International Immunopharmacology 11 (2011) 1907–1915

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International Immunopharmacology

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Schisandra chinensis α-iso-cubebenol induces heme oxygenase-1 expression throughPI3K/Akt and Nrf2 signaling and has anti-inflammatory activity in Porphyromonasgingivalis lipopolysaccharide-stimulated macrophages

Sun Young Park a,c, Da Jung Park b, Young Hun Kim a, YoungHee Kim c,Young-Whan Choi b,⁎, Sang-Joon Lee c,d,⁎⁎a Bio-IT Fusion Technology Research Institute, Pusan National University, Busan, Republic of Koreab Department of Horticultural Bioscience, Pusan National University, Miryang 627-706, Republic of Koreac Department of Molecular Biology, Pusan National University, Busan, Republic of Koread Department of Microbiology, Pusan National University, Busan, Republic of Korea

⁎ Correspondence to: Y.-W. Choi, Department of HNational University, Miryang 627-706, Republic of Kofax: +82 55 350 5529.⁎⁎ Correspondence to: S.-J. Lee, Department of MUniversity, Busan 609-735, Republic of Korea. Tel.: +8514 1778.

E-mail addresses: ywchoi@pusan.ac.kr (Y.-W. Choi),(S.-J. Lee).

1567-5769/$ – see front matter © 2011 Elsevier B.V. Aldoi:10.1016/j.intimp.2011.07.023

a b s t r a c t

a r t i c l e i n f o

Article history:Received 1 June 2011Received in revised form 28 July 2011Accepted 28 July 2011Available online 12 August 2011

Keywords:α-Iso-cubebenolHeme oxygenase-1Nrf-2Porphyromonas gingivalis LPSPro-inflammatory cytokineNF-κB

Heme oxygenase-1 (HO-1) is a potent anti-inflammatory molecule that regulates pro-inflammatorymediators. Several studies have indicated that HO-1 expression is induced by a variety of stimuli such aslipopolysaccharide (LPS), cytokines, oxidative stress, and antioxidant phytochemicals. In this study, weassessed the anti-inflammatory effects of a novel α-iso-cubebenol isolated from dried fruits of Schisandrachinensis in human macrophage THP-1 cells and investigated the involvement of HO-1 signaling. We firstobserved that α-iso-cubebenol induced HO-1 mRNA and protein expression in a dose- and time-dependentmanner via activation of erythroid-specific nuclear factor-regulated factor 2 (Nrf2). We also found that α-iso-cubebenol induced phosphorylation of phosphoinositide 3-kinase (PI3K)/Akt and extracellular-regulatedkinase (ERK) in a time-dependent manner. Furthermore, treatment of THP-1 cells with inhibitors and siRNAspecific for PI3K/Akt and ERK decreased the expression of HO-1. These results suggested thatα-iso-cubebenolinduced HO-1 expression through the activation of PI3K/Akt, ERK, and Nrf2 signaling. Next, α-iso-cubebenolstrongly inhibited Porphyromonas gingivalis LPS-stimulated pro-inflammatory cytokines (tumor necrosisfactor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-12). Moreover, we observed that α-iso-cubebenol treatmentinhibited nuclear levels and activity of NF-κB in a dose-dependent manner. Additionally, treatment with tin-protoporphyrin (SnPP), a selective inhibitor of HO-1, reversed the α-iso-cubebenol-mediated inhibition ofP. gingivalis LPS-induced pro-inflammatory cytokines. Hence, α-iso-cubebenol might induce anti-inflammatoryeffects on P. gingivalis LPS-stimulated human THP-1 macrophages by mediating the activation of PI3k/Akt andERK that leads to over-expression of HO-1 and Nrf-2. These findings suggest that α-iso-cubebenol may beconsidered as a novel therapeutic agent to ameliorate periodontitis.

orticultural Bioscience, Pusanrea. Tel.: +82 55 350 5522;

icrobiology, Pusan National2 51 510 2268; fax: +82 51

sangjoon@pusan.ac.kr

l rights reserved.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

The dried fruits of Schisandra chinensis have been traditionally usedto treat many types of inflammation such as chronic cough and asthma.Schisandra chinensis and its constituent dibenzo-cycloctadilene lignanshave been reported to have anti-oxidative, anti-inflammatory, anti-tumor, hepatoprotective, and neuroprotective effects [1–4]. Recently, by

using bioassay-guided fractionation, we identified a novel α-iso-cubebenol from the fruits of S. chinensis; thisα-iso-cubebenol markedlyinhibited lipopolysaccharide (LPS)-induced expression of induciblenitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in mousemacrophage RAW264.7 cells [5]. However, the anti-inflammatorysignaling pathways involved in theα-iso-cubebenol-induced activationof heme oxygenase-1 (HO-1) in periodontitis and chronic inflammationare not completely understood.

