Chemico-Biological Interactions Effect of mangiferin ...

Chemico-Biological Interactions 179 (2009) 344–350

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Effect of mangiferin on the development of periodontal disease: Involvement of lipoxin A4 , anti-chemotaxic action in leukocyte rolling Roney Rick Carvalho a , Claudia Helena Pellizzon b , Luis Justulin Jr. b , Sergio Luis Felisbino b , Wagner Vilegas c , Fernanda Bruni d , Mônica Lopes-Ferreira d , Clélia Akiko Hiruma-Lima a,∗ a

Department of Physiology, Biosciences Institute, São Paulo State University (UNESP), Rubiao Junior cp. 510, 18618-000 Botucatu, São Paulo, Brazil Department of Morphology, Biosciences Institute, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil Institute of Chemistry, São Paulo State University (UNESP), Araraquara, São Paulo, Brazil d Special Laboratory of Applied Toxinology (CAT/CEPID), São Paulo, Brazil b c

a r t i c l e

i n f o

Article history: Received 22 August 2008 Received in revised form 23 October 2008 Accepted 24 October 2008 Available online 5 November 2008 Keywords: Mangiferin Lipoxin Alveolar bone loss Leukocytes Inflammation

a b s t r a c t Mangiferin is a polyphenol compound obtained from mango and has been reported to possess antioxidant and anti-inflammatory properties. Aim: We propose to evaluate the influence of mangiferin in preventing and treating experimental periodontitis induced in Wistar rats. Main methods: Periodontitis was induced in rats by applying a ligature around the lower right first molar. After ligature, groups of animals were submitted orally to the following treatments: saline 10 mL/kg, piroxicam 20 mg/kg or mangiferin 100 mg/kg. On days 1, 4 or 7 after ligature application the alveolar bone loss (ABL) was determined. We evaluated the effect of mangiferin on ABL by histological techniques (alveolar bone loss and cellularity), enzyme immunoassay (lipoxin A4 ), intravital microscopy (rolling leukocytes and endothelial–leukocyte adhesion), zymographic analyses (metalloproteinases, MMPs 2 and 9), immunohistochemistry (PCNA, COX-2 and CXCR4) and toxicology. Key findings: Oral administration of mangiferin significantly reduced ABL. We also observed the reduction of cellularity in mangiferin-treated rats. Treatment with mangiferin inhibited COX-2 expression and the rolling and adhesion of leukocytes, while maintaining normal lipoxin A4 levels. The mangiferin did not interfere in the activity of MMP-2 or -9. The mangiferin-treated rats presented an earlier peak of cell proliferation and augmented angiogenesis in the injured region. Significance: Our results have demonstrated promising therapeutic potential of mangiferin both in the prevention and treatment of periodontitis. Published by Elsevier Ireland Ltd

1. Introduction Periodontitis is a chronic inflammatory disease whose pathogenesis is related to the formation of colonies of microorganisms present in subgingival plaque, including some gram-negative anaerobic rod bacteria, spirochetes, and viruses [1]. The disease may also be due to alterations in the immune response [2]. Among the gram-negative microorganism inducers of periodontitis, the most important are Porphyromonas gingivalis and Bacteriodes forsythus, which have virulence factors that increase their infectivity and provide conditions for their multiplication and persistence in periodontium [3]. The presence of these microorganisms leads to the formation of a biofilm that initiates a dental inflammatory

∗ Corresponding author. Tel.: +55 15143811 6077; fax: +55 15143815 3744. E-mail address: [email protected] (C.A. Hiruma-Lima). 0009-2797/$ – see front matter. Published by Elsevier Ireland Ltd doi:10.1016/j.cbi.2008.10.041

response in the individual. This inflammatory response results in the formation of edema, infiltration of leukocytes and release of inflammatory mediators. The final result of this process is the formation of an inflammatory periodontal pocket, destruction of the periodontal ligament, alveolar bone resorption and tooth loss [4]. Leukocytic infiltration is a process of many steps, characterized by the migration (rolling) of leukocytes which enables them to undergo the action of chemotaxins, endothelial adhesion and transmigration through the endothelium. The interruption of any of these steps is a potential target for anti-inflammatory drugs [5,6]. The various chemical mediators involved in the inflammatory process include prostaglandins, whose main activities are vasodilatation and increased capillary permeability, which in turn promotes leukocytic infiltration and consequent development of the inflammatory process. Prostaglandins are formed from the arachidonic acid pathway by the action of two cyclooxygenase enzymes (COX-1 and COX-2). The enzyme COX-1 is constitutive

