Influence of dealcoholized red wine on the healing of bone with induced apical periodontitis in rats

Influência do vinho tinto desalcoolizado na cicatrização de osso com periodontite apical induzida em ratos

Abstract

Apical periodontitis (AP) is an inflammatory disease influenced by local and systemic factors. Nutritional compounds can modulate the inflammation, helping the healing process. The aim of this study was to evaluate the local and systemic effects of dealcoholized red wine during the repair of alveolar bone with induced AP. Thirty-five male Wistar rats with AP that had their teeth extracted were arranged into five groups according to the supplementation: no supplementation (NC); red wine (RW); dealcoholized red wine (DRW); and alcohol (AL). The extraction of healthy teeth from rats with no supplementation served as control (C). The experimental protocol lasted 75 days with daily supplementation. At the 15th day, AP was induced in the four first molars, which were extracted 30 days later. After 30 days of alveolar bone healing, the animals were euthanized, blood and mandible samples were collected for hematological, ELISA, and histological analysis. Statistical analysis was performed at a significance of 5%. DRW, C, and ALC exhibited red blood cells count within the reference threshold while RW was above it (p<0.05). Hemoglobin in DRW and C were within the reference, but not in NC, RW, and AL (p<0.05). Hematocrit values were similar and remained within normal ranges for all groups. Platelet counts were elevated in all groups compared to C (p<0.05) but within reference. Medium platelet volume was lower in DRW, NC, and AL than in C (p<0.05) but within reference. No difference was found in MCV, MCHC, RDW, or leukocyte subsets (p>0.05). Alcohol supplementation increased serum IL-1β levels (p<0.05). In contrast, serum TNF-α levels were reduced in DRW and RW (p<0.05). No difference in IL-17A levels were observed (p>0.05). Bone repair was more advanced in C than in NC (p<0.05). More trabecular bone formation in DRW, C and RW (p<0.05). AL showed the highest inflammatory infiltration score, affecting both connective and bone tissue, whereas C and DRW had the lowest levels (p<0.05). Epithelial and connective tissue s scores were similar between C and DRW but significantly lower than NC, RW, and AL (p<0.05). It was concluded that DRW supplementation had a positive effect on the modulation of the inflammatory response and on the repair of bone tissue in the dental alveoli of teeth with AP. Supplementation with RW and AL resulted in changes in blood parameters, indicating a negative influence of alcohol.

Keywords:
apical periodontitis; tooth extraction; wine; alcoholism; bone remodeling

Resumo

A periodontite apical (PA) é uma doença inflamatória influenciada por fatores locais e sistêmicos. Compostos nutricionais podem modular a inflamação, auxiliando no processo de cicatrização. O objetivo deste estudo foi avaliar os efeitos locais e sistêmicos do vinho tinto desalcoolizado durante o reparo ósseo alveolar com PA induzida. Trinta e cinco ratos Wistar machos com PA que tiveram seus dentes extraídos foram divididos em cinco grupos de acordo com a suplementação: sem suplementação (NC); vinho tinto (RW); vinho tinto desalcoolizado (DRW); e álcool (AL). A extração de dentes saudáveis de ratos sem suplementação serviu como controle (C). O protocolo experimental teve duração de 75 dias com suplementação diária. No 15º dia, a PA foi induzida nos quatro primeiros molares, que foram extraídos 30 dias depois. Após 30 dias de cicatrização óssea alveolar, os animais foram eutanasiados, e amostras de sangue e mandíbula foram coletadas para análises hematológicas, de ELISA e histológicas. A análise estatística foi realizada com nível de significância de 5%. DRW, C e ALC apresentaram contagem de hemácias dentro do limite de referência, enquanto RW estava acima dele (p < 0,05). A hemoglobina em DRW e C estava dentro da referência, mas não em NC, RW e AL (p < 0,05). Os valores de hematócrito foram semelhantes e permaneceram dentro dos limites normais para todos os grupos. As contagens de plaquetas estavam elevadas em todos os grupos em comparação com C (p < 0,05), mas dentro da referência. O volume plaquetário médio foi menor em DRW, NC e AL do que em C (p < 0,05), mas dentro da referência. Nenhuma diferença foi encontrada nos subconjuntos de MCV, MCHC, RDW ou leucócitos (p> 0,05). A suplementação de álcool aumentou os níveis séricos de IL-1β (p < 0,05). Em contraste, os níveis séricos de TNF-α foram reduzidos em DRW e RW (p < 0,05). Nenhuma diferença nos níveis de IL-17A foi observada (p> 0,05). O reparo ósseo foi mais avançado em C do que em NC (p < 0,05). Maior formação óssea trabecular em DRW, C e RW (p < 0,05). AL apresentou a maior pontuação de infiltração inflamatória, afetando tanto o tecido conjuntivo quanto o ósseo, enquanto C e DRW apresentaram os níveis mais baixos (p < 0,05). As pontuações do tecido epitelial e conjuntivo foram semelhantes entre C e DRW, mas significativamente menores do que NC, RW e AL (p < 0,05). Concluiu-se que a suplementação de DRW teve um efeito positivo na modulação da resposta inflamatória e no reparo do tecido ósseo nos alvéolos dentais de dentes com PA. A suplementação com RW e AL resultou em alterações nos parâmetros sanguíneos, indicando uma influência negativa do álcool.

