Polyphenolic fraction of Lonicera caerulea L. fruits reduces oxidative stress and inflammatory markers induced by lipopolysaccharide in gingival fibroblasts

Food and Chemical Toxicology 48 (2010) 1555–1561

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Polyphenolic fraction of Lonicera caerulea L. fruits reduces oxidative stress and inflammatory markers induced by lipopolysaccharide in gingival fibroblasts A. Zdarˇilová a, A. Rajnochová Svobodová a,*, K. Chytilová b, V. Šimánek a, J. Ulrichová a a b

´ University, 775 15 Olomouc, Czech Republic Department of Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry, Hneˇvotínská 3, Palacky Department of Oral and Maxillofacial Surgery, I.P. Pavlova 6, University Hospital, 775 20 Olomouc, Czech Republic

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Article history: Received 4 November 2009 Accepted 17 March 2010

Keywords: Lonicera caerulea L. Antioxidant Anti-inflammatory activity Reactive oxygen species Cytokines Cyclooxygenase-2

a b s t r a c t The most common oral diseases have a microbial aetiology. Pathogenic bacteria liberate a number of irritating agents including a lipopolysaccharide (LPS) that activates pro-inflammatory cytokines promoting increased activity of polymorphonucleocytes (PMN). Release of PMN-derived free radicals into an infected gingival area affects gums, periodontal ligaments and alveolar bone. Berries of Lonicera caerulea L. (blue honeysuckle) are rich in phenolics, particularly phenolic acids, flavonoids and anthocyanins that have multiple biological activities in vitro and in vivo such as antiadherence, antioxidant and anti-inflammatory. Studies have shown that polyphenols suppress a number of LPS-induced signals and thus could be effective against gingivitis. Here we assessed effects of the polyphenolic fraction of L. caerulea fruits (PFLC; containing 77% anthocyanins) on LPS-induced oxidative damage and inflammation in human gingival fibroblasts. Application of PFLC (10–50 lg/ml) reduced reactive oxygen species (ROS) production, intracellular glutathione (GSH) depletion as well as lipid peroxidation in LPS-treated cells. PFLC treatment also inhibited LPS-induced up-regulation of interleukin-1b (IL-1b), interleukin-6 (IL-6) and tumour necrosis factor-a (TNF-a) and it suppressed expression of cyclooxygenase-2 (COX-2). The effects are presumably linked to its antioxidant and anti-inflammatory activities and suggest its use in attenuating the inflammatory process, including periodontal disease. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Periodontal disease is one of the most commonly reported chronic adult conditions. Gingivitis, the mild form of the diseases, is characterized by the soft and reversible inflammation of the gingiva (gum) caused by plaque formation. Untreated gingivitis may lead to the more serious and irreversible disease, periodontitis, which accompanied destruction of periodontal tissues (gingiva, periodontal ligament and alveolar bone) with eventual exfoliation of the teeth (Haffajee and Socransky, 1994). Periodontitis is the result of complex interactions between periodonto-pathogenic bacteria and host immunological responses (Page, 1999; Kinane and

Abbreviations: COX-2, cyclooxygenase-2; DHR, dihydrorhodamine 123; DMSO, dimethylsulfoxide; DMEM, Dulbecco’s modified Eagle’s medium; FCS, foetal calf serum; GSH, glutathione; HPLC–MS, high performance liquid chromatography with a mass spectrometry; iNOS, inducible nitric oxide synthase; IL-1b, interleukin-1b; IL-6, interleukin-6; IL-8, interleukin-8; LPS, lipopolysaccharide; MAPK, mitogenactivated protein kinases; NF-jB, nuclear factor kappa B; NR, neutral red; PBS, phosphate buffered saline; PMN, polymorphonucleocytes; PFLC, polyphenolic fraction of L. caerulea fruits; PVDF, polyvinylidene difluoride; PGE2, prostaglandin E2; ROS, reactive oxygen species; TBARS, thiobarbituric acid reactive substances; TNF-a, tumour necrosis factor-a. * Corresponding author. Tel.: +420 585 632 314; fax: +420 585 632 302. E-mail address: [email protected] (A.R. Svobodová). 0278-6915/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2010.03.024

