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Biomedicine & Pharmacotherapy
journal homepage:www.elsevier.com/locate/biopha
Tocoyena sellowiana
extract decreases bone loss in an experimental model of
periodontitis in rats: Putative role for cyclooxygenase-2 and IL-1
β
inhibition
Debora da Silva Freitas Ribeiro
a, Jordânia Marques de Oliveira Freire
b,
Alrieta Henrique Teixeira
b, Danielle Rocha do Val
b, Alice Ramos de Freitas
c,
Francisco Isaac Fernandes Gomes
c, Antonio Alfredo Rodrigues e Silva
c,i,
Paulo Nogueira Bandeira
d,i, Helcio Silva dos Santos
d, William Paulo dos Santos
d,
Fábio Nascimento Ávila
e, Karuza Maria Alves Pereira
f,i, Paula Goes
g,i,
Vicente de Paulo Teixeira Pinto
a,h,i, Gerardo Cristino-Filho
a,h,i,
Maria Rose Jane Ribeiro Albuquerque
d, Hellíada Vasconcelos Chaves
c,i,
Mirna Marques Bezerra
a,h,i,⁎aGraduate Program in Biotechnology, Medical School, Federal University of Ceará, Sobral, Ceará, Brazil b
Graduate Program in Biotechnology–RENORBIO–Ph.D. Program, Federal University of Ceará, Fortaleza, Ceará, Brazil cFaculty of Dentistry, Federal University of Ceará, Sobral, Ceara, Brazil
dDepartment of Chemistry, State University Vale do Acaraú, Sobral, Ceará, Brazil eDepartment of Chemistry, Federal University of Ceará, Fortaleza, Ceará, Brazil
fDepartment of Morphology, Faculty of Medicine, Federal University of Ceará, Fortaleza, Ceará, Brazil gDepartment of Pathology and Legal Medicine, Federal University of Ceará, Fortaleza, Brazil hFaculty of Medicine, Federal University of Ceará, Sobral, Ceará, Brazil
i
Graduate Program in Health Sciences, Medical School, Federal University of Ceará, Sobral, Ceará, Brazil
A R T I C L E I N F O
Keywords: Tocoyena sellowiana Periodontitis Sub-chronic toxicity
A B S T R A C T
Tocoyena sellowiana(Cham. & Schltdl.) K.Schum is one of the most important families of Brazilian medicinal plants. This study aimed to evaluate the effect ofTocoyena sellowiana(Cham. & Schltdl.) K.Schum ethanolic
extract in a pre-clinical trial of periodontitis and to investigate possible mechanisms underlying such effects. Periodontitis was induced in Wistar rats by placing a nylon thread ligature around second upper left molars for 11 days. Rats received (per os)Tocoyena sellowiana(0.1, 1 or 10 mg kg) or vehicle 1 h before ligature and daily until day 11. Macroscopic, histopathological, and COX-2 immunohistochemical analyses were performed to evaluate the periodontium. The gingival tissue was used to quantify the myeloperoxidase (MPO) activity and interleukin (IL)-1βlevels by ELISA. Blood samples were collected to evaluate bone-specific alkaline phosphatase (BALP), the dosage of creatinine, aspartate and alanine transaminases. The liver, kidneys, spleen, and body mass variations were also evaluated.Tocoyena sellowianadecreased bone loss, reduced MPO, IL-1βlevels as well as COX-2 immunostaining, and increased BALP activity. Moreover,Tocoyena sellowianadid not alter organs nor body weight.Tocoyena sellowianareduced bone loss in rats and its efficacy was at least partially dependent upon both IL-1βand cyclooxygenase-2 inhibition.
1. Introduction
Periodontitis is an inflammatory disease that causes damage to the connective tissue matrix and resorption of alveolar bone, culminating in tooth loss. It is a major public health problem due to its high prevalence and incidence worldwide [1].
A growing body of research suggests that the pathogenesis of peri-odontitis involves the presence of bacteria (oral biofilm), environ-mental factors, and the immune/inflammatory host response [2–4]. The
interplay between these events leads to the generation of inflammatory mediators such as cytokines (TNF-α, IL-6, IL-1β) and prostaglandins that are responsible for the amplification and maintenance of the
https://doi.org/10.1016/j.biopha.2018.01.011
Received 20 September 2017; Received in revised form 29 December 2017; Accepted 3 January 2018
⁎Corresponding author at: Universidade Federal do Ceará, Núcleo de Pesquisa e Desenvolvimento de, Medicamentos (NPDM), Rua Coronel Nunes de Melo, 1000 - Rodolfo, Teó
filo, Fortaleza - Ceará - Brasil, CEP - 60430-275.
E-mail address:[email protected](M.M. Bezerra).