HO-1 is a key enzyme in the defense against oxidative-stress, and itplays a substantial role in immunomodulatory and anti-inflammatoryfunctions. HO-1 is the rate-limiting enzyme in heme-catabolism,which produces biliverdin, free iron, and carbonmonoxide [6]. Recentreports have suggested that HO-1 is critical for inflammatoryresponses via the metabolic conversion of free heme and productionof anti-inflammatory compounds, carbon monoxide, and bilirubin.Exogenous carbon monoxide inhibits the LPS-induced production of

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Fig. 1. Chemical structure of α-iso-cubebenol (A) isolated from Schisandra chinensis:high-performance liquid chromatography (HPLC) profiles of the α-iso-cubebenol (B).HPLC profile showed that the active compound had a purity of N96%.

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pro-inflammatory cytokines and mediators via p38 MAPK. Further-more, bilirubin can block iNOS expression and NO production in LPS-induced macrophages [7,8]. HO-1 is shown to be activated by variousphytochemicals, which are reportedly involved in cytoprotective andbeneficial immune response [9,10].

The major signaling pathways that regulate anti-inflammatoryeffects and HO-1 expression are the phosphoinositide 3-kinase (PI3k)/Akt and MAPK signaling pathways [11,12]. Furthermore, HO-1expression is induced through the activation of transcription factors,including nuclear factor-κB (NF-κB), activator protein-1 (AP-1), anderythroid-specific nuclear factor-regulated factor 2 (Nrf2) and throughthe PI3K/Akt and MAPK signaling pathways [13–16]. HO-1 hasantioxidant response elements (AREs) that directly bind the Nrf2 andregulate the expression of many genes involved in adaptive responseand inflammation. Activation of Nrf2 by stimulators (such as phyto-chemicals) causes the dissociation of Nrf2 from Keap-1, which trans-locates to the nucleus, where itmodulates the expression of ARE-drivengenes such as NAD(P)H:oxidoreductase, thioredoxin reductase-1, andHO-1 [17–19].

Porphyromonas gingivalis LPS stimulates pro-inflammatory cyto-kine production in immune cells, which initiates the localizedinflammatory response in periodontitis. The high production of pro-inflammatory cytokines is a key factor in chronic inflammatorydestruction of the periodontal tissues [20,21]. Recently, HO-1signaling was shown to be involved in the suppression of P. gingivalisLPS-induced pro-inflammatory cytokines [22]. In the present study,we provide the first evidence that Nrf2 is required for α-iso-cubebenol-mediated HO-1 expression in human macrophage THP-1cells. The data also show that the PI3K/Akt and ERK signalingpathways are required for α-iso-cubebenol-induced expression ofHO-1 and nuclear accumulation of Nrf2. Furthermore, our resultssuggest that HO-1 up-regulation through the PI3K/Akt, ERK, and Nrf2signaling mediates the anti-inflammatory effect ofα-iso-cubebenol inP. gingivalis LPS-stimulated human macrophage THP-1 cells.

2. Materials and methods

2.1. Materials

The fruits of S. chinensis (Turcz.) Baill were collected in September2005 from Moonkyong, Korea. A voucher specimen (Accession No.,SC-PDRL-1) has been deposited in the Herbarium of Pusan NationalUniversity. The plant was identified by one of the authors (Y.W. Choi).Phorbol 12-myristate 13-acetate (PMA), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and other reagents notreferred were purchased from Sigma (St. Louis, MO, USA). Protopor-phyrin IX (SnPP); Nrf2; ERK1; Akt siRNA; and antibodies for HO-1,Nrf2, NF-κB, and TBP were purchased from Santa Cruz Biotechnology(Santa Cruz, CA, USA). LPS (phenol extracted from P. gingivalis) waspurchased from InvivoGen (San Diego, CA, USA). SB203580 (a specificinhibitor of p38), PD98059 (a specific inhibitor of ERK1/2), andSP600125 (a specific inhibitor of Jun kinase (JNK)) were purchasedfrom A.G. Scientific (San Diego, CA, USA).

2.2. Extraction of a new α-iso-cubebenol

The dried fruits of S. chinensis (2.5 kg) were ground to a finepowder and then successively extracted at room temperature with n-hexane, CHCl3, andMeOH. The hexane extract (308 g) was evaporatedin vacuo and chromatographed on a silica gel column (100×10 cm)(40 μm; Baker, Phillipsburg, NJ, USA) with a step gradient (0%, 5%, and20%) of EtOAc in hexane and 5% MeOH in CHCl3 to obtain 38 fractionsas described previously (Choi et al., 2009). Fraction 9 (KH9, 4866 mg)was separated on a silica gel column (100×3.0 cm) with a stepgradient (1%, 10%, and 15%) of acetone in CHCl3 to obtain 21 fractions.Next, fraction 2 (KH9IG, 529.9 mg) was separated on a silica gel

column (100×3.0 cm) with 5% acetone in CHCl3 to yield α-iso-cubebenol (162.9 mg) as previously described by [5]. Pure α-iso-cubebenol was identified by high-performance liquid chromatogra-phy (HPLC) on a Luna C18 column (150×4.6 mm internal diameter(ID); 5 μm particle size; Phenomenex, LA, CA, USA) with a methanol–acetonitrile gradient, at a flow rate of 1.0 mL/min. The structure of α-iso-cubebenol isolated from S. chinensis fruits was identified by the1H, 13C, nuclear magnetic resonance (NMR) spectra (including Dept,HSQC, and HMBC) in CDCl3, as described by [5].