Lappin, 2002). There are more than 300 different bacterial species present in the gingival area of the mouth. However, three species have been identified as the major pathogens of periodontitis, Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans, Tannerella forsythia (formerly Bacteroides forsythus) and Porphyromonas gingivalis (Jenkinson and Dymock, 1999). Bacteria produce a number of metabolites which affect gingival tissue. One irritating agent, a lipopolysaccharide (LPS), is a major constituent of the outer bacterial membrane and a critical determinant in gingivitis initiation and progression (Offenbacher, 1996). LPS induces generation of reactive oxygen species (ROS) from polymorphonucleocytes resulting in increased inflammation (Jenkinson and Dymock, 1999). The molecular mechanism of ROS-caused tissue damage includes peroxidation of membrane lipids, modification of proteins (enzymes) and stimulation of pro-inflammatory cytokine release. Imbalance between ROS generation and elimination also leads to alteration in cellular metabolism. Moreover, gingival fibroblasts exposed to LPS increase their release of pro-inflammatory cytokines (such as IL-1a, IL-1b, IL-6, IL-8 and TNF-a) (Lamont and Jenkinson, 1998) and also express inducible forms of nitric oxide synthase (iNOS) (Daghigh et al., 2002) as a part of the immune response. Although NO produced by iNOS has bactericide properties, it also plays a significant role in the pathophysiology of inflammatory diseases. Pro-inflammatory cytokines (IL-1, TNF-a)

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strongly induce cyclooxygenase-2 (COX-2), whose level is normally low. Elevated COX-2 expression also promotes the production of prostaglandin E2 (Huang et al., 2000). Several studies have shown that phenolic compounds which are widely distributed in plants suppress both inflammation and oxidative stress and thus could be effective against gingivitis. Berries of blue honeysuckle (Lonicera caerulea L.; Caprifoliaceae) are widely harvested and used in folk medicine in northern Russia, China and Japan. The fruits are rich in phenolics, particularly phenolic acids, flavonoids, anthocyanins and proanthocyanidins (Chaovanalikit et al., 2004; Svarcova et al., 2007). These compounds have been recognized to possess wide range of biological activities such as antimicrobial (Puupponen-Pimiä et al., 2001; Zhu et al., 2004), antioxidant (Kim et al., 2009; Gruia et al., 2008) and antiinflammatory (Park et al., 2004; Fawole et al., 2009). The freezedried fruit of L. caerulea and phenolic fraction of the fruit reduced the biofilm formation and adhesion to the artificial surface of several microbial strains (Palikova et al., 2008). Jin et al. also showed that ethanolic extract of L. caerulea fruit significantly suppressed the production of NO, PGE2 and TNF-a as well as the expression of iNOS and COX-2 by LPS-stimulated RAW264.7 cells (Jin et al., 2006). Considering the previous reports the present study was performed to evaluate potential of the polyphenolic fraction of L. caerulea fruit (PFLC; containing 77% anthocyanins) to suppress LPS-caused alterations to human gingival fibroblasts. We focused on oxidative damage and inflammation markers. 2. Materials and methods 2.1. Materials Dihydrorhodamine 123 was from Fluka Chemie (Germany). 2,20 -Dinitro-5,50 dithiobenzoic acid was purchased from Serva (Germany). Human IL-1b, IL-6 and TNF-a ELISA kits were obtained from R&D Systems (USA). COX-2 rabbit polyclonal antibody, iNOS rabbit polyclonal antibody, actin (1–19) goat polyclonal antibody, horseradish peroxidase conjugated goat anti-rabbit and rabbit anti-goat antibodies, Western Blotting Luminol Reagent were received from Santa Cruz Biotechnology (USA). Protease inhibitor cocktail tablets (Complete™) were purchased from Roche (Germany). Dulbecco’s modified Eagle’s medium, heat-inactivated foetal calf serum, stabilised penicillin–streptomycin solution, sterile dimethylsulfoxide (DMSO), lipopolysaccharide (LPS; from Escherichia coli 055:B5), neutral red (NR), Immun-Blot™ PVDF (polyvinylidene difluoride) membrane, KODAK BioMax light film, phenylmethylsulfonyl fluoride and all other chemicals were purchased from Sigma–Aldrich (USA).

