Quercetin Inhibits Inflammatory Bone Resorption in a Mouse

Article pubs.acs.org/jnp

Quercetin Inhibits Inflammatory Bone Resorption in a Mouse Periodontitis Model Marcelo H. Napimoga,*,† Juliana T. Clemente-Napimoga,‡ Cristina G. Macedo,‡ Fabiana F. Freitas,‡ Rafael N. Stipp,‡ Felipe A. Pinho-Ribeiro,§ Rubia Casagrande,⊥ and Waldiceu A. Verri, Jr.*,§ †

Laboratory of Immunology and Molecular Biology, São Leopoldo Mandic Institute and Research Center, 13045-755 Campinas, Brazil ‡ Laboratory of Orofacial Pain, Department of Physiology, Piracicaba Dental School, State University of Campinas, 13414-903 Campinas, Brazil § Departamento de Ciências Patológicas, Centro de Ciências Biológicas, Universidade Estadual de Londrina, 86051-990 Londrina, Brazil ⊥ Departamento de Ciências Farmacêuticas, Centro de Ciências de Saúde, Universidade Estadual de Londrina, 86038-350 Londrina, Brazil ABSTRACT: Periodontitis is a disease that leads to bone destruction and represents the main cause of tooth loss in adults. The development of aggressive periodontitis has been associated with increased inflammatory response that is induced by the presence of a subgingival biofilm containing Aggregatibacter actinomycetemcomitans. The flavonoid quercetin (1) is widespread in vegetables and fruits and exhibits many biological properties for possible medical and clinical applications such as its anti-inflamatory and antioxidant effects. Thus, in the present study, the properties of 1 have been evaluated in bone loss and inflammation using a mouse periodontitis model induced by A. actinomycetemcomitans infection. Subcutaneous treatment with 1 reduced A. actinomycetemcomitans-induced bone loss and IL-1β, TNF-α, IL-17, RANKL, and ICAM-1 production in the gingival tissue without affecting bacterial counts. These results demonstrated that quercetin exhibits protective effects in A. actinomycetemcomitans-induced periodontitis in mice by modulating cytokine and ICAM-1 production.

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over, the increased number of macrophages in periodontal lesions also interacts with leukotoxins and contributes to aggressive periodontitis by releasing IL-1β and inducing bone resorption.5 These cells are continuously recruited to the site of infection through cytokines (IL-1β, IL-17, TNF-α), chemokines, and adhesion molecules (expressed by activated endothelial cells), such as ICAM-1, leading to chronic inflammation and disease.6 Taken together, these events may explain why patients with aggressive periodontitis and infected with the JP2 clone have a more severe clinical course than those patients where this clone was not detected.3,7,8 Considering the reduced drug penetration in biofilms, it is conceivable that an enhanced efficacy of treatment might be provided by targeting the inflammatory host response in periodontitis. This new concept approach may lead to novel treatment protocols focused more on controlling, redirecting, and resolving the host response rather than on focusing solely on infection.2

eriodontitis is a chronic inflammatory disorder mediated by host and bacteria interactions and manifested by damage to the periodontal tissues that may progress to tooth loss. Paradoxically, the host inflammatory response primarily intended to eliminate the invading bacteria is responsible for the majority of the periodontal tissue destruction observed in aggressive periodontitis. However, the mechanisms related to the development of aggressive periodontitis remain incompletely known.1,2 The JP2 clone of the Gram-negative bacteria Aggregatibacter (Actinobacillus) actinomycetemcomitans is considered the major etiological agent of the aggressive form of periodontitis. In fact, the presence of subgingival biofilms containing A. actinomycetemcomitans has been related positively to increased inflammatory response in cases of periodontitis.3 The JP2 clone is characterized by a deletion in the promoter region responsible for regulating gene expression and production of leukotoxins, a powerful LFA-1 ligand and inducer of inflammation. When exposed to high concentrations of leukotoxin, polymorphonuclear leukocytes release proteolytic enzymes and die by necrosis, leading to a severe inflammatory response that contributes to the disease progression.4 More© 2013 American Chemical Society and American Society of Pharmacognosy

