Introduction
Numerous chronic diseases are known to involve the pathogenesis of inflammation, including atherosclerosis, Alzheimer's disease and Parkinson's disease (1). The development of atherosclerosis occurs as a response to injury of the vessel wall (2,3). Endothelial inflammation involves the recruitment of circulating monocytes by cells using a combination of chemokines and cell surface adhesion molecules, such as monocyte chemotactic protein-1 and intercellular cell adhesion molecule-1 (ICAM-1) (3). The inflammatory cytokine tumor necrosis factor (TNF)-α activates the nuclear factor-κB (NF-κB) pathway, which serves a key role in the expression of cell adhesion molecules in endothelial cells (ECs) (2,4). Following stimulation with TNF-α, a kinase cascade induces subsequent inhibitor of κB (IκB) phosphorylation, which rapidly degrades, resulting in the released NF-κB translocating to the nucleus to regulate gene transcription (5).
The prenylated stilbenoids arachidin-1 and arachidin-3 are produced in germinated peanut roots (6). They are prenylated analogs of piceatannol and resveratrol, respectively. Resveratrol and piceatannol are considered to have a number of beneficial effects, including anticancer, anti-atherogenic, anti-oxidative, anti-inflammatory, antimicrobial and estrogenic activity (7). Their prenylated analogs are less studied but display a wide spectrum of biological activity. Arachidin-1 has been reported to possess various pharmacological activities, including antioxidative, anti-inflammatory, anti-virus infection and antitumorigenic effects (7–10). However, to date it is not clear if arachidin-1 also has anti-inflammatory and anti-oxidative effects in ECs. Since arachidin-1 has been demonstrated to be pharmacologically active, the detailed mechanism by which arachidin-1-modulates inflammation in ECs requires further elucidation.
Numerous phytochemicals are electrophilic, a property that has been reported to result in nuclear factor-E2-related factor-2 (Nrf-2) activation and the induction of phase II detoxifying enzyme expression (11,12). Results from in vitro and in vivo studies have provided evidence that electrophilic stress induces the complexation of Nrf-2 with Kelch-like ECH-associated protein 1 (Keap1), enabling Nrf-2 to escape from Keap1-mediated ubiquitination and degradation (13). As a result, Nrf-2 translocates to the nucleus to form heterodimers with small Maf proteins and binds to the antioxidant response element (ARE) in the promoter region of phase II detoxifying enzymes and antioxidant enzymes, including quinone oxidoreductase and heme oxygenase-1 (HO-1) (11,13). In addition, Nrf-2 has been shown to control the expression of a number of thiol-regulating enzymes, including glutathione S-transferase (GTP), glutamyl-cysteine synthetase, thioredoxin and thioredoxin reductase (14). Increased levels of these intracellular thiol-containing proteins may provide cytoprotective effects under conditions of oxidative stress or inflammation. A previous study conducted by the present research team demonstrated that piceatannol, an analog of arachidin-1, induced the expression of the Nrf-2- associated gene HO-1 (15). This phenomenon has broad potential implications regarding the inhibitory effects of arachidin-1 upon inflammation in association with Nrf-2-associated enzymes. For these reasons, the present study investigated Nrf-2 associated pathways in order to elucidate the mechanisms underlying the cytoprotective effects of arachidin-1.
Materials and methods
Materials
The p3xARE/Luc vector was constructed as described previously (16). Luciferase assay kits were purchased from Promega Corporation (Madison, WI, USA). Antibodies against HO-1 (SPA-896) and p65 (KAS-TF110) were purchased from Stressgen Biotechnologies (San Diego, CA, USA). ICAM-1 (sc-7891), IκBα (sc-847), Lamin B1 (sc-56143), α-tubulin (sc-53646) and Nrf2 (sc-722) antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). TrxR1 antibody (07-613) was purchased from Upstate (Charlottesville, VA, USA). IκBα and Nrf-2 antibodies were obtained from Santa Cruz Biotechnology, Inc. Bacterially derived TNF-α was purchased from Calbiochem (EMD Millipore; Billerica, MA, USA). All other reagents were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany).