were trypsinized and transferred into 75 cm2 cultivation flasks. For all experiments the gingival fibroblasts were seeded in plates at a density of 1  105 cells/cm2. Cells were used between the 3 and 10 passages for experiments. 2.4. Cell viability Viability was assayed by neutral red (NR) uptake by lysosomes. The cells on 96well plates were treated with PFLC (10, 25, 50 lg ml1) in serum-free medium for 4 and 24 h. Control cells were treated with a medium containing dimethylsulfoxide (DMSO; 0.5%, v/v). After incubation period, NR (0.03%, w/v) in PBS was added to the cells (2 h; 37 °C). The fibroblasts were then washed with a mixture of formaldehyde (0.125%, v/v) and CaCl2 (0.25%, w/v) and the retained NR was dissolved using acetic acid (1%, v/v) in methanol (50%, v/v). The plates were read on a microplate reader (Sunrise, Tecan, Switzerland) at 540 nm. 2.5. Treatment of cells E. coli LPS is the most commonly used LPS in in vitro studies and it was therefore employed in the present experiments. LPS stock solution (0.2 mg ml1) was prepared in sterile water. Stock solutions of PFLC (2, 5, 10 mg ml1) were prepared in DMSO. The final concentration of DMSO in serum-free medium was 0.5% v/v. For the evaluation of PFLC effects on LPS-induced alterations, gingival fibroblasts were pre-treated with LPS (final concentration 1 lg ml1; 24 h) in serum-free medium. The cells were then washed with PBS and PFLC (final concentration 10, 25, 50 lg ml1) in serum-free medium applied for 4 h. Then the media were collected and immediately frozen (80 °C) for a measurement of IL-1b, IL-6 and TNF-a. Cells were washed with PBS, harvested and ROS generation, lipid peroxidation, level of intracellular GSH and expression of COX-2 and iNOS were evaluated. 2.6. Determination of ROS production Elimination of LPS-induced ROS by PFLC in living cells was monitored using the dihydrorhodamine 123 (DHR) assay (Royall and Ischiropoulos, 1993). After the incubation period, DHR (15 ll; 5 lmol l1; 15 min) was added to the cells. The cells were then thoroughly washed with PBS, scraped into 1 ml of PBS and sonicated. The cell lysates were centrifuged (10 min, 13,000 rpm, 4 °C) and the fluorescence was measured at 500/536 nm using a microplate spectrophotometer (INFINITE M200, Tecan, Switzerland). The protein concentration in lysates was determined by Bradford assay. 2.7. Intracellular GSH level GSH levels were measured according to Sedlak and Lindsay (1968). The cells were washed with cooled PBS, scraped into the cooled perchloric acid (1%, v/v) and sonicated. The aliquots were used for protein determination by Bradford assay. The suspension was centrifuged (10 min, 13,000 rpm, 4 °C) and the supernatant was used for estimation of GSH, based on the reaction with 2,20 -dinitro-5,50 -dithiobenzoic acid. The absorbance was read on a microplate reader at 412 nm. 2.8. Thiobarbituric acid reactive substances (TBARS) determination

2.2. Plant material L. caerulea berries (Lonicera caerulea L.; Caprifoliaceae) were grown and harvested in Central Moravia (Czech Republic; 2007). The phenolic fraction was prepared as described elsewhere (Palikova et al., 2008). The total phenolic content was determined using Folin–Ciocalteau reagent; total anthocyanins by pH differential absorbance method and single phenolics were identified and quantified by HPLC–MS. The phenolic fraction (0.4% of fresh fruits) contained 20.1% of phenolics including anthocyanins (77%), flavonoids and phenolic acids. Among non-condensed anthocyanins, cyanidin-3-glucoside dominated. Detailed analysis has been described previously (Palikova et al., 2008). 2.3. Cell culture Human gingival fibroblasts were harvested from medically healthy donors who were clinically free of periodontal disease. Samples of gingiva were obtained from patients undergoing surgical removal of third molars at the Department of Oral and Maxillofacial Surgery (University Hospital, Olomouc). The tissue acquisition protocol adhered to the requirements of the Ethics Committee of the University Hospital Olomouc and Faculty of Medicine and Dentistry, Palacky´ University in Olomouc. All patients had signed written informed consent. The gingival tissues were washed three times in phosphate buffered saline (PBS) containing antibiotics (pamycon and colinomycin). The excised gingiva were cut into 1 mm pieces, plated in Petri dishes (10 cm diameter) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated foetal calf serum (FCS), penicillin (100 mg ml1) and streptomycin (100 U ml1). Explants were incubated in humidified atmosphere with 5% CO2 at 37 °C. Cells were fed weekly until the fibroblasts reached confluence. After 4–6 weeks cell cultures