Received: August 22, 2013 Published: November 18, 2013 2316

dx.doi.org/10.1021/np400691n | J. Nat. Prod. 2013, 76, 2316−2321

Journal of Natural Products

Article

Plant-derived extracts or plant derivatives such as phenolic compounds (including flavonoids) show anti-inflammatory activity by controlling the levels of interleukins, chemokines, transcription factors, and adhesion molecules, among others,9,10 corroborating their use to inhibit bone loss caused by the immune-inflammatory response in periodontitis.11−14 It is known that quercetin (1) is an antioxidant flavonoid molecule with potential anti-inflammatory action.15−17 Treatment with 1 inhibits disease progression in experimental models of colitis,15 diabetes,16 steatosis,18 viral infection,19 and asthma,20 among other diseases. Its mechanisms of action are related to inhibition of cytokine production (IL-1β, TNF-α), reduced expression of inflammatory molecules, and inhibition of intracellular signaling pathways such as mitogen-activated protein kinases (MAPK) and NFκB,9,21 with consequent reduction of inflammation and oxidative stress. It has been demonstrated that the administration of 1 inhibits the bone loss in a rat lipopolysaccharide (LPS)-induced periodontitis model.22 However, there are no published in vivo data showing a possible therapeutic activity and the mechanisms of 1 in a model of periodontitis induced by A. actinomycetemcomitan. Thus, in the present study, the effect of 1 has been tested in bone loss and its immunoinflammatory mechanism of action using a mouse periodontitis model infected with A. actinomycetemcomitans. Figure 1. Quercetin (1) inhibits A. actinomycetemcomitans (A.a.infected)-induced bone loss. Mice were treated daily for 15 days with 1 (100 mg/kg, sc) or vehicle (20% Tween 80 in saline) after the last stimulus with a diluted culture of A. actinomycetemcomitans (1 × 109 CFU), and the bone loss was determined after the last treatment with 1. Representative images of negative control group (sham, panel A), A.a.-infected group treated with vehicle (panel B), A.a.-infected group treated with 1 (panel C), and morphometry analysis of bone loss (panel D) are shown. The samples were stained with methylene blue. Results are expressed as means ± SD of 5 mice per group per experiment [#p < 0.05 compared to the sham group; *p < 0.05 compared to the vehicle group (one-way ANOVA followed by Tukey’s test)].

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RESULTS AND DISCUSSION Quercetin (1) Inhibits A. actinomycetemcomitansInduced Bone Loss. Mice were stimulated with 1 × 109 colony-forming units (CFU) of a diluted culture of A. actinomycetemcomitans (A.a.) at days 0, 2, and 4 (A.a.-infected). Following the last stimulus, mice were treated daily for 15 days with vehicle (20% Tween 80 in saline) or 1 (100 mg/kg, sc). The dose of 1 was selected based on previous data showing its anti-inflammatory actions at 100 mg/kg and considering that 75 or 80 mg/kg used in periodontitis and other inflammation models is pharmacologically equivalent to 100 mg/kg.9,15,22−25 This protocol was used in all experiments. The values of the bone resorption for all groups are shown in Figure 1D. Sham (Figure 1A) animals presented the lowest distance between the cement−enamel junction and the alveolar bone crest during the experimental period. In contrast, animals infected orally with A. actinomycetemcomitans from the vehicle group (Figure 1B) showed significantly greater bone resorption when compared with noninfected sham animals. In addition, when A.a.-infected animals were treated daily with 1 (Figure 1C), bone resorption was abolished. This result corroborates evidence that 1 ameliorates periodontal damage by reducing osteoclast counts, periodontal inflammation, and bone loss in models of lipopolysaccharide of Gram-negative bacteria (LPS)- and

ligature (simulates an orthodontic device)-induced periodontitis.22 The present study indicates that 1 inhibits bone loss induced by a clinically relevant and viable bacterium. There were no A. actinomycetemcomitans in the oral cavities of the mice prior to deliberate infection. In contrast, persistent oral colonization by the pathogen was confirmed in all A.a.infected animals in the last day of experiment (Table 1). In Table 1. Quercetin (1) Treatment Does Not Affect A. actinomycetemcomitans Colony-Forming Units (CFU) before and after Oral Infection A.a-infected

noninfected untreated CFU before infection (× 106 colonies) CFU 14 days after infection (× 106 colonies)

vehicle

1 (100 mg/kg)

0

0

0

0

6.41 ± 0.96a,b

5.86 ± 0.76a,b

a p < 0.05 compared with the noninfected group. bp < 0.05 compared with samples collected from respective group before infection (oneway ANOVA followed by Tukey’s test).

