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RESEARCH REPORT |
1 Department of Clinical and Experimental Medicine and Pharmacology, Torre Biologica, Policlinico Universitario, Via C. Valeria, Gazzi, 98100 Messina, Italy;
2 Department of Clinical Veterinary Science, University of Teramo, Italy; and
3 Department of Clinical Veterinary Medicine and Pharmacology University of Messina, Italy
* corresponding author, salvator{at}unime.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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(PPAR-) receptor appears to play a pivotal role in the regulation of cellular proliferation and inflammation. Recent evidence also suggests that rosiglitazone, a PPAR- agonist, reduces acute and chronic inflammation. We hypothesized that rosiglitazone would attenuate periodontal inflammation. In the present study, we investigated the effects of rosiglitazone in a rat model of ligature-induced periodontitis. At day 8, ligation significantly induced an increase in neutrophil infiltration, as well as of gingivomucosal tissue expression of iNOS, nitrotyrosine formation, and poly (ADP-ribose) polymerase activation. Ligation significantly increased Evans blue extravasation in gingivomucosal tissue and alveolar bone destruction. Intraperitoneal injection of rosiglitazone (10 mg/kg 10% DMSO daily for 8 days) significantly decreased all of the parameters of inflammation, as described above. Analysis of these data demonstrated that rosiglitazone exerted an anti-inflammatory role during experimental periodontitis, and was able to ameliorate the tissue damage associated with ligature-induced periodontitis.
KEY WORDS: rosiglitazone • peroxisome proliferator-activated receptor- ligand • alveolar bone loss • reactive oxygen species • periodontal diseases
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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(Lehmann et al., 1995). All thiazolidinediones tested to date—i.e., rosiglitazone, pioglitazone, and troglitazone—bind to and activate the PPAR isotype with a receptor affinity (Kd) that parallels their anti-diabetic activity in vivo (Lehmann et al., 1995; Willson et al., 1996). In addition, recent studies have also shown that PPAR may participate in the control of inflammation, especially in modulating the production of inflammatory mediators (Cuzzocrea et al., 2004a,b). Human periodontal diseases are inflammatory disorders that give rise to damage of the surrounding cells and connective tissue structures, including alveolar bone, causing tooth loss (Lindhe and Nyman, 1987). It has also been demonstrated that the most frequent cause of periodontitis is bacteria. The toxins, enzymes, and metabolites of the bacteria present in the dental plaque play a key role in the initiation of the inflammatory process (Listgarten, 1987). Some reports have evaluated the functions of polymorphonuclear cells in patients affected by early-onset periodontitis, but the results were varied. Some authors reported defective chemotactic responses to formyl-met-leu-phe (Suzuki et al., 1984) and to complement-derived C5a (Genco et al., 1986). In contrast, chemotaxis of early-onset periodontitis polymorphonuclear cells has been reported to be normal or increased (Repo et al., 1990). Moreover, recent studies have clearly demonstrated that inducible nitric oxide synthase (iNOS) and reactive oxygen species (ROS) play an important role in the inflammatory process, as well as in bone resorption (Di Paola et al., 2004). In the present study, we investigated the effects of rosiglitazone in a rat experimental model of periodontitis, to test the hypothesis that this compound attenuates periodontal inflammation. |
MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Indirect Measurement of Arterial Blood Pressure in Conscious Rats
Mean arterial blood pressure in conscious rats was measured by a Blood Pressure Recorder (UGO BASILE, Biological Research Apparatus, 21025 Comerio, Italy). After 1 wk, rats were treated as described below, and blood pressure was measured 30 min before and after each IP injection, on each of the 8 days of treatment. For the measurement of arterial blood pressure, rats were housed for 30 min in a warm room (28–30°C). A tail cuff was placed about 2 cm from the base of the tail, and arterial blood pressure was measured. Heart rate was detected by a pulse rate counter placed after the tail cuff.
Experimental Groups
Rats were randomly allocated into the following groups:
At 8 days after the ligature induction of periodontitis, the rats (N = 10 from each group for each parameter) were killed for evaluation of the various parameters described below.
Measurement of Vascular Permeability by Evans Blue Extravasation
Vascular permeability was determined as previously described (Gyorfi et al., 1994). Briefly, animals received Evans blue (2.5% dissolved in physiological saline, at a dose of 50 mg/kg) via a femoral venous catheter. Extravasated Evans blue in the excised gingivomucosal tissue samples was extracted with 1 mL formamide for 48 hrs at room temperature for spectrophotometric determination at 620 nm, and results were expressed as µg/g gingivomucosal tissue (Gyorfi et al., 1994).
