Original Article http://dx.doi.org/10.1590/1678-7757-2021-0160 The effect of ellagic acid on the repair process of periodontal defects related to experimental periodontitis in rats Abstract Figen ÖNGÖZ DEDE¹ Objective: This study aims to evaluate the effect of ellagic acid (EA) by Şeyma BOZKURT DOĞAN2 measuring the levels of alveolar bone resorption and inflammatory and oxidative stress markers in the periodontal tissues and serum on the periodontal repair Umut BALLI³ process related to experimental periodontitis in rats. Methodology: Forty Wistar rats Mustafa Cenk DURMUŞLAR4 were divided into four study groups as follows: Group 1=healthy control (n=10); Bahattin AVCI⁵ Group 2=EA control (15 mg/kg)(n=10); Group 3=periodontitis (n=10); Group Kanat GÜLLE⁶ 4=periodontitis+EA (15 mg/kg) (n=10). The periodontitis model was established Meryem AKPOLAT FERAH⁷ by ligating bilateral mandibular first molars for 14 days. Then, rats were given normal saline or EA for another 14 days by gavage administration. Serum and gingiva myeloperoxidase (MPO) activity, 8-hydroxydeoxyguanosine(8-OHdG), and glutathione (GSH) levels were analyzed by ELISA. İmmunohistochemical analysis was used to detect Interleukin (IL)-6, IL-10, and tumor necrosis factor- alpha (TNF-α) immunoreactivities in the periodontal tissues. Alveolar bone loss (ABL) and attachment loss (AL) was evaluated by histomorphometry analysis. Results: ABL and AL were statistically higher in group 3 than in groups 1, 2 and 4 and in group 4 than in groups 1 and 2 (p<0.05). MPO activities in gingival tissue and serum were significantly increased in group 3 compared to groups 1 and 2 (p<0.05). Significantly higher serum GSH levels, lower gingiva, and serum 8-OHdG levels, and MPO activity were observed in group 4 compared to group 3 (p<0.05). Rats with periodontitis (group 3) expressed significantly higher immunoreactivities of IL-6 and TNF-α and lower IL-10 immunoreactivity compared to those other groups (p<0.05). IL-6 and TNF-α immunoreactivities significantly decreased and IL-10 immunoreactivity increased in group 4 after the use of EA compared to group 3 (p<0.001). Conclusions: Our findings showed that EA provides significant improvements on gingival oxidative stress and inflammatory markers and alveolar bone resorption in the repair process associated with experimental periodontitis. Therefore, EA may have a therapeutic potential on periodontitis. Submitted: March 24, 2021 Keywords: Ellagic acid. Periodontitis. Periodontal repair. Cytokines. Modification: July 5, 2021 Accepted: July 6, 2021 Antioxidant. Alveolar bone defect. Corresponding address: ¹Ordu University, Faculty of Dentistry, Department of Periodontology, Ordu, Turkey. Figen Öngöz Dede ²Yıldırım Beyazıt University, Faculty of Dentistry, Department of Periodontology, Ankara, Turkey. Ordu University - Faculty of Dentistry - Department of Periodontology - 52100 - Altınordu - Ordu - Turkey. ³Bezmialem Vakif University, Faculty of Dentistry, Department of Periodontology, İstanbul, Turkey. Phone: +90 506 7354313 ⁴Kent University, Faculty of Dentistry, Department of Oral and Maxillofacial Surgery, İstanbul, Turkey. e-mail: figen_ongoz@hotmail.com ⁵Ondokuzmayis University, Faculty of Medicine, Department of Biochemistry, Samsun, Turkey. ⁶Süleyman Demirel University, Faculty of Medicine, Department of Histology and Embryology, Isparta, Turkey. ⁷Bülent Ecevit University, Faculty of Medicine, Department of Histology and Embryology, Zonguldak, Turkey. J Appl Oral Sci. 1/12 2021;29:e20210160 The effect of ellagic acid on the repair process of periodontal defects related to experimental periodontitis in rats Introduction agent for the treatment of various chronic diseases, especially Alzheimer’s disease, ulcerative colitis, Crohn’s disease, and diabetes.16Periodontitis is one of the most widespread Moreover, Promsong, 17 infectious inflammatory diseases. It occurs due to an et al. (2015) showed that EA protects human gingival imbalance between dental plaque bacteria and the epithelial cells by reducing IL-2 and IL-8 levels and host’s inflammatory and immune responses.1 During suppressing defense factors, such as human beta- periodontitis, host cells produce excessive amounts of defensin 2 (hBD2) and secretory leukocyte protease 7 reactive oxygen species (ROS), such as those produced inhibitor (SLPI). Bakkiyaraj, et al. (2013) showed by myeloperoxidase (MPO), 8-hydroxydeoxyguanosine that the anti-biofilm activity of EA was greater in (8-OHdG) and inflammatory cytokines, such as tumor various bacterial pathogens (Staphylococcus aureus, necrosis factor-alpha (TNF-α) and interleukin (IL)-6, methicillin-resistant S. aureus [MRSA] compared to in response to bacterial infiltration. The release of Candida albicans. these markers leads to the destruction of periodontal Considering these observations, since ROS tissues.2–4 and