Lipid peroxidation was determined by measuring the TBARS in cells as described by Buege and Aust (1978). Fibroblasts rinsed with cooled PBS were scraped into trichloroacetic acid (2.8%, w/v), sonicated and aliquots were used for protein determination by Bradford assay. The suspension was mixed with thiobarbituric acid (1%, w/v) in a ratio 2:1. The mixture was heated (30 min, 95 °C), cooled (4 °C) and centrifuged (10 min, 13,000 rpm, 4 °C). The amount TBARS was determined spectrophotometrically at 535 nm. 2.9. Cytokine determination The effect of PFLC on LPS-induced production of IL-1b, IL-6 and TNF-a was determined using specific immunoassays (QuantikineÒ). The assays were performed according to the manufacturer’s protocols. Briefly, media were diluted (1:1–1:2) with the congruous assay diluents. Then media were applied on the 96well plate coated with a specific monoclonal antibody and incubated (2 h, room temperature). After incubation media were removed, wells were rinsed 3-times and conjugate solution was added (2 h, room temperature). Then the solution was removed, the wells were rinsed 3-times, substrate solution was applied. After incubation (30 min, room temperature, dark), stop solution was added and a yellow-coloured product was measured at 450 nm (Sunrise, Tecan, Switzerland). 2.10. COX-2 and iNOS expression (Western blot analysis) Cells were properly washed with cold PBS and scraped into the ice-cold lysis buffer (20 mM Tris, 5 mM ethylene glycol tetraacetic acid, 150 mM NaCl, 20 mM glycerol phosphate, 1 mM NaF, 1% Triton X-100, 1 mM Na3VO4, 0.1% Tween 20, protease inhibitor cocktail tablet). After incubation (15 min, 4 °C) the lysate was

A. Zdarˇilová et al. / Food and Chemical Toxicology 48 (2010) 1555–1561 cleared by centrifugation (14,000 rpm, 10 min, 4 °C). The supernatant protein concentration was determined by Bradford assay. Proteins were separated by 15% SDS– polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane. Residual binding sites on the membrane were blocked using blocking buffer (5% non-fat dry milk (w/v) in 100 mM Tris-buffered saline (pH 7.5) with Tween 20 (0.05%, v/v); 1 h; room temperature). The membrane was then incubated with a polyclonal primary antibody (rabbit anti-COX-2 or anti-iNOS or goat anti-actin (1–19), overnight, 4 °C) and then with a secondary horseradish peroxidase conjugated antibody (goat anti-rabbit or rabbit anti-goat, 2 h, room temperature). COX2, iNOS and actin expression was detected by chemiluminiscence using Western Blotting Luminol Reagent and autoradiography using a KODAK BioMax light film. 2.11. Statistical analysis Data are expressed as means ± SD of at least three independent experiments performed in triplicate for each sample. The Student’s t-test was used for statistical analysis. Statistical significance was determined at p = 0.01. Western blot data are representatives of three independent experiments.

3. Results 3.1. Cell viability Possible harmful effect of PFLC on gingival fibroblasts was excluded. No visible alterations to morphology of gingival fibroblasts treated with PFLC (10, 25, 50 lg ml1) was found using an inversion microscope (Olympus, Japan). Cytotoxic effects were not detected either by NR retention after 4 and 24 h treatments (data not shown). 3.2. Effect of PFLC on LPS-induced oxidative damage GSH is an ubiquitous and one of the most important cellular antioxidants. Therefore GSH levels were evaluated to determine the effect of PFLC on the redox status of cells affected by LPS. As shown in Fig. 1A, PFLC itself did not influence the GSH level. In cells treated with LPS (1 lg ml1, 24 h) GSH was markedly reduced