2.3. Cell culture

The human monocytic cell line THP-1 was obtained from theAmerican Type Culture Collection (ATCC, Rockville, MD, USA). Cellswere grown in RPMI 1640 medium (Invitrogen, Grand Island, NY,USA) supplemented with 10% heat inactivated fetal bovine serum(FBS) and 1% penicillin–streptomycin (BioSource International,Camarillo, CA, USA) at 37 °C in a humidified atmosphere of 5% CO2

and 95% air. Cellular differentiation into the macrophage-likephenotype was induced by the addition of PMA at a final concentra-tion of 200 nM. After incubation for 2 days, differentiation of THP-1cells into macrophages was confirmed by fluorescence-activated cellsorter (FACS) analysis of CD11b surface expression. The differentiatedcells were washed 3 times, incubated with RPMI 1640 mediumcontaining 0.5% FBS and 1% penicillin–streptomycin, and used insubsequent experiments.

2.4. Cell viability assay (MTT assay)

The cytotoxicity of α-iso-cubebenol was assessed using themicroculture tetrazolium (MTT)-based colorimetric assay. Cellswere incubated in 24-well plates at a density of 5×105 cells perwell. MTT solution (5 μL of 5 mg/mL) was added to each well (finalconcentration, 62.5 μg/mL). After incubation for 3 h at 37 °C in 5% CO2,the supernatant was removed and the formazan crystals produced inviable cells were solubilized with 150 μL of dimethylsulfoxide

1909S.Y. Park et al. / International Immunopharmacology 11 (2011) 1907–1915

(DMSO). The absorbance of each well was then read at 570 nm using amicroplate reader (Wallac 1420 Boston, MA USA).

2.5. Immunofluorescence confocal microscopy

THP-1 cells were cultured directly on glass coverslips in a 35-mmdish. Cells were fixed with 3.5% paraformaldehyde in phosphatebuffered saline (PBS) for 10 min at room temperature and permea-bilized with 100% MeOH for 10 min. To investigate the cellular local-ization of NF-κB and Nrf2, cells were treated with a polyclonal antibody(1:100) against either NF-κB or Nrf2 for 2 h. After extensive washingwith PBS, cells were incubated with a secondary fluorescein isothiocy-anate (FITC)-conjugated donkey anti-rabbit IgG antibody diluted at1:1000 in PBS for 1 h at room temperature. Nuclei were stained with1 μg/mL of 4′-6-diamidino-2-phenylindole (DAPI), and then analyzedby confocal microscopy using a Zeiss LSM 510 Meta microscope.

2.6. Preparation of cytosol and nuclear extract

THP-1 cells (3×106 cells) were washed 3 times with cold PBS, andthe cell pellets were suspended in hypotonic buffer (Active Motif,

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Fig. 2. Effects of α-iso-cubebenol on HO-1 expression in PMA-differentiated THP-1 cells. (A20 μMofα-iso-cubebenol for indicated periods, before measurement of HO-1mRNA using thα-iso-cubebenol and incubated with 20 μM α-iso-cubebenol for the indicated times, totadetection of α-tubulin was performed using a protein-loading control for each lane. (C)actinomycin D or cycloheximide. Subsequently, protein expression levels of HO-1 were detband (fold of control) estimated by using Image Quant TL software. Each bar represents the mcontrol group. (D) THP-1 cell were treated or untreated with 200 nM PMA for 48 h. Cells wereflow cytometry. Histograms show profiles of isotype controls (white) and CD11b antibody

California, USA) and incubated for 15 min on ice. Detergent (ActiveMotif, California, USA) was added to the cell extract, incubated on icefor 1 min, and centrifuged at 13,000 rpm for 1 min at 4 °C. Afterremoval of the supernatant containing cytosolic proteins, nuclearproteins were extracted by the addition of complete lysis buffer B(Active Motif) for 30 min at 4 °C with occasional vortexing. Aftercentrifugation at 13,000 rpm for 5 min at 4 °C, supernatants werecollected and stored at −70 °C.

2.7. Transient transfection with siRNA

Transfection of THP-1 cells with siRNA was performed using theX-treme GENE siRNA Transfection Reagent (Roche Applied Science),according to themanufacturer's instructions. Commercially availablehuman Nrf2, Akt, and ERK1 specific siRNAs (Santa Cruz, Heidelberg,Germany) and negative control siRNAs (Santa Cruz) were used fortransfection. In brief, X-treme GENE siRNA Transfection Reagent(10 μL) was added to 100 μL serum-free medium containing 2 μg ofeach siRNA oligo, andwas incubated for 20 min at room temperature.Gene silencing was measured after 48 h by western blotting.

