consistent with the RT–PCR data shown in Fig. 3. Then, we further examined whether these decreases in cellular protein expression led to a decline in cell surface expression. As shown in Figs. 4C and

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4D, AS extract at a concentration of 100 ␮g/mL significantly reduced the LPS-induced cell surface expressions of LFA-1 and Mac-1. Attenuation of LPS-induced I␬B-␣ degradation and p38 phosphorylation by AS extract To determine the downstream signal transduction pathways underlying the inhibition of LPS-induced cellular adhesion by exposure to AS extract, we examined the activity of MAPK family members, extracellular signal-regulating kinase (ERK), c-Jun N-terminal kinase (JNK), p38 MAPK, and I␬B-␣, key signaling molecules in the heterotypic adhesion pathway (Phillips et al. 1990; Aoudjit et al. 1997; Capodici et al. 1998; Hill et al. 2008). As shown in Fig. 5A, LPS-induced phosphorylation of p38 MAPK was decreased by the AS extract, whereas ERK and JNK phosphorylation was not altered. Additionally, it was shown that the level of total I␬B-␣ protein was markedly and transiently reduced by LPS, and this LPS-induced degradation of I␬B-␣ was dramatically blocked by treatment with AS extract (Fig. 5B). Degradation of I␬B-␣ is necessary to release NF-␬B from the cytoplasmic NF-␬B–I␬B-␣ complex and allow its subsequent translocation to the nucleus of the cell. Therefore, our data suggest that AS extract inhibits the NF-␬B pathway through inhibition of LPS-induced degradation of I␬B-␣. Inhibition of LPS-activated THP-1 cell adhesion to BAECs by AS extract occurs via inhibition of degradation of I␬B-␣ (NF-␬B activation) Finally, we investigated the signaling molecules that play a role in the inhibition of heterotypic adhesion by AS extract. Since we previously determined that AS extract inhibited LPS-activated p38 MAPK and NF-␬B, we examined which signaling molecules are important in LPS induced THP-1 cell adhesion through the use of inhibitors of p38 MAPK and NF-␬B. As shown in Figs. 6A and 6B, LPS-induced THP-1 cell adhesion was inhibited by pretreatment of cells with MG-132, which specifically blocks the activation of NF-␬B through inhibition of I␬B-␣ degradation (Zanotto-Filho et al. 2010), AS extract produced a similar effect. However the p38 MAPK inhibitor, SB202190 (SB), had no effect on LPS-induced THP-1 cell adhesion. LPS-induced I␬B-␣ degradation was returned to near control levels by pretreatment of cells with MG-132, while SB202190 had no effect (Fig. 6C). Taken together, our data conclude that AS extract inhibits the NF-␬B pathway, thereby downregulating the LPS-activated THP-1 cell adhesion to BAECs.

Discussion Much attention has recently been given to the use of plant extracts as therapeutic agents for a variety of human diseases (Dattner 2003; Huang et al. 2011). A series of studies have shown the beneficial functions associated with extracts of AS. AS extract, a traditional medicine with a long therapeutic history, has been used clinically for the treatment of several diseases including gastric ulcers, ischemic heart disease, hypertension, rheumatism, and allergy. The protective effects of AS extract such as anti-stress, anti-tumor, anti-hyperglycemia, and anti-arrhythmia have also been reported from animal experiments (Nishibe et al. 1990; Fujikawa et al. 1996; Davydov and Krikorian 2000; Yi et al. 2001). Despite these reported pharmaceutical efficacies, little is known about the detailed mechanisms by which AS extract dampens inflammatory diseases. Therefore, in this study, we explored the molecular and cellular mechanisms underpinning the therapeutic activity of AS extract in inflammatory diseases. Through a series of experiments using an AS extract, our present data have shown that AS extract crucially regulates antiinflammatory functions. Importantly, the AS extract-induced blockade of inflammatory pathways occurs in the initial stages of the inflammatory process, i.e., the adhesion of monocytes to endothelial cells. This inhibitory effect may be pharmaceutically significant, as interference with inflammatory pathways has drawn considerable attention as a potential therapeutic strategy Published by NRC Research Press

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Fig. 2. Inhibition of lipopolysaccharide (LPS)-induced adhesion of THP-1 to BAECs by Acanthopanax senticosus (AS) extract and eleutheroside E (EE). THP-1 cells were treated with 1 ␮g/mL LPS along with various doses of AS extract and EE for 24 h. Adherent THP-1 cells were observed under fluorescence microscopy. (A) Images are representative of at least 3 different observations. (B) Dose–response curve of LPS-induced cell adhesion in the absence or presence of AS extract and EE. Values are the mean ± SE, n = 3; *, P < 0.05; **, P < 0.001.

Fig. 3. Effect of Acanthopanax senticosus (AS) extract and eleutheroside E (EE) on mRNA expressions of cell adhesion molecules in lipopolysaccharide (LPS)-activated THP-1 cells. (A) THP-1 cells were treated with 1 ␮g/mL LPS alone, or in the presence 100 ␮g/mL AS extract, or 100 ng/mL EE for the indicated periods of time and subjected to RT–PCR analysis. (B) Each band was quantified by densitometry. Data are expressed as ratios of adhesion molecule mRNA normalized to GAPDH mRNA (mean ± SE, n = 3). *, P < 0.05; **, P < 0.001.

