inhibitory effects of ginkgo biloba extract on inflammatory mediator production by porphyromonas...

Inhibitory Effects of Ginkgo biloba Extract on InflammatoryMediator Production by Porphyromonas gingivalisLipopolysaccharide in Murine Macrophages via Nrf-2Mediated Heme Oxygenase-1 Signaling Pathways

Eun Yeon Ryu,1 Ah Jeong Park,2 Sun Young Park,1 Sung Hae Park,2 Hye Won Eom,2

Young Hun Kim,1 Geuntae Park,3 and Sang-Joon Lee2,4

Abstract—Periodontitis is an oral chronic inflammatory disease that influences systemic diseases.Heme oxygenase-1 has several beneficial abilities through Nrf-2 regulation. Ginkgo biloba has beenreported to have anti-inflammatory effects associated with heme oxygenase-1 (HO-1) expression. Inthis study, we investigated whether the anti-inflammatory effects of G. biloba were involved withNrf-2-mediated HO-1 expression in Porphyromonas gingivalis LPS-stimulated RAW264.7 macro-phage cells. G. biloba was extracted with ethyl acetate (EGB). EGB exhibited anti-inflammatoryactivities, which suppressed the production of pro-inflammatory mediators, the activation of mito-gen-activated protein kinases, and nuclear translocation of transcription factors. EGB also up-reg-ulated the HO-1 expression, and the Nrf-2 level in the nucleus and its transactivity. Furthermore,reduced pro-inflammatory mediator levels by EGB were inverted in the presence of SnPP. Thecollective results suggest that the anti-inflammatory effects of EGB are due to the HO-1 expressionvia up-regulation of Nrf-2 in RAW 264.7 cells stimulated by P. gingivalis LPS.

KEY WORDS: Ginkgo biloba extract; heme oxygenase (HO)-1; inflammation; NF-E2 related factor (Nrf)-2;Porphyromonas gingivalis lipopolysaccharide.

INTRODUCTION

Periodontitis is a chronic periodontal disease in thehuman oral cavity that is caused by bacterial infection.Periodontal bacteria, such as Porphyromonas gingivalis,secrete a number of virulence factors like lipopolysac-charide (LPS), attack the tissue enclosing the teeth, andultimately lead to tooth loss [1]. These oral pathogenicbacteria or secreted pro-inflammatory cytokines and

mediators circulate throughout the body in the bloodstream[2]. The resulting disruption of vascular endothelium anddistant organ tissues underlies the association of chronicperiodontal disease as one of the major risk factors forsystemic inflammations, cardiovascular disease (CVD),diabetes, atherosclerosis, coronary heart disease, and stroke[3]. The extract of Ginkgo biloba leaves has been reportedto have therapeutic benefits in these conditions by virtue ofimproved blood circulation, restricted clot development,and cell protection from oxidative stress [4–6].

G. biloba is a valuable herb that has been used as atraditional medicine in the Orient. The medicinal use ofginkgo dates back to at least 2,800 B.C. In thesetraditional formulations, ginkgo leaves provide relieffrom asthma and cough symptoms, and have brain-related benefits [6]. Research concerning its medicinaleffects, and structure, ingredient, and biological activi-ties has been conducted for a very long time. Variouseffects of G. biloba extract have being revealed,

E.Y. Ryu and A.J. Park have contributed equally to this work.

1 BIO-IT Fusion Technology Research Institute, Pusan NationalUniversity, Busan 609-735, South Korea

2 Department of Microbiology, Pusan National University, Busan609-735, South Korea

3 Institute for Research & Industry Cooperation, Pusan NationalUniversity, Busan 609-735, South Korea

4 To whom correspondence should be addressed at Department ofMicrobiology, Pusan National University, Busan 609-735, SouthKorea. E-mail: [email protected]