Palavras-chave:
periodontite apical; extração dentária; vinho; alcoolismo; remodelação óssea

1. Introduction

Apical periodontitis (AP) is an inflammatory disease caused by microbial infection affecting the dental pulp (Park et al., 2020). In response to the microbial infection, the host immune system leads an inflammatory process with bone resorption in the apical region (Nair, 2004; Subramanian and Mickel, 2009; Cintra et al., 2018). Inflammation disrupts bone turnover, increasing resorption and reducing bone formation (Torres-Monjarás et al., 2023). The severity of inflammation and the extent of bone resorption are modulated by local and systemic factors, including cytokines and hormones (Veiga Vasques et al., 2024; Holland et al., 2017; Nair, 2004; Kajiya et al., 2010).

The presence of AP foci also elevates systemic levels of pro-inflammatory mediators (Cintra et al., 2016), increases total leukocyte counts (including lymphocytes, monocytes, and eosinophils), and indicates chronic inflammation characterized by mononuclear cell predominance and reduced neutrophil levels (Azuma et al., 2021). Evidence suggests that AP promotes increased inflammatory cells and mediators (Cintra et al., 2014a, b).

Once AP is established, root canal treatment or surgical procedures, including tooth extraction, are employed to eliminate the infection (Karamifar et al., 2020). However, endodontic treatment is not always effective, as bacteria may persist within the complex root canal system (Ricucci and Siqueira Junior, 2008). Tooth extraction involves an active process of inflammation resolution, driven by a cascade of anti-inflammatory mediators aimed at clearing inflammatory cells from the site but not a mere cessation of pro-inflammatory pathways (Georgiou et al., 2020). Inflammation modulation, bone resorption downregulation, osteoclastogenesis, and osteogenesis occur to give place for the repair phase to restore homeostasis (Ricucci et al., 2014; Paula-Silva et al., 2021).

The treatment outcome of endodontic infections can be influenced by general health and nutrition once some substances that contribute for general health have also positive effects on apical periodontitis, such as omega-3 fatty acids, curcumin, lactobacilli, and red wine with its phenolic compounds (Holland et al., 2017; Azuma et al., 2021; Cosme-Silva et al., 2021; Dal-Fabbro et al., 2021; Justo et al., 2022).

Red wine is associated with various health benefits, including the prevention and management of chronic diseases, cardiovascular conditions, metabolic syndrome, and cancer (Pavlidou et al., 2018). It contains polyphenols that are believed to underpin its health-promoting properties (Haseeb et al., 2017; Snopek et al., 2018). However, excessive RW consumption can be detrimental to health once excessive alcohol intake can exacerbate inflammation and induce immunosuppression (Rotondo et al., 2001; Walzem, 2008; Dal-Fabbro et al., 2019).

To mitigate the adverse effects of alcohol while preserving red wine health benefits, dealcoholization methods have been developed to produce a dealcoholized red wine (Belisário-Sánchez et al., 2009; Pham et al., 2019). Administration of dealcoholized red wine significantly reduced inflammation in apical periodontitis as prophylactic supplementation or even when it is already established (Ricci et al., 2025; Oliveira Sales-Junior et al., 2025). However, it was not yet evaluated during the repair process of the alveolar bone with AP.

Thus, this study aimed to evaluate the local and systemic effects of dealcoholized red wine supplementation during the repair of alveolar bone with induced AP.

2. Material and Methods

2.1. Ethical approval

The in vivo experimental procedures proposed in this study were submitted to and approved by the Ethics Committee on Animal Use (CEUA – FOA/UNESP) (Protocol 0221-2022) of São Paulo State University, São Paulo, Brazil. The study strictly adhered to the ARRIVE guidelines and followed the national regulations for the ethical use and care of laboratory animals.

2.2. Animals and housing conditions

A total of 35 male rats (Rattus albinus, Wistar), weighing approximately 350 g, were used. The animals were obtained from the vivarium of the School of Dentistry, Araçatuba – UNESP. They were housed in isolators (Alesco, Monte Mor, São Paulo, Brazil) under controlled temperature conditions (22–24°C) and a 12-hour light/dark cycle. Throughout the experimental period, the animals were fed a solid diet and had access to water ad libitum, except during the first 24 hours following the intervention.

2.3. Anesthesia and postoperative care

For surgical procedures, anesthesia was administered intramuscularly using a 2% xylazine-based sedative (Dopasere, Calier S.A., Barcelona, Spain; 10 mg/kg) and a 10% ketamine hydrochloride anesthetic (Vetanarcol, Konig S.A., Avellaneda, Argentina; 80 mg/kg).

Following the induction of periapical lesions and tooth extraction, the analgesic dipyrone (Medley® Farmacêutica Ltda., Campinas, Brazil; 150 mg/kg) was administered intravenously every 12 hours for 3 days.