R.R. Carvalho et al. / Chemico-Biological Interactions 179 (2009) 344–350

while COX-2 is inducible and found abundantly in periodontal diseases [7–9]. Besides prostaglandins, arachidonic acid provides other chemical mediators such as leukotriene B4 (LTB4 ) responsible for leukocyte chemotaxis, and lipoxin A4 (LXA4 ) which acts as an endogenous anti-inflammatory mediator by presenting anti-chemotaxic action to inhibit the rolling of leukocytes. The relation of lipoxins to periodontitis has been demonstrated by various authors, including their action in blocking inflammation by reducing the recruitment of leukocytes, and in targeting potential new strategies for treatment [10,11]. Concomitant with periodontitis, the metalloproteinases (MMPs) use various components of the extracellular matrix and basement membrane as substrates. These enzymes are essential to the growth, repair and remodeling of tissues, but are also involved in the destruction of tissues in pathological cases including periodontal disease. In particular, MMP-2 and MMP-9 belong to the group of gelatinases identified as prevalent in both rat and human periodontitis, and are notable for their association with bone resorption in the inflammatory periodontal procedure [12]. In contrast to the harmful effects of these inflammatory process, other events such as the formation of new blood vessels in the injured region (angiogenesis) and cell proliferation assist in the regeneration of injured periodontal tissues [6,13]. Against periodontitis, various drugs, mainly non-steroidal anti-inflammatory drugs (NSAIDs) are used as an adjunctive therapy option to reverse the damage caused by periodontal disease, but have proven unsatisfactory due to various side effects such as gastrointestinal tract injuries, bleeding, perforations, obstructions, ulcerations and symptomatic diverticular disease, while increasing the risk of myocardial infarction [14,15]. Mangiferin is the major component (10%) of Mangifera indica L. (mango) which belongs to the family Anacardiaceae. This plant, which grows in tropical and subtropical regions, is widely used in folk medicine for various therapeutic indications [16]. Mangiferin is a glycosylated xanthone (1,3,6,7-tetra-hydroxyxanthone-C2-␤-d-glucose) of natural origin [17], that has been described as presenting the following effects: immunomodulation [18,19], anti-inflammatory inhibition of COX-2 expression [20,21], as well as LTB4 [22], NF-␬ B (nuclear transcription factor-kappa B), TNF-␣ (tumor necrosis factor-alpha) and IL-1 (interleukin-1), which are very important cytokines in the inflammatory process and in bone resorption [19,21,23,24]. Its bone anti-resorption activity in lumbar vertebrae was demonstrated in the experimental model using a dose of 100 mg/(kg day) [25]. This plant species also shows antibacterial activity in vivo against specific periodontal pathogens including P. gingivalis and Prevotella intermedia [26]. Based on all these activities already described for mangiferin, the present study aims to evaluate its pharmacological effects on alveolar bone loss in experimental periodontitis in rats.

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2.2. Animals Adult male rats (n = 88, Rattus norvegicus albinus, Wistar, 180 g), obtained from the UNESP Breeding Center (Botucatu, São Paulo, Brazil), were fed food and water ad libitum. The rats were divided randomly into groups that orally received saline, piroxicam or mangiferin. After treatment, the rats were anesthetized with intraperitoneal injection of 10 mg/kg of ketamine (Dopalen® , Vetbrands, SP, Brazil) and 10 mg/kg of xylazine (Anasedan® , Vetbrands, SP, Brazil). 2.3. Induction of periodontal disease After anesthesia, the animals received a cotton ligature (Coats Corrente Ltda., SP, Brazil) around the lower right first molar in a submarginal position to induce experimental periodontitis, according to the method described by Johnson [28]. The lower first molars on the left side did not receive the ligature, and served as the unligated control. The ligature was maintained for 1, 4 or 7 days after its placement, and then the rats were killed by decapitation. Treatment prior to induction of periodontitis (1 day) provides an indication of preventive action at the onset of the disease. This experimental protocol was submitted to the Animal Experimentation Ethics Committee at the Biosciences Institute of State University of São Paulo (CEEA/IB/UNESP) and was approved as no. 43/06. 2.4. Obtainment of the histological slide The mandible of each animal was removed and fixed in buffered formalin (pH 7.2) 10% for 48 h. Then, the mandibles were decalcified in a solution of 50 mL of 50% formic acid and 50 mL of 20% sodium citrate [29] which was replaced three times a week. After decalcification, the mandibles were washed in running water, dehydrated in increasing alcohol series and diaphanized in xylol. Then the mandibles were enclosed in paraffin, in order to obtain standardized serial 5-␮m thick histological section. 2.5. Measurement of alveolar bone loss The slides were stained with hematoxylin/eosin (HE) after which alveolar bone loss (ABL) was determined by measuring in micrometers (␮m) the distance from the cemento-enamel junction (CEJ) to the alveolar bone crest on the distal face of the first lower molars, with the aid of a Leica DM microscope (5× magnification) coupled with the image-capturing software Leica QWin Standard Version 3.1.0. The ABLs were obtained by comparing the different values on the side with ligature (first lower right molar) and the unligated side (first lower left molar), respecting the individual variation of each animal, from which we obtained three images of each slide that were counted manually.