0360-3997/12/0400-1477/0 # 2012 Springer Science+Business Media, LLC

Inflammation, Vol. 35, No. 4, August 2012 (# 2012)DOI: 10.1007/s10753-012-9461-6

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although information on periodontal disorders is sparse.One study reported a G. biloba extract-related inhibitoryeffect of collagenolytic activity of P. gingivalis [7].Broad medicinal properties of G. biloba leaf extractinclude antioxidant, anti-inflammation, and neuroprotec-tive effects against several cardiovascular and neuronaldiseases [5, 8]. The pharmacological effects of G. bilobaextract may be related to its free radical scavengingability [8]. Furthermore, G. biloba extract induces theexpression of genes encoding proteins with anti-inflam-matory and antioxidant activity, such as heme oxygen-ase-1 (HO-1), through the nuclear factor erythroid-derived 2-related factor (Nrf-2)-antioxidant responseelement (ARE) signaling pathway [9].

HO-1 is a rate-limiting catabolic enzyme of heme.Presence of stimulatory compounds such as its substrate,heme, reactive oxygen species (ROS), LPS, cytokines,and nitric oxide (NO) leads to degradation of heme intocarbon monoxide (CO), iron, and biliverdin. The latter israpidly reduced to bilirubin [10]. HO-1 and its by-products have anti-inflammatory properties [11, 12].Induction of HO-1 drives an adaptive survival responsethat protects cells from oxidative stress and reducesexpression of pro-inflammatory compounds [12]. Theho-1 gene has an ARE sequence in its promoter regionand its expression is regulated by Nrf-2. Under normalconditions, Nrf-2 is combined with actin-bound Kelch-like ECH-associated protein 1 (Keap1) in the cytosol[13]. In response to stimulators, however, Nrf-2 sepa-rates from Keap1 and translocates into the nucleus,where it activates transcription of antioxidant genes forcytoprotection through binding to ARE [14].

The collective data to date support the view that G.biloba leaves play a crucial role in the control ofperiodontitis. This is important, given the link betweenperiodontitis and the aforementioned CVDs. The mech-anism of G. biloba extract in inflammation induced byoral bacterial infection is unknown. The present studyaddressed this shortcoming, and was grounded in thepostulation that G. biloba extract is involved, at leastpartially, in the Nrf-2 mediated HO-1 signaling pathway.

MATERIALS AND METHODS

Materials

G. biloba leaves were purchased in March 2006. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbro-mide (MTT), and other reagents were purchased from

Sigma-Aldrich (St. Louis, MO, USA). ProtoporphyrinIX (SnPP) and antibodies were purchased from SantaCruz Biotechnology (Santa Cruz, CA, USA). LPS(phenol extract of P. gingivalis) was purchased fromInvivoGen (San Diego, CA, USA).

Preparation of EGB

The dried leaves of G. biloba was purchased from alocal herb store, Kwang Myoung Herb Medicine (Pusan,South Korea) in March 2006. The dried leaves of G.biloba were homogenized and meshed. To prepare astock solution, 100 g of G. biloba leaves was added to85% ethanol (3×1 l) or dichloromethane (3×1 l). Eachextract was shaken for at least 6 h and after that eachextract was filtered through a 0.2-μm filter. The filtratewas evaporated to dryness under reduced pressure. Thedried extract was dissolved in water and furtherpartitioned in succession with ethyl acetate, n-butanoland distilled water. The ethyl acetate fraction wasconcentrated by rotary vacuum evaporation (Eyela,Tokyo, Japan). The active ethyl acetate fraction(12.5 g) was used as the EGB (Fig. 1).

Cell and Measurement of Concentration of Cytokines

The murine macrophage cell line, RAW 264.7, wasobtained from American Type Culture Collection (Rock-ville, MD). The cells were grown in Dulbecco’s modifiedEagle’s medium (DMEM; Gibco, Franklin Lakes, NJ)supplemented with 10% fetal bovine serum (FBS), andincubated at 37 °C in a humidified atmosphere of 5% CO2

and 95% air. The pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) levels were quantified in the culture mediausing an enzyme-linked immunosorbent assay (ELISA) kit(R&D Systems, Minneapolis, MN) according to themanufacturer’s instructions.