0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
inflammatory response, leading ultimately to the destruction of the alveolar bone [5–7].
To develop new therapies, there has been an increase in the use of natural products. The Rubiaceae family, mainly concentrated in the tropics, includes about 637 genera and 13,000 species [8]. It represents one of the most important medicinal plant families in the Brazilianflora [9]. Some authors suggested that this family presents a large diversity of substances such as iridoids, indole alkaloids, anthraquinones, ter-penoids (diterpenes and triterpenes), flavonoids and other phenolic derivatives, with emphasis on production of bioactive alkaloids [10]. Previous studies onT. sellowianabark resulted in the isolation of 10-O-feruloygeniposidic acid, 7-hydroxy-6-methoxy-coumarin, 1-ethoxy-glycopyranosyl and 3-β-O-D-glycopyranosylquinovic acid. The fruit
ethanolic extract led to the isolation ofβ-gardiol [11].
Particularly, our group reported a phytochemical assay on Rubiaceae, particularly, Tocoyena sellowiana (Cham. & Schltdl.) K.Schum. Mannitol, 3-β-O-D-glycopyranosylquinovic acid and its
gly-coside were isolated from the root ethanolic extract [12].
This plant has been chosen to be studied because its bark is popu-larly used as an anti-inflammatory agent for bruises and no previous pharmacology study was reported. Therefore, the present study aimed to investigate the unexplored anti-inflammatory efficacy of Tocoyena sellowiana(Cham. & Schltdl.) K.Schum in a ligature-induced model of periodontitis in rats. Additionally, we investigated whether Tocoyena sellowiana (Cham. & Schltdl.) K.Schum efficacy depends on cycloox-ygenase-2 and IL-1βinhibition. Further, a systemic evaluation of the sub-chronic toxicity ofTocoyena sellowianawas carried out.
2. Materials and methods
2.1. Animals
Female Wistar rats from the Federal University of Ceará, weighing 200–220 g were housed in temperature-controlled rooms under 12-h light-dark cycles and received water and food ad libitum. Surgical procedures and animal treatments were conducted in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and with the approval of the Local Ethics Committee (CEPA Number 52/2012). All efforts were made to minimize suffering.
2.2. Experimental model of periodontitis
The animals were allotted tofive groups of six animals. The ligature-induced periodontitis model used consisted of insertion of a sterilized nylon (3.0) thread ligature around the cervix of the second left upper molars of animals anesthetized with ketamine (90 mg/kg in-traperitoneally) and xylazine (10 mg/kg inin-traperitoneally). The ligature was knotted on the vestibular side of the tooth. The ligature-induced periodontitis was made randomly. The contralateral right side was used as the non-ligated control. After induction of periodontitis, animals weight was daily assessed for the construction of a body mass variation curve. On day 11, animals were euthanized with an overdose of keta-mine and xylazine (300/30 mg/kg; i.p.) when we removed the hemi-arcades for morphometric, histopathological, and im-munohistochemical analysis. The gingival tissue was collected to measure the levels of PGE2 and IL-1β. For the measurement of MPO, the gingival tissue was collected 6 h after induction of periodontitis. Blood samples were also collected for serum biochemical levels (ALT, AST, TALP, BALP, creatinine) and organs removed for histopathological analysis (stomach, liver, kidney, and heart).
2.3. Experimental groups
2.3.1. Naive group
This group consisted of six mice not subjected to periodontitis.
2.3.2. Saline group (non-treated group)
This control group consisted of six animals subjected to period-ontitis. The animals received 0.9% sterile saline solution orally, 30 min before ligature and, after that, daily, for an 11-day period, when they were then euthanized.
2.3.3. Tocoyena sellowiana treated (Ts) groups
The animals were subdivided into three groups of six animals each, which received Ts orally dissolved in 0.9% sterile saline solution at the doses of 0.1, 1 or 10 mg/kg, respectively, 30 min before ligature, and daily until day 11.
2.4. Bone tissue morphometric study
On day 11, animals were sacrificed, and their maxillae were re-moved andfixed in 10% neutral buffered formalin (Reagen®, Rio de Janeiro, RJ, Brazil) for 24 h. Following that, the maxillae were dissected and stained with 1% aqueous methylene blue (Vetec®, Duque de Caxias, RJ, Brazil) and placed on microscope slides. Then, they were followed by photographic registration using a digital camera, Nikon (D40, Melville, NY, USA). The bone loss measurement was assessed by a de-fined region, including the occlusal border of the buccal side of the hemimaxilla until the alveolar bone border. These areas were evaluated by ImageJ software (Software ImageJ 1.32j, National Institutes of Health; EUA) as previously described [13].