2. Materials and methods

2.6. Total cell count

2.1. Drugs

Through the images captured by the program Leica QWin Standard Version 3.1 of the histological slides stained with HE, the cellularity of the injured region was determined by the program AVSoft BioView/Seeker 4, utilizing an area of 5 cm2 (40× magnification) from the distal region of the first lower molars.

Mangiferin (Sigma–Aldrich, USA) was administered at the dose of 100 mg/(kg day). This dose was selected based on its bone anti-resorption activity in the lumbar vertebra model described by Li et al. [25]. Piroxicam (Feldene® , Sao Paulo, Brazil) was selected at 20 mg/(kg day) on the basis of the alveolar bone antiresorption activity of meloxicam [27], which is a non-steroidal anti-inflammatory drug with effects similar to piroxicam. The piroxicam dose was selected on the basis of effective doses marketed for the two drugs. As the vehicle, we used saline (distilled water + 0.9% NaCl) at 10 mL/(kg day).

2.7. Immunohistochemical analysis A representative slide from each treatment was deparaffinized, rehydrated and designed by the immunohistochemical method to reveal peroxidase (PCNA, Nova Castra; COX-2, Cayman Chemical and CXCR4, Nova Castra). The blockade of nonspecific reaction

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was accomplished with skimmed milk and whey from normal sheep. After the recovery, the antigen and samples were incubated overnight, with specific antibodies, in blocking solution. Subsequently, the sample was washed in phosphate buffer (0.01 mol/L, pH 7.4) and incubated in secondary antibody (Vector Kit ABC) and revealed with Avidin-biotin associated with 3-3 diaminobenzidine tetrahydrochloride (DAB, Sigma), and examined in a Leica DM microscope coupled with Leica QWin Standard Version 3.1.0. image-capturing software, obtaining three images of each slide that were counted manually [30–32]. 2.8. Intravital microscopy Mice (Swiss, male, 18–22 g) provided by the Butantan Institute (Brazil) were divided into two groups of five animals each and maintained fasting for 2 h. After that, one group received mangiferin (100 mg/kg) orally and 30 min later all mice were anesthetized with sodium pentobarbital (Hypnol® , Cristália; 50 mg/kg, intraperitoneally) and underwent surgery to expose the cremaster muscle [33]. The animals were kept on a platform thermally controlled at 37 ◦ C. An initial leukocyte count was performed, and then 1 ␮g/20 ␮L LPS was applied topically to the cremaster muscle. The number of rolling leukocytes in post-capillary venules was recorded every 5 min for a period of 30 min to surpass a predetermined fixed point. The number of leukocytes adhering to the endothelial wall for a period of 1 min or more at a pre-set distance of 100 ␮m was assessed every 5 min for a period of 30 min [34,35]. This microvascular study by transillumination of tissue was completed with the aid of an optical microscope (Imager A1, CarlZeiss, Oberkochen, DE) coupled to a camera (AxionCam, ICc1) using an objective opening/longitudinal numerical distance × 10/0.3 and optovar 1.6. 2.9. Determination of lipoxin A4 concentration in gingival tissues Seven days after the induction of periodontitis in rats, the animals were killed and the gingival tissues surrounding the lower molars were removed. A homogenate was obtained from these tissues using a tissue macerator (Ultra-turbax® T 25 basic IKA® – Werke) and added along with 1 mL of PBS (phosphate buffered saline), pH 7.4, to each sample, maintained at 37 ◦ C for 20 min, then centrifuged at 9000 × g for 25 min, after which the supernatants were extracted and diluted 10 times with a phosphate buffer (ELISA kit BioAssayTM , USBiological). The lipoxin A4 concentration of these samples, expressed in picograms (pg) of lipoxin per microgram (␮g) of protein, was determined using the kit, following the manufacturer’s recommendations supplied in the manual. The amount of protein present in the sample was determined by the BCA method [36]. 2.10. Determination of the activity of MMPs Zymographic analysis was used to evaluate the effect of mangiferin on gelatinolytic activity of MMPs 2 and 9. Samples of recombinant MMPs (4 ng of MMP-2 and 40 pg of MMP-9) (Calbiochem, CA, USA) were subjected to electrophoresis, under non-reducing conditions, in 8% polyacrylamide gel, co-polymerized with 0.1% gelatin. After electrophoresis, the gels were washed twice (15 min) in a solution of 2.5% Triton X-100 to remove the sodium dodecyl sulfate (Bio-Rad, CA, USA), followed by two washes for 5 min in buffer solution of 50 mM Tris–HCl, pH 8.0. Then the gels were incubated overnight in Tris–HCl buffer, containing 5 mM CaCl2 and 1 ␮M ZnCl2 and increasing concentrations of mangiferin (10 ␮g/mL, 100 ␮g/mL, 500 ␮g/mL and 1 mg/mL), at 37 ◦ C. Finally, the gels were stained with Coomassie Brilliant Blue R-250 [37]. The

