What is the effectiveness of beta-glucan for treatment of acute otitis media?

Introduction As a supplement, beta–glucan has various therapeutic healing effects generated by the immune cells. It has been scientifically approved and proven to be a biological defense modifier. The aim of this study was to investigate the effects of beta–glucan on treatments administered in an acute otitis media model Objectives This study investigated the effect of beta–glucan on the treatment of acute otitis media in an acute otitis media -induced animal model. Efficacy was evaluated both immunologically and histologically. Methods The study sample comprised 35 adult rats, randomly separated into 5 groups of 7: Group 1 (control), Group 2 (acute otitis media, no treatment), Group 3 (acute otitis media + antibiotic), Group 4 (acute otitis media + beta–glucan) and Group 5 (acute otitis media + beta–glucan + antibiotic). Analyses were made of the histopathology and immunology examination results in respect of thickening of the tympanic membrane, epithelium damage, inflammation, and sclerosis. In all groups the serum levels of TNF-α, IL-4, IL-6 and IL-1β were evaluated. Results All serum cytokine levels were significantly lower in the beta–glucan and antibiotic-treated groups compared to the acute otitis media Group. Significant differences in tympanic membrane thickness, inflammation, epithelium damage, and sclerosis values were observed between the acute otitis media + antibiotic and acute otitis media + beta–glucan Groups. According to these parameters, the values in aute otitis media + antibiotic + beta–glucan Group were markedly lower than those of the other groups. There was a significant difference in the acute otitis media + antibiotic + beta–glucan Groups compared to acute otitis media Group (p < 0.001). Conclusions Both antibiotic and beta–glucan treatment reduced acute otitis media signs of inflammations in an acute otitis media-induced rat model, decreasing histological damage and cytokine levels. Co-administration of antibiotic and beta–glucan led to a significant reduction in tympanic membrane thickness, inflammation, and epithelium damage. Antibiotic + beta–glucan treatment resulted in a greater decrease in tympanic membrane thickness, inflammation, and epithelium damage than in the other groups. From these results, it can be suggested that beta–glucan, in combination with antibiotics may provide an alternative for the treatment of acute otitis media.


Introduction
Acute otitis media (AOM) is seen extremely frequently during childhood and 75% of pre-school age children have been reported to have suffered at least one episode. Intracranial spread of infection is among the complications that may develop. Within the middle ear cavity (MEC), the mechanisms of AOM in the MEC which could make an individual susceptible to the development of inflammation are only partially known. 1,2 The 5 most common bacteria associated with AOM are Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pyogenes, and Staphylococcus aureus. The most common viruses that can contribute to AOM are human rhinovirus, influenza viruses, respiratory syncytial virus, adenovirus, and enterovirus. Patients with AOM suffer a reduced quality of life because of pain, hearing loss, headaches, and cognitive impairment. 3,4 A major contributing factor to AOM pathophysiology is the impaired function of the middle ear mucosa and subsequent stasis of infected secretions; this has been associated with local inflammation. 4,5 As AOM pathogens can be resistant to commonlyused antibiotics, proper treatment is challenging. 6 Some AOM patients are prescribed supplementary and alternative therapies, particularly medicinal herbs, in addition to conventional treatments. Non-prescribed use of medicinal herbs for upper respiratory tract infection is common around the world. Beta-Glucan (␤---G) is a wide-spread plant constituent, which has been studied for decades in investigation of beneficial effects on humans. It has been scientifically approved and proven to be a biological defense modifier. 6,7 As a supplement, ␤---G has various therapeutic healing effects generated by the immune cells. It can trigger development of a group of immune cells including macrophages, neutrophils, monocytes, natural killer cells, and dendritic cells, and it can inhibit tumor growth in the promotion stage. 7---9 The aim of this study was to investigate the effects of ␤---G on treatments administered in an experimentally-induced AOM rat model and to evaluate the results both immunologically and histologically.

Animals and experimentally-induced AOM
The groups were separated for treatment as follows: to Groups 2, 3 and 4, Staphylococcus aureus strain ATCC 25923 in sterile solution (0.5---1 × 10 8 CFU/mL) was administered bilaterally using a dental needle (0.1 mL) via the transtympanic route. AOM developed within 48 h. Hyperemia of the tympanic membrane was observed macroscopically on day 2, and S. aureus growth was confirmed by culture. (Approval number of the ethics committee: 2015/A-56).