inflammatory cytokines are involved in the The elimination of microbial dental biofilms is pathogenesis of periodontitis, we theorize that EA essential to the treatment of periodontal diseases.1,5 exhibits its anti-inflammatory and antioxidant effects Mechanical removal (scaling and root planning) is not in periodontal lesions by scavenging radicals and sufficient for the complete elimination of periodontal suppressing cytokine production. However, no studies microflora.1,5 Therefore, pharmacologic agents have investigated the effect of EA in periodontitis. (antiseptics, nonsteroidal anti-inflammatory drugs, Therefore, our study aimed to evaluate the therapeutic and antibiotics) are used as adjunctive therapy to effects of EA administered orally, measuring gingiva ensure the destruction of microorganisms.6 When these and serum MPO, 8-OHdG, and glutathione (GSH) drugs are used systemically with high doses, they can levels, expression of IL-6, IL-10, and TNF-α in gingiva lead to complications such as antibiotic resistance and and alveolar bone loss on repair process associated other side effects.1 This is the reason why alternative with experimental periodontitis (EP) in rats. natural products are needed for therapeutic use.1,6,7 For such purpose, polyphenolic compounds, which have antioxidant, anti-cancer, and anti-inflammatory Methodology effects have been suggested as potential candidates.8,9 Polyphenols have been reported to have several Animals biological activities; they prevent oral disease, Forty systemically and periodontally healthy adult inactivate bacterial toxins, promote the antioxidant (8 weeks old) male Wistar albino rats weighing an activity of oral fluids, exhibit antibacterial activity average of 220 to 250 kg were used in our study. against periodontal pathogens, and can inhibit the Rats were placed in separate plastic cages, provided proteolytic activity of Porphyromonas gingivalis.3,9,10 food and water being ad libitum, housed at a room Ellagic acid (EA), a polyphenol, is found in many temperature of 22±1°C within 50% humidity fruits, including strawberries, walnuts, pomegranates, conditions in a 12-hour light/dark cycle. All animal and grapes, and many medicinal plants.11,12 EA has care and experimental protocols were approved by been reported to exhibit antioxidant, anti-cancer, the Ethical Committee of Animal Research of Bulent anti-allergic, antiproliferative, and anti-inflammatory Ecevit University in Zonguldak, Turkey (Protocol activities, as well as radical scavenging activity and number 2013-29-02/10) in accordance with both inhibition of lipid peroxidation.11–14 Ogawa, et al.15 the Guide for Care and Use of Laboratory Animals (2002) reported that EA eliminated superoxide and (National Institute of Health, Bethesda, MD, USA) and hydroxy anions. They stated that EA’s effect was the ARRIVE guidelines.18 stronger than that of α-tocopherol and it was as Sample size could be estimated before the study potent as superoxide dismutase (SOD). EA has been due to the lack of precise information available suggested to suppress the production of various regarding ellagic acid effects in experimental cytokines, such as IL-1β, IL-8, and TNF-α.15 A recent periodontitis. We, therefore, based our estimates on review concluded that EA could be a promising the pilot study, which suggested 6 rats in each group. J Appl Oral Sci. 2/12 2021;29:e20210160 ÖNGÖZ DEDE F, BOZKURT DOĞAN Ş, BALLI U, DURMUŞLAR MC, AVCI B, GÜLLE K, AKPOLAT FERAH M The sample size was estimated based on the results mg\kg) was once-daily administered by gavage for 14 of biochemical biomarkers levels in gingival tissue days, starting after the ligature removal in group 4. between ellagic acid application groups and their control groups. A sample size of 10 per group was Sample collection required for detection of a significant difference (80% All rats were anesthetized and 5mL of venous blood power, two-sided 5% significant level). was drained out through cardiac punctures for serum The animals were randomly divided into four groups analyses. Blood samples were centrifuged (Shimadzu of ten rats each. 1) Group 1=periodontally healthy UV160A, SNo:28006648, Kyoto, Japan) at 3000 g control (n=10), in which each rat was gavaged daily and room temperature for 10 minutes, enabling the with 2 mL of saline by gastric intubation for 14 days; collection of serum, which were then placed at −70°C 2) Group 2=periodontally healthy rats+ EA control (15 before biochemical analysis. Block biopsy samples, mg/kg)(n=10), in which rat was gavaged daily with including the gingiva and alveolar bone tissue, were 2 mL of saline containing EA by gastric intubation for removed from the molar regions of the mandibles. 