For euthanasia, sodium thiopental (Thiopentax® Cristália – Produtos Químicos Farmacêuticos Ltda., Itapira, SP, Brazil; 150 mg/kg) and lidocaine hydrochloride (20 mg/mL; Novafarma Indústria Farmacêutica Ltda., Anápolis, GO, Brazil; 10 mg/kg) were administered intraperitoneally.

2.4. Sample size determination

The sample size was calculated using prior studies with comparable methodologies, applying a 5% alpha error and 95% statistical power. This calculation yielded a minimum of six animals per group (Cintra et al., 2014a; Cosme-Silva et al., 2021; Dal-Fabbro et al., 2021). To account for potential complications associated with the extended experimental duration, one additional animal was included per group, resulting in a total of seven animals per group.

2.5. Experimental groups

The animals were randomly arranged into four groups:

• · Control (C): Rats subjected to tooth extraction of healthy teeth.

• · Negative Control (NC): Rats subjected to tooth extraction of teeth with induced apical periodontitis (AP).

• · Red Wine (RW): Rats supplemented with RW and subjected to tooth extraction of teeth with induced AP.

• · Alcohol (AL): Rats supplemented with AL and subjected to tooth extraction of teeth with induced AP.

• · Dealcoholized Red Wine (DRW): Rats supplemented with DRW and subjected to tooth extraction of teeth with induced AP.

2.6. Administration of supplementation diet

The solutions were administered therapeutically via gavage, beginning 15 days before the induction of periapical lesions, continuing for 30 days during lesion development, and extending for an additional 30 days following tooth extraction, totaling 75 days (Figure 1).

Figure 1
Experimental timeline.

Both the dealcoholized red wine (DRW) and red wine (RW) used in this study (Vinoh, Bento Gonçalves, RS, Brazil) were produced from Merlot grapes. Merlot and Cabernet Sauvignon grapes are known for their high polyphenol concentrations (Pervaiz and Holme, 2009). For the alcohol group (AL), a solution of water with 12.5% alcohol was administered, matching the alcohol content in the same volume as the RW group.

The administration was performed daily in the morning, with a volume of 4.28 mL/kg of body weight for all groups, as established in previous studies (Schmatz et al., 2013; Dal Fabbro et al., 2021). This dosage corresponds to a daily human consumption of approximately 300 mL (two glasses) of DRW for a 70 kg individual.

In the control(C) and negative control (NC) groups, sterilized potable water was administered via gavage in the same volume as DRW to simulate the stress experienced by the other animals. For all groups, water and solid feed (Labina Purina®, Paulínia, Brazil) were freely available throughout the experiment.

2.7. Animal weight variation

The animals were weighed at the beginning and end of the experiment (75 days). The weight change was obtained as a percentage by calculating the difference between the final weight and the initial weight.

2.8. Induction of periapical lesions

The induction of periapical lesions was performed under anesthesia, as described previously. Coronary access was made to the first upper and lower molars on both the right and left sides of the C, RW, AL, and DRW groups (4 teeth per animal) (Cintra et al., 2016).

The procedure was conducted using a carbon steel drill (Ln Long Neck Bur - Maillefer, Dentsply) with a 0.05 mm diameter, ensuring standardized pulp exposures with a uniform diameter of 0.05 mm.

2.9. Tooth extraction

As previously described, tooth extractions were performed on all animals from all groups under sedation. Thirty days after the induction of periapical lesions, the animals were positioned on the surgical table. Oral antisepsis was conducted using 10% povidone-iodine (PVPI 10%, Riodene Degermante, Rioquímica, São José do Rio Preto, Brazil).

The procedure involved detaching the soft tissues adjacent to the tooth (syndesmotomy), luxating the tooth, and extracting the first upper and lower molars on both the right and left sides. Specialized surgical dental instruments adapted for animal use were employed for the procedure (Okamoto and Russo, 1973).

2.10. Blood analysis

At the end of the experimental period, on the day of euthanasia, the animals were anesthetized again using the same protocol described previously. Cardiac puncture was performed to collect 5 mL of blood from each animal.

The samples were placed in EDTA tubes and homogenized for processing. The following hematological parameters were analyzed using an automated analyzer (ABX Micros ABC et; Horiba ABX Diagnostics, Montpellier, France): leukocytes, platelets, red blood cell count (RBC), hemoglobin, hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width-coefficient of variation (RDW-CV), neuthophils, lymphocytes, monocytes and eosinophils (Azuma et al., 2021). The reference values come from previous studies establishing standard values for Wistar rats (Patel et al., 2024; He et al., 2017).

2.11. Plasma analysis for interleukin IL-1β, IL-17A and tumor necrosis factor (TNF)-α levels by ELISA

The cytokines and proteins levels were assessed by ELISA using the MILLIPLEX® Rat Myokine Magnetic Bead Panel (RMYOMAG-88K, Merck Life Science, LLC, Darmstadt, Germany) and MILLIPLEX® Rat Cytokine/Chemokine Magnetic Bead Panel - Immunology Multiplex Assay (RECYTMAG-65K, Merck Life Science, LLC, Darmstadt, Germany) according to the manufacturer's instructions. Overnight incubation at 4°C and a handheld magnet were used. The plates were analyzed on a Luminex MAGPIX System (Luminex Corporation, Austin, TX, USA), and the data were generated with xPONENT 4.3 software. Luminex uses proprietary techniques to encode microspheres with two fluorescent dyes internally. Using the concentration of these dyes, sets of microspheres coated with capture antibodies were created. After capturing the analyte sample by the microsphere, a biotinylated detection antibody was added. The reaction mixture was then incubated with streptavidin-PE conjugate to complete the reaction on the surface of each microsphere. Each microsphere was identified, and the result of its bioassay was quantified based on reporter fluorescent signals. The results were presented in picograms per milliliter (pg/mL) (Vasyukova et al., 2023).