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(Fig. 1B). When PFLC (10, 25, 50 lg ml1; 4 h) was applied to LPS-treated cells, depletion of intracellular GSH decreased (Fig. 1B). The PFLC effect was not concentration-dependent and the maximal protection was observed at the concentration of 25 lg ml1. Development of LPS-induced lipid peroxidation in cell biomembranes was measured as TBARS level. PFLC itself (10, 25, 50 lg ml1) did not increase TBARS level after 4 h treatment (Fig. 2A). LPS application (1 lg ml1, 24 h) obviously enhanced lipid peroxidation (Fig. 2B). Treatment with PFLC significantly reduced the quantity of TBARS in LPS-exposed fibroblasts. The reduction approached the level of non-LPS-treated cells for all tested concentrations (Fig. 2B). LPS is known to stimulate ROS generation. To investigate whether treatment with PFLC influences LPS-induced ROS production, a dihydrorhodamine 123 assay was performed. As evident from Fig. 3A, PFLC itself (10, 25, 50 lg ml1; 4 h) caused no increase in basal ROS generation in gingival fibroblasts, while LPS (1 lg ml1, 24 h) stimulated production of intracellular ROS (Fig. 3B). Application of PFLC reduced the level of ROS in LPS-treated cells in a concentration-dependent manner (Fig. 3B). In cells treated with PFLC at concentrations of 25 and 50 lg ml1 ROS levels were comparable with those in non-LPS-treated cells. 3.3. Effect of PFLC on LPS-stimulated IL-1, IL-6 and TNF-a production PFLC application (10, 25, 50 lg ml1; 4 h) showed no effect on the basal level of IL-1b (Fig. 4A) and TNF-a (Fig. 5A) in fibroblasts. However, treatment with PFLC (25 lg ml1) significantly decreased the amount of IL-6 (Fig. 6A). Incubation of cells with LPS (1 lg ml1) markedly increased IL-1b, TNF-a and IL-6 release into the medium after 24 h compared to the basal levels in untreated cells (Figs. 4B, 5B and 6B). LPS-stimulated expression of IL-1b

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Fig. 5. Effect of LPS-induced TNF-a production by PFLC. Cells were treated with PFLC for 4 h (A) or were pre-treated with LPS (24 h) and then incubated with PFLC for 4 h (B). Control cells were incubated with DMSO (0.5%, v/v). Data are expressed as mean ± S.D. *p < 0.01 and #p < 0.05 statistically different from LPS-treated cells, respectively.

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Concentration (µg.ml ) Fig. 6. Effect of LPS-induced IL-6 expression by PFLC. Gingival fibroblasts were treated with PFLC for 4 h (A) or were pre-treated with LPS (24 h) and then incubated with PFLC for 4 h (B). Control cells were incubated with DMSO (0.5%, v/v). Data are expressed as mean ± S.D. *p < 0.01 and #p < 0.05 statistically different from LPStreated cells, respectively.

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COX-2 Actin iNOS Actin Fig. 7. Effect of PFLC on LPS-induced COX-2 and iNOS expression. Cells were treated with PFLC for 4 h or were pre-treated with LPS (24 h) and then incubated with PFLC for 4 h. Control cells were incubated with DMSO (0.5%, v/v). Data are representatives of three independent experiments.

(1.5-fold increase in comparison to control cells, Fig. 4B), TNF-a (1.3-fold increase in comparison to control cells, Fig. 5B) and IL-6 (10-fold increase in comparison to control cells, Fig. 6B) was significantly reduced only at the highest PFLC concentration (50 lg ml1). 3.4. Effect of PFLC on LPS-induced COX-2 and iNOS expression To acquire more evidence that PFLC is capable of suppressing inflammation in LPS-stimulated gingival fibroblasts, the PFLC effect on COX-2 expression was assessed. We found that the LPS-stimulated expression of COX-2 protein in gingival fibroblasts (Fig. 7, line 2) while PFLC application had no effect on COX-2 expression (Fig. 7, line 3–5). We further discovered that PFLC (10, 25, 50 lg ml1) suppressed LPS-induced COX-2 expression. The maximal COX-2 reduction was found in the cells treated with PFLC at the concentration of 25 mg ml1. iNOS is another enzyme involved in inflammatory processes. As shown in Fig. 7, treatment with PFLC did not induce iNOS expression (line 3–5). LPS caused significant induction of this protein (line 2) compared to untreated cells (line 1). However, PFLC application (10, 25, 50 lg ml1) showed no effect on the LPS-stimulated iNOS expression (line 6–8) at any used concentration. 4. Discussion In vitro and in vivo reports indicate that natural compounds may be useful in the treatment of periodontal disease (Zdarilova et al., 2009; Gutiérrez-Venegas et al., 2007, 2006; Bodet et al., 2007a,b). In this study we tested the ability of the polyphenolic fraction of L. caerulea fruit to suppress cellular changes induced by LPS (E. coli) in human gingival fibroblasts. We found that PFLC significantly decreased ROS and TBARS production. It also reduced GSH depletion as well as IL-1b, IL-6 and TNF-a release and COX-2 induction. However, no effect was observed on iNOS expression. The release of bacterial components including LPS activates cells to produce pro-inflammatory mediators, cytokines and chemokines that lead to the expression of adhesion molecules and the recruitment of inflammatory cells (Okada and Murakami, 1998). Several bacterial types of LPS have been used in research. However, cell responses to different LPS may be controversial. For example, the LPS isolated from the periodontopathogen P. gingivalis has demonstrated different chemical and biological characteristics than classical LPS from E. coli (Hamada et al., 1990). The differences involve variations in phosphorylation and acylation patterns in the biologically active component lipid A (Ogawa, 1994; Nair et al., 1983). LPS of P. gingivalis did not stimulate epithelial cells to express E-selectin or promote neutrophil adhesion, which may facilitate bacterial colonization of the gingival tissue and persistence, in comparison to E. coli LPS (Darveau et al., 1995). Furthermore, P. gingivalis LPS compared to E. coli LPS induced higher levels of cytokine amount (TNF-a and IL-1) by mono-