14 days; 3) Group 3=experimental periodontitis group For histological analysis, the left mandibular molar (n=10), in which each rat was gavaged daily with 2 mL regions were resected en bloc from each rat and of saline by gastric intubation for 14 days; 4) Group were fixed in 4% paraformaldehyde in 0.1 mol/L 4=experimental periodontitis group + EA (15 mg/kg) phosphate buffer (pH 7.4) for 1 day. Gingival biopsy (n=10), in which rat was gavaged daily with 2 mL of samples of the right mandibular molar regions were saline containing EA by gastric intubation for 14 days.19 collected and immediately frozen and kept at −70°C The dosage and administration form of drugs were until biochemical analysis. determined based on the literature.19 Drug treatment began after periodontitis was induced. Biochemical analysis The gingival tissue was blotted before being weighed Experimental periodontitis and repair protocol upon a microbalance. The tissues were cryogenically A ligature-induced periodontitis model was created frozen using liquid nitrogen. Subsequently, the tissues due to the induction of EP.20 With the rats under were manually grounded, by placing them within general anesthesia through intraperitoneal injection of Eppendorf tubes containing a required volume of PBS ketamine (100 mg/kg of body weight) and xylazine1 (pH 7.4, 10mM), diluted to 10 mg. tissue/mL PBS. (10 mg/kg of body weight), EP was induced by the This was sonicated (METU Electromechanical, Serial placement of sterile 3–0 silk ligatures in a subgingival No.30607, Berlin, Germany) for 10 minutes at 4°C position around the mandibular first molars (left and with 220V. On the day of evaluation, homogenates right) in all groups for 14 days, except groups 1 and defrosted within the room from the samples were 2. The ligatures contributed to periodontal diseases, centrifuged (SIGMA 3K30, Serial No.76262, Osterode facilitating the movement and passage of bacteria am Harz, Germany) at 4°C and 15000g for 5 minutes within the gingival cavities.21 All ligatures in groups 3 and the supernatants were arranged for subsequent and 4 were removed to allow periodontal repair after GSH, MPO, and 8-OHdG analysis. Gingival tissue and 14 days of periodontitis induction22. Then, rats started serum MPO activity, GSH, and 8-OHdG levels were receiving once-daily normal saline or EA for 14 days evaluated using commercially marketed enzyme- by gavage administration. The rats were euthanized linked immunosorbent assay (ELISA) kits (Cayman on the 28th day. Chemical Company, Item No. 589320 Ann Arbor, MI, USA). The quantum of protein present within the Treatment with ellagic acid tissues was observed by the Lowry method,23 with the The EA (Sigma Chemical Company, St Louis, MO, results expressed as mg per protein. The conclusions USA) treatment in rats was conducted as described were expressed in microgram (µg) of cytokine\ by Kannan, Quine, and Sangeetha19 (2012). As soon mg of protein (µg\mg.protein) within the gingival as EA display poor solubility in water, the treatment tissues, and in ng\mL within the serum, barring GSH in each animal was carried out with a suspension of concentrations. The GSH concentration was expressed ellagic in water at a dose of 15mg/kg. The suspension as mg\ mg prot within the gingival tissues, and as was homogenized with the syringe used in the oral mg\L within the serum. administration before each animal treatment. EA (15 J Appl Oral Sci. 3/12 2021;29:e20210160 The effect of ellagic acid on the repair process of periodontal defects related to experimental periodontitis in rats Immunohistochemical analyses (HScore).24 Staining intensity was semiquantitatively Avidin-biotin peroxidase method was used for the scored according to the following categories: 0, absent; immunohistochemical studies to investigate the anti-IL 1, weak; 2, moderate; and 3, intense. The formula was 10, IL-6, and TNF-α activities. Cross-sections prepared HScore = S Pi (i + 1), in which i represents the intensity from the mandible tissue blocks in 4 μm thickness were scores: Pi, the percentage of stained cells; and 1,the incubated at 60°C. Tissues were rinsed with sequential correction factor. In total, 100 positive stained cells xylol and alcohol solutions for deparaffinization and in the field were evaluated according to the staining distilled water was used to remove the alcohol from intensity and formulated as follows [1×(%cells1+)+ dehydrated tissues. To expose the receptor areas 2×(%cells2+)+3×(%cells 3+)]. within the tissue blocked by formaldehyde, citrate buffer (pH 6.0) (Lab Vision, Fremont, USA) was applied Histomorphometric analysis to the tissues under high temperatures. Following The left side of the mandible detached from within the antigen retrieval procedure, the tissues were left the gingiva was fixated with 10% neutral buffered in the room to cool down for 20 minutes and then formalin. The samples collected were decalcified in rinsed with distilled water to remove the citrate. Each 8% formic acid (14 days) and