2.12. Histological analysis

At the conclusion of the experimental period (75 days), the animals were anesthetized as per the previously described protocol and euthanized using 150 mg/kg of thiopental. The left mandible of each animal was collected (Cintra et al., 2016).

The left mandibles were fixed in 4% neutral-buffered formalin for 24 hours and subsequently washed in running water for 12 hours. Following fixation, the mandibles were decalcified in 10% EDTA solution, washed in running water, dehydrated in ethanol, cleared in xylene, and embedded in paraffin. Semi-serial sections of 5 µm thickness were obtained using a microtome (Leica - RM 2045).

For histological staining, sections were deparaffinized in xylene, rehydrated through a graded ethanol series, and washed in running water. For hematoxylin and eosin (H&E) staining, the sections were immersed in Harris hematoxylin solution, washed in running water, stained in eosin solution, and washed again. Sections were then dehydrated in a graded ethanol series, cleared in xylene, and mounted on slides with Permount.

Descriptive and qualitative analyses were performed. Slides containing representative sections of each specimen were evaluated under light microscopy. The descriptive analysis involved detailing histopathological phenomena to globally characterize them based on experimental variables (Gomes-Filho et al., 2015).

The qualitative analysis used a scoring system to grade the magnitude of histopathological phenomena. This histological evaluation of the mandibles served to characterize the inflammatory profile of the alveoli undergoing repair (Gomes-Filho et al., 2015; Ervolino et al., 2019). The left lower first molar alveolus was selected as the region of interest (ROI) for this analysis. The histological evaluation was based on the method described by Statkievicz et al. (2018). The following parameters were assessed in the ROI according to the scores ranging from 1 to 4, being 1 the best one:

1. Intensity of local inflammatory response.

2. Extent of the inflammatory process.

3. Cellular and structural pattern of epithelial tissue.

4. Cellular and structural pattern of connective tissue.

5. Cellular and structural pattern of bone and related tissues.

2.13. Statistical analysis

The statistical analysis was performed using the SigmaPlot 14.0™ software (Chicago, IL, USA), with a significance level of 5% (p<0.05) applied to all comparisons. Initially, the data were subjected to the Shapiro-Wilk test to assess normal distribution.

For data that did not follow a normal distribution, the Kruskal-Wallis test was applied for comparative analyses among groups.

The hematological parameters with a normal distribution, analysis of variance (ANOVA) was performed, followed by Tukey's test for multiple comparisons. In cases of non-parametric data, the Dunn test was used.

For the histological analysis, qualitative scores were assigned to observed phenomena. When significant differences were detected, the groups were compared using the Student-Newman-Keuls multiple comparisons test, ensuring the identification of specific differences between experimental groups.

3. Results

3.1. General health condition and control of weight variations in the animals

During the experiment, the general health status of the animals remained adequate. During the experiment, all groups had the expected weight gain and there was no statistically significant difference in the comparison among the groups (Table 1) (p>0.05).

Table 1
Weight of animals in each group (mean and standard deviation) at the beginning and end of the experiment and percentage variation.

Group	Initial (g)	Final (g)	Difference (%)
C	315.5±15.10a	564.0±21.72a	78.9±7.05a
NC	319.7±22.53a	559.0±54.90a	74.8±12.98a
RW	314.7±21.84a	529.5±39.13a	68.2±6.28a
AL	312.7±25.23a	547.9±43.45a	75.5±10.25a
DRW	309.2±14.88a	533.4±32.37a	73.4±9.68a

• Mean ± standard deviation obtained in the ANOVA test and the different lowercase letters in the same row show the statistical differences found in the Tukey test (p < 0.05) among the groups.

3.2. Histological analysis

The results of the histological analysis are presented in Table 2, while representative images of hematoxylin-eosin staining for each group can be observed in Figure 2. Histological confirmation of dental extractions was evidenced by the absence of dental elements and the periodontal ligament, which were replaced by bone tissue. Additionally, differences in the repair process were observed between healthy alveoli (C) and those previously affected by apical periodontitis (NC), once C had a more advanced healing process than NC (p<0.05).

Table 2
Histological Parameters, Scores, and Specimen Distribution Based on the Pattern of Inflammatory Infiltration and Bone Tissue After Tooth Extraction.