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Fig. 4. Effect of Acanthopanax senticosus (AS) extract on the protein levels and surface expressions of LFA-1 and Mac-1 in LPS activated THP-1 cells. THP-1 cells were incubated for 24 h with 1 ␮g/mL lipopolysaccharide (LPS) alone or in the presence of 100 ␮g/mL AS extract. (A) Cellular protein levels of LFA-1, Mac-1, and tubulin were determined by Western blotting analysis. (B) The values for the densitometry data are the mean ± SE, n = 3; *, P < 0.05; **, P < 0.01. (C) Cells were treated with LPS alone or in the presence of 100 ␮g/mL AS extract and LFA-1 and Mac-1 surface expression was detected by FACS analysis. (D) The values for the FACS data are the mean ± SE, n = 3; *, P < 0.001.

Fig. 5. The inhibitory effect of Acanthopanax senticosus (AS) extract on phosphorylation of p38 and degradation of I␬B-␣ in lipopolysaccharide (LPS) activated THP-1 cells. Serum-depleted THP-1 cells were treated with 1 ␮g/mL LPS along with AS extract for various periods of time. Western blotting was performed with p-ERK, ERK, p-JNK, JNK, p-p38 MAPK, and p38 MAPK antibodies (A) as well as I␬B-␣ antibodies (B). Each band was quantified by densitometry. Line graphs show the mean ± SE, n = 3; *, P < 0.05; **, P < 0.001.

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Fig. 6. Effect of NF-␬B inhibitor on lipopolysaccharide (LPS)-induced THP-1 adhesion to BAECs and degradation of I␬B-␣. (A) THP-1 cells were pretreated with SB202190 or MG-132 for 30 min before LPS treatment (1 ␮g/mL LPS for 24 h). Stimulated cells were then incubated with BAEC for an additional 1 h and adherent THP-1 cells were observed under a fluorescent microscope. (B) Adherent THP-1 cells were quantified and plotted; values are the mean ± SE, n = 3; *, P < 0.01. (C) Western blotting was performed with I␬B-␣ antibodies. Each band was quantified by densitometry and plotted; values are the mean ± SE, n = 3; *, P < 0.01.

(Riedemann and Ward 2002). Mechanistically, it is established that expression of CAMs such as L-selectin, LFA-1, and Mac-1 on monocytes plays an important role in this inflammatory cascade (Bevilacqua et al. 1987; Butcher 1991; Hmama et al. 1999; Kounalakis and Corbett 2006). Based on this established concept, we assessed the expression levels of CAMs known to be crucial for monocytic adhesion to endothelial cells. By this assessment, we found that AS extract inhibits expression of LFA-1 and Mac-1, thereby diminishing LPS-induced THP-1 cell adhesion. Given that LFA-1 and Mac-1 are essential for the interaction between monocytes and endothelial cells, these findings are consistent with known mechanisms. It is also important to note that the whole AS extract, containing many constituents, provides a superior inhibitory effect when compared with EE, an active lignan component of AS. It remains to be identified whether other components synergistically promote the inhibitory activity of EE, or whether the AS extract may contain other unknown active compound(s). In this study, we further determined the specific signaling pathways that are responsible for AS-mediated down-regulation of LFA-1 and Mac-1 expression. Among various MAPK pathways, LPS induction of p38 MAPK was inhibited by AS extract, whereas ERK and JNK induction were not affected. However, it was found that p38 MAPK does not play a role in the LPS-induced THP-1 cell adhesion. To further explore this issue we focused on another target signaling molecule, NF-␬B, in part due to our finding that AS extract blocked LPS-induced degradation of I␬B-␣. Given that LPS is known to induce degradation of I␬B-␣ and consequently activate NF-␬B (Hayden and Ghosh 2004), the AS extract-induced inhibition of I␬B-␣ degradation thus promotes the inhibition of LPS-induced activation of NF-␬B. Consistently, we further determined that an inhibitor of NF-␬B markedly reversed LPS-promoted THP-1 cell adhesion to endothelial cells. These data suggest that AS extract plays a critical role in LPS-induced THP-1 cell adhesion by

regulating the activity of NF-␬B; however, regulation of P38 MAPK by AS extract may be significant in other cellular functions. Finally, it is critical to note that the range (10⬃100 ␮g/mL) of AS extract concentrations employed in this study were physiologically relevant. A recent pharmacokinetic study in rats demonstrated that plasma concentrations of EE were in the range of 20⬃70 ng/mL up to 8 h, and 500⬃5000 ng/mL up to 2 h after a single intravenous administration (Ma et al. 2013). Based on this and other data showing that the dried stem of AS contains less than 0.1% (w/w) EE (Kang et al. 2001), the effective concentrations of the AS extract used in this study are comparable with in-vivo studies. In conclusion, this study provides evidence for the following mechanisms: (i) AS extract inhibits LPS-induced THP-1 adhesion to BAECs; (ii) AS extract reduces the expression levels of LFA-1 and Mac-1 in LPS activated-THP-1; and (iii) AS extract inhibits NF-␬B activity, thereby preventing LPS-induced THP-1 cell adhesion. Taken together, there is strong evidence to suggest that AS extract could be utilized as a phytotherapeutic agent for the prevention of inflammatory diseases.

Acknowledgements This study was carried out with the support of “Forest Science & Technology Projects (Project No. S111113L010110)” provided by the Korean Forest Service.

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