Seventy-two male Wistar rats (±200 g) (Rattus novergicus), from our own facilities, were used in this study. The animals were kept in cages with temperature-controlled rooms, with food and water ad libitum throughout the experiment. In accordance to the Ethical Principles for Animal Research, all efforts were done in order to reduce pain or discomfort to the animals. All procedures and animal treatments were conducted after approval by the institutional Ethical Committee for Animal Research from Federal University of Ceará (UFC) (number 38/15).

For the induction of experimental periodontitis (EP) it was used the model of ligature-induced periodontitis (Goes et al., 2010). The animals were anesthetized with ketamine (70 mg/kg administered i.p., 10% Quetamina, Vetnil, São Paulo, SP, Brazil) and xylazine (10 mg/kg administered i.p., 2% Calmium, São Paulo, SP, Brazil). Later, a sterilized nylon (000) thread ligature was placed around the cervix of the second left upper molar. The ligature was then knotted on the vestibular side of the referred tooth. Eleven days after ligature placement the animals were euthanized with 20 mg/kg thiopental (0.5 g Thiopentax, Cristália, São Paulo, SP, Brazil). The ligatures were blinded to the group.

The animals (n = 6 in each group) were randomly assigned into 3 groups: SAL, CLO and Naïve. The rats received, according to the group, either 0.9% of Saline solution (SAL) at the dose of 2 ml/kg or 90 mg/kg of aqueous flower extract of Calendula officinalis (CLO) (Batch number PROD004257) purchased from Mapric Cosmetic and Pharmaceutical Products, Brazil (chemical abstract service—CAS – number 64-17-5; 7732-18-5; 99-76-3), by gavage, 30 min before EP and daily for 10 days. The Naïve group experienced no manipulation.

On the 11th day, animals were euthanized under anesthesia and had their maxillae removed and fixed in 10% neutral formalin for 24 h. Following, maxillae were divided in half, dissected and stained with 1% methylene blue (Goes et al., 2010). For the measurement of ABL, hemi-maxillae were placed in microscope slides and photographed with a digital camera (Nikon® D40, Melville, NY, USA). The acquired image was analyzed using the IMAGE J® software (ImageJ 1.32j; National Institute of Health, Bethesda, MD, USA), according to the methodology described by Goes et al. (2010).

Two additional groups of 6 animals submitted to EP, which received SAL or CLO (90 mg/kg), were euthanized and had their maxillae removed. The specimens were fixed in Karnovisky for at least 6 h, then they were held in a Cacodylate buffer. The maxillae were cut in a diamond blade cutter, in a mesial-distal plane, to obtain the maxilla fragment (0.5 × 0.2 cm and 0.5 mm thick). The fragment was placed in an eppendorf tube and left in the desiccator drying for 24 h. The fragments were assembled into stubs for metallization with gold powder (Quorum Metallizer QT150ES, Quorum Technologies, Laughton, England) for the analysis by a scanning electron microscopy (SEM inspect-50, FEI, Hillsboro, Oregon, USA). It was evaluated the bone topography of the interproximal region between the first and second maxillary left molars (Lu et al., 2014).

In addition, it was used the Energy Dispersive Spectroscopy (EDS), which is a detector (Oxford Instruments, Abingdon, Oxfordshire, UK) installed in the vacuum chamber of the MEV. The elemental and chemical analysis of the sample were performed using the manufacturer's software (AztecEnergy, Oxford Instruments, Abingdon, Oxfordshire, UK).

Two additional groups of 6 animals submitted to EP, which received SAL or CLO (90 mg/kg), were euthanized and had their maxillae removed. The specimens were fixed in 10% neutral buffered formalin for 24 h, and then demineralized in 10% EDTA for 30 days. Following this, the specimens were dehydrated, embedded in paraffin and sectioned (4 μm) along the molars in a mesiodistal plane for H&E staining (Goes et al., 2012). Considering that collagen is a structural protein that presents a natural phenomena of self-fluorescence, the images were obtained using the Confocal LSM 710 microscope (Zeiss, Jena, Germany) and analyzed by manufacture's software (Zen 2.1 lite black, 64-bit version, 758 MB, Zeiss, Jena, Germany). In order to evaluate the presence and organization of collagen fibers in periodontium, between the first and second left upper molar, it was used 488 nm wavelength laser and emission channel for FITC-green fluorescence (Gonçalves et al., 2014).

In order to identify the presence and type of fibrilar collagen, histological sections (4 μm) were obtained from the previously prepared paraffin blocks, and were stained with Picrosirius Red. The slides were evaluated under a polarized light filter. Collagen birefringence, as yellow-red for type I collagen and green for type III collagen (Junqueira et al., 1979), was evaluated between the first and second maxillary left molars.

The streptavidin–biotin–peroxidase method was used for Immunohistochemistry assay, in paraffin-embedded tissue sections 4 μm thick. Sections of the excised maxillae, demineralized in a 10% EDTA solution were used for DKK1 (antibody rabbit policlonal IgG, Santa Cruz Biotechnology), WNT 10b (antibody rabbit policlonal IgG, Abcam), and β-catenin (antibody goat policlonal IgG, Santa Cruz Biotechnology) (Sousa et al., 2016). Five microscopic fields (400x) were used to count osteoblasts exhibiting cytoplasmic positivity for DKK1, WNT 10b and β-catenin (de Barros Silva et al., 2016).

Reduced glutathione (GSH), catalase (CAT) superoxide dismutase (SOD) and malondialdehyde (MDA) were performed to evaluate oxidative stress. For this, the gingival tissue was removed 11 days after EP, then stored at −80°C. The level of GSH in gingival tissue was estimated according to the methods described previously (Sedlak and Lindsay, 1968). The GSH concentration was expressed as micrometers of GSH per gram of wet tissue.