Histopathological Analysis of the Tooth Extraction Site							p value
Parameters and respective scores		Experimental Groups (n=7)
		C	NC	RW	AL	DRW
Intensity of local inflammatory response	1 - Absence of inflammation (presence of rare inflammatory cells)	1	0	0	0	2	p<0.05
	2 - Small quantity of inflammatory cells (< 1/3 of cells are inflammatory cells)	8	0	2	0	5
	3 - Moderate quantity of inflammatory cells (from 1/3–2/3 of cells are inflammatory cells)	0	4	5	2	0
	4 - Large quantity of inflammatory cells (over 2/3 of cells are inflammatory cells).	0	3	0	5	0
	Median	2c	3a	3b	4a	2c
Inflammation extension	1 - Absence of inflammation	1	0	0	0	2	p<0.05
	2 - Partial extension of connective tissue	6	0	3	0	5
	3 - Entire extension of connective tissue, without reaching bone tissue	0	6	4	3	0
	4 - Entire extension of connective tissue and bone tissue;	0	1	0	4	0
	Median	2c	3a	3b	4a	2c
Cellular pattern and epithelial tissue structure	1 - Epithelial tissue with moderate thickness (larger than half the surgical wound epithelium border thickness) and completely covering extraction site	6	2	3	0	6	p<0.05
	2 - Epithelial tissue with thin thickness (smaller than half the surgical wound epithelium border thickness) and completely covering extraction site	1	5	4	7	1
	3 - Thin layer of epithelial tissue (smaller than half the surgical wound epithelium border thickness) and only in edges of open surgical wound	0	0	0	0	0
	4 - Absence of epithelial tissue and open surgical wound.	0	0	0	0	0
	Median	1b	2a	2a	2a	1b
Cellular pattern and connective tissue structure	1 - moderate quantity of fibroblasts and large quantity of collagen fibers (dense connective tissue);	1	0	0	0	2	p<0.05
	2 - moderate quantity of both fibroblasts and collagen fibers	6	0	3	2	5
	3 - small quantity of both fibroblasts and collagen fibers;	0	7	4	5	0
	4 - severe tissue disruption, with possible areas of tissue necrosis.	0	0	0	0	0
	Median	2b	3a	3a	3a	2b
Cellular pattern and bone tissue structure	1 - presence of bone tissue composed of thick bone trabeculae filling more than half of the dental alveolus;	0	0	0	0	2	p<0.05
	2 - presence of bone tissue composed of thin bone trabeculae filling more than half of the dental alveolus	7	3	4	2	5
	3 - presence of bone tissue composed of thin bone trabeculae filling less than half of the dental alveolus;	0	4	3	5	0
	4 - absence of bone tissue inside the dental alveolus.	0	0	0	0	0
	Median	2b	3a	2a	3a	2b

• The obtained data were subjected to statistical analysis, considering a significance level of 5%, using the Shapiro-Wilk, Kruskal-Wallis, and Student-Newman-Keuls tests. Different lowercase letters in the same row indicate statistical differences.

Figure 2
Histological Analysis. Histological aspects of the tooth extraction site and adjacent area. Photomicrographs showing histological characteristics of connective tissue and bone tissues at the extraction site and adjacent area 30 days postoperatively. Staining: HE. Original magnification: 400×.

Mild inflammatory infiltration, restricted to connective tissue (median = 2) was observed in C and DRW, which was lower than that observed in AL, where intense inflammatory infiltration was present, affecting both connective and bone tissue (median = 4, p<0.05). An intermediate pattern was displayed in NC and RW, with moderate inflammation confined to connective tissue and without bone involvement (median = 3, p<0.05).

The cellular pattern and epithelial response during extraction site healing were similar between C and RW, both with a median of 1 (p>0.05), whereas NC, RW, and AL presented a median of 2, with no differences among them (p>0.05). Regarding connective tissue, C and DRW exhibited similar characteristics, with a median of 2 (p>0.05), showing a moderate presence of fibroblasts and collagen fibers. These values were statistically lower than those observed in NC, RW, and AL, which presented a median of 3 (p<0.05).

Bone tissue analysis revealed that the alveoli in C, RW, and DRW exhibited thick trabecular bone, filling more than half of the dental alveolus, corresponding to a median of 2. This pattern was statistically different from that observed in NC and AL, where bone tissue consisted of thin trabeculae filling less than half of the alveolus (median = 3, p<0.05).

3.3. Blood analysis

The results of the Blood Analysis are presented in Table 3.

Table 3
Mean and standard deviation of blood cell parameters.