Dried Ginkgo biloba sample (100 g)

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Fig. 1. Schematic diagram of the extraction process of Ginkgo bilobaleaves extract.

1478 Ryu, Park, Park, Park, Eom, Kim, Park and Lee

HO-1 Activity Assay

HO-1 activity was determined by measuring theamount of bilirubin as described previously [15]. Briefly,cells were pre-incubated with hemin and treated withEGB, SnPP or CoPP. To determine the bilirubin levels,barium chloride dehydrate and benzene was added toeach culture supernatant. The amount of bilirubin inupper benzene layer was observed at an absorbance of450 nm by an Ultrospec 6300 Pro UV/visible spectro-photometer (Amersham Biosciences, Piscataway, NJ).The quantity of bilirubin was calculated using anextinction coefficient (ε450=27.3 mM/cm).

Transient Transfection and Dual Luciferase Assay

Cells were transfected with a κB-luc reporterplasmid consisting of three κB concatamers from theIgγ chain and an ARE reporter plasmid (Stratagene,Grand Island, NY) using FuGENE-HD reagent (RocheApplied Science, Basel, Switrzerland) according to themanufacturer’s instructions. Luciferase activity wasassayed with a dual-luciferase assay kit (Promega), andluminescence was measured with a Wallac 1420 micro-plate luminometer.

Western Blot Analysis

Cells were harvested in ice-cold lysis buffer, andthe protein content of the cell lysates was thendetermined using Bradford reagent (Bio-Rad, Hercules,CA). The protein in each sample were resolved by 8.5%SDS-polyacrylamide gel electrophoresis (PAGE), trans-ferred to a polyvinylidene fluoride membrane, andexposed to the appropriate antibody. The proteins werevisualized using an enhanced chemiluminescence detec-tion system (Amersham Biosciences).

Reverse Transcription (RT) Real-Time PolymeraseChain Reaction (PCR)

Total cellular RNA was isolated using a RNAspinmini RNA isolation kit (GE Healthcare, Buckingham-shire, UK) according to the manufacturer’s instructions.Total RNA (1 μg) was reverse-transcribed using MaximeRT PreMix (Intron Biotechnology, Seongnam, SouthKorea) and anchored oligo-dT15 primers. Real-time PCRwas performed using a Chromo4 ™ instrument (Bio-Rad) with the SYBR Green Master Mix (AppliedBiosystems, Foster City, CA).

Statistical Analyses

Data are expressed as mean ± standard error (SE).Each experiment was repeated at least three times.Statistical analysis was performed with SPSS version16.0 software (SPSS, Chicago, IL) to determine significantdifferences. We used either one- or two-way analysis ofvariance (ANOVA), followed by Dunn’s post hoc tests foranalyses. *P<0.05 was considered statistically significant.

RESULTS

Fractionation of G. biloba and Selection of EffectiveFraction

G. biloba leaves were extracted and fractionated withEtOAc, n-BuOH, and water to dissolve the active compo-nents. Each fraction was pre-tested with 1–50 μg/mlconcentrations for screening cytotoxicity and inhibition ofNO production. Judging by MTTassay results, all fractionswere not cytotoxic under the given conditions (data notshown). However, the inhibition activity against NOproduction differed in the fractions. The EtOAc fractionreduced NO reduction in a dose-dependent manner(Fig. 2a). The others did not display any effect on NOproduction (data not shown). The screening steps revealedthat the EtOAc extract from G. biloba leaves has anti-inflammatory activity. This fraction was subsequently used.