Superoxide dismutase (SOD) activity was assayed as described previously (Beauchamp and Fridovich, 1971). In a dark room, the gingival samples were homogenized in 20 μl of ice-cold phosphate buffer at 15,000 G for 20 min. The supernatants were mixed with a solution comprised of phosphate buffer (50 nM), EDTA (100 nM) and L-methionine (19.5 mM) in a pH of 7.8. Then, 150 ml of a solution of riboflavin (10 nM) and NBT (750 nM) as added and the mixture was exposed to light (20 W) for 15 min. The absorbance of the samples was measured at 560 nm. The results are expressed as grams of SOD per ml.

CAT activity has as principle the measurement of O₂ production rate and H₂O in proportion of H₂O₂ (Maehly and Chance, 1954). Briefly, 20 μl of gingival homogenate was mixed with a solution comprised of 3% H₂O₂ and Tris-HCl EDTA buffer (5 nM, pH 8.0). The absorbance at a 230 nm wavelength was measured immediately and 6 min after preparing the samples.

Malondialdehyde (MDA) indicates lipid peroxidation based on the reaction of this substance with thiobarbituric acid, in the gingival tissue of rats. Briefly, 250 μl of 10% homogenate of gingival tissue were mixed with 1.5 ml of 1% H3PO4 and 0.5 ml of 0.6% thiobarbituric acid aqueous solution and the mixture was stirred and heated in boiling water for 45 min. After cooling, 2 ml of n-butanol were added and the mixture was homogenized. The butanol layer was separated, and the difference between the optical densities at 535 was used for calculating the MDA concentrations, which were expressed as nanomol of MDA per gram of gingival tissue (Mihara and Uchivam, 1978).

Data are presented as mean ± SEM. In order to compare means it was used Analysis of variance (ANOVA) followed by Bonferroni test. P < 0.05 was set to indicate significant differences among groups. All analyses were performed using GraphPad Prism 6 software, San Diego, CA, USA.

Morphometric study demonstrated that the experimental periodontitis caused intense bone resorption (Figure 1D) compared to the normal hemi-maxillae from Naïve group (Figure 1A). Rats from SAL group presented intense alveolar resorption, root exposure and furcation lesion (Figure 1B). Ninety mg/kg of CLO prevented bone loss (Figure 1C), by 42.8%, when compared to SAL (p < 0.05). The treated animals showed greater preservation of bone tissue.

Experimental Periodontitis (Figure 2B) caused important destruction of bone tissue when compared to the normal tissue from Naïve group (Figure 2A). CLO, at 90 mg/kg, prevented bone loss (Figure 2C). In 800x magnification, it was possible the observe that the bone tissue from an animal of SAL group (Figure 2E) presented an irregular topography, when compared to the bone tissue of an animal from Naïve group (Figure 2D). The treatment with CLO (Figure 2F) kept the tissue topography more regular than the one seen on SAL group.

The EDS analysis allowed the immediate identification of minerals and the chemical elements distribution on the samples (Figures 2G–I). It was possible to see elevated peaks of calcium and phosphorus on bone tissue, without any difference between groups.

The confocal analysis of periodontium of animals from SAL group (Figure 3B) demonstrated great destruction and derangement of collagen fibers in periodontal ligament compared to the Naïve group (Figure 3A). The treatment with 90 mg/kg CLO (Figure 3C) preserved these collagen fibers.

Using picrosirius red staining under polarized light filter, it was possible to observe that the majority of collagen fibers from periodontal ligament from animals of Naïve group showed green color (Figure 3E) The collagen fiber of periodontium of animals from SAL group presented reduction on the birefringencewhen compared to Naïve (Figure 3D) The treatment with CLO caused a slight increase on birefringence when compared to SAL, showing fibers in greenish color (Figure 3F).

Considering the canonical WNT pathway, the animals submitted to EP that received SAL showed reduction on the amount of immunopositive cells to WNT 10b (8.50 ± 1.25 positive cells/mm²) (Figures 4A, 5C) (p < 0.05), β-catenin (12.80 ± 1.20) (Figures 4B, 5G) (p > 0.05), and an increase to DKK-1 (58.00 ± 11.03) (Figures 4C, 5K) (p < 0.05) when compared to Naïve [WNT 10b = 22.44 ± 2.82 (Figure 5B); β–catenin = 15.80 ± 1.77 (Figure 5F); DKK-1 = 17.00 ± 3.21 (Figure 5J)]. The treatment with CLO 90 mg/kg increased the count of immunopositive cells for WNT 10b (23.89 ± 2.41) (Figure 5D) and β-catenin (22.25 ± 3.16) (Figure 5H) (P < 0.05) when compared to SAL (WNT 10b = 8.50 ± 1.25 and β-catenin = 12.80 ± 1.20). There was a significant decrease on the count of immunopositive cells for DKK-1 in CLO group (22.20 ± 5.55) (Figure 5L), compared to SAL (58.00 ± 11.02). The negative controls for WNT 10b, β-catenin, and DKK-1 can be seen on Figures 5A,E,I, respectively.

The experimental periodontitis caused significant reduction of the GSH (Figure 6A), SOD (Figure 6B) and CAT (Figure 6C), as well as an increase on MDA (Figure 6D) gingival levels (P < 0.05) when compared to the Naïve group, suggesting that oxidative stress is observed in gingival tissue subjected to periodontitis. Administration of 90 mg/kg CLO increased gingival GSH, SOD and CAT concentration, and reduced MDA levels compared with SAL group.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.