bands were analyzed in an ImageQuant 300 gel image-capturing equipment employing ImageQuant 300 image-analysis software (GE Healthcare, NJ, USA). The values are expressed as integrated optical density. 2.11. Development of body weight and vital organs The rats submitted to periodontal disease and treated with saline, piroxicam or mangiferin were weighed daily for 7 days to assess the possible body weight alterations, a potential indicator of toxicity in these animals. After the sacrifice of the rats, the heart, lungs, liver, kidney and spleen were removed and weighed (in g) on a precision analytical balance. The ratios between organ weights and body weights were converted into arcsine for statistical analysis, since this parameter may indicate drug toxicity in these organs [38]. 2.12. Biochemical evaluation of serum Blood samples were obtained from all animals submitted to treatment with saline, piroxicam or mangiferin, which were then centrifuged for serum removal to determine the concentrations of urea, creatinine, ␥-GT (gamma glutamyltransferase), ALT (alanine aminotransferase), AST (aspartate aminotransferase) and glucose. These serum biochemical parameters were quantified by the automatic biochemical analyzer Cobas Mira S® (Roche) with colorimetric and kinetic kits (CELM® , Brazil). 2.13. Statistical analysis The data were presented as the mean ± standard error (S.E.). The means obtained were submitted to the analysis of variance (ANOVA), followed by Tukey or Dunnett’s test. Differences were considered significant at P < 0.05. 3. Results and discussion Periodontitis is a chronic inflammatory disease that leads to the loss of alveolar bone [4], and can be induced in rats in a form reproducible in humans [39]. Previous studies under the lumbar vertebra model have demonstrated that mangiferin, a xanthone of natural origin, presents anti-resorption activity in bone [25]. The mangiferin dose–response curve obtained in the same study determined the dose of 100 mg/kg to be significant [25]. The latter work aimed to evaluate the effect of mangiferin on ABL in the experimental periodontitis model and also proposed the action mechanism of this polyphenol compound. Table 1 shows the result 1 day after the induction of periodontitis. Only the group of rats treated with mangiferin was able to significantly reduce (P < 0.05) ABL (105.5 ± 33.1 ␮m) when compared to the vehicle-treated group (217.6 ± 18.9 ␮m). Piroxicam at the dose of 20 mg/kg was unable to prevent the appearance of periodontitis 24 h after its induction. We could observe that mangiferin administered prior to the induction of periodontitis induction was already displaying a preventive action at the disease onset. Our results also showed that mangiferin has a progressive healing effect on periodontal disease after the onset. Mangiferin significantly reduced ABL 4 and 7 days after induction when compared to saline-treated animals (Table 1). After 4 days of daily treatment, both the groups treated with piroxicam and mangiferin significantly reduced (P < 0.001) the ABL (167.2 ± 13.4 ␮m vs. 162.2 ± 11 ␮m, respectively) when compared to saline-treated animals (290.4 ± 20 ␮m). We concluded that the mangiferin treatment was able to reduce ABL in rats submitted to experimental periodontitis after 1, 4 or 7