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reducing IL-1β-induced nuclear translocation of NFκB p65 in human primary keratinocytes,21 LPS-induced degradation of IκB and cytokine production in dendritic cell culture,17 colitisinduced IL-1β production,15 and myocarditis-induced TNF-α and IL-17 production.28 Targeting IL-1β, TNF-α, or IL-17 reduces inflammation and bone resorption in vitro as well as in vivo.29−31 IL-17 enhances the effects of IL-1β and TNF-α, leading to exacerbation of the inflammatory response, an elevated number of neutrophils, and increased bone resorption in both experimental models32 and patients with periapical lesions.33 In fact, the higher levels of IL-17 found in periodontal tissues and in the gingival crevicular fluid of patients with chronic periodontitis suggest that IL-17 (and also IL-1β and TNF-α) is involved in periodontitis.34,35 The inhibition of cytokine production by 1 in the present study is consistent with other models of inflammation.15,17,21,28 Therefore, considering that 1 inhibits cytokine production and the role of cytokines in bone resorption, it seems that this is a relevant mechanism to its action. Corroborating this hypothesis, there is an association between reduction of IL-17 levels and resolution of periodontitis in patients after mechanical removal of dental plaque.36 Furthermore, biological therapies using antibodies, receptor antagonists, and soluble receptors targeting cytokines are expensive, and over a period of time the host produces antibodies against these compounds. Considering the issues with these therapies, treatment with 1 could represent a possible therapeutic approach with lower cost and presumably no induction of antibodies. Quercetin (1) Inhibits A. actinomycetemcomitans (A.a.)-Induced ICAM-1 Expression. Representative images (Figure 3A) and image analysis (Figure 3B) demonstrated that ICAM-1 expression was up-regulated in the A.a-infected vehicle group in comparison to noninfected sham mice, and treatment of A.a.-infected mice with 1 (100 mg/kg, sc) reduced this expression. This result corroborates the notion that during infection, cytokines orchestrate the recruitment of inflammatory cells by increasing the expression of adhesion molecules by endothelial cells and leukocytes.37 In infection-induced periodontitis, an increased expression of ICAM-1 has been demonstrated (via NF-κB activation) as observed in the present study and also that inhibition of ICAM-1 expression correlates with diminished bone loss and inflammation.35,37,38 Quercetin (1) Inhibits A. actinomycetemcomitansInduced Receptor Activator of NF-κB Ligand (RANKL) Expression. Representative images (Figure 4A) and image analysis (Figure 4B) demonstrated that there was an increase of RANKL expression as determined by Western blotting in the A.a-infected vehicle group when compared with noninfected sham animals. On the other hand, treatment with 1 (100 mg/ kg, sc) reduced the expression of RANKL (Figure 4). Bone resorption is regulated by the levels of RANKL, RANK, and OPG (osteoprotegerin). RANKL binds to its receptor RANK to induce osteoclast-mediated bone resorption, and OPG inhibits the binding of RANKL to RANK, therefore inhibiting bone resorption. RANKL is an osteoclastogenesis-related factor induced in periodontitis.39 Thus, 1 acts by reducing RANKL expression without affecting OPG expression (data not shown) in periodontitis induced by A.a. The inhibition of RANKL expression lines up well with the inhibition of cytokine production since IL-1β, TNF-α, and IL-17 induce the expression of RANKL.40 To our knowledge, this is the first study to evaluate the molecular mechanisms by which 1 inhibits A. actinomycetemcomitans-induced alveolar bone loss.