Hematologic Parameters	Reference Values	Groups (mean±SD)			AL	DRW
		C	NC	RW
RBC 1012/µL	7.2 – 9.1	7.8±0.4b	8.8±0.3ab	10.0±1.3a	8.6±0.5b	8.5±0.9b
Hemoglobin (g/dL)	12.8 – 15.8	15.0±0.4b	16.2±0.5ab	16.6±0.7a	16.4±1.1a	15.1±0.4b
Hematocrit (%)	40.5 – 49.8	44.7±3.6b	47.2±2.3ab	48.0±2.8ab	48.1±3.1b	42.7±1.2b
VCM (f)	49.2 – 60.6	53.4±3.3a	53.3±1.4a	54.1±2.2a	54.1±2.5a	52.1±1.8a
CHCM (g/dL)	30.5 – 35.3	34.9±0.5a	34.3±0.9a	34.1±1.1a	34.4±0.9a	35.4±0.4a
RDW-CV (%)	13.0 – 18.4	13.4±0.2a	14.3±0.5a	14,1±0.8a	14,3±1.1a	13,8±0.4a
Platelets (103/μL)	412.2–849.2	631.4±116.3b	841.4±74.4a	854.2±87.7a	802.8±67.0a	740.0±150.6a
VPM (fL)	6.9 – 8.0	7.7±0.4a	6.8±0.4b	7.2±0.2b	7.0±0.2b	7.1±0.1b
Neutrophils (%)	17.1 – 47.9	30.8±4.2a	31.1±4.5a	32.1±4.3a	33.1±4.4a	30.5±2.5a
Lymphocytes (%)	48.7 – 78.1	61.8±6.4a	63.0±4.8a	62.0±5.2a	58.7±3.4a	62.1±3.2a
Monocytes (%)	1.0 – 6.5	4.0±2.3a	2.2±1.8a	2.4±1.9a	3.8±2.1a	2.4±1.9a
Eosinophils (%)	0.3 – 4.0	3.2±1.4a	3.5±0.9a	3.2±1.7a	3.5±1.1a	5.2±1.9a

• Mean ± standard deviation obtained in the ANOVA test, and the different lowercase letters in the same row show the statistical differences found in the Tukey test (p < 0.05) between the groups.

All groups exhibited RBC values within the reference range, except for the RW group, which showed an RBC count of 10.0 (10¹²/µL), a value significantly higher than those observed in groups C, DRW, and AL (p<0.05).

Hemoglobin levels were within the recommended standards with no statistically significant difference between C and DRW. In contrast, NC, RW, and AL exhibited elevated hemoglobin levels above the recommended thresholds, without significant differences among them (p>0.05).

The lowest hematocrit values were observed in C (44.7%) and DRW (42.7%), whereas NC (47.2%), RW (48%), and AL (48.1%) showed higher values. However, all groups remained within the normal reference range for hematocrit without significant differences (p>0.05).

Platelet counts were significantly higher in NC, RW, and AL compared to C (p<0.05). Nevertheless, all groups exhibited values below the reference range, except for the RW group, which presented slightly above-normal values.

Although all groups remained within the reference range for MPV, NC, AL, and DRW displayed a significantly lower value compared to C (p< 0.05).

Finally, no statistically significant differences were observed among MCV, MCHC, RDW, neutrophils, lymphocytes, monocytes, or eosinophils (p> 0.05).ELISA assay for IL-1β, IL-17A and TNF-α levels

The results for ELISA can be observed in Figure 3. Alcohol supplementation increased serum IL-1β levels in AL compared to C, NC, RW, and DRW (p<0.05). In contrast, serum TNF-α levels were reduced in DRW and RW compared to NC (p<0.05). No significant difference in IL-17A levels were observed between groups (p>0.05).

Figure 3
Values found in the quantification of IL-1β(A), IL-17A(B) and TNF-α(C) by ELISA. Values are expressed as mean and standard deviation. For IL-1β, ^&statistically significant difference for the C; ^§statistically significant difference for the NC; *statistically significant difference for the RW; ^#statistically significant difference for the DRW. For TNF-α, ^§statistically significant difference for the NC. No significant differences were found in the serum amounts of IL-17A.

4. Discussion

This study presents an innovative approach by investigating, in an experimental rat model, the effect of daily DRW supplementation on the repair of dental alveoli affected by AP. The analysis was conducted both locally and systemically, considering histological and hematological parameters of the extraction sites and blood. The results showed that daily DRW supplementation reduced the local and systemic inflammatory response and improved the quality of alveolar bone tissue in teeth affected by AP. These effects may be associated with the synergistic action of polyphenols present in DRW, which modulate the inflammatory response and promote bone regeneration (Ricci et al., 2025; Oliveira Sales-Junior et al., 2025).

The animal model used is widely accepted in alcohol-related research, as rats exhibit physiological and neurobiological characteristics similar to humans, easily handled, cost-effective, and well-documented in the literature (Malherbe & Messaoudi, 2022; McBride et al., 2014; Ponnappa and Rubin, 2000).

The experimental design followed previous studies (Gomes-Filho et al., 2015; Tobias Duarte et al., 2014), which demonstrated that 30 days are sufficient for the development of periapical bone lesions, ensuring an adequate timeframe for tooth extraction. Moreover, 28 days are enough to observe bone consolidation and repair (Hassumi et al., 2018). Thus, it was established a total experimental period of 75 days, evaluating bone tissue through histological analysis of inflammatory, epithelial, connective, and bone parameters.