Decreasing Effects of EGB on the Pro-inflammatoryCytokines Secreted from P. gingivalis LPS-StimulatedRAW 264.7 Cells

Immune cells protect an organism from severalstimulations activating many intracellular signal pathwaysincluding inflammation [16]. P. gingivalis LPS also plays apathogenic role on periodontitis and generates macro-phages to secrete pro-inflammatory cytokines such asTNF-α, IL-1β, and IL-6 [17]. We first investigated theanti-inflammatory activities of EGB against the productionof pro-inflammatory cytokines by P. gingivalis LPS. Thecells were exposed to P. gingivalis LPS for 1 h and weretreated with 5, 10, and 20 μg/ml of EGB. TNF-α, IL-1β,and IL-6 elaborated in the medium were measured. Asshown in Fig. 2, the production levels of pro-inflammatorycytokines were significantly reduced in a dose-dependentmanner compared to those of cells not treated with EGB.EGB inhibited the production of pro-inflammatory cyto-kines induced by P. gingivalis LPS without destruction ofRAW 264.7 cells.

1479Anti-Periodontal Inflammatory Effects of EGB

Down-regulation of MAPK Signaling by EGB in RAW264.7 Cells Stimulated with P. gingivalis LPS

Mitogen-activated protein kinases (MAPKs) are thehigher signal pathway molecules of pro-inflammatorycytokines, and are activated in response to P. gingivalisLPS [16, 18]. To determine which signal pathway wasassociated with the anti-inflammatory effects of EGB,the phosphorylation levels of ERK, JNK, and p38 weredetected. The normal forms of MAPKs were detected asits reference proteins. Phosphorylation of ERK, JNK,and p38 were clearly increased under LPS stimulatedconditions, however, these increases were reduced underEGB treatment conditions in a dose-dependent fashion(Fig. 3). The results indicated that the anti-inflammatoryactivities of EGB arose from the down-regulation ofMAPK (ERK, JNK, and p38) signaling pathways.

Inhibitory Effects of EGB on NF-κB and AP-1Nuclear Translocation in RAW264.7 Cells Stimulatedby P. gingivalis LPS

P. gingivalis LPS activates transcription factors(TFs) such as NF-κB and activator protein-1 (AP-1)[18]. Both of these TFs are involved in MAPK signalingpathways and, under a variety of stimulations, they

control gene expression in cell differentiation, proliferation,and apoptosis [19]. We detected the existing level of p65,

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Fig. 2. Inhibitory effects of EGB on production of pro-inflammatory mediators in Porphyromonas gingivalis LPS-stimulated RAW 264.7 cells. Thecells were incubated with EGB in various doses for 1 h and treated with P. gingivalis LPS (1 μg/ml) for 24 h. The secreted volume of nitrite (a), TNF-α (b), IL-1β (c), and IL-6 (d) were measured in the collected culture supernatant by Griess method and ELISA, respectively. Each bar represents themean ± SE from three independent experiments in each group. *P<0.05 vs. the LPS-treated group.

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Fig. 3. Suppression activity of EGB on MAPK activation in RAW264.7 murine macrophage cells treated with P. gingivalis LPS. Thecells were treated with EGB in indicated doses for 1 h and exposed toP. gingivalis LPS (1 μg/ml) for 15 min. The cell extracts were subje-cted to Western blot for ERK, phosphorylated ERK, JNK, phosphor-ylated JNK, p38, and phosphorylated p38 expression.


c-jun, and c-fos in nuclei; p65 is a major subunit of NF-κB,c-jun and c-fos are components of AP-1. First, the RAW264.7 cells were treated with 1 μg/ml of P. gingivalis LPSfor different times (0.25, 0.5, 1, 2, and 4 h) to choose theoptimal time of translocation of TFs for the followingstudies. The nuclear translocation of p65 was occurred in30 min and those of c-fos/c-jun were translocated in 1 h(Fig. 4a). On the basis of this result, both TFs nucleartranslocations were estimated under different concentra-tions of EGB. As shown in Fig. 4b, the nuclear transloca-tion of p65 was completely blocked at a low concentration.The phosphorylation degree of IκBα also significantlydecreased (Fig. 4b); IκBα forms the complex with p65 toinhibit the activation of NF-κB. To confirm the effects ofEGB on p65 translocation, the promoter activity wasassessed by a luciferase assay. EGB blocked the luminance

of luciferase in a dose-dependentmanner (Fig. 4c). The dataindicated that EGB inhibited p65 nuclear translocation andtransactivity. The inhibitory effect of EGB on the activationof another TF, AP-1, was examined. The existing levels ofc-fos and c-jun in nucleus were gradually reduced accordingto the concentrations of EGB (Fig. 4d). The observationwas consistent with the EGB-mediated inhibition of AP-1nuclear translocation. The collective data support the viewthat the anti-inflammatory effects of EGB are regulated atthe transcriptional level, blocking the P. gingivalis LPS-induced nuclear translocation of NF-κB and AP-1.