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cytes (Roberts et al., 1997). In contrast, other investigators have shown similarity of P. gingivalis and E. coli LPS in terms of mitogenicity, polyclonal B cell activation, and stimulation of IL-1 production in BALB/c mice (Koga et al., 1985) and the ability to induce expression of IL-1b and IL-6 in gingival fibroblasts (Tardif et al., 2004; Kent et al., 1998). Nevertheless LPS from E. coli is predominantly used for in vitro experiments and therefore it was employed in the present study. The correlation between the periodontitis and increased levels of pro-inflammatory cytokines such as IL-1b, IL-6 and TNF-a has been well demonstrated (Hou et al., 1995). TNF-a is generated in early stage of inflammation (Driscoll, 2000) and plays a major role in the inflammatory process, mostly via induction of other cytokines (IL-1, IL-6, IL-8) and prostaglandins (Matsuzaki et al., 2004). IL-1b elicits a great number of physiological activities and plays role in the immunity of periodontal tissue such as stimulation of IL-6 production. However, the local unlimited production of IL-1b could lead to initiation and progression of connective tissue breakdown, by production of other cytokines, matrix-degrading enzymes and prostaglandins (Boch et al., 2001). Our results showed that application of PFLC (50 lg ml1) to LPS-treated gingival fibroblasts significantly reduced release of all monitored cytokines, IL1b, IL-6 and TNF-a. Effect on IL-1b and TNF-a was more powerful than on IL-6 (Figs. 4–6). Due to irritation by bacterial antigens such as lipopolysaccharides, periodontal cells increase production of ROS (Sculley and Langley-Evans, 2002). Some reports indicate that released IL-1 and TNF-a are also able to promote ROS generation and increase inflammation. Excess ROS requires greater use of intracellular antioxidants. A key cell redox state regulator GSH is rapidly oxidized and its extensive depletion may break down the oxidation–reduction balance in cells and increase ROS-caused damage (Hirano et al., 1990). ROS-related cell/tissue injury can be observed as DNA lesions, protein modifications or lipid peroxidation products (Sculley and Langley-Evans, 2002). Lipid peroxidation, monitored as the final product, malondialdehyde, has been shown to be increased in chronic apical periodontitis tissue in comparison to healthy human tissue (Tsai et al., 2005). A recent study in our laboratory has revealed that the polyphenolic fraction of L. caerulea fruits effectively scavenges DPPH and superoxide radical (Palikova et al., 2008) and, protects against chemically induced lipid peroxidation and LDL oxidation in vitro (Palikova et al., 2009). Here we demonstrated that treatment of LPS-stimulated gingival fibroblasts with PFLC significantly prevented GSH depletion as well as decreased lipid peroxidation and ROS generation. Moreover, PFLC at concentrations of 25 and 50 lg ml1 eliminated the effects of LPS to the level of non-LPS-treated cells (Figs. 1–3). Exposure of gingival fibroblasts to LPS further leads to expression of inducible form of NO synthase (iNOS). This enzyme participates in NO generation that has many physiological functions including bactericidal properties. However, high concentrations of NO are toxic to exposed cells (Gutiérrez-Venegas et al., 2007). iNOS expression is induced by several factors including cytokines (IL-1b, IL-6 and TNF-a) and bacterial LPS. These factors trigger off a cascade of events that lead to nuclear factor kappaB (NF-jB) activation mediated by mitogen-activated protein kinases family (Kleinert et al., 2004). Many different compounds have been shown to induce or inhibit iNOS expression by activating or blocking a wide variety of signal transduction pathways (Kleinert et al., 2004). Under our conditions, treatment of gingival fibroblasts with PFLC (10–50 lg ml1) had virtually no effect on LPS-induced iNOS protein level. In contrast, Jin et al. showed that ethanolic extract of L. caerulea fruits (1–100 lg ml1; containing 9.2% anthocyanins) inhibited NO production and iNOS expression in LPS-stimulated mouse macrophage cell line (RAW264.7 cells). However, pure cyaniding-3-O-glucoside (100 lg ml1) did not suppress iNOS