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Table 1 Alveolar bone loss (␮m) in rats submitted to experimental periodontitis and treated with vehicle (saline), piroxicam or mangiferin. Groups (dose)

Treatment periods

Saline Piroxicam (20 mg/kg) Mangiferin (100 mg/kg)

1 day

4 days

7 days

217.67 ± 18.95 138.14 ± 18.68 105.56 ± 33.12*

290.47 ± 20.00 167.29 ± 13.48*** 162.26 ± 11.03***

620.98 ± 103.37 298.53 ± 30.99** 220.32 ± 15.79***

Data are means ± S.E. (n = 7). ANOVA, followed by Tukey’s test. * P < 0.05 vs. saline group within the same treatment period. ** P < 0.01 vs. saline group within the same treatment period. *** P < 0.001 vs. saline group within the same treatment period.

days of treatment. These results point out the promising effect of mangiferin in the treatment of ABL. Therefore, we investigated the possible action mechanisms of this natural xanthone. ABL is caused by an inflammatory process, whose main features include the recruitment of defense cells, a process which results in a large number of cells at the inflamed site [35]. Based on this observation, we chose to quantify the cells present in injured region, as shown in Table 2. One day after periodontitis induction there was significant increase (P < 0.001) in cellularity of the injured region in rats treated with vehicle (221.8 ± 4.59 cells) when compared to the unligated control group (155.17 ± 3.64 cells). Both the piroxicamand mangiferin-treated groups presented significantly diminished (P < 0.01 and P < 0.001, respectively) cellularity in the injured region (196 ± 5.12 cells vs. 187.6 ± 1.88 cells, respectively) in relation to animals treated only with saline. Four days after periodontitis induction, the cellularity in rats treated with saline (221 ± 4.14 cells) had risen significantly (P < 0.001) compared to the unligated control group (155.6 ± 6.35 cells), but had dropped significantly in animals treated with piroxicam or mangiferin (P < 0.05 and P < 0.001, respectively, 191.6 ± 4.63 cells vs. 173.8 ± 7.87 cells) when compared to the rats treated with vehicle. The mangiferin-treated group showed the lowest reduction of cellularity 4 days after periodontitis induction. After 7 days of periodontitis both treated groups (piroxicam and mangiferin) also displayed significantly reduced (P < 0.001) cellularity (204.25 ± 3.09 cells vs. 181.5 ± 2.78 cells, respectively) in comparison to vehicle-treated rats (249.4 ± 9.04 cells). These results demonstrate that the least recruitment of defense cells occurred in mangiferin-treated rats. We also observed that, after 1, 4 or 7 days of mangiferin treatment, all the periodontitis carriers presented lower cellularity in the injured region. The literature reported that cell recruitment is directly related to vasodilatation and increased capillary permeability, effects that result largely from the action of prostaglandins synthesized from cyclooxygenases (COXs) 1 and 2 [7,8]. COX-2 is found abundantly in periodontitis [9], and our results showed greater inhibition of COX2 expression in mangiferin-treated rats than in those treated with saline (Table 3). The immunohistochemical analysis showed that the saline-treated group showed diminished COX-2 expression in

Table 3 Immunohistochemical analysis of COX-2 in rats with experimental periodontitis. Groups (dose)

Unligated control Saline Piroxicam (20 mg/kg) Mangiferin (100 mg/kg)

Treatment periods 1 day

4 days

7 days

0 48 0 33

0 84 3 2

0 58 7 9

Data represent the sum of cells stained in three images obtained from a slide representative of each group.