Effects of EGB on HO-1 Expression and Its Activity

HO-1 is a key enzyme that exerts anti-inflammatoryeffects with its by-products carbon monoxide (CO), iron,

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Fig. 4. The attenuation effects of EGB on TFs nucleus translocation related with pro-inflammatory mediators expression in P. gingivalis LPS-stimulated RAW 264.7 murine macrophage cells. a Cells were treated with 1 μg/ml of P. gingivalis LPS for the indicated lengths of time and weresubjected to Western blot for p65, c-jun, and c-fos. TBP was detected to prove the same amount of protein for each lane. b Nuclear translocation ofNF-κB was detected by Western blotting. The cells were incubated with EGB for 1 h and treated with P. gingivalis LPS for (1 μg/ml) 30 mindepending on result A. Nuclear extracts were prepared and analyzed as verification of the volume of p65. Cytosolic extracts were also analyzed byWestern blotting with phosphorylated (p)-IκB-α antibody. (C) Cells were co-transfected with kB-luc reporter and control Renilla luciferase plasmidpRL-CMV for 24 h. Then EGB was treated in indicated concentrations for 1 h, and stimulated with P. gingivalis LPS 1 μg/ml for 24 h. Equal amountsof cell extracts were assayed for dual-luciferase activity. kB-luciferase activity was normalized to control Renilla luciferase expression. Each barrepresents the mean ± SE from three independent experiments in each group. *P<0.05 vs. the LPS-treated group. d Nuclear translocation of AP-1 wasdetected by Western blotting. The cells were incubated with EGB for 1 h and treated with P. gingivalis LPS (1 μg/ml) for 2 h depending on result A.Nuclear extracts were prepared and analyzed as verification of the volume of c-jun and c-fos.


and biliverdin/bilirubin [11, 12]. We investigated theeffects of EGB on induction of HO-1 mRNA and proteinexpression using real-time RT-PCR and Western blot.EGB induced mRNA and protein levels of HO-1expression in a dose-dependent manner (Fig. 5a and b).The optimal time of HO-1 mRNA and protein expres-sion was 6 and 12 h, respectively, in RAW 264.7 cells.EGB-mediated induction of HO-1 expression wasconfirmed using actinomycin D (Act. D) and cyclohex-imide (CHX). Act. D is a transcription inhibitor thathinders the action of DNA-dependent RNA poly-merases. CHX is a translation inhibitor that exerts itsaction by disturbing the ribosomal protein synthesis. ThemRNA and protein levels of HO-1 expression inducedby EGB were significantly reduced in the presence ofAct. D and CHX treated conditions (Fig. 5c). These datasupport the suggestion that EGB induces HO-1 expres-sion at the transcriptional and translational level, whichproduces anti-inflammatory activities.

HO-1 degrades free-heme to equimolar amounts ofCO, ferrous iron, and biliverdin, which is altered tobilirubin [11, 12]. Hemin was presently used as thesource of free-heme to determine HO-1 activity bymeasuring the amount of bilirubin production. As shownin Fig. 5d, EGB increased the production of bilirubin byover 2-fold. Understandably, there was no difference insamples pre-incubated with the HO-1 inhibitor, SnPP,compared with normal cells. EGB accentuated HO-1activity more than the HO-1 inducer, CoPP. The resultsverified that EGB revitalized not only the mRNA andprotein expression of HO-1 but also its activity.