2.5. Alveolar bone histological analysis
All groups were euthanized as described above and had their maxilla excised. The specimens were fixed in 10% neutral buffered formalin and were demineralized in 7% formic acid (Dinâmica Química Contemporânea, Diadema, SP, Brazil) for 3 days. The H&E-stained maxillae were semi-quantitatively evaluated for cell infiltration, os-teoclast number, alveolar bone and cementum integrity. Sections of 4-mm thickness, corresponding to the area between thefirst and second molars, were evaluated by a pathologist unaware of the treatment protocol by using light microscopy.
2.6. Plant materials and extraction
Tocoyena sellowiana was collected in April 2010, from Ibiapaba Mountain, State of Ceará, Northeast of Brazil. A voucher specimen (N° 33.739), corresponding to the collection is deposited in the Herbarium Prisco Bezerra of the Federal University of Ceará. The isolation of the extract was carried out at the Laboratory of the Organic Chemistry from the Department of Chemistry (State University Vale do Acaraú, Sobral, Ceará, Brazil). The stem bark (1.900 g) was air-dried and the powdered botanical material was extracted with n-hexane (2 × 6 L) followed by EtOH, with same amounts of solvent at room temperature to yield 65.4 g and 41.1 g of the respective extracts, after evaporation of the solvents under reduced pressure. The EtOH extract was partitioned between methanol/water (7:3) and CHCl3 (3 × 100.0 mL). After re-moving CHCl3 fraction, the aqueous layer was partitioned again using AcOEt (5 × 100.0 mL), followed by n-BuOH (2 × 100.0 mL) to yield the fractions CHCl3 (7.3 g), AcOEt (4.2 g), n-BuOH (9.0 g) and water-soluble fraction (20.0 g).
2.7. Serum dosage of BALP
was in accordance with the manufacturer’s directions (Labtest®, Lagoa Santa, MG, Brazil).
2.8. Dosage of prostaglandin E2 (PGE2) and Interleukin 1β(IL-1β) levels
In another series of experiments, the gingivae surrounding maxillary left molars were excised on the 11th day after periodontitis challenge and were homogenized in a solution of RIPA Lysis Buffer System (Santa Cruz Biotechnology, USA). The samples were centrifuged at 10,000 rpm for 15 min at 4 °C. The supernatants were stored at−80 °C for posterior
analysis to evaluate the levels of PGE2 and IL-1β. The PGE2 dosage was performed by ELISA, using R&D Systems®, Kit ParameterTMPGE2 Assay (catalog PKGE004B, USA kit) following the company's protocol. Similarly, IL-1βdosage was performed by ELISA, using R&D Systems® Kit Quantikine rat IL-1βimmunoassay (catalog RLB00, USA) following the company's protocol. The absorbance was measured at 450 nm. Results were shown as picogram/ml (pg/ml).
2.9. Immunohistochemistry
To perform immunohistochemical analysis, the samples were sec-tioned into 4-mm thickness specimens and mounted on glass slides previously prepared with organosilane -based adhesive aminopropyl-triethoxi 3-silane (Sigma®Chemical Co., St. Louis, MO, USA).
The sections underwent anti-COX-2 immunostaining using the streptavidin-biotin method. The technique consisted of the following steps: histological sections were deparaffinized, rehydrated in xylene (3 baths for 5 min each), immersed in absolute alcohol (3 passages), wa-shed in tap water and underwent a transition in distilled water.
Antigen retrieval was performed in citrate at pH 6.0 pressurized pan (Pascal) is within ± 22–25 psi for 30 s at a temperature of 126 °C. After returning to room temperature, it was carried out the blockade of en-dogenous peroxidase with hydrogen peroxide 3% –10 min. The sec-tions were incubated with primary rabbit anti-COX-2 (Abcam®) over-night at room temperature at a dilution of 1:300 in PBS with bovine serum albumin (PBS-BSA) and then washed with solution PBS.
The sections were then incubated with the secondary antibody LSAB kit (DAKO®, Carpinteria, CA) for 10 min at room temperature. Then, incubation was carried out in chromogen solution prepared with 3,3′ diaminobenzidine (DAB) for 5 min in a dark chamber. Negative controls were carried out simultaneously as described above, but the primary antibody was replaced with PBS-5% BSA. The sections were then wa-shed in running water and then in distilled water. Counterstaining was performed with hematoxylin, followed by dehydration in a series of concentrations of alcohol, diaphanized in xylene, and, finally, the coverslip was placed. The slides were evaluated NIKON Eclipse E200 microscope and photographed under a microscope LEICA coupled to a computer.
The parameters of positive immunohistochemical staining of the antigen in all sections consisted of cells that exhibited brown staining in the cytoplasm, regardless of the intensity of immunostaining.