Measurement of Alveolar Bone Loss
In the same set of experiments, the distance from the cementoenamel junction of the first lower molars to the alveolar crest was measured with a modification of the method of Crawford et al.(1978). Recordings were made along the median axis of the lingual surfaces of the mesial and mediolingual roots of the lower first left and right molars, as previously described (Di Paola et al., 2004). These measurements were performed by an independent investigator who was unaware of the treatment regimens. The alveolar bone loss induced by the ligature was expressed as a difference between the left and the right sides.
Histological Examination
For histopathological examination, biopsies of gingival and mucosal tissue from the buccal and lingual aspects of the teeth were taken 8 days after the ligature induction of periodontitis. The tissue slices were fixed in 10% neutral-buffered formaldehyde for 5 days, embedded in paraffin, and sectioned. The sections, oriented longitudinally from the crowns, were stained with trichrome stain. We quantitatively assessed the total number of infiltrating leukocytes (e.g., neutrophils and mononuclear cells) in cortical interstitial spaces from gingival and mucosal tissues by counting the number of polymorphonuclear cells in 20 high-power fields.
Radiography
Mandibles were placed on a radiographic box at a distance of 90 cm from the x-ray source. Radiographic analysis of normal and legated mandibles was performed by an x-ray machine (Philips X12, Munich, Germany) with a 40-kW exposure for 0.01 sec. A radiographic examination 8 days after ligature placement revealed bone matrix resorption in the lower first left molar, as previously described (Di Paola et al., 2004).
Myeloperoxidase Activity
Myeloperoxidase activity, an indicator of polymorphonuclear leukocyte (PMN) accumulation, was determined as previously described (Mullane et al., 1985). Gingivomucosal tissue samples, collected at the specified time, were homogenized in a solution containing 0.5% hexa-decyl-trimethyl-ammonium bromide, dissolved in 10 mM potassium phosphate buffer (pH 7) and centrifuged for 30 min at 20,000 x g at 4°C. An aliquot of the supernatant was then allowed to react with a solution of tetramethyl-benzidine (1.6 mM) and 0.1 mM H2O2. The rate of change in absorbance was measured spectrophotometrically at 650 nm. Myeloperoxidase activity was defined as the quantity of enzyme degrading 1 µmol/min of peroxide at 37°C and was expressed in milliunits/g of wet tissue.
Immunohistochemical Localization of iNOS, Nitrotyrosine, and Poly (ADP-ribose) Polymerase
After deparaffinization, endogenous peroxidase was quenched with 0.3% (v/v) H2O2 in 60% (v/v) methanol for 30 min. The sections were then incubated overnight with primary anti-nitrotyrosine antibody (1:1000 dilution), primary anti-PAR (1:500 dilution), or primary anti-iNOS (1:500 dilution), with control solutions including buffer alone or non-specific purified rabbit IgG. Specific labeling was detected with a biotin-conjugated goat anti-rabbit IgG and avidin-biotin peroxidase complex (DBA, Milan, Italy). Immunohistochemistry photographs (n = 5 photos from each sample collected from all rats in each experimental group) were assessed by densitometric analysis with Optilab Graftek software on a Macintosh personal computer.
Materials
Primary anti-nitrotyrosine antibody was obtained from Upstate Biotech (DBA, Milan, Italy). All other reagents and compounds used were obtained from Sigma Chemical Company (Sigma, Milan, Italy).
Data Analysis
All values in the Figs. and text are expressed as mean ± standard error of the mean of n observations, where n represents the number of animals studied. Datasets were examined by one- and two-way analyses of variance, and individual group means were then compared with the Bonferroni or Student’s unpaired t test. A P-value less than 0.05 was considered significant. In the experiments involving histology or immunohistochemistry, the Figs. shown are representative of at least 3 experiments performed on different experimental days.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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, 2a), nitrotyrosine (Figs. 1c, 2a), and anti-poly (ADP-ribose) polymerase (Figs. 1e1, 2a) was found in the gingivomucosal tissues from ligature-operated rats. Rosiglitazone (10 mg/kg, i.p.) abolished the staining for iNOS, nitrotyrosine, and anti-poly (ADP-ribose) polymerase (Figs. 1b, 1d, 1f, 2a, respectively).
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). Rosiglitazone treatment prevented this increase in Evans blue extravasation, but did not change the Evans blue content of the contra-lateral side (Fig. 2b). Myeloperoxidase activity was significantly elevated (P < 0.001) at 8 days after ligation (Fig. 2c), and rosiglitazone treatment significantly reduced these levels (Fig. 2c). No significant changes of myeloperoxidase activity were observed in the gingivomucosal tissues from the contralateral side (Fig. 2c).