Effects of EGB on Up-regulation of Nrf-2-ARESignaling Pathway in RAW 264.7 Cells

HO-1 gene has ARE sequence in its promoterregion, which is the binding site for Nrf-2 as a TF [9].To investigate the influence of the Nrf-2-ARE signalingpathway on the expression and augmentation of HO-1,we examined the accumulation of the Nrf-2 in nuclei ofRAW 264.7 cells treated with EGB. The existing level ofNrf-2 in nuclei climbed steadily in proportion to EGBconcentrations, and the maximum translation levelappeared 30 min after treatment (Fig. 6a). To confirmthese results, the binding activity of Nrf-2 on AREsequence was measured using a luciferase assay method.As shown in Fig. 6b, the transactivity of Nrf-2 wassimilar to that found in the previous experiment. Theresults of Figs. 5 and 6 indicate that EGB could induce

HO-1 expression at both the transcriptional and transla-tional level through the Nrf-2-ARE signaling pathway.

Involvement of HO-1 Expression on Anti-inflammatoryActivity of EGB

EGB has anti-inflammatory effects on P. gingivalisLPS-stimulated RAW 264.7 cells, and it can induce Nrf-2-mediated HO-1 expression. To verify the relationshipsbetween these two findings, the effects of EGB wereobserved on secreted pro-inflammatory mediators in thepresence of SnPP. The secreted levels of pro-inflamma-tory mediators (nitrite, TNF-α, IL-1β, and IL-6)increased significantly after P. gingivalis LPS stimula-tion, but application of 20 μg/ml EGB produced similaranti-inflammatory activity (Fig. 7a,c,d, and e) as docu-mented in Fig. 2. However, these reductions werereversed by SnPP co-treatment (Fig. 7). To supportthese data, we also examined the alteration of iNOS andCOX-2 expression with SnPP treatment. SuppressediNOS and COX-2 protein expressions were reversed onaccount of HO-1 activity blocking in the same manner(Fig. 7b). The collective data are consistent with theview that the anti-inflammatory effects of EGB are dueto its induction of HO-1 expression.

DISCUSSION

We assumed that the dissolved active componentfrom G. biloba leaves obtained using EtOAc has anti-inflammatory effects on murine macrophage cells stim-ulated with P. gingivalis LPS and performed a series ofexperiments to reveal the inhibitory mechanisms ofinflammation involving Nrf-2-mediated HO-1 expres-sion. This study investigated three aspects: (1) anti-inflammatory activity of EGB against provoked inflam-matory reactions in RAW 264.7 cells by P. gingivalisLPS, (2) inductive ability of EGB on protein and mRNAexpressions of HO-1 via Nrf-2 regulation, and (3) aconnection between inhibitory effects on inflammatorystatus and inducible effects on HO-1 expression of EGB.

P. gingivalis is a well-known gram-negative oralbacterium, which has LPS on its surface. It stimulatesthe macrophages, induces the immune responses, andproduces inflammatory mediators [20]. It is functionallyand structurally different with enterobacterial LPS. P.gingivalis LPS has a unique form of lipid A, accordingto previous report, it seems that the concentration ofhemin is the key factor to construction of lipid A


structure [21]. P. gingivalis LPS is also known to usedifferent strategy to attack the macrophages in comparedwith those of enterobacteria. Several reports haveindicated that for host cell activation, P. gingivalis LPS

interacts with TLR2, not TLR4 [22, 23], but it producesthe same activation of the inflammatory response in thehost. Presently, P. gingivalis LPS induced severalinflammatory responses, which echoed other studies. P.