Purification of arachidin-1 and arachidin-3 from peanut
Arachidin-1 and arachidin-3 were isolated using the method originally described by Chang et al (17). Briefly, slices of Tainan 14 peanut kernel (1.2 kg; Fengxiang Seed Co., Ltd., Chiayi, Taiwan) were artificially aerated at 25°C and under 65l/min air flow rate for 16 h. Following pretreatment and freeze drying, the slices of peanut kernel were pulverized into a powder and extracted with methanol. The methanol extract was suspended in water and partitioned with an equal volume of ethyl acetate. The ethyl acetate extract was passed through Sephadex LH-20 and eluted with methanol. The fractions were chromatographed using a LiChroprep RP-18 (2.5×52 cm) column eluted with 0.05% trifluoroacetic acid-CH3CN (60:40) to obtain arachidin-1 and arachidin-3. The yields of arachidin-1 and arachidin-3 were 0.0045 and 0.0087% and the purities were 96.82 and 98.10%, respectively.
EC cultures
The human umbilical vein cell line EA.hy926 (ATCC CRL-2922; ATCC, Manassas,VA, USA) was cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco-BRL, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (Gibco-BRL, Carlsbad, CA, USA) at 37°C under 5% CO2 in air. When the ECs were grown to confluence, the culture medium was replaced with serum-free DMEM and the cells were incubated for 12 h prior to experimental treatments.
Cell adhesion assay
The THP-1 cells (ATCC) were labeled with 1 µM calcein-AM (Trevigen, Gaithersburg, MD, USA) in PBS for 40 min at 37°C, and then washed twice with PBS. After treatment, ECs were washed with DMEM and co-cultured with calcein-labeled THP-1 cells for 30 min. After washing twice with RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA), adherent cells were examined using an enzyme-linked immunosorbent assay plate reader (FLx800; Bio-Tek Instruments, Inc., Winooski, VT, USA) at 485 nm excitation and 538 nm emission wavelengths.
Cytotoxicity
The resazurin reduction test, an index of the metabolic activity of living cells, was carried out using an AlamarBlue® assay kit according to the manufacturer's protocol (Serotec; Bio-Rad Laboratories, Inc., Hercules, CA, USA). ECs were plated into 96-well microtiter plates (Falcon; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 20,000 cells/well and incubated with AlamarBlue® reagent for 2 h at 37°C. Fluorescence was then measured at excitation and emission wavelengths of 570 and 600 nm, respectively.
Western blotting
A total of 106 cells were lysed on ice in lysis buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and a protease inhibitor mixture). The total protein concentrations were determined with the use of the Bio-Rad DC protein assay kit (Bio-Rad Laboratories, Inc.). Equal amounts (10–20 µg) of whole-cell extracts were boiled for 5 min prior to separation using 10% SDS-PAGE. The proteins were then transferred to a nitrocellulose membrane (EMD Millipore) in Tris-glycine buffer (25 mM Tris-HCl, 192 mM Glycine, pH 8.3) at 10 V for 1.5 h. The membranes were then blocked with PBS containing 5% non-fat milk for 2 h at 4°C and were subsequently probed with the appropriate primary antibody overnight at 4°C, with gentle shaking. The next day, following PBS washes, the membranes were incubated with secondary antibodies [horseradish peroxidase-conjugated goat anti-rabbit (34083) or anti-mouse (34081) antibody; Thermo Fisher Scientific, Inc.] for 1 h at room temperature. The results were visualized by chemiluminescence using ECL SuperSignal West Pico kit (Thermo Fisher Scientific, Inc.) in accordance with the manufacturer's protocol.
Preparation of nuclear fractionation for immunoblotting
ECs were collected by scraping and lysed with cell lysis buffer (10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride and 0.3% NP-40). The cell lysate was separated into nuclear fractions by centrifugation at 500 × g for 5 min at 4°C. The nuclei were washed using nuclei washing buffer (320 mM sucrose, 5 mM MgCl2, 10 mM HEPES at pH 7.4) and the nuclear protein was extracted using a buffer containing 25% glycerol, 20 mM HEPES, 0.6 M KCl, 1.5 mM MgCl2 and 0.2 mM EDTA for 15 min at 4°C.
Luciferase reporter assays
ECs were subcultured at a density of 1×106 cells in 60-mm dishes. ECs were subsequently co-transfected with 100 ng p3xARE/Luc with pSV-β-galactosidase using Lipofectamine-2000 (Invitrogen). Luciferase activity was detected using a luciferase reporter assay system (Promega), and the resulting luciferase activity was measured using a luminometer. For each experiment, luciferase activity was determined in triplicate and normalized with β-galactosidase activity.
RNA interference by small interfering RNA (siRNA) of Nrf-2
The siRNA nucleotide sequence for human Nrf-2 was as follows: 5'-UCCCGUUUGUAGAUGACAA-3' (18). The effects of this siRNA molecule have been demonstrated in a previous study (16). A non-targeting siRNA, 5-GCAAGCUGACCC UGAAGUUCAU-3, was purchased from Ambion (Thermo Fisher Scientific, Inc.). Cells were transfected with Nrf-2 siRNA or non-targeting siRNA using Lipofectamine 2000 reagent according to the manufacturer's protocol. Following 24 h of transfection, the ECs were cultured in medium without serum for another 12 h prior to treatment.
RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA isolated from the ECs using TRIzol was reverse transcribed using SuperScript II reverse transcriptase (both Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The cDNA was subjected to PCR amplification using the following forward and reverse primer sets: GAPDH, 5'-TAT CGT GGA AGG ACT CAT GACC-3' and 5'-TAC ATG GCA ACT GTG AGG GG-3'; GST-Pi, 5'-CCC TCA CTG TTT CCC GTT GC-3' and 5'-TGA ATG ACG GCG TGG AG-3'; thioredoxin-1 (Trx1), 5'-GGC ATG CAT TTG ACT TCA-3' and 5'-ACG TGA TAT TCC TTG AAG TAG-3'; glutamate-cysteine synthetase regulatory subunit (GCLM), 5'-CAG CGA GGA GCT TCA TGA TTG-3' and 5'-TGA TCA CAG AAT CCA GCT GTGC-3'; glutamate-cysteine synthetase catalytic subunit (GCLC), 5'-GTT CTT GAA ACT CTG CAA GAG AAG-3' and 5'-ATG GAG ATG GTG TAT TCT TGT CC-3'; HO-1, 5'-GGT AAG GAA GCC AGC CAA GAG-3' and 5'-GCC AGC AAC AAA GTG CAA GAT-3'. The cDNA samples were amplified using Taq DNA polymerase (Thermo Fisher Scientific, Inc.) for 17–25 cycles of 94°C (30 sec), 60°C (30 sec), and 72°C (30 sec) using Mastercycler personal (Eppendorf, Hamburg, Germany). The PCR products were separated on 1.5% agarose gels and visualized by ethidium bromide staining.
Measurement of intracellular reactive oxygen species (ROS)
The fluorescence probe 5-(and-6)-carboxy-2,7,dichlorodihydro fluorescein diacetate (carboxy-H2DCFDA; Molecular Probes; Thermo Fisher Scientific, Inc.) was used to detect the cellular production of ROS. The final treatment concentration of carboxy-H2DCFDA was 20 µM, and the cells were incubated with this for 30 min in dark at 37°C. Following two further washes with PBS, the cells were solubilized with 1% SDS and 5 mM Tris HCl (pH 7.4). H2DCF reacts with ROS to form the green fluorescent compound DCF, which is detected by spectrofluorophotometry (Rf-5301PC; Shimadzu Corporation, Kyoto, Japan) with excitation and emission wavelengths of 475 and 525 nm, respectively.
Statistical analysis
Results are expressed as the mean ± standard error of the mean of at least three independent experiments. Statistical significance was assessed by one-way analysis of variance followed by Tukey's test using SigmaPlot version 12 (Systat Software Inc., San Jose, CA, USA). P<0.05 was considered to indicate a statistically significant difference.
Results
Arachidin-1 inhibits monocyte/EC adhesion and ICAM-1 expression
The anti-inflammatory potential of two peanut-derived prenylated stilbenoids, arachidin-1 and arachidin-3 (Fig. 1A and B), was evaluated via assessment of their effects on the adhesion of monocytes to ECs. The results demonstrated that TNF-α significantly increased monocyte adhesion to the ECs and this adhesion was attenuated by treatment of the ECs with arachidin-1 or arachidin-3 (Fig. 1C and D). In another experiment, the cytotoxic effects of 24 h incubation with 5–50 µM concentrations of arachidin-1 and arachidin-3 in ECs were assessed (Fig. 1E and F). Arachidin-1 exhibited no toxic effects in ECs at dosages ≤25 µM. However, a cytotoxic effect was detectable following treatment with 5 µM arachidin-3. These data suggest that the reduction in monocyte adhesion observed following treatment with arachidin-3 may result from cell damage. These results also indicate that the 3'-hydroxyl group of the treatment agent is critical for determining the levels of cytotoxicity. To further test whether arachidin-1 is a modulator of ICAM-1 induction, ECs were stimulated with TNF-α, with or without arachidin-1 pretreatment. Arachidin-1 appeared to have a dose-dependent inhibitory effect on the TNF-α-induced ICAM-1 expression (Fig. 2A). The pretreatment of ECs with arachidin-1 also appeared to inhibit TNF-α-induced ICAM-1 expression in a time-dependent manner (Fig. 2B). This functional assay implies that arachidin-1 exerts an anti-inflammatory effect via the inhibition of adhesion molecule expression.