Effect of Rosiglitazone on Tissue Damage and Bone Destruction
When compared with gingivomucosal tissue sections taken from the contralateral side (Fig. 3a), histological examination of gingivomucosal tissue sections of ligature-operated rats showed edema and tissue injury, as well as infiltration of the tissue with inflammatory cells (Fig. 3c1). Rosiglitazone treatment reduced the degree of gingivomucosal tissue injury (Fig. 3b). Quantification of polymorphonuclear cells infiltrating gingivomucosal tissue showed that there was only a minimal number of polymorphonuclear cells in tissue from the contralateral side (Figs. 4a, 4d). However, a large number of infiltrating polymorphonuclear cells were observed in the gingivomucosal tissue of ligated rats (Fig. 3d). Rosiglitazone administration significantly reduced the numbers of polymorphonuclear cells infiltrating gingivomucosal tissue (Fig. 3d). A radiographic examination of the mandibles, 8 days after ligature placement, revealed bone matrix resorption in the lower left first molar region (Fig. 4a). There was no evidence of pathology in right first molars (data not shown). Rosiglitazone markedly reduced the degree of post-ligation bone resorption in the lower left first molar region (Fig. 4b). In addition, significant alveolar bone loss, between the lower first left and the right first molars, induced by the left-side ligature, was observed in vehicle-treated rats (Fig. 4c). Rosiglitazone treatment resulted in a significant inhibition of post-ligation alveolar bone loss (Fig. 4c).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Our results demonstrated that rosiglitazone exerted a significant inhibitory effect on plasma extravasation during periodontitis. Our study also confirmed earlier findings, that one of the characteristic signs of inflammation, Evans blue extravasation, was higher on the ligated side than on the opposite side on the 8th day (Gyorfi et al., 1994). In addition, we also report, in the present study, that ligature-induced peridontitis in the rat resulted in a significant infiltration of inflammatory cells into the gingivomucosal tissues, and we also demonstrated that treatment with rosiglitazone reduced this inflammatory cell infiltration, as assessed by myeloperoxidase and by the moderation of tissue damage as evaluated by histological examination. Neutrophils are recruited into the tissue and can then contribute to tissue destruction by the production of reactive oxygen metabolites that further amplify the inflammatory response by their effects on macrophages and lymphocytes (Salvemini et al., 2001). A possible mechanism by which rosiglitazone attenuates polymorphonuclear cell infiltration is by down-regulating adhesion molecules ICAM-1 and P-selectin, as previously demonstrated (Cuzzocrea et al., 2000). These findings are in accordance with those of Berglundh and Lindhe (1993), who also found a significant increase in inflammatory cell infiltration in inflamed compared with healthy gingiva.
Several cellular mechanisms, including the mode of gene regulation and signal transduction, may account for the anti-inflammatory effect of PPAR- ligands. Recently, it has been shown that PPAR- ligands may act at the transcriptional level, at least in part, through inhibition of AP-1 and NF-
B activity (Fahmi et al., 2001).
Although the exact mechanisms remain unclear, activated PPAR could down-regulate AP-1, NF-B, and STATs activity by nitration of essential transcription co-factors, such as CBP/p300 and SRC-1 (Staels et al., 1998). PPAR may also antagonize AP-1 and NF-B activity through protein-protein interaction (Karin and Delhase, 1998).
NF-B has been shown to activate, via transcription, the genes encoding pro-inflammatory cytokines and iNOS. Several studies also support the conclusion that NO from iNOS plays an important role in the pathogenesis of periodontitis (Di Paola et al., 2004). The present study demonstrates that rosiglitazone attenuates the expression of iNOS in periodontal tissue. Our finding of reduced iNOS expression by rosiglitazone in vitro is also in accordance with our recent reports clearly demonstrating that rosiglitazone inhibits the expression of iNOS in another model of inflammation. Thus, the reduction of the expression of iNOS by rosiglitazone may contribute to the attenuation by this agent of the formation of nitrotyrosine in the periodontal tissues from ligature-treated rats. Increased nitrotyrosine staining is an indication of "increased nitrosative stress".
ROS and peroxynitrite produce cellular injury and necrosis via several mechanisms, including peroxidation of membrane lipids, protein denaturation, and DNA damage. ROS produce strand breaks in DNA that trigger energy-consuming DNA repair mechanisms and activate the nuclear enzyme PARP, resulting in the activation of ’the PARP Suicide Hypothesis’. There is recent evidence that the activation of PARP may also play an important role in experimental periodontitis (Di Paola et al., 2004). We demonstrate here that rosiglitazone attenuates the increase in PARP activity in periodontal tissue.
In conclusion, this study provides the first evidence that rosiglitazone causes a substantial reduction of ligature-induced periodontitis in the rat. This study also demonstrated that rosiglitazone, a drug affecting a fundamental body process, is well-tolerated in vivo at the dose and for the time of treatment used in this experimental setting. Finally, our findings suggest that interventions that may reduce the generation or the effects of ROS may be useful in conditions associated with local or systemic inflammation.
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ACKNOWLEDGMENTS |
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Received March 7, 2005; Last revision September 2, 2005; Accepted September 22, 2005
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