2.10. Myeloperoxidase activity evaluation
Myeloperoxidase (MPO) is an enzyme predominantly present in the azurophilic granules of neutrophils, which has been used as a quanti-tative marker of the infiltration of neutrophils in inflammatory pro-cesses in multiple tissues. For this, 50 mg of gingival tissue was placed in a buffer with 0.5% potassium bromide hexadecyl-trimethylammonium (pH 6.0, 50 mg tissue per ml) and subsequently homogenized in a Polytron®. Next, the homogenate was centrifuged at 4500 rpm for 12 min at 4 °C, and the supernatant was collected. The MPO activity per mg of tissue was measured using 0.0005% hydrogen peroxide as a substrate for MPO. The unit of MPO activity was defined as being able to convert 1μmol of hydrogen peroxide in water for 1 min at 22 °C. During the test, as the hydrogen peroxide was degraded
occurred the production of superoxide anion, responsible for the con-version of a compound in dianisidine - brown color. The results were expressed as MPO activity/mg tissue [14].
2.11. Serum dosage of transaminases (AST and ALT), TALP and Creatinine
Blood samples were collected before periodontitis-induction (day zero) and on day 11 of the assay from anesthetized rats (naive, saline and Ts). Liver function parameters (AST and ALT) were evaluated. TALP serum levels were also evaluated. Measuring serum creatinine assessed renal function. Specific kits were used, and methodology fol-lowed the manufacturer’s instructions (Labtest®, Lagoa Santa, MG, Brazil).
2.12. Body mass variation
The body mass variation was evaluated before periodontitis-induc-tion (day zero) and on day 11. Values were expressed as body mass variation (g) compared with the initial body mass.
2.13. Histological analysis of the liver, the kidney, and the heart
To assess possible detrimental effects on the liver, kidney, and heart tissue parenchyma, the animals received Ts (0.1, 1, 10 mg/kg) and animals from the naive group and non-treated group had their organs removed for subsequent histological analysis. These organs werefixed in 10% buffered formalin for 24 h and then embedded in paraffin. Sections 5 mm thick were deparaffinized stained with hematoxylin-eosin (H&E) and examined under an optical microscope.
2.14. Analysis of the gastric mucosa
The H&E-stained glandular portion of the stomach was semi-quan-titatively evaluated according to the following criteria, as previously described [15]: loss of epithelial cells (Score: 0–4), mucosal edema (Score: 0–4), hemorrhagic injury (Score: 0–4) and cellular infiltration (Score: 0–3).
2.15. Statistical analysis
The data are presented as mean ± standard error of the mean (SEM) or median (and range), where appropriate. Analysis of variance (ANOVA) followed by Bonferroni’s test was used to compare means, and Kruskal–Wallis and Dunn tests were used to compare medians. P < 0.05 was considered significant. All analyses were performed using Graph Pad Prism 5 software, San Diego, CA, USA.
3. Results
3.1. Measurement of alveolar bone loss
Surgical placement of a nylon ligature around the cervix of the second upper molars of the animals could reproduce the main clinical signs of periodontitis, such as loss of alveolar bone, furcation exposure, and interdental contact loss, when compared to animals without peri-odontitis (naive) (Fig. 1). Rats subjected to periodontitis and treated withTs(0.1, 1 or 10 mg kg) exhibited a dose-dependent inhibition of alveolar bone loss that was significantly different from the rats sub-jected to periodontitis and receiving the vehicle only (saline 0.9%) (saline-treated rats) (P < 0.05) (Fig. 1).
3.2. Alveolar bone histological analysis
inflammatory cell infiltrates in the periodontal tissue, coupled with severe destruction of both cementum and alveolar process, so that the animals from this group (NT group) were assigned a median score of 2 (range 1–3) (P < 0.05) (Table 1andFig. 2C and D).
Further, histopathological analysis from the groups treated withTs (0.1; 1 or 10 mg/kg) depicts discrete cellular infiltration and a pre-served alveolar process and cementum in the periodontal tissue, so that the animals from these groups were assigned a median score of 2 (1–2), 1 (1–2), 1 (0-2), respectively (Table 1andFig. 2E and F).
3.3. Plasma bone alkaline phosphatase (BALP)
The treatment withTs(10 mg/kg) resulted in a significant increase in the BALP serum levels variation when compared with the saline-treated rats (Fig. 3).
3.4. Gingival MPO assay
Six hours after periodontitis induction, there was a significant in-crease (P < 0.05) in the MPO activity in the saline-treated rats (NT group) compared with unchallenged rats (naive group).Ts(10 mg kg) significantly (P < 0.05) reduced gingival MPO levels when compared with saline-treated rats. Further, there was no significant (P > 0.05) difference between theTs-treated group and the naive group (Fig. 4).