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) Cells were cultured with increasing concentrations of α-iso-cubebenol for 12 h ande real time RT-PCR. (B) Cells were incubated for 24 hwith the indicated concentration ofl cellular extracts were prepared, and western blotting was performed. Western blotTHP-1 cells were treated with 20 μM of α-iso-cubebenol for 24 h in the presence ofermined by western blotting. Numbers at bottom are expressed as relative intensity ofean (SD) from 3 independent experiments in each group. *Pb0.05, with respect to eachharvested for staining with FITC-conjugatedmonoclonal antibodies and analyzed using(black).

1910 S.Y. Park et al. / International Immunopharmacology 11 (2011) 1907–1915

2.8. Transient transfection and dual luciferase assay

THP-1 cells were transfected with the κB-luc reporter plasmidconsisting of 3 κB concatemers from the Igγ chain and firefly luciferasegene and ARE reporter plasmid (Stratagene, Grand Island, NY) usingFuGENE-HD reagent (Roche Applied Science) according to the manu-facturer's instruction. Renilla luciferase control plasmid pRL-CMV(Promega) was cotransfected as an internal control for transfectionefficiency. After 24 h of transfection, the cells were incubated withindicated reagents for 1 h and then treated with LPS (1 μg/mL) for 24 h.Luciferase activity was assayed using the dual-luciferase assay kit(Promega) according to the manufacturer's instruction. Luminescencewas measured using microplate luminometer (Wallac 1420).

2.9. Western blot analysis

Cells were harvested in ice-cold lysis buffer consisting of 1% TritonX-100, 1% deoxycholate, and 0.1% sodium dodecyl sulfate (SDS). Theprotein content of the cell lysates was then determined using Bradfordreagent (Bio-Rad; Hercules, CA, USA). The proteins in each sample

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Fig. 3. Effects ofα-iso-cubebenol on HO-1 expression through Nrf2 activation in PMA-differecubebenol for 3 h. The total cellular extracts (T), cytosolic extracts (C), and nuclear extractswith 20 μM of α-iso-cubebenol for 3 h. Fixed cells were stained with DAPI and anti-Nrf2 antmicroscopy. (C) The THP-1 cells were transfected with control siRNA (si con) and Nrf2 siRNA48 h after transfection, the cells were treated with 20 μM α-iso-cubebenol for 24 h, and theexpressed as relative intensity of band (fold of control) estimated by using Image Quant TLconstruct (ARE) and then treatment with indicated concentrations of α-iso-cubebenol. Equarenilla luciferase control was used to normalize ARE–luciferase activity. Each bar represent

(50 μg total protein) were resolved by 7.5% SDS-polyacrylamide gelelectrophoresis (SDS-PAGE), transferred to a polyvinylidene difluoride(PVDF) membrane, and incubated with the appropriate antibodies. Theproteins were visualized using an enhanced chemiluminescencedetection system (Amersham Biosciences, Piscataway, NJ, USA) withhorseradish peroxidase-conjugated anti-rabbit or anti-mouse second-ary antibodies. Imageswereacquiredusingan ImageQuant 350 analyzer(Amersham Biosciences).

2.10. Reverse transcription real-time polymerase chain reaction

The total cellular RNA was isolated using an RNA spin mini RNAisolation kit (GE Healthcare) according to the manufacturer's in-structions. Total RNA (1 μg) was reverse-transcribed using Maximereverse transcriptase (RT) PreMix (Intron Biotechnology) andanchored oligo-dT15-primers. Real-time polymerase chain reaction(PCR) was performed on a Chromo4 instrument (Bio-Rad) using theSYBR Green Master Mix (Applied Biosystems, CA, USA). The relativeamount of target mRNA was determined using the comparativethreshold (Ct) method by normalizing target mRNA Ct values to those

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ntiated THP-1 cells. (A) Cells were incubated with the indicated concentrations ofα-iso-(N) were prepared, and western blotting was performed. (B) THP-1 cells were treatedibody and FITC-conjugated anti-rabbit IgG antibody. Images were obtained by confocal(siNrf2) using the X-treme GENE reagent according to themanufacturer's instruction. Atexpression of HO-1 protein was examined by western blotting. Numbers at bottom aresoftware. (D) Cells were transfected with the antioxidant response elements–luciferasel amounts of cell extract were assayed for dual-luciferase activity. Expression from thes the mean (SD) from 3 independent experiments in each group.

1911S.Y. Park et al. / International Immunopharmacology 11 (2011) 1907–1915

for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ΔCt).Relative HO-1 mRNAs expression (2−Δct) was assessed by real-timePCR and calculated by subtracting the Ct value for GAPDH from that forHO-1, which were determined by real-time RT-PCR relative to GAPDHmRNA; ΔCt=Ct HO-1−Ct GAPDH. The real-time PCR cyclingconditions were as follows: 95 °C for 5 min, followed by 40 cycles of30 s at 95 °C, 20 s at 55 °C, and 30 s at 72 °C; this was followed byfluorescence measurement. The primer sequences were as follows:HO-1-sense (5′-cgggccagcaacaaagtg-3′), HO-1-antisense (5′-agtg-taaggacccatcggaga-3′), GAPDH-sense (5′-aggtggtctcctctgacttc-3′),and GAPDH-antisense (5′-taccaggaaatgagcttgac-3′).