subsequently embedded Tissue was rinsed with phosphate buffer saline (PBS, within paraffin. Serialized paraffin sections (5 µm) Ph: 7.4) three times for 3 minutes, exposed to 3% were concluded from within the mesiodistal aspects hydrogen peroxide (Lab Vision, Fremont, USA) for within the mandibular first molars. Three of the 15 minutes, and the endogenous peroxidase activity sections, reflective of the central parameters of the was blocked and rinsed with PBS. Ultra V block (Lab individual tooth, were observed and thereafter stained Vision, Thermo Scientific) was applied for 5 minutes with hematoxylin and eosin (H&E). to prevent nonspecific binding. Following the blocking Individual sections were stained with H&E and the stage, the sections were left at room temperature for parameters assessed included: 1) the percentage 45 minutes without being washed and were exposed of alveolar bone in the furcation area, 2) alveolar to anti-IL-10 antibody (ab9722, Abcam, UK), anti-IL-6 bone loss (ABL), and 3) attachment loss (AL). The antibody (ab6672, Abcam, UK), anti-TNF-α antibody percentage ratios of alveolar bone area upon individual (NB600-587, Novus biological, USA) and primer specimens were concluded as a ratio of the alveolar antibodies that were prepared 1/100 proportion. All bone area versus the furcation area. The alveolar primer antibodies were incubated for an hour at 40ºC. bone area was concluded as a mix of the trabecular Then they were rinsed with PBS following the primer bone area and the bone marrow area in furcation. The antibody. A secondary antibody (Lab Vision, Thermo levels of the alveolar bone were concluded through a Scientific) was applied for 10 minutes. They were measure of the distances within the cementoenamel rinsed with PBS and were exposed to streptavidin junction (CEJ) and the alveolar bone crest. AL was peroxidase enzyme (Lab Vision, Thermo Scientific) concluded to be the distance within the CEJ versus complex for 10 minutes. Each tissue was rinsed once the coronal extent of the connective tissue attached to more with PBS. Finally, chromagen DAB (Spring the cementum. ABL and AL values were obtained from Bioscience) containing the diaminobenzidine substrate within mesial and distal regions of the mandibular first (Spring Bioscience) was added to the medium and molars. All averages of the measurements performed was put aside for about 5-10 minutes to ensure the were used to analyzing the data. immune reaction. Mayer’s hematoxylin was used as the background strain. The slides were rinsed with serial Intra-examiner reproducibility alcohol solutions with diminishing concentrations. They Before histomorphometric and immunohistochemical were kept in xylol for 20 minutes and were coated analysis, the examiner (K.G.), who was blinded to with entallan. All sections were evaluated in the light the groups and treatments, evaluated 20 specimens microscope Axio Scope A1 Imager Microscope (Carl twice, with one-week interval between the measures. Zeiss, Oberkochen, Germany). Bland-Altman plots along with intraclass correlation The immunoreactivities of IL-6, IL-10, and TNF-α coefficients were used to perform the Intra examiner 25 proteins in the periodontal ligament were scored by agreement and reliability measures. Bland– one researcher (KG), using a histologic scoring method Altman plots reflected the agreements within the two values obtained within one-week interval in J Appl Oral Sci. 4/12 2021;29:e20210160 ÖNGÖZ DEDE F, BOZKURT DOĞAN Ş, BALLI U, DURMUŞLAR MC, AVCI B, GÜLLE K, AKPOLAT FERAH M the histomorphometric and immunohistochemical Immunohistochemical findings parameters. Figure 2 shows Immunoreactivity findings of IL-10, IL-6, and TNF-α in gingival tissues. IL-6 and TNF-α Statistical analysis immunoreactivities were greater in group 3 among The Shapiro–Wilk test was used to determine if the the groups (p<0.001). TNF-α immunoreactivity was data were normally distributed. Comparisons of the statistically decreased after EA administration in biochemical parameters, histomorphometric data, and group 4 when compared with group 3 (P<0.001). IL-6 immunohistochemical scores were analyzed using the immunoreactivity was statistically lower in group 4 Kruskal–Wallis nonparametric test, followed by post- than those in other groups (P<0.001). hoc group comparisons with the Bonferroni-adjusted IL-10 immunoreactivity was significantly greater Mann–Whitney U test. The Spearman’s rank correlation in group 2 than group 1 (p<0.01), and its level was test was used to detect the relationships among lower in group 3 than in groups 1, 2, and 4 (p<0.001). biochemical parameters, histomorphometric data, and Figure 3 shows immunohistochemical images. immunohistochemical scores. All tests were performed using statistical software (SPSS Inc., version 19.0, Biochemical findings Chicago, IL, USA). p<0.05 was considered