the injured region on days 1 (48 cells), 4 (84 cells) and 7 (58 cells) after periodontitis induction when compared to the unligated control group (0 cells). Yet mangiferin-treated rats showed inhibited COX-2 expression in 33 cells 1 day after treatment. But the drop in COX-2 expression was more evident after 4 (2 cells) and 7 days (9 cells) of mangiferin treatment than it was under vehicle treatment. These data are consistent with the literature in which COX-2 inhibition by mangiferin was proven in other experimental models [19,21]. Leukocytes are among the defense cells recruited in the inflammatory process of periodontitis. These cells, when attracted by the action of chemotaxins, increase their capacities for rolling through vessels and for adhering to the endothelium in order to cross it and migrate toward the inflammation site [5,40]. Therefore, the rolling and endothelial–leukocyte adhesions are the important parameters for evaluating the action mechanism of anti-inflammatory substances such as mangiferin. Our results showed that mangiferintreated animals presented significant inhibition of the rolling and endothelial–leukocyte adhesion compared with the control group (Figs. 1 and 2, respectively). As shown in Fig. 1, mangiferin previously administered to the mice inhibited the rolling of leukocytes induced by topical application of LPS at all experimental moments evaluated. According to Fig. 2, the mangiferin-treated mice also presented significant inhibition of the endothelial–leukocyte

Table 2 Total cell counts in rats with experimental periodontitis. Groups (dose)



155.17 221.8 196 187.6

4 days ± ± ± ±

3.64 4.59*** 5.12++ 1.88+++

155.6 221 191.6 173.8

7 days ± ± ± ±

6.35 4.14*** 4.63+ 7.87+++

153 249.4 204.25 181.5

± ± ± ±

Data are means ± S.E. (n = 4–6). ANOVA, followed by Tukey’s test. *** P < 0.001 vs. unligated control group within the same treatment period. + P < 0.05 vs. saline group within the same treatment period. ++ P < 0.01 vs. saline group within the same treatment period. +++ P < 0.001 vs. saline group within the same treatment period.

3.5 9.04*** 3.09+++ 2.78+++

Fig. 1. Effect of mangiferin on leukocyte rolling in post-capillary venules of the cremaster muscle of mice induced by topical application of LPS. Data are means ± S.E. (n = 6). ANOVA, followed by Tukey’s test. ***P < 0.001 within the same experimental period.

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R.R. Carvalho et al. / Chemico-Biological Interactions 179 (2009) 344–350 Table 4 Immunohistochemical analysis of angiogenesis in rats with experimental periodontitis treated with saline, piroxicam or mangiferin for 1, 4 or 7 consecutive days. Groups (dose)

Unligated control Saline Piroxicam Mangiferin


4 days

7 days

10 8 13 14

6 9 29 27

15 14 26 38

Data represent the sum of cells stained in three images obtained from a slide representative of each group. Fig. 2. Effect of mangiferin on the endothelial–leukocyte adhesion of post-capillary venules of the cremaster muscle of mice induced by topical application of LPS. Data are means ± S.E. (n = 6). ANOVA, followed by Tukey’s test. ***P < 0.001 within the same experimental period.

Table 5 Immunohistochemical analysis of PCNA in rats with experimental periodontitis treated with saline, piroxicam or mangiferin for 1, 4 or 7 consecutive days. Groups (dose)

adhesion induced by topical LPS application after 15, 20, 25 and 30 min. Our results agree closely with those of Beltrán et al. [20,41] who reported that mangiferin inhibited adhesion molecules such as ICAM-1, ICAM-2, ICAM-3, ELAM-1 and VCAM-1, all adhesion molecules involved in leukocyte–endothelial adhesion. As mentioned previously, the rolling is stimulated by the action of chemotaxins such as LTB4 and inhibited by the action of antichemotaxic mediators such as LXA4 [10,16]. The inhibitory effect of mangiferin on LTB4 has already been demonstrated [22], though there were no reports of a mangiferin effect on LXA4 . Our results (Fig. 3) showed that both vehicle- and piroxicam-treated rats presented significantly reduced LXA4 levels when compared to unligated animals. Nevertheless, the mangiferin-treated specimens displayed significantly higher LXA4 levels than those treated with vehicle. We also observed that there was no significant difference between the mangiferin-treated group and the unligated controls. This result clearly indicates the maintenance of normal lipoxin A4 levels in rats treated with mangiferin, unlike those treated with saline or piroxicam which were shown to be ineffective in maintaining normal lipoxin A4 levels (Fig. 3). Our results demonstrate that the administration of mangiferin maintained normal LXA4 levels in rats. Therefore, the joint action of mangiferin on LTB4 and LXA4 , shown in the present study, confirmed the anti-chemotaxic action of mangiferin on leukocyte rolling. It is well known that MMP-2 and MMP-9 are found abundantly in periodontitis and are involved in alveolar bone resorption [12]. Thus, we evaluated the effect of mangiferin in relation to the activity of these enzymes. As shown in Fig. 4, different mangiferin concentrations did not alter the gelatinolytic activity of MMP-2 or MMP-9. On the other hand, we cannot discard a possible inhibitory action of