2.11. Statistical analysis

Data are expressed as the mean (standard deviation) (SD). Eachexperiment was repeated at least 3 times. Statistical analysis wasperformed using SPSS software (version 16.0) to determine signifi-cant differences. We used either one-way or two-way analysis ofvariance (ANOVA) followed by Dunn's post hoc tests for analyses. Pvaluesb0.05 were considered statistically significant.

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Fig. 4. Effects ofα-iso-cubebenol on HO-1 expression and Nrf2 nuclear accumulation via PI3/20 μM of α-iso-cubebenol for 24 h in the presence of LY294002 (PI3K/Akt inhibitor, 20 μM)inhibitor, 20 μM). Subsequently, HO-1 protein levels were determined by western blotting. (LY294002 and PD98059 at the indicated concentrations andwere examined by western blottand ERK siRNA (si ERK) using X-treme GENE reagent according to the manufacturer's instru24 h, and HO-1 protein expression was examined by western blotting. (D) Cells were treatblotting for Akt, phosphorylated Akt, ERK, and phosphorylated ERK. Numbers at the bottomQuant TL software.

3. Results

3.1. Isolation of α-iso-cubebenol from Schisandra chinensis

In order to prepare α-iso-cubebenol for the study of their anti-inflammatoryeffects, various compoundswereextracted from S. chinensisby n-hexane, EtOAc and MeOH. Subsequently, α-iso-cubebenol that wasextracted in largequantities compared to theother lignanswasharvested.The structure of α-iso-cubebenol was determined by NMR analysis, andidentified as α-iso-cubebenol (Fig. 1). α-Iso-cubebenol that was morethan 95% in chromatographic verification was used in these experimentsto determine their anti-inflammatory effects.

3.2. α-Iso-cubebenol induced HO-1 mRNA and protein expression inhuman macrophage THP-1 cells

The cytotoxicity of α-iso-cubebenol on PMA-differentiated THP-1cells was initially determined using the MTT assay. The PMA-differentiated THP-1 cells were treated with α-iso-cubebenol atvarious concentrations (1, 5, 10, 20, 40, and 60 μM) for 24 h. The

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Akt and ERK signals in PMA-differentiated THP-1 cells. (A) THP-1 cells were treated with, PD98059 (ERK inhibitor, 20 μM), SB203580 (p38 inhibitor 20 μM), or SP600125 (JNKB) Cells were incubated for 24 h with 20 μM α-iso-cubebenol in presence or absence ofing. (C) The THP-1 cells were transfected with control siRNA, (si con) Akt siRNA (si Akt),ction. At 48 h after transfection, the cells were treated with 20 μM α-iso-cubebenol fored with 20 μM α-iso-cubebenol for the indicated times and were subjected to westernare expressed as relative intensity of band (fold of control) estimated by using Image

1912 S.Y. Park et al. / International Immunopharmacology 11 (2011) 1907–1915

results showed that treatment with α-iso-cubebenol at differentconcentrations ranging from 1 to 40 μM exhibited no cytotoxic effectson the cells (data not shown). To explore the potential effects of α-iso-cubebenol on HO-1 expression, real time RT-PCR and westernblotting were performed. Treatment of cells with α-iso-cubebenol atvarious concentrations (1–20 μM) for 2–24 h induced HO-1 mRNAand protein expression in a time- and dose-dependent manner(Fig. 2A and B). To confirm whether α-iso-cubebenol-induced HO-1expression in PMA-differentiated THP-1 cells was mediated bytranscription and translation, we used actinomycin D (Act D), aninhibitor of DNA-dependent RNA polymerase, and cycloheximide(CHX), an inhibitor of ribosomal protein synthesis. Treatment withboth Act D and CHX reduced HO-1 expression induced by α-iso-cubebenol, suggesting that induction of HO-1 expression occurs at thetranscription level (Fig. 2C).

3.3. α-Iso-cubebenol augments HO-1 expression via Nrf2 nucleartranslocation and transactivity

The transcription factor Nrf2 is known to regulate the antioxidantresponse element-dependent transcription ofHO-1 [16]. Several reportshave shown that the phytochemical-mediated HO-1 induction occursvia Nrf2 stabilization and activation by directly binding to Keap1through a covalent linkage [9,23]. To determine whether Nrf2 plays arole inα-iso-cubebenol-mediated HO-1 expression, we determined theeffect ofα-iso-cubebenol on cytosolic and nuclear levels of Nrf2 in PMA-differentiated THP-1 cells. As shown in Fig. 3A and B, treatment withα-iso-cubebenol completely induced Nrf2 stabilization and nuclearaccumulation in a dose-dependent manner. To elucidate, we examinedthe application of the luciferase reporter gene driven by Nrf-2 bind to