The present methodological design simulates the removal of infection and inflammation controlling associated with AP. Tooth extraction allowed a direct assessment of inflammation down-regulation and subsequent alveolar bone repair, as evidenced in the histological analysis of C and NC. Bone repair in AP extraction sites may be significantly delayed compared to the process observed after the extraction of healthy teeth (Ahn and Shin, 2008). In diseased sites, the formation of fibrous scar tissue instead of bone may be an inevitable outcome, even under a rigorous protocol involving careful debridement and healing periods exceeding 12 weeks (Kim et al., 2014). These findings align with a previous study, which also evaluated bone repair patterns in extraction sites with a history of periodontitis and AP in a canine model, demonstrating that AP significantly affects alveolar repair post-extraction, delaying and impairing bone formation and maturation (Kim et al., 2017; Pietrokovski and Massler, 1967). A history of periodontal and endodontic pathology leads to the presence of inflammatory granulation tissue, delayed bone marrow formation, and altered periodontal ligament fiber orientation, all of which affect the healing process (Amler, 1969).

The dose selection for the beverages used in this study was based on recommendations suggesting a daily intake of 300 mL of red wine for a 70 kg human, with adjustments according to the animals' body weight (Kikura et al., 2004; Pavlidou et al., 2018). Red wine supplementation demonstrated a positive effect on reducing inflammation and improving the quality of alveolar bone tissue. Previous studies, also reported that prophylactic red wine supplementation reduced inflammation in rats with induced AP (Dal-Fabbro et al., 2021).

In the present study, red wine supplementation improved the cellular and structural patterns of both epithelial and bone tissue, achieving a score of 2 for both parameters. These findings align with previous study that highlighted the anti-inflammatory activity of red wine in apical periodontitis (Dal-Fabbro et al., 2021). However, for other histological parameters, the performance of the RW was limited, with a score of 3 for local inflammatory response intensity, inflammation extent, cellular pattern, and connective tissue structure, similar to group NC. This result suggests that, despite the beneficial effects of RW, the presence of alcohol may have negatively influenced tissue repair in certain aspects.

The negative effect was even more evident in the results of the AL, which showed the worst performance among the groups even in comparison with NC, indicating compromised recovery and an environment less favorable for tissue regeneration. These findings align with an in vivo study on rats with apical periodontitis, which demonstrated that alcohol consumption reduces cortical bone area, bone formation rates, and bone deposition, especially at concentrations above 8.1% v/v (Hogan et al., 1997), in addition to exacerbating the inflammatory response and bone resorption (de Almeida et al., 2020). Similarly, rats exposed to alcohol at concentrations of 14%, 25%, and 36% showed increased local inflammation and higher bone resorption (de Almeida et al., 2020), while at 20%, an alcoholic diet significantly worsened the severity of apical periodontitis (Dal-Fabbro et al., 2019). Moreover, even at lower concentrations (5% and 10%), no protective effects on the immune response were observed when compared to the control group (Dal-Fabbro et al., 2019), reinforcing that, regardless of the dose, alcohol consumption compromises bone repair and aggravates inflammation.

In contrast to the compromised performance of the AL group, the group supplemented with dealcoholized red wine stood out in the histological analysis, highlighting its potential in alveolar repair. The animals that received the phenolic compound exhibited scores comparable to the repair of alveoli without apical lesion regarding local inflammatory response intensity, inflammation extent, cellular pattern, and the structure of epithelial, connective, and bone tissues. Similar to previous studies that reported significant reduction in the inflammation in apical periodontitis when dealcoholized red wine was used as prophylactic supplementation or even when the lesion was already established (Ricci et al., 2025; Oliveira Sales-Junior et al., 2025).

The hematological results indicate that RW and AL significantly altered some blood parameters compared to the C, with increased hemoglobin and hematocrit levels, but no statistical differences from the NC. These alterations are associated with chronic alcohol consumption, which compromises the production of hematopoietic precursors in the bone marrow, leading to defective erythrocytes in circulation. Additionally, it disrupts iron homeostasis, potentially causing either deficiency or excessive accumulation of this mineral in the body (Ballard, 1997).

This effect was particularly evident in the RW group, which exhibited a significantly higher red blood cell count than the other groups. In alcohol-dependent individuals, gastrointestinal mineral absorption is altered, favoring iron uptake, as observed in RW. Chronic alcohol consumption can thus increase RBC levels, contributing to hemochromatosis, macrocytosis, and hemolysis (Ballard, 1997). This increase may be related to the presence of the 12.5% alcohol component in the red wine, which influences ferritin levels, a protein responsible for iron storage, potentially enhancing its dietary absorption (Charlton et al., 1964; Milman & Pedersen, 2009). Furthermore, the inflammatory process of AP may exert a systemic effect on iron availability, impacting erythropoiesis due to bacterial activity (Ross, 2017).

A significant increase in platelet parameters was also observed in the NC, RW, and AL compared to the C. Excessive alcohol consumption raises leukocyte, platelet, and mean corpuscular volume (MCV) levels, linked to the acute-phase inflammatory response and neutrophilic calprotectin release (Niemelä et al., 2022). This alteration may be related to the effect of alcohol on tissue plasminogen activator, which promotes partial platelet degradation, leading to the circulation of altered-function platelets (Bau et al., 2007). Additionally, polyphenols present in RW and DRW can inhibit platelet-fibrinogen binding, interfering with coagulation (Rabai et al., 2010), which may explain the slightly elevated platelet values in the RW group.