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expression and only partially reduced NO generation (Jin et al., 2006). Pergola et al. found that treatment of murine monocyte/ macrophage cell line with anthocyanin fraction of blackberry extract (88.0% of cyaniding-3-O-glucoside) and pure cyaniding-3-Oglucoside resulted in inhibition of NO synthesis and iNOS expression. The inhibition was mediated via suppression of NF-jB and/ or mitogen-activated protein kinases (MAPK) activation. However, these effects were obvious at high concentrations (45 and 90 lg ml1 of the extract and 20–80 lg ml1 of cyaniding-3-O-glucoside) (Pergola et al., 2006). Studies showed that pathways associated with the induction/suppression of iNOS expression may vary in different cells and thus treatment with natural compounds including polyphenols often results in contradictory data (Kleinert et al., 2004). Apart from interleukins, gingival fibroblasts secrete another group of inflammatory mediators, prostaglandins. Their production is catalyzed by COX. The normally low level of inducible isoform COX-2 is strongly induced by inflammatory agents such as TNFa, IL-1 or lipopolysaccharides (Gutiérrez-Venegas et al., 2005). The results of Western blot analysis have demonstrated that PFLC application effectively decreased LPS-caused COX-2 expression, mainly at concentrations of 25 and 50 lg ml1. Similar results were also reported by Jin et al. who used ethanolic extract of L. caerulea fruits (1–100 lg ml1) (Jin et al., 2006). It has been suggested that controlling the cellular reactions of fibroblast, the most abundant structural cell in periodontal tissue, to bacterial insult leading to inflammation may be one method for management of periodontitis (Paquette and Williams, 2000). Polyphenols exhibit a wide range of biological activities such as antimicrobial (Puupponen-Pimiä et al., 2001), anti-viral (Kaul et al., 1985), radical scavenging (Cao et al., 1997), anti-inflammatory (Gutiérrez-Venegas et al., 2007; Kim do et al., 2007; Sautebin et al., 2004) and wound healing (Phan et al., 2001) that may be useful for the remedial treatment of the disease. Studies have also shown that use of polyphenol rich extracts/fractions that contain various phytochemicals, have a number of advantage than a single compound e.g. extracts/fractions are cheaper and easier to prepare, individual components may provide additive and/or synergistic effects, and lower concentration of constituents lessen their possible toxic effects. For example effect of L. caerulea fruit extract (10 lg ml1) on LPS-stimulated production of pro-inflammatory mediators was stronger than effect of its three major phenolic components at a 10-fold higher concentration (cyanidine-3-O-glucoside, cyanidine-3-O-rutinoside, chlorogenic acid; 100 lg ml1) (Jin et al., 2006). For this reason polyphenol mixtures are of great interest. Here we demonstrated that the polyphenolic fraction of L. caerulea fruit was also able to reduce most studied alterations induced by LPS in gingival fibroblasts, particularly markers related to oxidative stress and inflammation. In addition to our findings, a recent study has shown that the freeze-dried fruit of L. caerulea and its phenolic fraction (the same as we used in this study) was able to reduce the biofilm formation and adhesion to the artificial surface of some bacteria such as E. coli and Staphylococcus epidermis (Palikova et al., 2008). Taken together, the phenolic fraction of L. caerulea fruit may be beneficial for the adjunctive treatment of periodontitis as an agent for attenuation the inflammatory process. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgements This work was supported by Ministry of Education of the Czech Republic (MSM 6198959216) and Ministry of Trade and Commerce

(FT-TA3/024). We thank to Dr. Katerˇina Valentová and Anna Vojteková (Department of Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry, Palacky´ University, Olomouc, Czech Republic) for providing the phenolic fraction from L. caerulea fruits.

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