4 days

7 days

0 0 0 0

23 48 69 88

37 98 90 72

Data represent the sum of cells stained in three images obtained from a slide representative of each group.

Fig. 4. Effects of mangiferin on gelatinolytic activity of MMPs 9 and 2 determined by Zymography assay. Data are means ± S.E. (n = 6–7). ANOVA, followed by Dunnett’s test vs. control group. P > 0.05.

mangiferin on the expression of these enzymes, since the literature reports that mangiferin acts on the cytokines TNF-alpha and IL-1 [16,20,23,24], which have the ability to activate the expression of MMPs [42,43]. In this context, inflammatory processes lead to the destruction of periodontal tissues such as alveolar bone resorption but,

Fig. 3. Determination of lipoxin A4 concentration in gingival tissues 7 days after inducing periodontitis in rats. Data are means ± S.E. (n = 4–7). ANOVA, followed by Tukey’s test. *P < 0.05 vs. unligated control group. + P < 0.05 vs. saline group.


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Table 6 Ratio between body and organ weights of rats 1, 4 and 7 days after the induction of periodontitis and treated with vehicle (saline), piroxicam or mangiferin. Periods

Treatments (dose)

Heart

Lungs

Liver

Kidneys

Spleen

1 day


3.72 ± 0.01 3.70 ± 0.04 3.72 ± 0.03

4.37 ± 0.05 4.50 ± 0.06 4.47 ± 0.06

12.63 ± 0.13 12.14 ± 0.17 12.16 ± 0.16

5.30 ± 0.02 5.27 ± 0.04 5.22 ± 0.04

3.60 ± 0.08 3.53 ± 0.07 3.40 ± 0.07

4 days


3.82 ± 0.04 3.95 ± 0.08 3.96 ± 0.05

4.74 ± 0.14 5.05 ± 0.14 4.97 ± 0.19

12.29 ± 0.24 12.50 ± 0.09 11.96 ± 0.07

5.30 ± 0.04 5.31 ± 0.06 5.36 ± 0.05

3.63 ± 0.08 3.60 ± 0.06 3.56 ± 0.07

7 days


3.65 ± 0.07 4.17 ± 0.27 3.83 ± 0.05

4.42 ± 0.06 4.82 ± 0.34 4.22 ± 0.06

11.62 ± 0.19 11.80 ± 0.13 11.68 ± 0.11

5.18 ± 0.05 5.32 ± 0.04 5.07 ± 0.04

3.15 ± 0.14 3.32 ± 0.05 3.11 ± 0.08

Data are means ± S.E. (arcsine) (n = 6–7). ANOVA, followed by Dunnett’s test vs. saline group within the same experimental period. P > 0.05.

Table 7 Biochemical evaluation of serum 1, 4 and 7 days after treatment with saline, piroxicam or mangiferin in rats submitted to periodontitis. Urea (mg/dL)

Creatinine (mg/dL)

Gamma-GT (u/L)

ALT (u/L)

AST (u/L)

1 day Saline Piroxicam Mangiferin

Glucose (mg/dL)