Arachidin-1 inhibits TNF-α-induced NF-κB activation and blocks the degradation of IκBα
Since the NF-κB pathway is a well-known inflammatory pathway (5), whether arachidin-1 regulates TNF-α-induced NF-κB activation was examined in the present study. ECs were pretreated with arachidin-1 to examine whether this agent regulates p65 nuclear translocation in TNF-α-treated ECs. Western blotting demonstrated that TNF-α-induced p65 nuclear translocation decreased following pretreatment with 5 µM arachidin-1 from 6 to 12 h (Fig. 3A). Following pretreatment at concentrations of 0.1–10 µM for 12 h, it was observed that TNF-α-induced p65 nuclear translocation was inhibited by ≥1 µM arachidin-1 pretreatment (Fig. 3B). To clarify the inhibitory mechanisms of arachidin-1 in TNF-α-induced NF-κB activation, the degradation of IκBα was determined over time. TNF-α alone induced a marked degradation of IκBα following a 1-h treatment. Pretreatment with 5 µM arachidin-1 for 6 and 12 h inhibited the TNF-α-induced degradation of IκBα in ECs (Fig. 3C). These results indicate that arachidin-1 inhibits NF-κB nuclear translocation and transactivation in a time- and dose-dependent manner.
Arachidin-1 induces Nrf-2 activation
It has previously been reported that Nrf-2-induced phase II detoxifying and antioxidant enzymes provide cytoprotective effects in ECs (13). In the present study, ECs treated with arachidin-1 exhibited a continuous increase of Nrf-2 nuclear accumulation (Fig. 4A). Nrf-2 regulates the ARE that drives the expression of specific genes (11). The ability of arachidin-1 to increase Nrf-2 transcriptional activity was demonstrated by transfecting ECs with an ARE-luciferase reporter construct (Fig. 4B). To investigate whether Nrf-2 contributes to the protective effects of arachidin-1, experiments using Nrf-2 siRNA were performed. Transfection with Nrf-2 siRNA suppressed the inhibitory effects of arachidin-1 on ICAM-1 expression (Fig. 4C), NF-κB nuclear translocation (Fig. 4D) and IκBα degradation (Fig. 4E). These data indicate that arachidin-1 induces Nrf-2 activation and that this mechanism contributes to the anti-inflammatory effects of this compound.
Arachidin-1 induces Nrf-2-associated phase II enzyme expression
The effect of arachidin-1 on phase II enzymes was investigated using ECs in the current study. Cells were treated with arachidin-1 at a concentration of 5 or 10 µM for 12 h. RT-PCR analysis indicated that the mRNA expression levels of GST-Pi, Trx1, HO-1, GCLC and GCLM were increased (Fig. 5A). The protein levels of HO-1 and thioredoxin reductase-1 (TrxR-1) were evaluated by western blot analysis (Fig. 5B). The results suggest that arachidin-1 activates phase II enzymes through increasing their expression at the mRNA and protein levels.
Arachidin-1 reduces H<sub>2</sub>O<sub>2</sub>-induced ROS levels
To evaluate the antioxidant property of arachidin-1, the effects of arachidin-1 in H2O2-treated cells were examined. It was observed that the exposure of cells to 0.5 mM H2O2 for 1 h increased the ROS level. Pretreating cells with 5 or 10 µM arachidin-1 for 12 h significantly reduced the intracellular ROS level (Fig. 6A). In addition, the protective effects of arachidin-1 under oxidative stress were examined by subjecting the cells to treatment with 0.5 mM H2O2 for 1 h. The results demonstrate that H2O2 increases cytotoxicity, and arachidin-1 did not prevent this oxidative stress-induced cell death (Fig. 6B).
Discussion
Arachidin-1 is an electrophilic phytochemical present in germinated peanut roots and the present study provides the first evidence that this compound, at non-cytotoxic concentrations, enhances the suppression of TNF-α-induced monocyte adhesion to ECs by downregulating the expression of ICAM-1. The present data also demonstrate that the inhibitory effects of arachidin-1 on the activation of the inflammatory transcriptional factor, NF-κB, are mediated via blocking the degradation of IκBα, and exerted through the induction of Nrf-2-associated phase II detoxifying and antioxidant enzymes. The Nrf-2-associated protective mechanisms may be the major effecter of the anti-inflammatory and anti-oxidative effects arachidin-1.
