Dietary supplementation with multi-strain formula of probiotics modulates inflammatory and immunological markers in apical periodontitis

Abstract Objective The aim of this study was to evaluate whether probiotics multi-strain formula affects the development of apical periodontitis (AP) induced in rats. Methodology 16 Wistar rats were divided in two groups (n=8): rats with AP fed with regular diet (Control-C (CG)); rats with AP, fed with regular diet and supplemented with multi-strain formula (one billion colony-forming units (CFU)): GNC Probiotic Complex (PCG) ( Lactobacillus acidophilus, Lactobacillus salivaris, Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium bifidum, Bifidobacterium animalis subs. lactis and Streptococcus thermofilus ). AP was induced in the upper and lower first molars by dental pulp exposure to the oral environment. PCG was administered orally through gavage for 30 days during the AP development. After this period the animals were euthanized and the mandibles were removed and processed for histologic analysis, and immunochemical assays for interleukin (IL)-6, IL-10, IL-1β, RANKL, OPG, and TRAP. The Mann–Whitney U test and Student’s t test were performed (P<.05). Results The CG showed more intense inflammatory infiltrate than the PCG group (P<.05). IL-1β, IL 6 and RANKL decreased in the PCG group compared with CG (P<.05). The IL-10 level increased in the PCG group (P<.05). The OPG level was similar in both groups (P>.05). The number of mature osteoclasts (TRAP-positive multinucleated cells) was lower in PCG group when compared to the CG (P<.05). Conclusion Probiotic Complex modulates inflammation and bone resorption in apical periodontitis.


Introduction
Despite recent modernization and mechanization in endodontic therapy, conventional treatments for periaptical periodontitis still demand new adjunctive therapies. However, beneficial microorganisms has arisen as a potential concept. According to the World Health Organization, probiotics are "live microorganisms that can confer health benefits to the host when consumed in adequate amounts", modulating host immunoinflammatory response and influencing the structure and function of microbiota. 1 On the other hand, the concept of probiotics use in addressing endodontic disease is new and has not been studied adequately. 2 Recently, several studies assessing the effects of probiotics on periodontal disease showed encouraging results regarding the properties of these beneficial strains. 3,4 In an investigation, patients with chronic periodontitis were selected and received Bifidobacterium animalis subsp. lactis as supplementary treatment (probiotic group). This group presented a decrease in probing pocket depth and a clinical attachment gain significantly higher than the control group. 4 Also, the probiotic group displayed lower proinflammatory cytokine levels when compared to the control group, proving that this supplementation promotes additional clinical, microbiological, and immunological benefits in the treatment of chronic periodontitis. 3 In endodontics, a preliminary study indicated that probiotic organisms of the species Lactobacillus and Bifidobacterium are effective for preventing the growth of E. faecalis and C. albicans in vitro. 2 Chronic periodontitis and apical periodontitis are highly prevalent oral diseases in the world population. 5,6 The pathogenic process of apical periodontitis can be initiated by an injury (usually bacteria) evoking an acute immune-inflammatory host response in the periapical region. Once the immune system cannot eliminate the causative agent itself, a succession of events occurs to restrict the infected area (forming a circumscribed barrier) and prevent its spread. [5][6][7] The effect of probiotics on the immune response has been well investigated. [8][9][10][11][12] Much of these investigations in animal and human models suggest that probiotics may stimulate both nonspecific and specific immune responses. These positive effects on the immune system occur without triggering a harmful and potentially inflammatory response, being mediated by macrophages activation, an increase in antiinflammatory cytokine levels, an increase in natural killer cell activity and an increase in immunoglobulin levels. [8][9][10][11][12] It has been shown that, in isolation, probiotics Lactobacillus rhamnosus and Lactobacillus acidophilus had a significant effect on inflammation reduction and bone resorption in apical periodontitis development in rats. 9,13 However, the immune response can be increased when one or more probiotics are administered in association and act synergistically, as appears to be the case with Lactobacillus administered in association with Bifidobacterium. 14,15 Nowadays, although some studies are suggesting the use of single-strain probiotics in endodontics, 9,13 none of the studies addresses the most common commercial form in the world: the administration of probiotics through mixtures of strains. 1, 14,15 Especially when in association with mixed strains result in an additive or even synergistic outcomes in terms of bioactivity by the component strains. 1,14,15 To the best of our knowledge, no investigations assessed the effects of a probiotic complex on the apical periodontitis development. Therefore, the purpose of this study was to evaluate the histopathological and immunohistochemical outcomes following the administration of the probiotics multi-strain formula of 10%, the sample size calculated before was divided by 0.9, resulting in eight animals per group.

Induction of AP
The rats were anesthetized via intramuscular injections of ketamine (87 mg/kg, Francotar; Virbac do Brazil Ind e Com Ltda, Roseira, São Paulo, Brazil) and xylazine (13 mg/kg, Rompum, Bayer AS, São Paulo, Brazil). Coronal pulp tissue was exposed and disorganized in the animals through an access cavity in the crown of the upper and lower first molar first molars using a spherical carbide bur with 0.5 mm diameter (Jet Carbide ¼, Kavo Kerr Group, Orange, CA, USA) with a surgical microscope (Decius, DF Vasconcelos, Rio de Janeiro, Brazil). The coronal pulp tissue was exposed and kept open to the oral cavity until euthanasia. 16,17 Probiotic therapy The animals were randomly assigned into two groups Histopathological analyses using a light microscope (DM 4000 B; Leica) were performed by a certified histologist (E.E) who was blinded to the experimental groups and used a color camera (DFC 500; Leica, Wetzlar, Germany). The distal root was standardized for all analyses. 17 Histopathological analysis was conducted using the quality of inflammation and the cellularity pattern of dental and periodontal tissues as guidelines to score the inflammatory infiltrate as follows: absent (0 to few inflammatory cells, score = 1), mild (<25 cells, score=2), moderate (25-125 cells, score=3), and severe (>125 cells, score=4). 19 The presence or absence of necrosis was also recorded. 9, 17,20 Immunohistochemical analysis

Statistical Analysis
The data were analyzed using GraphPad Prism 7 software (La Jolla, CA, USA). After the Shapiro-Wilk test of normality, the Mann-Whitney test was performed for nonparametric data, and unpaired t-test was performed for parametric data. The level of significance was 5%. Figure 1 shows representative histologic images of AP in different experimental groups Necrosis was observed in the CG and PCG at 30 days after exposure.

Results
In AP, CG group showed more intense inflammatory infiltrates compared with that in the PCG group ( Figure   1). The median scores assigned to the CG group were significantly more severe than those in the PCG group (P<.05).   In our study, the AP model employed is supported by the scientific literature, and the authors have extensive practice with their management. 9, 13,16,17,20 The apical periodontitis induction in rats allows researchers to detach extrinsic factors that would be difficult to manage if the investigation was performed as a clinical study. 16,17,19,20,24 The probiotics can kill or inhibit the bacteria, modify

Conclusion
Supplementation with multi-strain probiotics formula had a significant effect on reducing inflammation and bone resorption in apical periodontitis.

Conflict of interest
The authors deny any conflict of interests to this study.