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Fig. 5. Effects of α-iso-cubebenol on pro-inflammatory cytokines (TNF-α, IL-1β, IL-6 andconcentrations of α-iso-cubebenol for 1 h, and then treated with LPS (1 μg/mL) for 24 h. TNFusing an ELISA kit. Each bar represents the mean (SD) of 3 independent experiments in eac

antioxidant response elements (AREs) for Nrf-2 transactivity analysis.As shown in Fig. 3C, treatment of THP-1 cells with α-iso-cubebenolincreased ARE promoter activity by 4.8-fold. Moreover, transfection ofPMA-differentiated THP-1 cells with Nrf2 siRNA reduced α-iso-cubebenol-mediated nuclear Nrf-2 and HO-1 expression, whereas acontrol siRNA had no effect (Fig. 3D). Taken together, these resultsshowed that α-iso-cubebenol mediates HO-1 expression through Nrf2nuclear accumulation and transactivity.

3.4. α-Iso-cubebenol-mediated HO-1 expression and Nrf2 nuclearaccumulation involves PI3K/Akt and ERK MAP kinase

HO-1 gene expression can be induced bymany signal transductionpathways, including PI3K/Akt and MAPK [11,12]. Subsequent exper-iments were performed to determine which of these signalingpathways are involved inα-iso-cubebenol-mediated HO-1 expressionand Nrf2 nuclear accumulation. First, the effect of pharmacologicalinhibitors of PI3K/Akt (LY294002), ERK (PD98059), JNK (SB203580),and p38 (SP600125) on the α-iso-cubebenol-mediated induction ofHO-1 expression in PMA-differentiated THP-1 cells was analyzed bywestern blotting. Pre-incubation of PMA-differentiated THP-1 cellswith pharmacological inhibitors of PI3K/Akt and ERK resulted in areduction of α-iso-cubebenol-mediated HO-1 expression and Nrf2nuclear accumulation. However, JNK and p38 inhibitors had no effect.These findings implicate that both PI3K/Akt and ERK signals areinvolved in the α-iso-cubebenol-induced HO-1 expression and Nrf2nuclear accumulation. Since pharmacological inhibitors have a non-specific effect, we knocked down Akt and ERK by applying Akt andERK siRNA systems. Transfection of PMA-differentiated THP-1 cellswith PI3K/Akt and ERK siRNAs reduced α-iso-cubebenol-mediated

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Fig. 6. Effect of α-iso-cubebenol on NF-κB signaling in P. gingivalis LPS-stimulated THP-1cells. (A) Nuclear translocation of NF-κB was assessed by confocal microscopy. The THP-1cellswere pre-treatedwithα-iso-cubebenol for 1 h and stimulatedwith LPS (1 μg/mL) for30 min. Fixed cells were stained with DAPI or anti-NF-κB p65 antibody, followed byincubation with FITC-conjugated anti-rabbit IgG antibody. Images were obtained using aconfocal microscope. (B) Nuclear translocation of NF-κB was confirmed by westernblotting. The nuclear extracts were prepared and analyzed by western blotting. Thecytosolic extracts were analyzed by western blotting by using anti-phospho-IκB-αantibody. For western blot detection of histone H1, α-tubulin was used as a protein-loading control for each lane. Numbers at the bottom are expressed as relative intensity ofband (fold of control) estimated by using Image Quant TL software. (C) Cells were co-transfected with the κB-luc reporter and the control renilla luciferase plasmid, pRL-CMV.After 24 h, cells were incubated with the indicated concentrations of α-iso-cubebenol for1 h, and then stimulated with LPS (1 μg/mL) for 24 h. Equal amounts of cell extract wereassayed for dual-luciferase activity. Expression from the renilla luciferase controlwasusedto normalize κB-luciferase activity. Eachbar represents themean (SD) from3 independentexperiments in each group. *Pb0.05 vs. the LPS-treated group.

1913S.Y. Park et al. / International Immunopharmacology 11 (2011) 1907–1915

HO-1 expression and Nrf2 nuclear accumulation, whereas controlsiRNA had no effect (Fig. 3C). Furthermore, exposure of PMA-differentiated THP-1 cells to α-iso-cubebenol increased Akt and ERKphosphorylation. Immunodetection of normal Akt and ERK served asinternal controls. These results showed that PI3K/Akt and ERKsignaling have important roles in regulating α-iso-cubebenol-medi-ated HO-1 expression and Nrf2 nuclear accumulation in PMA-differentiated THP-1 cells (Fig. 4).