statistically Serum and gingival tissue MPO activities were significant. higher in group 3 than in groups 1 and 2 (p<0.05). After EA administration in group 4, MPO activities in the gingival tissue and serum significantly decreased Results when compared with groups 1, 2, and 3 (p<0.05). On the other hand, no significant difference was Histomorphometric findings found in gingival 8-OHdG levels between group 3 Table 1 shows the alveolar bone area in the and the control groups (p>0.05). However, after furcation region, alveolar bone level, and attachment EA administration in group 4, 8-OHdG levels in the loss values. The alveolar bone area of furcation gingival tissue significantly decreased when compared was greater in control groups (p<0.001). However, with groups 2 and 3, and its serum level in group no significant differences in the amount of alveolar 4 presented the lowest value when compared with bone in the furcation area in experimental groups those of other groups (p<0.05). EA administration in were observed (p>0.05). ABL and AL were higher in groups 2 and 4 significantly increased in serum GSH groups 3 and 4 when compared with groups 1 and levels compared to groups 1 and 3 (p<0.05), but not 2 (P<0.001). On the other hand, ABL and AL values in gingival tissue (p>0.05). Figures 4 and 5 show the decreased in group 4 after EA administration than in biochemical findings for gingival tissue and serum group 3 (p<0.01). No significant differences in the values, respectively. alveolar bone area of furcation, ABL, and AL values between groups 1 and 2 were observed (p>0.05). Correlations Figure 1 shows histologic images. Table 2 shows the correlation coefficients. When all groups were examined together, a statistically Table 1- Percentage of Alveolar Bone in Furcation Area, Alveolar Bone Level, and Attachment Level Groups Group 1 Group 2 Group 3 Group 4 (n=10) (n=10) (n=10) (n=10) Alveolar Bone Area (%) 64.08±5.57 64.26±4.55 46.70±4.37*† 51.47±5.71*† Attachment level (μm) 127.79±18.64 128.64±13.52 525.57±23.64*† 407.25±64.30*†‡ Alveolar Bone Level (μm) 436.26±21.45 479.19±35.60 1205.47±71.40*† 1085.63±90.47*†‡ Data are expressed as the mean ± standard deviation. p<0.05 was considered statistically significant. Group 1: control-systemic saline, Group 2: control-systemic ellagic acid, Group 3: experimental periodontitis-systemic saline, Group 4: experimental periodontitis-systemic ellagic acid. * Statistically significant difference from Group 1 (p<0.001). † Statistically significant difference from Group 2 (p<0.001). ‡ Statistically significant difference from Group 3 (p<0.01). J Appl Oral Sci. 5/12 2021;29:e20210160 The effect of ellagic acid on the repair process of periodontal defects related to experimental periodontitis in rats positive correlation was observed in levels of MPO, H-score values (p=0.018). Furthermore, TNF-α and 8-OHdG in the gingival tissue, and H-score values IL-10 H-score values were correlated with the alveolar of IL-6, TNF-α (p<0.001). Also, we found a positive bone level, alveolar bone area, and attachment level correlation between gingival GSH levels and IL-10 (p<0.001). In contrast, we found significant negative CEJ: Cemento-enamel junction, CTA: Connective tissue attachment, AC: Alveolar crest Figure 1- (A) Sections from the mesiodistal aspects throughout the mandibular first molars in the control group (Group 1) (H&E, 4×), (B) Sections from the mesiodistal aspects throughout the mandibular first molars in Group 2 (H&E, 4×), (C) Sections from the mesiodistal aspects throughout the mandibular first molars in Group 3 (H&E, 4×), (D) Sections from the mesiodistal aspects throughout the mandibular first molars in Group 4 (H&E, 4×) Data are expressed as the mean ± standard deviation. p<0.05 was considered statistically significant. Group 1: control-systemic saline, Group 2: control-systemic ellagic acid, Group 3: experimental periodontitis-systemic saline, Group 4: experimental periodontitis-systemic ellagic acid. * Statistically significant difference from Group 1 (p<0.01). † Statistically significant difference from Group 2 (p<0.001) ‡ Statistically significant difference from Group 3 (p<0.001) Figure 2- HSCORE Values of IL-10, IL-6 and TNF-α Immunoreactivity in the periodontal ligament for all groups (A) TNF-α immunoreactivity; (B) IL-6 immunoreactivity; (C) IL-10 immunoreactivity J Appl Oral Sci. 6/12 2021;29:e20210160 ÖNGÖZ DEDE F, BOZKURT DOĞAN Ş, BALLI U, DURMUŞLAR MC, AVCI B, GÜLLE K, AKPOLAT FERAH M Group 1: control-systemic saline, Group 2: control-systemic ellagic acid, Group 3: experimental periodontitis-systemic saline, Group 4: experimental periodontitis-systemic ellagic acid. Figure 3- TNF-α, IL-6, and IL-10 immunoreactivity in the periodontal tissues of the rats for all groups. A) Group 1; B) Group 2; C) Group 3; D) Group 4 Data are expressed as the mean ± standard