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Fig. 5. The inductive effects of EGB on HO-1 expression and its activity in RAW 264.7 cells. a The cells were cultured with increasing concentr-ations of EGB for 6 h or 20 μg/ml of EGB for the indicated times. Relative HO-1 mRNA expression (2−Δct) was determined by real-time PCR andcalculated by subtracting the Ct value for GAPDH from the Ct value for HO-1. The mRNA relative content was indicated as fold change over control.b The cells were incubated for 12 h with the indicated concentration of EGB and incubated for various times with 20 μg/ml EGB. Total cellularextracts were prepared and examined by Western blot. α-Tubulin was detected to prove the same amount of protein for each lane. c The cells weretreated with 20 μg/ml of EGB for 12 h in the presence of Act D or CHX (1 μg/ml). We determined HO-1 protein expression by Western blot. d Thecells were co-incubated with 50 μM hemin for 2 h and thereafter exposed to EGB (20 μg/ml), SnPP (20 μM), or CoPP (20 μM) during 12 h. Thequantity of bilirubin production in the culture media was measured spectrophotometically and calculated using a molar extinction coefficient ofbilirubin dissolved in benzene. Each bar represents the mean ± SE from three independent experiments in each group. *P<0.05 with respect to eachcontrol group.


gingivalis LPS stimulated induction of the MARKsignaling pathway in RAW 264.7 cells, which precededthe nuclear translocation the TFs, NF-κB and AP-1,involved with expression of pro-inflammatory mole-

cules, and the secretion of nitrite and the pro-inflamma-tory cytokines TNF-α, IL-1β, and IL-6. However, EGBwas consistently suppressive, decreasing the amount ofthose pro-inflammatory mediators (Fig. 2), inhibiting

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Fig. 6. Effects of EGB on Nrf-2 nuclear accumulation in RAW 264.7 murine macrophage cells. a The cells were incubated with 20 μg/ml of EGB forthe indicated lengths of time, and were incubated with the indicated concentration of EGB for 1 h. The nuclear extracts were prepared and examinedby Western blot. Western blot detection of TBP was used as a protein loading control for each lane. b The cells were transfected with the ARE-luciferase construct and treated with indicated concentration of EGB. Equal amounts of cell extract were assayed for dual-luciferase activity. Expr-ession from the renilla luciferase control was used to normalize ARE-luciferase activity. Each bar represents the mean ± SE from three independentexperiments in each group.

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Fig. 7. Anti-inflammatory effects of EGB involved with HO-1 expression in RAW 264.7 murine macrophage cells stimulated with P. gingivalis LPS.The cells were pretreated with 20 μg/ml EGB in the presence of SnPP (20 μM) for 1 h and then stimulated with P. gingivalis LPS (1 μg/ml) for 24 h.We measured the secreted amounts of nitrite (b), TNF-α (c), IL-1β (d), and IL-6 (e) in the culture supernatant by Griess method and ELISA,respectively. cell extracts were subjected to Western blot for iNOS, COX-2, and HO-1 expression (b).


phosphorylation of the MAPKs (Fig. 3), and reducingTF nuclear translocation and transactivity (Fig. 4). Theresults demonstrated that the soluble active componentsof G. biloba leaves in EtOAc have the controllingactivity against several inflammatory responses occurredby P. gingivalis LPS in RAW264.7 cells.

Several reports have described that standard G.biloba extracts induce HO-1 expression to protect cells.Kotakadi et al. [24] showed that HO-1 expression byEGB761 decreases the activation of mouse macrophagesassociated with colon cancer in vitro, and Yao et al. [25]suggested that EGB761-mediated HO-1 expression hashepatoprotective effects and suppression effects againstethanol-induced oxidative damages. Moreover, Shah etal. [5] reported that EGB761-induced HO-1 expressionand production of its by-products have antioxidanteffects in mouse primary cortical cells involved withischemia and Alzheimer’s disease. However, most priorstudies used standardized extracts of G. biloba leaves(EGB 761) and mainly assessed its antioxidant proper-ties. EGb761 is composed of 24% flavonoid glycosidesand 6% terpenoid including ginkolides and bilobalide.Flavonol glycosides have free radical-scavenging effect,G. biloba leaves may have inhibitory effects on thedamages resulting from oxidative stresses. Terpenoid hassuperoxide-scavenger activity, inter alia, bilobalide wasnoted that have protective ability against neuronal deathin global brain ischemia and in glutamate-inducedexcitotoxicity [26].