vitro, it has been demonstrated that 1 presents antimicrobial effects against A. actinomycetemcomitans at a concentration of 100 g/L.26 When considering 1 L equivalent to 1 kg, it could be extrapolated that a dose of 100 g/kg would be necessary to achieve antimicrobial effects, which is 100-fold greater than the dose used in the present study. Thus, it is unlikely that 1 is acting by an antimicrobial effect, which was also supported by the results of Table 1, indicating no difference in bacteria load on the last day of experiment. In this sense, it is also unlikely that 1 would modify the microbiome. Emerging evidence suggests that the early host immune-inflammatory response to oral bacteria leads to the tissue changes noted in gingivitis.27 Thus, searching for molecules to control the exacerbated host response is a promising approach to avoid disease progression. Quercetin (1) Inhibits A. actinomycetemcomitansInduced IL-1β, TNF-α, and IL-17 Production. The levels of some cytokines expressed in the gingival tissue of the animals were also investigated. The IL-1β (Figure 2A), TNF-α (Figure

Figure 2. Quercetin (1) inhibits A. actinomycetemcomitans (A.a.infected)-induced cytokine production. A.a.-infected mice were treated daily for 15 days with 1 (100 mg/kg, sc) or vehicle (20% Tween 80 in saline) after the last stimulus with A. actinomycetemcomitans. Fifteen days after the last stimulus with a diluted culture of A. actinomycetemcomitans (1 × 109 CFU), mice were terminally anesthetized and gingival samples were collected for the determination of levels IL1β (panel A), TNF-α (panel B), and IL-17 (panel C). Results are presented as means ± SD of 5 mice per group per experiment [#p < 0.05 compared with the sham group and *p < 0.05 compared to the vehicle group (one-way ANOVA followed by Tukey’s test)].

2B), and IL-17 (Figure 2C) levels were statistically higher in the A.a.-infected vehicle group than in the noninfected sham group, while treatment with 1 (100 mg/kg, sc) statistically inhibited the A.a.-infection-induced increase of all evaluated cytokines. These data are in line with the demonstration that 1 inhibits ultraviolet radiation-induced NF-κB DNA binding and the consequent production of IL-1β and TNF-α as well as 2318



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Figure 3. Quercetin (1) inhibits A. actinomycetemcomitans (A.a.infected)-induced ICAM-1 expression. Mice were treated daily for 15 days with 1 (100 mg/kg, sc) or vehicle (20% Tween 80 in saline) after the last stimulus with A. actinomycetemcomitans. Fifteen days after the last stimulus with a diluted culture of A. actinomycetemcomitans (1 × 109 CFU), mice were terminally anesthetized and gingival samples were collected for the determination of ICAM-1 protein expression by Western blotting. The optical densities of the ICAM-1 (panel A) bands were normalized to those of the α-tubulin bands (panel A) and are represented as arbitrary units of an optical density ratio (panel B). Results are presented as means ± SD of 5 mice per group per experiment [#p < 0.05 compared with the sham group, and *p < 0.05 compared to the vehicle group (one-way ANOVA followed by Tukey’s test)].

Figure 4. Quercetin (1) inhibits A. actinomycetemcomitans (A.a.infected)-induced receptor activator of NF-κB ligand (RANKL) expression. Mice were treated daily for 15 days with 1 (100 mg/kg, sc) or vehicle (20% Tween 80 in saline) after the last stimulus with A. actinomycetemcomitans. Fifteen days after the last stimulus with a diluted culture of A. actinomycetemcomitans (1 × 109 CFU), mice were terminally anesthetized and gingival samples were collected for the determination of RANKL protein expression by Western blotting. The optical densities of the RANKL (panel A) bands were normalized to those of the α-tubulin bands (panel A) and are represented as arbitrary units of an optical density ratio (panel B). Results are presented as means ± SD of 5 mice per group per experiment [#p < 0.05 compared with the sham group, and *p < 0.05 compared to the vehicle group (one-way ANOVA followed by Tukey’s test)].

Importantly, several studies have evaluated possible acute and chronic toxic and genotoxic effects of quercetin (1), leading to the consensus that it is a safe molecule.41−45 Furthermore, 1 is not classifiable as carcinogenic to humans, which is in agreement with the daily intake of this flavonoid in the human diet and the absence of revealed cases of adverse effects for human health.46 These data support the possible clinical usefulness of 1. In conclusion, the present study has demonstrated that prolonged treatment in mice with quercetin (1) reduces the A. actinomycetemcomitans-induced alveolar bone loss by mechanisms involving the reduction of pro-inflammatory cytokine production (IL-1β, TNF-α, and IL-17) and down-regulation of the adhesion molecule ICAM-1 and the osteoclastogenic cytokine RANKL. Therefore, treatment with 1 seems a promising approach to inhibit the development of alveolar bone loss during infection-induced periodontitis, which merits further investigation preclinically and possibly in a clinical setting.