deviation. p<0.05 was considered statistically significant. Group 1: control-systemic saline, Group 2: control-systemic ellagic acid, Group 3: experimental periodontitis-systemic saline, Group 4: experimental periodontitis-systemic ellagic acid. * Statistically significant difference from Group 1 (p<0.05). † Statistically significant difference from Group 2 (p<0.05). ‡ Statistically significant difference from Group 3 (p<0.05). Figure 4- Levels of GSH, MPO, and 8-OHdG in rat gingival tissue correlations between the TNF-α and IL-10 H-score Discussion values (p<0.001). Over the past decade, polyphenols have received increasing attention from researchers concerning human health, especially regarding dental caries J Appl Oral Sci. 7/12 2021;29:e20210160 The effect of ellagic acid on the repair process of periodontal defects related to experimental periodontitis in rats Data are expressed as the mean ± standard deviation. p<0.05 was considered statistically significant. Group 1: control-systemic saline, Group 2: control-systemic ellagic acid, Group 3: experimental periodontitis-systemic saline, Group 4: experimental periodontitis-systemic ellagic acid. * Statistically significant difference from Group 1 (p<0.05). † Statistically significant difference from Group 2 (p<0.05). ‡ Statistically significant difference from Group 3 (p<0.05). Figure 5- Levels of GSH, MPO, and 8-OHdG in rat serum Table 2- The Spearman's rank correlation (r) among groups regarding MPO, 8-OHdG, GSH levels, H-Score values of TNF-α, IL-6, IL-10 and alveolar bone loss, alveolar bone area, and attachment level in all groups G_MPO G_8OHdG G_GSH IL-10 IL-6 TNF-α ABA AL ABL G_MPO r .511** -.155 -.285 .756** .453** -.269 .302 .265 p .001 .339 .075 .000 .003 .093 .058 .099 G_8OHdG r .511** -.268 -.039 .411** .283 -.051 -.048 -.032 p .001 .094 .810 .008 .077 .753 .768 .846 G_GSH r -.155 -.268 .372* -.139 -.246 .137 -.227 -.213 p .339 .094 .018 .392 .125 .399 .160 .187 IL-10 r -.285 -.039 .372* -.270 -.649** .784** -.825** -.765** p .075 .810 .018 .093 .000 .000 .000 .000 IL-6 r .756** .411** -.139 -.270 .514** -.140 .292 .243 p .000 .008 .392 .093 .001 .389 .068 .131 TNF-α r .453** .283 -.246 -.649** .514** -.605** .601** .549** p .003 .077 .125 .000 .001 .000 .000 .000 ABA r -.269 -.051 .137 .784** -.140 -.605** -.693** -.688** p .093 .753 .399 .000 .389 .000 .000 .000 AL r .302 -.048 -.227 -.825** .292 .601** -.693** .851** p .058 .768 .160 .000 .068 .000 .000 .000 ABL r .265 -.032 -.213 -.765** .243 .549** -.688** .851** p .099 .846 .187 .000 .131 .000 .000 .000 ** Correlation is significant at the 0.01 level. *Correlation is significant at the 0.05 level. G, gingival tissue; MPO, myeloperoxidase; 8-OHdG, 8-Hydroxydeoxyguanosine; IL, interleukin; TNF-α, Tumor necrosis factor-alpha; ABA, alveolar bone area; AL, attachment level; ABL, alveolar bone level. and periodontal disease.3,10 The protective effects of best knowledge, our study is the first to show that polyphenols have been investigated in many animal EA may provide curative effects on the periodontal studies4,7–9, however, few studies focusing on the tissues by reducing gingival oxidative stress and pro- therapeutic effects of polyphenols in periodontal inflammatory markers, and alveolar bone loss in the lesions are available.1 In our study, we focused on periodontal repair process after EP in rats. the antioxidative and inflammatory effects of EA on In a previous study, daily oral administration of 7.5 periodontal repair related to experimentally induced mg/kg and 15 mg/kg EA effectively reversed abnormal periodontitis in rats and investigated their effects on changes in biochemical and hematological parameters the alveolar bone loss after EA administration. To the in isoproterenol (ISO)-treated rats, returning them to J Appl Oral Sci. 8/12 2021;29:e20210160 ÖNGÖZ DEDE F, BOZKURT DOĞAN Ş, BALLI U, DURMUŞLAR MC, AVCI B, GÜLLE K, AKPOLAT FERAH M near-normal levels.26 The study showed that the higher levels depending on the dose.6 Moreover, Favarin, et dose of EA (15 mg/kg) was more effective in restoring al.27 (2013) stated that EA decreased IL-6 levels and biochemical parameters to normal than the lower dose elevated IL-10 levels in bronchoalveolar lavage fluid. (7.5 mg/kg).26 In another study of Favarin, et al.11 Umesalma and Sudhandiran32 (2010) observed that (2013), they evaluated the anti-inflammatory effect EA regulates the inflammatory process by reducing of EA on acute lung injury and reported that 10mg\kg the production of inflammatory cytokines through EA application had positive results on inflammatory inhibition of NF-kB expression.32 Additionally, Usta, cytokine levels. Since an effective dose has not yet et al.33 (2013) suggested that the anti-inflammatory been investigated in the treatment of periodontal effect of EA may occur due to the suppression of diseases, in our study, we