Distinct from those studies, the present studyexamined for the first time the anti-inflammatory effectsin macrophage cells using EtAOc extract of G. bilobaleaves, which exhibited low toxicity and which can bemanufactured on a large scale for use as a solvent [27].The obtained fraction displayed HO-1 inductive abilityvia regulation of Nrf-2 nuclear translocation. The mRNAand protein levels of HO-1 were expressed in dose-dependent manner at 6 and 12 h, respectively (Fig. 5aand b). These results were confirmed using the inhibitorsAct.D and CHX, which disturb transcription andtranslation. The protein expression of HO-1 was re-pressed in inhibitor-treated cells (Fig. 5c). EGB affectedthe HO-1 mRNA and protein expression levels as wellas its activity (Fig. 5d). Nrf-2 plays a crucial role inregulating ARE related genes such as HO-1 [3, 4]. Ourcrude extract also mediated Nrf-2 nuclear translocationand its activity. Nrf-2 nuclear accumulation increased ina dose-dependent manner 30 min after EGB treatmentand promoter activity also steadily increased (Fig. 6a).EGB also was effective in inducing HO-1 expression,

regardless of the oxidative stress changes (Fig. 7b),similar to data regarding HO-1 expression usingEGB761 [4]. Throughout the present study, we wereconfident that the crude extract of G. biloba leaves couldeffectively induce HO-1 expression regulating theactivation of Nrf-2 TF.

The protective activity of HO-1 is related with thedepletion of the production of pro-inflammatory cyto-kines [18]. We designed an experiment using a HO-1inhibitor (SnPP) to assess whether the inhibitory effectsof EGB could be connected with the induction of HO-1expression on P. gingivalis LPS-related inflammatoryresponses in RAW 264.7 cells. The decreased levels ofpro-inflammatory mediators were reversed due to theblocking of the HO-1 expression by SnPP (Fig. 7). Theresults support the conclusion that EGB induces HO-1expression by regulating Nrf-2-mediated transcriptionand exhibiting anti-inflammatory effects. A recent reportconsidered that p65 interrupts the Nrf-2-ARE signalingpathway. Further studies will be necessary to reveal therelationship between p65 and transactivity of Nrf-2,which influences the expression of one of the major anti-inflammatory factor, HO-1 [28]. For the present, ourresearch marks the first attempt to study the anti-inflammatory effects of a crude extract of G. bilobaleaves on oral inflammation using P. gingivalis LPS-stimulated murine macrophage cells.

Inflammation is a kind of defensive response of aliving organism, and is a protective reaction againstseveral aggressions. However, dysregulation owing tocontinuous inflammatory activity can lead to seriousconditions. Periodontal diseases arise from local chronicinflammation in gingival soft tissue. For these reasons,periodontal diseases must be treated early. Severalprevious reports showed that P. gingivalis is the majormicroorganism in periodontitis, and its destructive effecton the periodontium has been researched in several clinicaland laboratory studies. Salari and Kadkhoda [29] havereported the isolates ratio from the dental with periodontaldisease: P. gingivalis (21.9%), Fusobacterium nucleatum(0.4%), Peptostreptococcus micros (1.3%) and Prevotellaintermedia (10.5%). In another study, P. gingivalis wasdetected in only 25% (46 of 181) of healthy subjects butwas detected in 79% (103 of 130) of individuals in theperiodontitis group (P<0.0001). The possible ratio forbeing infected with P. gingivalis was 11.2 times greater inthe periodontitis group than that in the healthy group [30].Our results revealed that EGB has anti-inflammatory effectthrough Nrf-2-mediated HO-1 signaling pathway againstP. gingivalis LPS-induced inflammatory responses from


murine macrophages. In this light, the EtAOc extract of G.biloba leaves has merit as a commercial additive to tea, atoothpaste, mouthwash and as a dental floss coating to helpprotect from oral disease. The present study may be thebeginning of the refinement and use of preventive orcurative natural substances for several P. gingivalis LPS-induced inflammatory diseases.

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