36.14 ± 3.00 34.57 ± 2.12 29.28 ± 2.39

0.39 ± 0.01 0.43 ± 0.01 0.38 ± 0.01

14.85 ± 3.85 21.57 ± 4.14 17.85 ± 5.33

70.14 ± 2.12 79.57 ± 3.49 68.57 ± 5.19

256.86 ± 5.91 235.43 ± 6.02 204.43 ± 7.78**

70.57 ± 4.63 85.57 ± 2.67 84.57 ± 5.85

4 days Saline Piroxicam Mangiferin

35.57 ± 1.88 35.42 ± 2.28 31 ± 0.75

0.4 ± 0.03 0.37 ± 0.01 0.35 ± 0.00

12.57 ± 4.29 23.85 ± 6.60 10.71 ± 4.78

94.57 ± 6.89 77.14 ± 7.18 93.28 ± 3.68

280.14 ± 8.48 247.7 ± 13.26 220.4 ± 9.25**

76.71 ± 5.30 81.85 ± 2.87 88.14 ± 2.72

7 days Saline Piroxicam Mangiferin

36.16 ± 2.45 34.14 ± 3.81 31.71 ± 1.68

0.41 ± 0.01 0.39 ± 0.01 0.37 ± 0.00

23 ± 8.48 41.28 ± 7.64 26.14 ± 6.28

87.16 ± 2.42 85.71 ± 8.28 87.57 ± 3.58

252 ± 17.50 239.86 ± 34.40 203.29 ± 15.47

92.83 ± 1.24 88.42 ± 5.81 103.43 ± 2.75

Data are means ± S.E. (n = 6–7). ANOVA, followed by Dunnett’s test. ** P < 0.01 vs. saline group within the same experimental period.

at the same time, events such as the formation of new blood vessels (angiogenesis) and cell proliferation are important events in the recovery of the injured area [6,13]. Thus, we investigated the possible role of mangiferin in the formation of new blood vessels. Table 4 shows that rats treated with saline did not show a tendency to increase the formation of new blood vessels 1 (8 vessels), 4 (9 vessels) or 7 (14 vessels) days after periodontitis induction when compared to the unligated control group 10 vessels, 6 vessels and 15 vessels, respectively. In contrast, we observed that rats treated with mangiferin tended to increase angiogenesis 1 (14 vessels), 4 (27 vessels) and 7 (38 vessels) days after periodontitis induction in relation to saline-treated rats. Our results showed that the mangiferin-treated rats tended to demonstrate augmented angiogenesis compared to those treated with saline, and an earlier peak of cell proliferation in relation to animals treated with saline or piroxicam (Tables 4 and 5, respectively). Proliferating cell nuclear antigen (PCNA) is a nuclear protein associated with the cell cycle. The nuclear PCNA immunoreactivity is found in the proliferative compartment of normal tissues. Celenligil-Nazliel et al. [44] indicated that inflammation caused increased proliferative activity in periodontitis. As Table 5 shows, the rats treated with saline and mangiferin tended to increase proliferation of cells on days 4 (48 and 88 cells, respectively) and 7 (98 and 72 cells, respectively) after periodontitis induction in relation to unligated controls (23 and 37 cells, respectively). The mangiferintreated rats presented a sharper rise after 4 days of treatment, which shows an earlier commencement of repair and healing than saline-treated or even piroxicam-treated animals. Thus, these results indicate that, in rats, mangiferin treatment accelerates the process of repair and healing of periodontal tissue injured by the inflammatory process.

Mangiferin administration at a dose of 100 mg/(kg day) showed no apparent toxic effect at the experimental moments observed with respect to serum parameters, organ weights (Tables 6 and 7) or body weight development. Data in Table 6 indicate that rats treated with piroxicam or mangiferin showed no significant changes in the weights of organs 1, 4 and 7 days after periodontitis induction compared to the animals that received only the vehicle. The results (Table 7) show a significant drop (P < 0.01) in AST levels on days 1 (204.43 ± 7.78 u/L) and 4 (220.4 ± 9.25 u/L) after periodontitis induction in the mangiferin-treated rats compared to those treated with saline (256.86 ± 5.91 u/L) and piroxicam (280.14 ± 8.48 u/L), respectively. The decreases in AST levels caused by mangiferin (Table 7) constitute evidence of a hepatoprotective effect, thus confirming the data in the literature on this natural xanthone [45]. It has long been known that NAIDs such as piroxicam have serious adverse effects on the gastrointestinal tract [14] and increased the risk of myocardial infarction [15]. Though the mangiferin and piroxicam treatments were not significantly different in reducing alveolar bone loss, mangiferin has demonstrated a great advantage in gastroprotective effect which did not occur with piroxicam [46]. 4. Conclusions Our results clearly demonstrate that mangiferin reduced alveolar bone loss in rats submitted to experimental periodontitis. The action mechanism is related to its anti-inflammatory property and its ability to accelerate the processes of repairing and healing of injured areas. Therefore, mangiferin can be regarded as a promising target for the development of a new drug for both the prevention and the treatment of periodontitis.

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