applied a 15 mg/kg dose cyclooxygenase (COX) protein activation. Granica, et of EA,14,26,27 reporting both its anti-inflammatory and al.34 (2016) stated that Geum urbanum L. roots, which anti-oxidant effects. is the rich source of EA derivatives, have a beneficial In our study, we induced experimental periodontitis effect on gingival inflammation because it decreases for 14 days. Observational periods of 15 days or TNF-α levels due to its effect on neutrophils and can fewer are recommended in the EP model because be used in inflammation of mucositis, gingivitis, and rats have a large capacity to adapt to inflammatory periodontitis. Therefore, consistent with previous stimulation.2,21 In our study, clinical symptoms such as study findings,6,11,30–32 our results indicate that edema, redness, bleeding, and mobility in the teeth anti-inflammatory activity of EA in periodontium is were observed in the gingival tissues of the rats due evidenced by reducing proinflammatory cytokines to EP on the 14th day following the ligature placement. and increasing anti-inflammatory cytokine activities Moreover, after the rats were euthanized on the 28th in gingival tissue. day of study, the histomorphometric analysis showed Several studies have proved that periodontal tissue that periodontitis was successfully induced in the loss is associated with increased reactive oxygen experimental groups over the experimental 14-day species and decreased antioxidant levels.35–37 Akman, period. These outcomes are in accordance with several et al.38 (2013) stated that MPO is the marker most previous studies.2,21 Herein, histologic assessments commonly used for determining increased oxidative also showed greater degrees of alveolar bone stress in periodontal diseases. Moreover, various resorption and periodontal tissue destruction in the studies found that rats with experimental periodontitis rats of group 3 when compared with those of control had high MPO activities in the gingival tissue compared groups. Nevertheless, our study demonstrated that to the control group.28,29,38,39 In accordance with these rats treated with EA provided significant attachment studies,28,29,38,39 our results showed that gingival and alveolar bone level gain. However, no previous tissue and serum MPO levels were higher in group studies have looked at the effects of EA on periodontal 3 compared to the control groups. These results tissue destruction. The results of our study indicate indicate that MPO activity measurement may be a that ellagic acid may reduce alveolar bone loss during useful marker for periodontitis. On the other hand, periodontal repair after EP in the rat model. we determined that 8-OHdG levels, which are the Similar to previous studies,28,29 immunohistochemical markers of oxidative DNA damage, were higher in results of our study showed that group 3 had the the gingival tissue and serum in group 3 compared lowest IL-10 level and highest IL-6 and TNF-α levels to the control groups, although it was not statistically in gingival tissues compared to the control groups. significant. Previous experimental studies have shown According to our findings, rats treated with EA (15 that 8-OHdG levels are positively correlated with mg/kg) had significantly reduced IL-6 and TNF-α periodontal tissue loss.40,41 The difference of our study levels and increased IL-10 levels in gingival tissues. may be related to the treatment with saline after A previous review reported that ellagic acid reduced ligature removal, improving the periodontal lesion TNF-α and IL-6 levels in vivo and in vitro.30 A study and some reducing 8-OHdG levels. The findings of has reported that TNF-α levels, which increase with the this study showed that, although no difference was development of colonic inflammation, decreased after observed in the control group treated with EA in the administration of EA.31 Another study observed that gingival tissue and serum MPO and 8-OHdG levels, EA efficiently suppressed LPS-induced increased IL-6 these levels decreased in group 4 treated with EA J Appl Oral Sci. 9/12 2021;29:e20210160 The effect of ellagic acid on the repair process of periodontal defects related to experimental periodontitis in rats when compared with the control groups. Moreover, in the periodontal repair process after experimental analysis between experimental groups showed that periodontitis within the limits of the study. These EA significantly increased serum GSH levels. Many effects could be attributed to the antioxidative and experimental studies have shown that EA significantly anti-inflammatory nature of ellagic acid. Thus, EA reduced the levels of MPO and increased the levels may have therapeutic potential on periodontitis. of GSH.13,42 Tomofuji, et al.43 (2009) stated that the However, studies should further explore and show the production of ROS in the periodontal inflammation