Resveratrol and piceatannol are dietary polyphenols that are recognized to have numerous health-promoting effects, including anti-oxidative, anti-inflammatory and anticancer properties (19). Although arachidin-1 and arachidin-3 are naturally occurring prenylated analogs of piceatannol and resveratrol, respectively, the biological activity of these prenylated stilbenoids is less studied. The addition of a prenyl group to these compounds increases their hydrophobicity, which may facilitate their attachment to cell membranes or translocation into cells. Theoretically, prenylated stilbenoids should have much more potent biological activity and bioavailability than stilbenoids. Dietary resveratrol has been shown to have a low bioavailability; a previous study revealed that for a dietary 25-mg oral dose of resveratrol, only <5 ng/ml amounts of unchanged resveratrol were detected in plasma (20). Therefore, arachidin-1 may have greater potential applicability than resveratrol. A previous study by the present research team demonstrated that piceatannol induced Nrf-2-related HO-1 expression in ECs, which peaked at 6 h with 25 µM piceatannol treatment (15). Other researchers demonstrated that 30 µM piceatannol treatment significantly upregulated the expression of HO-1 at 6 h in human breast epithelial cells (21). In the present study, it was demonstrated that prenylated piceatannol, arachidin-1, induced HO-1 expression when applied to ECs at a concentration of 5 µM for 3–12 h. A comparison of these findings indicates that the prenylated stilbenoid has a greater efficiency in cell protection than the unprenylated molecule.
The present study provides the first evidence in ECs that arachidin-1 is anti-inflammatory at non-cytotoxic concentrations. The results show that the inhibitory effect differed between arachidin-1 and arachidin-3, indicating that even subtle changes of the chemical structure significantly affect the potency. Notably, the only structural difference between these two prenylated stilbenoids is the 3'-hydroxyl group present in arachidin-1 (Fig. 1A and B). It is notable that arachidin-3 was cytotoxic. Thus, the reduction of cytotoxicity is a crucial factor in the modification of the structure of prenylated stilbenoids.
The redox-sensitive transcription factor Nrf-2 serves a pivotal role in the induced expression of a number of cytoprotective enzymes (13). The present study findings demonstrate that arachidin-1 induces Nrf-2 translocation and activates ARE-luciferase promoter activity, indicating that it directly induces Nrf-2 via its ARE (Fig. 4A). They also indicate that the anti-inflammatory effects of arachidin-1 exerted through protection against ICAM-1 expression and IκBα degradation were reduced by the transfection of Nrf-2 siRNA (Fig. 4C and D). Furthermore, arachidin-1 was found to induce Nrf-2-associated phase II enzyme expression. These data suggest that arachidin-1 induces Nrf-2-activated phase II enzymes involved in the anti-inflammatory effect in ECs. Although the exact mechanisms by which arachidin-1 activates the induction of Nrf-2-associated genes remain unknown, arachidin-1 may contribute to the activation of Nrf-2 through its catechol moiety (22). Although arachidin-1 possesses reducing properties, it may also act as an electrophile. The catechol moiety of arachidin-1 can be oxidized to the electrophilic o-quinone form (23). The electrophilicity of the quinone may lead to the activation of the Nrf-2/Keap1 pathway due to the modification of specific cysteine residues of Keap1, which allows Nrf-2 to translocate to the nucleus and bind to ARE, leading to an increased expression of Nrf-2-associated genes. The characteristic catechol moiety of arachidin-1 appears to be critical for the induction of Nrf-2 activation and subsequent upregulation of phase II enzymes.
The induction of Nrf-2-associated enzymes has been recog-nized as a mechanism underlying the antioxidant properties of phytochemicals (24). A previous study demonstrated that piceatannol protects cells from glutamate-mediated oxidative injury by upregulating cellular Nrf-2-associated HO-1 expression (25). In the present study, arachidin-1 was revealed to induce the expression of HO-1, GST and other thiol-containing enzymes in ECs, indicating a protective effect of arachidin-1 against oxidative stress. Although arachidin-1 did not prevent H2O2-induced cell damage, it significantly reduced the H2O2-induced increase in ROS levels (Fig. 6). These findings suggest that the anti-inflammatory effect of arachidin-1 was in accordance with its Nrf-2-associated anti-oxidative property. However, whether the reduction in oxidative stress was dependent on phase II enzyme induction requires further investigation.
In conclusion, the present study demonstrates that an Nrf-2-regulated antioxidant response serves a pivotal role in the anti-inflammatory mechanism underlying the inhibition of NF-κB activation in arachidin-1-treated ECs. A clearer understanding of the mechanisms of action of arachidin-1 in the future may contribute to a potential therapeutic application for the treatment of pathological inflammation.