3.5. Effect ofTs on gingival PGE-2 and IL-1βlevels
There was a significantly (P < 0.05) higher gingival PGE-2 levels in saline-treated rats when compared with the naive group (Fig. 5a). Furthermore, gingival IL-1βlevels were also increased in saline-treated rats compared with the naive group (Fig. 5b). Ts(10 mg kg) reduced both PGE-2 and IL-1βgingival levels. It is worth to note that there was no significant difference (P > 0.05) between theTs-treated group and the naive group (Fig. 5a and b).
3.6. Effect of Ts on COX-2 immunostaining
There was a marked COX-2 immunolabeling in the periodontal tissue of the saline-treated rats, especially within fibroblasts (white arrow) in comparison with healthy animals (naive group) (Fig. 6a and b).Ts(10 mg kg) reduced COX-2 immunolabeling (Fig. 6c).
3.7. Systemic evaluation of rats treated with Ts
Ts(0.1, 1 or 10 mg/kg) did not produce any signs of toxicity or Fig. 1.(a) Illustrative photography of the maxilla (buccal surface) from rats subjected to periodontitis and treated withTocoyena sellowiana(Cham. & Schltdl.) K.Schum (Ts). A: rat not subjected to periodontitis (naive); B: rat subjected to periodontitis and treated with the vehicle (saline) for 11 days (NT group); C, D and E rats subjected to periodontitis and treated with Ts(0.1; 1 or 10 mg/kg) for 11 days, respectively. White arrows indicate alveolar bone resorption. (b) Morphometric analysis of the alveolar bone loss in rats subjected to periodontitis and treated withTocoyena sellowiana(Cham. & Schltdl.) K. Schum (Ts0.1; 1 or 10 mg/kg). Data are shown as mean ± SEM (n = 6 for each treatment). Naive: rats without periodontitis (unchallenged group); NT (saline-treated group): Rats subjected to periodontitis and treated with the vehicle (saline); 0.1; 1 and 10: rats subjected to periodontitis and treated withTs (0.1, 1 or 10 mg kg), respectively.#P < 0.05 compared to the naive group. *P < 0.05 compared to saline-treated group (NT) (ANOVA, Bonferroni Test). (c) Schematic diagram showing how the bone loss area is calculated.
Table 1
Effect ofTocoyena sellowiana(Cham. & Schltdl.) K.Schum on histopathology (H&E) in the maxilla of rats subjected to periodontitis.
Tocoyena sellowiana
Naive NT 0,1 1 10
Scores 0 (0–0) 2 (1–3)* 2 (1
–2)* 1 (1
–2)* 1 (0
–2)**
Kruskal-Wallis, Dunnʼs Test.
mortality during the experimental period. Plasmatic levels of enzymatic markers of hepatic function (ALT and AST) did not differ from the re-spective controls. Plasmatic levels of creatinine, an indicator of renal function, also did not differ from the controls (Table 2).
Moreover,Ts(0.1, 1 or 10 mg/kg) did not alter the wet weight of the heart, liver, and kidney (Table 3). Histopathological analysis of these organs revealed no differences between animals treated with Ts (10 mg/kg) and those of the saline-treated group (Fig. 7). There were no significant lesions in the organs analyzed. Thus, no consistent signs of systemic damage were observed.
Concerning the body mass, during thefirst 2 days after periodontitis induction, all experimental groups lost body weight when compared with the naive group (Fig. 8). Animals treated withTs(0.1, 1 or 10 mg/ kg) tended to recover the body mass after this time point with no sta-tistical significance if compared to saline-treated rats. Further, while
the corporal mass curve from the saline-treated rats showed the same pattern of weight gain, it was below the values observed in the rats treated withTs (0.1, 1 or 10 mg/kg) during the experiment (Fig. 8). Lastly, histopathological analysis of the gastric mucosa of animals treated with Ts (10 mg/kg) revealed no alterations, when compared with naive group (Fig. 9andTable 4).
4. Discussion
causing systemic alterations. We also found evidence that, at least in part, the effectiveness ofTs in periodontitis depended upon cycloox-ygenase-2 and IL-1βinhibition. To the best of our knowledge, this is the first report evaluating both the efficacy and the safety ofTsin a pre-clinical trial of periodontitis in rats.
The pharmacological activity reported for various species of Rubiaceae reveal that this family may be a promising source of new bioactive products. In fact, many of these plants have widespread use in folk medicine and some showed anti-inflammatory, analgesic, anti-bacterial, mutagenic, antiviral, antioxidant, and effect on vascular diseases as well as activity on the central nervous system [10].