Treatment groups
The 35 rats were randomly separated into 5 groups of 7. Group 1 (control) was administered 0.1 mL saline via the intratympanic route, followed by 0.01% Carboxymethyl Cellulose (CMC) via gavage. For Group 2 (AOM), 0.1 mL solution of S. aureus was administered via the intratympanic route to induce AOM, followed by 0.01% CMC via gavage. In Group 3 (AOM + antibiotic), following AOM induction, 50 mg/kg ampicillin was administered via gavage. In Group 4 (AOM+␤---G), following AOM induction, 50 mg/kg ␤---G was administered via gavage. In Group 5 (AOM+␤-G + antibiotic) following AOM induction, 50 mg/kg antibiotic and 50 mg/kg ␤---G were administered via gavage. Ampicillin was administered twice a day for 14 days, and the ␤---G was also administered for 14 days. On day 14, all the rats were weighed, then ketamine hydrochloride(75 mg/kg) and xylazine (8 mg/kg) anesthesia was applied and the animals were sacrificed under general anesthesia. Blood samples were syringed from the left ventricle for immunological analysis. After whole blood centrifugation (3,000×g, 20 min, at 4 • C) sera were obtained. Tissue and sera samples were stored at −45 • C until further analysis.

Histopathological analysis
After sacrifice of the rats, the temporal bone was dissected from the skull. After opening the bullae, tissues were fixed in 10% buffered formalin, then incubated in 5% formic acid and routine tissue processing was performed for decalcification. Sections 4 m in thickness were cut from the paraffin blocks and stained with hematoxylin and eosin (H&E). All the slides were examined under light microscopy (Olympus BX-51; Olympus, Tokyo, Japan) and images were captured using a digital DP70 camera attached to the microscope (Olympus). In the histological examination, evaluation was made of thickening of the tympanic membrane (ThicTM), damage to the epithelium (DamEpith), inflammation (Inf), and sclerosis (Sc). The severity of changes was scored as follows: none (-), mild (+), moderate (++), and severe (+++) 10,11 The score of none (-) was defined as normal epithelial and connective tissue; mild (+) as mild infiltration of individual inflammatory cells or their clusters, mild degeneration of epithelial cells, mild connective tissue fibroblastic cell proliferation; moderate (++) as moderate infiltration of inflammatory cells, focal epithelial loss and moderate connective tissue fibroblastic cell proliferation; and severe (+++) as dense infiltration of inflammatory cells and loss of epithelial integrity and marked connective tissue fibroblastic cell proliferation.
For statistical analysis these scores were equated to numerical scores (-)/0, mild (+)/+1, moderate (+ +)/+2, severe (+++)/+3. Quantitative assessment was made of the Thic TM thickness from measurement of the width of the tympanic membrane at the external edge facing the external auditory canal space and the width of the tympanic cavity facing the tympanic cavity at 10 different points. 11 Epithelial damage was assessed by loss and erosion of the epithelium lining the tympanic membrane surface. Inflammatory cell increase was examined to evaluate inflammation. There was an increase in capillary vessels, congestion and edema. In the process of sclerosis; increased fibroblastic cells and myxoid degeneration were observed.

Statistical analysis
Statistical analysis of the study data was made using SPSS for Windows software (ver. 18.0; SPSS Inc., Chicago, IL, USA). The results were stated as mean ± standard deviation (SD) values. The TNF-␣, IL-4, IL-6, and IL-1␤ levels were compared between the treatment groups using Oneway analysis of variance (ANOVA) and the post-hoc Duncan test. Kruskal-Wallis variance analysis was applied to the histological results. When differences were detected between the groups, the groups mean values were compared using the Mann-Whitney U test. A value of p < 0.01 was considered statistically significant.

Results
The serum levels of TNF-␣, IL-4, IL-6 and IL-1␤ for all the treatment groups are shown in Table 1. In the AOM Group, serum levels of TNF-␣, IL-4, IL-6 and IL-1␤ increased significantly compared to the other groups (p < 0.01). The serum levels of TNF-␣, IL-4, IL-6 and IL-1␤ were significantly decreased in the ␤---G-treated and antibiotic-treated groups compared to the AOM Group. No significant differences were determined between the ␤---G, antibiotic, and ␤-G+antibiotic-treated groups in respect of cytokine levels. The cytokine levels in these groups were observed to be similar to those of the control group.

Histopathological results
In the histological examination, the control group was observed to have a clear MEC, external auditory Canal (eAC) and a thin, single cell mucosal lining of the cavity. In the AOM groups, normal EAC was observed, but there was effusion within the MEC and a thickened mucosa, with lamina propria including polymorphonuclear leukocytes, histiocytes, fibroblasts and granulation tissue. Cavity effusion in affected rats contained foamy macrophages and neutrophils.
The histopathological examination results of changes in the tympanic membrane in respect to thickening of the tympanic membrane (ThicTM), damage to the epithelium (DamEpith), inflammation (Inf), and sclerosis (Sc) are presented in Table 2 and Figs. 1---5 .
H&E stained sections demonstrated that the values of ThicTM, DamEpith, Inf, and Sc were significantly increased in the AOM group compared to the control group (Table 2 and Figs. 1---2; p < 0.001).   The values of these parameters in the AOM + antibiotic group were markedly lower than those in the AOM Group (Table 2 and Figs. 2---3; p< 0.001). In the AOM Group, ThicTM was increased with the migration of inflammatory cells and edema in the subepithelial layer of the bulla mucosa. The highest levels of DamEpith and Sc were determined in the AOM Group compared to all the other groups (Fig. 2).
No significant difference was determined between the AOM and AOM+ ␤---G Groups in respect of the Sc values, and a significant difference was determined in respect of ThicTM, DamEpith and Inf values (  Semi-quantitative evaluation was made of the changes observed. The arithmetic mean was obtained based on the AOM and control groups. In the control group, DamEpith, Inf, and Sc were not observed. An evident increase in ThicTM in the AOM group was observed due to inflammation and edema effect. Dense inflammation was caused by infiltration of "neutrophils and lymphohistiocytic cells" (2.83 ± 0.40). As a result of inflammation, degenerative changes and cellularity increase were observed in the connective tissue of the membrane wall. These changes occurred secondary to the inflammation in otitis media.