3.5. α-Iso-cubebenol suppresses P. gingivalis LPS-induced pro-inflammatory cytokines in human macrophage THP-1 cells

P. gingivalis LPS is known to be associatedwith periodontal diseasesby producing consistently high pro-inflammatory cytokines inimmune cells [20,21]. In general, host cellular inflammatory responsestarget the elimination and regulation of periodontal pathogens.However, consistent challenges from periodontal pathogens such asLPS result in dysregulated production of pro-inflammatory cytokines.We attempted to determine whether α-iso-cubebenol affectsP. gingivalis LPS-stimulated pro-inflammatory cytokines in THP-1cells. By using an enzyme-linked immunosorbent assay (ELISA) thatmeasures pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-12),we found that exposure of PMA-differentiated THP-1 cells toP. gingivalis LPS increased pro-inflammatory cytokine production(Fig. 5). Treatment with α-iso-cubebenol obviously inhibited theP. gingivalis LPS-stimulated pro-inflammatory cytokines (TNF-α, IL-1β,IL-6, and IL-12) in a dose-dependent manner. α-Iso-cubebenol alonehad no effect on pro-inflammatory cytokine production in THP-1 cells.These results showed that α-iso-cubebenol attenuated the productionof pro-inflammatory cytokines in P. gingivalis LPS-stimulated PMA-differentiated THP-1 cells.

3.6. α-Iso-cubebenol inhibits P. gingivalis LPS-stimulated NF-κB nucleartranslocation and activity in human macrophage THP-1 cells

Because transcription factor NF-κB-regulated genes have beenlinked to periodontal chronic inflammation disease, we assessed theα-iso-cubebenol-mediated anti-inflammatory effects by modulatingNF-κB signaling in P. gingivalis LPS-stimulated THP-1 cells. Todetermine whether α-iso-cubebenol could regulate P. gingivalis LPS-stimulated NF-κB, PMA-differentiated THP-1 cells were treated withα-iso-cubebenol for the indicated concentrations, followed byanalysis of NF-κB levels in the cytosolic and nuclear fractions bywestern blotting and immunocytochemical analysis. As shown inFig. 6A and B, treatment withα-iso-cubebenol suppressed P. gingivalisLPS-activated NF-κB nuclear accumulation. Furthermore, nucleartranslocation of NF-κB occurred simultaneously with the phosphor-ylation and degradation of IκBα. Therefore, our next aim was todetermine whether α-iso-cubebenol inhibited IκBα phosphorylationin P. gingivalis LPS-stimulated THP-1 cells. We pretreated PMA-differentiated THP-1 cells with α-iso-cubebenol and then exposedthem to P. gingivalis LPS. The results showed that α-iso-cubebenolinhibited the phosphorylation of IκBα in P. gingivalis LPS-stimulatedTHP-1 cells. Although we showed that α-iso-cubebenol inhibitedP. gingivalis LPS-induced nuclear translocation of NF-κB using westernblotting and immunocytochemical analysis, nuclear translocationdoes not always correlate with NF-κB-mediated gene regulation.Therefore, we also investigated whether α-iso-cubebenol regulatedthe P. gingivalis LPS-induced NF-κB activation. PMA-differentiatedTHP-1 cells were transiently transfected with a luciferase reportergene driven by NF-κB binding consensus concatemers (κB-Luc). Wethen pretreated these cells with α-iso-cubebenol and then exposedthem to P. gingivalis LPS. The results showed that α-iso-cubebenolinhibited P. gingivalis LPS-induced transactivation of NF-κB in a dose-dependent manner. We found that P. gingivalis LPS induced nucleartranslocation and transactivation of NF-κB, and that pretreatment

with α-iso-cubebenol inhibited these events, leading to the suppres-sion of P. gingivalis LPS-induced pro-inflammatory cytokine produc-tion (Fig. 7).

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Fig. 7. Effect of HO-1 inhibitor on the α-iso-cubebenol-mediated inhibition of pro-inflammatory cytokine production in P. gingivalis LPS-stimulated THP-1 cells. Cell were pretreatedwith 20 μM of α-iso-cubebenol in the presence of SnPP for 1 h and then stimulated with LPS (1 μg/mL) for 24 h. The amounts TNF-α (A), IL-1β (B), IL-6 (C), and IL12 (D) in thecultured supernatant were measured by using an ELISA kit.

1914 S.Y. Park et al. / International Immunopharmacology 11 (2011) 1907–1915

3.7. Suppressive effect of HO-1 inhibits α-iso-cubebenol-induced anti-inflammatory functions in P. gingivalis LPS-stimulated humanmacrophageTHP-1 cells

To further elucidate whether α-iso-cubebenol-mediated HO-1 ex-pression played a role in the inflammatory response induced byP. gingivalis LPS in PMA-differentiated THP-1 cells, we used SnPP as apotentHO-1 inhibitor [24].Wepretreated PMA-differentiatedTHP-1 cellswith α-iso-cubebenol and SnPP and then exposed them to P. gingivalisLPS. We found that treatment with SnPP inhibited α-iso-cubebenol-mediated suppression of P. gingivalis LPS-stimulated pro-inflammatorycytokine (TNF-α, IL-1β, IL-6, and IL-12) production. However, treatmentof the cells with SnPP alone did not affect the pro-inflammatory cytokineproduction. These results indicated that α-iso-cubebenol-induced HO-1expression inhibited P. gingivalis LPS-stimulated pro-inflammatorycytokines (TNF-α, IL-1β, IL-6, and IL-12).