The alkaline phosphatase (TALP) is a metalloenzyme present on the cell membrane, which is distributed in the liver, in the bowel, in the placenta, and bones [17]. Among the biochemical markers of bone formation, total alkaline phosphatase (TALP) is widely employed, but it lacks specificity. However, an isoform of TALP, the bone-specific al-kaline phosphatase (BALP), is considered a specific indicator of osteo-blastic activity and bone formation. BALP acts specifically as a bone formation marker. In this regard, our group demonstrated that the measurement of BALP could provide information on the response to a
selected treatment [17,19]. In the present study, we performed this assay, providing a simple, accessible and accurate method of assessing alveolar bone loss during ligature-induced periodontitis in rats. We verified that the administration ofTsprevented the reduction of BALP serum levels when compared with the saline-treated rats, suggesting thatTsprevents bone resorption and induces bone formation. Like our results, some authors observed a decreased BALP activity and increased of IL-1βlevels in the gingival tissue during ligature-induced period-ontitis in rats [20]. Further, it was verified a relationship between the loss of attachment in periodontal disease and a drop in BALP activity in serum from patients with chronic periodontitis [18].
The role of neutrophils in periodontitis has been addressed and some authors have presented the current knowledge on neutrophil-modulating agents as a potential therapeutic approach for periodontitis [21]. Neutrophils infiltration into inflamed tissues is a hallmark of acute inflammatory reactions and reflects a primary immunological response to invading pathogens. Through the release of several in-flammatory mediators, such as eicosanoids, cytokines, and reactive oxygen/nitrogen species, our group showed that neutrophils can am-plify and sustain the inflammatory response by activating and re-cruiting more polymorphonuclear cells [22,23]. Some authors have confirmed that the recruitment and activation of neutrophils constitute the front line of the acute host inflammatory response, represent the main source of PGE2, and promote the initiation of bone metabolism breakdown by stimulating osteoclasts. Hence, the ability to decrease Fig. 3.Effect ofTocoyena sellowiana(Cham. & Schltdl.) K.Schum on the plasma bone
alkaline phosphatase (BALP) in rats subjected to periodontitis. Data represent the mean ± SEM of six animals for each group. Naive: unchallenged rats; NT (not-treated): rats subjected to periodontitis and receiving the vehicle (saline 0.9%);Ts: rats subjected to periodontitis and treated withTs(10 mg/kg) for 11 days. *P < 0.05 was considered significantly different compared to the group of animals subjected to periodontitis that receivedTs(10 mg/kg) for 11 days. (ANOVA, Bonferroni Test).
Fig. 4.Gingival myeloperoxidase (MPO) activity assayed on the 6th hour in rats sub-jected to periodontitis and treated withTocoyena sellowiana(Cham. & Schltdl.) K.Schum (10 mg/kg). The bars represent mean ± SEM of MPO units/mg tissue. Naive: un-challenged rats; NT: rats subjected to periodontitis and receiving the vehicle (saline 0.9%);Ts: rats subjected to periodontitis and treated withTs(10 mg/kg) for 11 days. #P < 0.05 compared to naive group (NT).*P < 0.05 compared to the saline-treated
group.&P > 0.05 compared to naive group (ANOVA, Bonferroni Test).
recruitment and activation of neutrophils seems to be critical to redu-cing inflammatory bone loss.
Myeloperoxidase (MPO) is an enzyme found in azurophilic granules of neutrophils and other inflammatory cells [14]. MPO tissue activity is used as a marker of inflammatory cell infiltrates (especially neu-trophils) in the inflamed tissue [24]. In the present study, we observed a markedly increase in gingival MPO activity 6 h after periodontitis challenge, which was notably abrogated byTs treatment. These data support the idea thatTstreatment, in addition to reducing the alveolar bone resorption, could also reduce the local inflammation.
Epidemiological and microbiological studies have suggested that periodontitis is a multifaceted condition in which several factors si-multaneously play a role [25]. Periodontitis pathogenesis highlights the presence of infectious agents (multi-species oral biofilm), and the host inflammatory response, in which the release of a plethora of mediators such as prostaglandins and cytokines are primary regulators of alveolar bone destruction in periodontitis [25]. Although, the mechanisms by which the host inflammatory response towards microbial challenges in periodontitis interrupt the highly coordinated process of bone re-modeling in favor of bone resorption remain to be established [26].
Inflamed gingiva significantly synthesizes larger amounts of pros-taglandins (mainly the E series) and this secretion is enhanced by pro-inflammatory cytokines (IL-1β, TNF-α) [27]. These mediators together
promote osteoclast activity, particularly in states of inflammatory os-teolysis such as those found in periodontitis [28].