therapeutic mechanism of ellagic acid in periodontal gradually reduced after ligature removal. Consistent inflammation and to examine its clinical effectiveness with the study of Tomofuji, et al.43 (2009), our study in human periodontal diseases, thus eliminating the indicated that EA administered systemically for 14 limitations. days had sufficient effect on the serum of experimental periodontitis rats but could not increase GSH levels in Conflict of interest and source of funding the gingival tissue adequately. Based on the results of The authors declare that they have no financial this study, we deduce that EA may improve gingival relationships related to any products involved in this oxidative stress and increase systemic antioxidant study. The study was self-funded by the authors. levels. However, there is no study in the literature to which we could compare our results. Authors’ contributions However, our study has some limitations. The first Öngöz Dede, Figen: Conceptualization (Lead); limitation was the effect of EA on bone resorption Data curation (Equal); Formal analysis (Supporting); based on the evaluation of biomarker levels and that Funding acquisition (Equal); Investigation (Equal); the exact mechanism was not fully demonstrated. Methodology (Equal); Project administration (Lead); Second, ellagic acid levels were not evaluated in body Resources (Equal); Software (Equal); Supervision fluids, so we could not determine how much systemic (Equal); Validation (Lead); Visualization (Equal); administered ellagic acid reached the gingival tissue. Writing-original draft (Lead); Writing-review & editing Third, our study is not a dose-dependent experimental (Lead). Dogan, Seyma Bozkurt: Conceptualization study. Therefore, the effective and non-toxic dose (Supporting); Data curation (Equal); Formal analysis of EA can be determined in periodontal tissues by (Equal); Funding acquisition (Equal); Investigation experimental studies with different doses. Fourth, (Equal); Methodology (Equal); Project administration our study did not evaluate the effect of EA application (Supporting); Resources (Equal); Software (Equal); on microbial dental biofilm in the periodontium. Supervision (Equal); Validation (Equal); Visualization However, we reported that EA had anti-biofilm (Equal); Writing-original draft (Supporting); Writing- activity against bacterial pathogens.7 In addition to review & editing (Supporting). Balli, Umut: systemic administration, if EA was applied locally, Conceptualization (Supporting); Data curation (Equal); EA’s anti-biofilm activity would be observed better Formal analysis (Equal); Funding acquisition (Equal); in periodontal tissues. On the other hand, our study Investigation (Equal); Methodology (Equal); Project suggests that EA healed gingival oxidative stress and administration (Supporting); Resources (Equal); periodontal inflammation unconnected to the biofilm Software (Equal); Supervision (Equal); Validation activity. Additionally, the experimental periodontitis (Equal); Visualization (Equal); Writing-original draft model is not directly equivalent to chronic disease in (Supporting); Writing-review & editing (Supporting). humans. Future studies should further evaluate how Durmuşlar, Cenk: Formal analysis (Equal); Funding EA administration affects gingival oxidative stress and acquisition (Equal); Project administration (Equal); periodontal inflammation in periodontitis patients. Resources (Equal); Validation (Equal); Visualization (Equal).Avci, Bahattin: Data curation (Equal); Formal analysis (Equal); Project administration Conclusions (Equal); Writing-original draft (Equal). Gülle, Kanat: Conceptualization (Equal); Data curation (Equal); Formal analysis (Equal); Methodology (Equal); Project Our study showed that EA provides significant administration (Equal). Akpolat Ferah, Meryem: improvements on gingival oxidative stress and Conceptualization (Equal); Data curation (Equal); inflammatory markers and alveolar bone resorption Formal analysis (Equal); Methodology (Equal). J Appl Oral Sci. 10/12 2021;29:e20210160 ÖNGÖZ DEDE F, BOZKURT DOĞAN Ş, BALLI U, DURMUŞLAR MC, AVCI B, GÜLLE K, AKPOLAT FERAH M References 18- Kilkenny C, Browne W, Cuthill IC, Emerson M, Altman DG. Animal research: Reporting in vivo experiments: the ARRIVE guidelines. Br J Pharmacol. 2010;160(7):1577-9. doi: 10.1111/j.1476- 1- Thangavelu A, Elavarasu S, Sundaram R, Kumar T, Rajendran 5381.2010.00872.x D, Prem F. Ancient seed for modern cure - Pomegranate review 19- Kannan MM, Quine SD, Sangeetha T. Protective efficacy of ellagic of therapeutic applications in periodontics. J Pharm Bioallied Sci. acid on glycoproteins, hematological parameters, biochemical changes, 2017;9(Suppl 1):S11-S14. doi: 10.4103/jpbs.JPBS_101_17 and electrolytes in myocardial infarcted rats. 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