4. Discussion

This study provides evidence for the mechanism by which α-iso-cubebenol contributes to the anti-inflammatory responses by inducingHO-1 in human macrophages. HO-1 and the products of it enzymaticactivity (biliverdin, carbonmonoxide, and free iron) have cyto-protectiveproperties, including anti-oxidant, anti-inflammatory, and anti-apoptoticactivities [24–26]. In recent reports, HO-1 deficiency has been suggested

to be associated with the development of several chronic inflammatorydiseases by inducing oxidative stress and inflammatory mediators [27].Therefore, targeted overexpression of HO-1 has potential for thetreatment of inflammatory disorders. In this regard, HO-1 inducershave been implicated in anti-inflammatory mechanisms. Many phyto-chemicals in plants are accepted as HO-1 inducers in therapeuticstrategies for chronic inflammatory diseases [15,22]. We previouslyshowed that α-iso-cubebenol from S. chinensis exerts anti-inflammatoryactivity through the inhibition of iNOS and COX-2 expression inSalmonella enteritidis LPS-stimulated macrophages [5]. Similarly, resultsreported in this study support the HO-1 anti-inflammatory-relatedmechanism mediated by α-iso-cubebenol in P. gingivalis LPS-stimulatedmacrophages. The results show that α-iso-cubebenol induced HO-1mRNA and protein expression. Moreover, the results in humanmacrophage THP-1 cells showed that HO-1 is induced via the Nrf2pathway, and that treatment with α-iso-cubebenol resulted in thestabilization and nuclear accumulation of Nrf2. The fact that HO-1induction by α-iso-cubebenol was also observed in murine macrophageRAW264.7 cells and BV2 microglia cells (data not shown) suggests theexistence of a common mechanism in response to α-iso-cubebenol inthese cells.

Many kinase signaling pathways, including PKC, PI3K/AKT, ERK,JNK, and p38, may regulate the Nrf2 activation that facilitates itsaccumulation in the nucleus to promote ARE-related gene expression[28]. The data presented here provides evidence that PI3K/AKT and

1915S.Y. Park et al. / International Immunopharmacology 11 (2011) 1907–1915

ERK signaling plays a pivotal, mediatory role in the overexpression ofHO-1 and subsequent nuclear accumulation of Nrf2 in response to α-iso-cubebenol.

The induction of HO-1 signaling is an important event inmediatingthe anti-inflammatory response to natural compounds [15]. In recentreports, HO-1 induction has been shown to inhibit pro-inflammatorymediator expression through the inactivation of NF-κB [29]. Weinvestigated the anti-inflammatory effects of α-iso-cubebenol inP. gingivalis LPS-stimulated humanmacrophage THP-1 cells and foundthat up-regulation of HO-1 by α-iso-cubebenol mediated theinhibition of P. gingivalis LPS-stimulated pro-inflammatory cytokines(TNF-α, IL-6, IL-1β, and IL-12). Numerous studies on P. gingivalis LPShave been performed, which show that it activates the host'sinflammatory response and produces pro-inflammatory cytokinesthrough NF-κB signaling activation [30]. There is recent reportconcerning that p65 repress the Nrf-2 transcription activity [31].Therefore, we can infer throughout the results that the anti-inflammatory effects of α-iso-cubebenol via induction of HO-1 arepossible due to inhibition of p65 activation. Our data showed that α-iso-cubebenol attenuated NF-κB transactivation and nuclear translo-cation in P. gingivalis LPS-stimulated human macrophage THP-1 cells.These data imply that the ability of α-iso-cubebenol to mediate anti-inflammatory functionsmay be achieved through the up-regulation ofHO-1 expression via Nrf2, PI3K/Akt, and ERK activation. The pro-inflammatory cytokine IL-12 plays a central role in driving thedevelopment of T-cell stimulated responses [13,32]. Our data showthat IL-12 secretion was inhibited by α-iso-cubebenol through HO-1signaling. Clearly, further studies are needed to clarify which α-iso-cubebenol-mediated HO-1 signals affect T-cell activation and antigenpresentation mechanisms.

In conclusion, in this study, we isolated α-iso-cubebenol fromS. chinensis and clearly showed that α-iso-cubebenol induced HO-1expression, which is mediated by Nrf2, PI3K/Akt, and ERK activation.Furthermore, the biological relevance of HO-1 signaling for the anti-inflammatory activity of α-iso-cubebenol was also demonstrated inP. gingivalis LPS-stimulated human macrophage THP-1 cells, in whichblocking HO-1 signaling with pharmacological inhibitors led to areduction in the inhibitory effect of α-iso-cubebenol on P. gingivalisLPS-induced pro-inflammatory cytokines. Our findings suggest thatan HO-1 signaling pathway exists by which α-iso-cubebenol andpossibly other natural compounds can prevent periodontitis andchronic inflammatory diseases.

Acknowledgments

This study was supported by the Technology DevelopmentProgram for Agriculture and Forestry, Ministry for Food, Agriculture,Forestry and Fisheries, Republic of Korea (106048031SB010).

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