To further investigate the inflammatory response and identify potential biomarkers associated with systemic alterations, an ELISA assay was performed using serum samples from the animals. IL-17A is a pro-inflammatory cytokine involved in the pathogenesis of various inflammatory diseases, as well as in bone formation and remodeling, primarily through the upregulation of osteoclast differentiation factor (Osta et al., 2014; Kotake et al., 1999; Moseley et al., 2003). Although levels remained elevated, no statistically significant differences were observed among groups for IL-17A after four weeks of healing. Aligning with previous studies that demonstrated elevated serum IL-17A level in rats with multiple AP foci, notably, the number of infectious foci (1, 2, or 4) did not significantly influence IL-17A concentrations, suggesting that its systemic expression remains stable regardless of the extent of local infection or supplementation (Cintra et al., 2014b, 2016). Despite these similarities, the present study is the first to assess IL-17A levels during the repair of alveoli of teeth with AP.

In contrast, IL-1β level was significantly lower in DRW and RW than in AL, highlighting the favorable effect of polyphenols as down-regulating but negative of alcohol as up-regulating the inflammatory response. The use of a red wine extract also reduced IL-1β levels of inflamed macrophages in vitro, showing convergence with the findings of the present study (Chalons et al., 2018). Moreover, previous studies showed that alcohol presented higher levels of IL-1β regardless of the alcohol volume used (Pinto et al., 2024; Dal-Fabbro et al., 2019). Frequently detected in high concentrations in persistent periapical periodontitis, IL-1β also plays an essential role in the host response to microbial infection (Yang et al., 2018). IL-1β is one of the primary mediators of bone resorption and periodontal tissue degradation, being primarily secreted by macrophages. Its inflammatory effect involves the induction of proteinases and matrix metalloproteinases, which degrade the extracellular matrix and intensify tissue destruction (Cheng et al., 2020). Additionally, it plays an active role in initiating and regulating the inflammatory response in AP, promoting the increase in IL-6 and prostaglandin E2 levels (Martinho et al., 2012).

Beyond its involvement in AP, IL-1β and TNF-α modulate several systemic conditions by influencing both inflammatory and bone metabolic pathways (Cheng et al., 2020). In the present study, the NC group exhibited the highest TNF-α levels, consistent with its role in sustaining inflammatory activity and impairing bone repair, since this cytokine is mainly produced by macrophages and acts as a key mediator of bone resorption associated with periodontitis (Torres-Monjarás et al., 2023), stimulating the expression of prostaglandins and matrix metalloproteinases (Graves & Cochran, 2003; Almeida-Junior et al., 2023). However, as previously demonstrated by Wang and Stashenko (1993), TNF-α may be present in periapical lesions at insufficient levels to stimulate significant bone resorption, which may explain the absence of an alcohol-induced increase in this cytokine in the present study. Interestingly, the administration of RW and DRW resulted in lower TNF-α levels compared with the NC group, suggesting that polyphenols present in red wine may exert an anti-inflammatory effect capable of downregulating systemic TNF-α expression regardless of the inflammatory status. This interpretation is supported by recent evidence showing that systemic administration of dealcoholized red wine reduced TNF-α, IL-1β, and TRAP immunoreactivity while improving bone volume and decreasing inflammation in established apical periodontitis in rats (Oliveira Sales-Junior et al., 2025), reinforcing the potential of red wine polyphenols to modulate inflammatory pathways and favor bone repair. This modulatory effect may be related to the ability of polyphenols to inhibit NF-κB activation and downstream proinflammatory cytokine expression (Calabriso et al., 2022).

The findings of this study demonstrate that apical periodontitis delays bone repair, affects hematological homeostasis, and alters systemic inflammatory responses. Moreover, alcohol may exacerbate inflammation and interfere with bone regeneration, whereas dealcoholized red wine improved alveolar repair and systemic parameters.

A limitation of the present study is that histomorphometric analysis was performed exclusively on sections stained with Hematoxylin and Eosin (H&E) that although it is widely used for morphological evaluation, it does not allow precise identification of specific inflammatory cell populations, collagen fiber organization, or the differentiation between osteoid and mature bone tissue. Future studies should consider the use of complementary techniques, such as special immunohistochemistry, which could provide a more detailed understanding of the cellular and matrix events underlying the inflammatory and bone repair processes associated with apical periodontitis (Ervolino et al., 2016; Di Carlo et al., 2018).

It was concluded that DRW supplementation had a positive effect on the modulation of the inflammatory response and on the repair of bone tissue in the dental alveoli of teeth with AP. Supplementation with RW and AL resulted in changes in blood parameters, indicating a negative influence of alcohol.

Acknowledgements

This study was supported by the Coordination for the Improvement of Higher Education Personnel (CAPES), process-88887.817415/2023-00, São Paulo State Research Support Foundation (FAPESP), process-2022/05023-8, and National Council for Scientific and Technological Development (CNPq), process-302124/2022-5.

Data Availability Statement

The research data analyzed in this study are not publicly available by any means.

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Edited by

• Editor:
  Marcelo A. M. Esquisatto

Publication Dates

• Publication in this collection
  26 Jan 2026
• Date of issue
  2025

History

• Received
  19 June 2025
• Accepted
  13 Nov 2025