Alveolar bone loss is the most noticeable feature of periodontitis. There is a great number of evidence that correlates PGE2levels within periodontal tissues to the periodontal damage [25]. Cytokine involve-ment in periodontitis is well known, and these molecules, particularly TNF-α and IL-1β, may amplify the inflammatory response, causing tissue destruction and bone loss. The release of proinflammatory cyto-kines (TNF-αand IL-1β) during periodontitis activates osteoblasts and osteoclasts to produce COX2-mediated prostaglandin E2 (PGE2), trig-gering bone resorption [29].
Apparently, the administration of aspirin-like drugsor nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit prostaglandin synth-esis by inhibiting the cyclooxygenase (COX) pathway, have proven beneficial in controlling periodontitis due to the reduction of acute inflammatory changes, such as local edema, cell migration, and release of free radicals [30,31]. Some authors have demonstrated a therapeutic effect of NSAIDs in experimental periodontitis, as well as in human patients [32]. In this regard, our group demonstrated that selective cyclooxygenase-2 inhibition prevents alveolar bone loss in rats [16]. Also, some authors verified the effect of COX-2 selective inhibitor as an adjunct to the treatment of periodontal disease in a clinical double-blind study in humans [33].
In the present study, we demonstrated that the ligature-induced periodontitis was associated with both increased gingival levels of PGE2 and increased immunostaining for COX-2 in thefibroblasts. These data are in accordance with the current literature showing that levels of PGE2 and its processing enzyme, prostaglandin-endoperoxide-synthase-2 or cyclooxygenase-prostaglandin-endoperoxide-synthase-2 (PTGSprostaglandin-endoperoxide-synthase-2 or COX-prostaglandin-endoperoxide-synthase-2), are elevated in progressing periodontal lesions [34]. The treatment of rats with Ts (10 mg/kg) significantly reduced both the gingival levels of PGE2 and the im-munostaining for COX-2, when compared with the control group. Our results suggest thatTsprevented alveolar bone resorption and reduced inflammatory changes in the rat ligature-induced model of periodontitis through COX-2 inhibition.
Selective COX-2 inhibitors display a better risk/benefit clinical anti-inflammatory ratio when compared with classical NSAIDs. Aiming to investigateTssafety, we evaluated the gastric mucosa of rats for he-morrhagic lesions. We observed that the Ts-treated rats had no Fig. 6.Representative immunohistochemistry of the maxilla from rats subjected to periodontitis and treated withTocoyena sellowiana(Cham. & Schltdl.) K.Schum during 11 days. (A) Negative control (sections in the absence of anti-COX-2 antibody) (400×). (B) Rats subjected to periodontitis and receiving only the vehicle (saline 0.9%) showing increased COX-2 immunolabeling offibroblasts (white arrow) that was reduced byTstreatment (C) (400×).
Table 2
Evaluation of blood biochemical parameters from rats subjected to periodontitis and treated withTocoyena sellowiana(Cham. & Schltdl.) K.Schum.
Parameters Experimental Groups
Naive Saline-treated Tocoyena sellowiana(0.1 mg/kg) Tocoyena sellowiana(1 mg/kg) Tocoyena sellowiana(10 mg/kg)
Creatinine 0.52 ± 0.01 0.54 ± 0.02 0.67 ± 0.01 0.64 ± 0.02 0.55 ± 0.02
AST 102.9 ± 8.27 88.3 ± 4.08 81.69 ± 6.32 88.69 ± 4.49 98.80 ± 5.25
ALT 54.1 ± 2.01 47.63 ± 1.36 42.41 ± 1.69 47.67 ± 0.45 55.24 ± 4.43
Naive: unchallenged rats; Saline-treated: rats subjected to periodontitis and receiving the vehicle (saline 0.9%);Tocoyena sellowiana(Cham. & Schltdl.) K.Schum: rats subjected to periodontitis and treated withTocoyena sellowiana(Cham. & Schltdl.) K.Schum (0.1; 1 or 10 mg/kg) during 11 days.
Table 3
Evaluation of wet weight (g) of organs (liver, kidney, and heart) from rats subjected to periodontitis and treated withTocoyena sellowiana(Cham. & Schltdl.) K.Schum (10 mg/ kg).
Treatments
Wet weight (g) Naive Saline-treated Tocoyena sellowiana(10 mg/kg)
Liver 9.9 ± 0.35 9.92 ± 0.37 10.81 ± 0.28
Kidney 1.82 ± 0.04 1.84 ± 0.05 1.99 ± 0.05 Heart 0.88 ± 0.03 0.87 ± 0.04 0.92 ± 0.10
significant macroscopic and microscopic alterations. These data are relevant since the side-effects resulting from NSAIDs administration are a severe limitation to the routine use of these drugs in the treatment of periodontitis.