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micropipet. This procedure was repeated after 48 and 96 h. Treatment with quercetin (1) was initiated after the third bacterial inoculation and consisted of daily subcutaneous (sc) injections (15 days) of 100 mg/kg of 1 diluted in 20% Tween 80 in saline. The dose of 1 was based on previous studies demonstrating that a dose of 100 mg/kg was able to inhibit inflammation in varied models.9,15,23 It was also taken into account that a dose of 75 mg/kg has been effective in other models of periodontitis.22 The control groups consisted of noninfected mice (sham) or infected mice treated with 20% Tween 80 in saline (vehicle).15 Test Compounds. Quercetin (1) at a 95% purity was purchased from Acros (Fair Lawn, NJ, USA). Cytokine ELISA kits for murine IL1β, TNF-α, and IL-17 were obtained from R&D Systems (Minneapolis, MN, USA), protease inhibitor cocktail, tryptic soy broth agar, nonfat dried milk, Gram stain, methylene blue, carboxymethylcellulose, Tris-buffered saline−Tween, DMSO, and Tween 80 were obtained from Sigma-Aldrich (St. Louis, MO, USA), and formalin was purchased from Merck (Darmstadt, Germany). A micro BCA protein assay kit was obtained from Pierce Chemical Co. (Rockford, IL, USA), sodium dodecyl sulfate−polyacrylamide was obtained from Amersham Biosciences (Piscataway, NJ, USA), nitrocellulose membrane and molecular weight standard were obtained from Bio-Rad Laboratories (Hercules, CA, USA), hydrogen peroxide and the antibodies antireceptor activator of NFκB ligand (RANKL), anti-CD55 (1:1000), and anti-α-tubulin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and the secondary antibody conjugated with peroxidase was obtained from Vector Laboratories Inc. (Burlingame, CA, USA).

EXPERIMENTAL SECTION

General Experimental Procedures. Animals received an oral delivery of 1 × 109 CFU of a diluted culture of JP2 A. actinomycetemcomitans grown anaerobically in supplemented agar medium (kindly provided by Dr. Rafael N. Stipp, State University of Campinas, Brazil), in 100 μL of phosphate-buffered saline (PBS) with 2% carboxymethylcellulose, placed into the oral cavity with a 2319