Discussion
AOM is a common upper respiratory tract infection for which antibiotics are prescribed. It can affect all ages, with the greatest frequency reported in children. Moreover, AOM is the most common complaint associated with medical therapy for children < 5 years of age. 3,4 Antibiotic resistance has guided researchers to investigate new options. Medicinal herbs have been used for a long time, and in fact, continue to be used more often than prescribed medications. 8---10 Current information proposes that these medicinal herbs exert a biological effect on immune regulation or cytotoxic mechanisms. 7---12 One of the immune-effective herbal products is in the form of complex polysaccharides known as ␤-glucans. 12,13 These biologically active polysaccharides found in both bacterial or fungal cell walls increase host immune defense by activating complementary systems and enhancing the functions of natural killer cells and macrophages. According to in vitro studies, ␤---G interacts with several immune cell surface receptors, including Complement Receptor 3 (CR3; CD11b/CD18), dectin-1 (bGR), Toll-Like Receptors 2 and 6 (TLR-2/6), selected macrophage scavenger receptors and lactosylceramide. In addition, ␤---G can inhibit tumor growth in the promotion stage. 8,9,12,14 In this study, the efficacy was evaluated of the use of a conventional antibiotic (ampicillin) and an herbal medication (␤---G), used in the management of anti-inflammatory and antimicrobial activities, in the treatment of AOM. The four histopathological examination parameters used to evaluate the effects of treatment were ThicTM, DamEpith, Inf, and Sc. 9 It was seen that with the migration of inflammatory cells and edema into the subepithelial layer of the bulla mucosa, ThicTM was increased in the AOM Group. The highest DamEpith and Sc values were obtained in the AOM group. Compared to the AOM Group, all four values were significantly decreased in the AOM+␤---G + antibiotic group. According to these parameters, the values in the AOM+␤---G + antibiotic group were markedly lower than those of the other groups. It can be considered that this effect may be due to synergistic effects occurring through the combined use of the antibiotic and ␤---G.
In previous experimental animal models, the role of cytokines in AOM has been well studied. 8---11 TNF-␣, IL-1␤ IL-4 and IL-6 are known to be important local mediators associated with acute inflammation. For the evaluation of disease pathogenesis, the cytokine expression profile during AOM episodes is useful. 14---16 In this study, the serum levels were measured of all four of these cytokines in the different treatment groups. The results showed that cytokine levels were significantly decreased in the ␤---G-treated and antibiotic-treated groups compared to the AOM Group.
Several studies have revealed that ␤---G regulates the production of various inflammatory cytokines. In addition, ␤---G regulates host immune defence by activating the complementary system and enhancing the functions of natural killer cells and macrophages. These regulatory activities shed light on the potent effects of this herbal remedy. 8,17,18 In addition to reducing the risk of (coronary) heart disease and anticancer actions, ␤---G has also been reported to have a role in infection healing. ␤---G has been approved as safe for us by the Food and Drug Administration. Furthermore, at all doses tested, no toxicity has been observed. 19---21 In this prospective animal study, the effect of ␤---G on the treatment of AOM was investigated in an AOM -induced rat model. Histopathological and immunological analyses were performed and the results were analyzed. To the best of our knowledge, there has been no previous study that has histologically and immunologically investigated the impact of ␤---G on AOM.

Conclusions
Animal model experiments related to otitis media have great benefits, such as subject manipulation and controlled disease/cure experimentation. The collective benefits of otitis media experimentation on animals has expanded the field for continuous and improved models on these subjectsFrom the results of this study, it can be suggested that ␤---G, in combination with antibiotics, may provide a successful alternative treatment for AOM compared with antibiotics used alone. The favorable effects observed using ␤---G are related to its immunomodulating activities. Therefore, the potential use of ␤---G for the treatment of AOM should be further investigated. Future studies are required, which should focus on the use of ␤---G in the treatment of AOM using randomized controlled studies in humans.