Cytokines are low-molecular-weight proteins involved in the in-itiation and further stages of inflammation. These proteins play a well-documented role in the pathogenesis of periodontitis [35]. Particularly
interleukin-1β(IL-1β) and tumor necrosis factor-α(TNF-α) are thefirst to appear in periodontitis pathways and are associated with in-flammatory cell migration and osteoclast activity [36]. Levels of IL-1β and TNF-αare elevated in gingival tissue in periodontitis and they are involved in the induction of new inflammatory mediators such as other cytokines (IL-6) and PGE2, which may amplify the inflammatory re-sponse, causing tissue damage and bone loss [36,37]. Pre-clinical and Fig. 7.Photomicrographs of organs from rats subjected to periodontitis and treated withTocoyena sellowiana(Cham. & Schltdl.) K.Schum (10 mg/kg). Heart (A–C), liver (D–F) and kidney (G–I) (400×). A, D, G: unchallenged rats (Naive group); B, E, H: rats subjected to periodontitis and receiving the vehicle (saline 0.9%) (Saline-treated group); C, F, I: rats subjected to periodontitis and treated withTs(10 mg/kg) for 11 days. Animals displayed no changes worthy of note.
clinical trials have proven the protective effect of both IL-1βand TNF-α inhibitors on periodontitis [13,28].Our present data are in accordance with the current literature, showing that the induction of periodontitis in rats is associated with increased levels of IL-1βin gingival tissue. Further, the treatment withTs(10 mg/kg) reduced these increased le-vels.
The scientific literature during the last years has seen an ex-ponential increase in the number of reports concerning the use of nat-ural products, especially those derived from plants. However, local populations have used most of these products without adequate in-formation on efficacy and safety. Thus, research concerning these issues must be largely encouraged [38]. Therefore, in the present study, we
also evaluated the sub-chronic toxicity ofTsadministration.
To evaluate the safety ofTs, we performed biochemical analysis in peripheral blood from rats 11 days after daily injection (per os) ofTs (0.1, 1 or 10 mg/kg). This study also evaluated the integrity of the heart, liver, and kidneys in rats that receivedTs.
Biochemical analyses revealed no changes in the enzymatic activity of transaminases in the serum of treated mice. Similarly, histopatho-logical analysis of the heart and liver revealed no changes among groups. Further, serum dosages of creatinine and histopathological analysis of kidneys, which were used as parameters of renal function, revealed no changes.
5. Conclusion
In conclusion, we demonstrated the resorptive and the anti-inflammatory efficacy ofTsin the rat ligature-induced model of peri-odontitis. Although the mechanisms of Tocoyena genus remain to be determined, our results strongly suggest thatTsefficacy, at least in part, depends upon both IL-1βand COX-2 inhibition. Albeit the attention on COX-2 has raised the possibility of new uses for selective COX-2 in-hibitors,Ts may be a promising source of new bioactive substances, which may give rise to the designing of alternative compounds to classical anti-inflammatory agents.
Conflict of interest
The authors declare that there is no conflict of interest regarding this publication.
Fig. 9.Photomicrographs of gastric mucosa from rats subjected to periodontitis and treated withTocoyena sellowiana(Cham. & Schltdl.) K.Schum during 11 days. (A) Rats subjected to periodontitis and receiving the vehicle (saline 0.9%) for 11 days (100× and 400×); (B) Rats subjected to periodontitis and treated withTs(10 mg/kg) for 11 days (100× and 400×) showing preservation of the morphologic characteristics of the mucosa.
Table 4
Effects ofTocoyena sellowiana(Cham. & Schltdl.) K.Schum on gastric histological as-sessment after periodontitis induction.
Experimental groups (n = 6)
Epithelial cell loss
Edema Hemorrhagic damage
Inflammatory cells
Naive 0 (0-0) 0 (0-1) 0 (0-0) 0 (0-0)
Saline-treated 0 (0-0) 0 (0-0) 0 (0-0) 0 (0-0) Tocoyena
sellowiana (0.1 mg/kg)
0 (0-0) 0 (0-1) 0 (0-0) 0 (0-0)
Tocoyena sellowiana (1 mg/kg)
0 (0-1) 0 (0-2) 0 (0-0) 0 (0-0)
Tocoyena sellowiana (10 mg/kg)
0 (0-2) 1 (0-2) 0 (0-0) 0 (0-0)
Acknowledgments
This study was supported by Brazilian grants from Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico (FUNCAP), Conselho Nacional de Pesquisa (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). During this study, Prof. Mirna M. Bezerra was a senior investigator of CNPq/Brazil. The authors thank Adalberto Nascimento de Lima Júnior, Sthefane Gomes Feitosa, and Conceição S. Martins for the technical assistance provided.
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