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Animals. This study was carried out with Balb/c mice (20−25 g) maintained in a temperature-controlled room (23 ± 1 °C) with a 12 h light−dark cycle. Animals were age- and strain-matched males (5 mice per group), 6 to 7 weeks old. All animals were manipulated in accordance with the Guiding Principles in the Care and Use of Animals, approved by the Council of the American Physiological Society. This animal study was deemed to be ethical according to Brazilian guidelines (Federal Law 11794/2008) and was approved by the Committee on Animal Research of the Faculty São Leopoldo Mandic (#2012/038). The suffering of the animals and the number per group were kept at a minimum, and each animal was used only once. Oral Colonization with A. actinomycetemcomitans. To confirm A. actinomycetemcomitans colonization, the oral cavity of each animal was sampled on day 0, before A. actinomycetemcomitans inoculation, and on the last day of treatment, using a sterile cotton swab immersed in sterile saline. Aliquots of 50 μL were plated onto tryptic soy broth agar plates, in triplicate, and incubated anaerobically for 1 week to determine the number of colony-forming units by Gram staining. Quantification of Alveolar Bone Loss. Evaluation of the extent of alveolar bone loss was performed as described previously.47 Fifteen days after the third inoculation, the animals were sacrificed, and the jaws were removed, autoclaved, and defleshed, then immersed overnight in 3% hydrogen peroxide, and stained with 1% methylene blue in PBS. Horizontal bone loss was assessed morphometrically by measuring the distance between the cement−enamel junction and the alveolar bone crest of the first and second molars. Measurements at 14 sites per mice (seven sites each on the left and right maxillary molars) were made under a microscope (Zeiss Axiostar Plus, Carl Zeiss GmbH, Germany) fitted with an Axion Vision LE imaging measurement system (Carl Zeiss). Random and blinded bone measurements were taken by the same person. Intraexaminer reproducibility of the measurements achieved ≥90% by intraclass correlation. Gingival Tissue Analyses. The whole buccal and palatal periodontal tissues of the upper molars were collected and weighed, and the portions of gingival tissue obtained for protein extraction were triturated and homogenized in 300 μL of the appropriate buffer containing protease inhibitors (Sigma-Aldrich), followed by centrifugation for 10 min at 10000g. The total amount of extracted proteins was measured by colorimetric analysis, using a micro BCA protein assay kit from Pierce Chemical Co. (Rockford, IL, USA). The supernatant was rapidly frozen and stored at −70 °C. Enzyme Linked Immunosorbent Assay (ELISA). Aliquots of each gingival sample were assayed by ELISA to determine the levels of IL-1β, TNF-α, and IL-17 according to the manufacturer’s recommendations (R&D). Briefly, 100 μL of detection antibody was added to all wells, except the blank, mixed gently, and incubated overnight (16−24 h) at room temperature. Plates were washed three times, and standards or supernatants were added in the respective wells in duplicate. After the incubation time, the plates were washed again and incubated with 200 μL of the respective conjugate for 60 min at room temperature. Plates were washed three times again, and 200 μL of substrate was added and incubated for 15 min at room temperature in the dark. The reaction was stopped by the addition of 50 μL of stop solution, and the color produced was measured in an automated microplate spectrophotometer (Epoch, Biotek, Winooski, VT, USA). The total amounts of cytokines were determined as picograms/mg of gingival tissue. Results were calculated using the standard curves created in each assay. The ELISA assays were carried out in a blind fashion in duplicate. Western Blotting. Equal amounts of protein (20 μg) from the gingival tissue were separated by 10% sodium dodecyl sulfate− polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. A molecular weight standard was run in parallel to estimate the molecular weight. Membranes were blocked overnight at 4 °C in Tris-buffered saline−Tween (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.1% Tween 20; TBST) containing 5% nonfat dried milk. After blocking, the membranes were incubated at 4 °C overnight with

antireceptor activator of NF-κB ligand (RANKL) (1:1000), anti-CD55 (1:1000), or anti-α-tubulin diluted in TBST containing 5% nonfat dried milk, for the analysis of the gingival proteins. The membranes were then incubated with appropriate secondary antibody conjugated with peroxidase (1:5000) diluted in TBST containing 5% nonfat dried milk, at room temperature for 60 min. Finally, the bands recognized by the specific antibody were visualized using a chemiluminescence-based ECL system and exposed to an X-ray film for 30 min. A computerbased imaging system (Image J, National Institute of Health, Bethesda, MD, USA) was used to measure the optical density of the bands. It is important to mention that Western blotting membranes were stripped for reprobing; thus, the same representative membrane for α-tubulin was used for analysis and presentation in Figures 3 and 4. Statistical Analysis. The statistical analysis was performed using Prism 4.0 software (GraphPad, La Jolla, CA, USA). The data were first examined for normality using the Kolmogorov−Smirnov test and then analyzed using one-way ANOVA followed by the Tukey test. Data are presented in the figures as means ± SD.

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +55 19 3211-3627. Fax: +55 19 3211-3712. E-mail: [email protected] or [email protected]. *Tel: +55 43 3371 4979. Fax: +55 43 3371 4387. E-mail: [email protected] or [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

This work was supported by Brazilian grants from Fundaçaõ de Amparo à Pesquisa do Estado de São Paulo (FAPESP), ́ Coordenadoria de Aperfeiçoamento de Pessoal de Nivel Superior (CAPES), Conselho Nacional de Desenvolvimento ́ Cientifico e Tecnológico (CNPq), Ministério da Ciência, Tecnologia e Inovaçaõ (MCTI), SETI/Fundaçaõ Araucária, and Governo do Estado do Paraná.

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