Beta-defensins-2 expressions in gingival epithelium cells after probiotic Lactobacillus reuteri induction

Background: Beta-de Beta-defensins (BD) are expressed by a variety ct. BD-1 is expressed constitutively, while BD-2 a nsal bacteria, thus L. reuteri as probiotic bacteria ing dental caries and sensitive to BD-2. Purpo may improve BD-2 expressions in the gingiva us norvegicus Wistar strains aged 10-12 weeks egative control group (not induced with L. reuteri group 1 (induced with L. reuteri for 14 days an mutans for 14 days concurrently). The concent 1010cfu/ml. 0.1 ml of each was dropped in the r hemical method. The difference of BD-2 express Results: There were significant differences in B nova test (p=0.001). Conclusion: The administ lls.


INTRODUCTION
Gingival epithelium is a defense against bacteria in oral cavity, not only physically but also chemically.Defense function of the gingival epithelium is characterized by their unique structure and integrity of the anti-microbial peptide (AMP), such as human beta defensins (hBDs). 1 Defensins are antimicrobial peptides first discovered in mammals.Defensins in humans consist of two sub-families, namely alpha and beta-defensins.
Alpha-defensins are produced by polymorphonuclear leukocytes and panet cells, while beta-defensins are produced by epithelial surface of skin, intestine, trahea, and oral cavity. 2 Defensins have broad activities against bacteria, fungi, and viruses.In the optimal conditions, antimicrobial activities of defensins work at low concentration of 1-10mg /mL. 3 Human beta-defensins (hBDs) are widely expressed in tissues of the oral cavity, including gingival epithelium, salivary glands, saliva and salivary duct.These peptides are involved in defense against bacteria that colonizes in the oral cavity.
Defensins in the oral cavity have an important role to protect the structure of the teeth from bacteria causing caries.Human beta-defensins have broad antimicrobial activity against oral microorganisms, such as Streptococcus mutans, Porphyromonas gingivalis, and Actinobacillus actinomycetemcomitans. 3Changes in lifestyle and diet can affect the composition and amount of the normal flora in someone's oral cavity.occurrence of dental caries are physical factors, biological factors, environmental factors, habits, and life style. 4ental caries is an example of a disease that one of causes is too often consuming sucrose being offset by the improper process of oral cavity cleaning.Dental caries is an infectious disease caused by an imbalance of homeostasis between host and microbe. 56][7] Excessive amount of S. mutans in the oral cavity can be considered as an early occurrence of dental caries.
Recently, there have been many outstanding supplement products containing probiotics.According to the WHO / FAO, probiotics are life micro-organisms which, when administered in adequate amount, Confer a health benefit on the host.Meanwhile, International Life Science Institute (ILSI) Europe defines probiotics as a life of microbial food ingredient that, when ingested in sufficient quantities, exerts health benefits on the consumer. 8Based on both definitions, it can be concluded that probiotics are live microorganisms causing an effect on health.One of the many probiotics often used in dentistry is Lactobacillus reuteri (L.reuteri).Probiotics including L. reuteri are commensal bacteria.Commensal bacteria are excellent inducers to BD-2 in epithelial cells of the oral cavity, so it is possible for probiotic bacteria to act as an inducer for BD-2. 9There are two species of bacteria causing dental caries, namely S. mutans and S. sobrinus, sensitive to BD-2. 10 Thus, it indicates that BD-2 can be used as an alternative material for preventive and therapeutic potential against dental caries.Therefore, this research was aimed to determine whether the administration of probiotic L. reuteri, induced into gingival mice could increase BD-2 expressions in the gingival epithelium.

MATERIALS AND METHOD
This research was approved by Komisi Kelaikan Etik Penelitian Kesehatan-KKEPK (the Commission on Health Research Ethics Airworthiness), Faculty of Dental Medicine, Universitas Airlangga.
Twenty-four male rats (Rattus norvegicus wistar strain) aged 10-12 weeks with body weight of 120-150 grams were divided into four groups, namely negative control group, positive control group, treatment group 1, and treatment group 2. There were five rats in the negative control group, which were not induced by S. mutans or L. reuteri.In the positive control group, there were five rats induced with S. mutans for 14 days.In the treatment group 1, furthermore, there were seven rats induced with L. reuteri from day 1 to day 14 (14 days), and S. mutans from day 8 to day 14 (7 days).On the other hand, in the treatment group 2, there were seven rats induced with L. reuteri and S. mutans from day 1 to day 14 simultaneously.On day 15, those rats were sacrificed, and then their jaws and gingival tissue were cut for the preparation of paraffin blocks for immunohistochemical examination (IHC).
The concentration of the bacterial suspension used was 4 x 10 8 cfu/ml of L. reuteri, and 1 x 10 10 cfu / ml of S. mutans. 9DSM 17 938 + ATCC PTA 5289 was added to brain heart infussion (BHI) liquid, and then incubated in anaerobic condition for 1x24 hours using gas generating kit with Oxoid brand.After removing from the incubator, turbidity and sediment will appear, indicating L. reuteri growth.Planting to MRS agar (De Man, ROGOSA and Sharpe; Merck GmbH, Damrstadt, Germany) was performed, and then incubated for 2x24 at a temperature of 37 0 C.After that, several colonies were taken and planted into a liquid BHI medium, and then incubated for1 x 24 hours at a temperature of 37 0 C.After removing from the incubator, bacterial density were observed to know whether it had reached 4 x 10 8 cfu / ml, and then examined using spectrophotometry with a wavelength lambda of 625 nm and an optical density of 0.08-0.10identical to the Mc Farland 0.5. 9. mutans used in this research were S. mutans serotype c taken from the stock in the form of freeze dry.S. mutans were taken about 1 ose, added to liquid BHI medium, and then incubated for 1 x 24 hours at a temperature of 37 0 C.After removing from the incubator, turbidity and sediment will appear, indicating S. mutans growth.Planting to a blood agar medium was performed, and then incubated for 1x24 at a temperature of 37 0 C.After that, several colonies were taken and planted into a liquid BHI medium, and then incubated for1 x 24 hours.After removing from the incubator, bacterial density were observed to know whether it had reached 1 x 10 10 cfu / ml, and then examined using spectrophotometry with a wavelength lambda of 625 nm and an optical density of 0.08-0.10identical to the Mc Farland 0.5.9 Examination of beta-defensin-2 (BD-2) expressions was performed.There are several preparation stages of histopathology conducted in this research as stated in Humason's method cited in Sudiana, 2005.Calculating method of the results of immunohistochemical staining, according to Soini et al. 11,12 consists of several stages.First, gingival tissue was fixated in paraformaldehid 4% for 4 hours at a temperature of 4 0 C.After washed in PBS, the tissue was planted in OCT compound, and then immediately frozen.Then the tissue was cut into a thickness of 6 μm, and then incubated with primary antibody anti BD-2 (Lab Vision) for 24 hours at a temperature 4 0 C. The tissue sections were incubated with secondary antibody rat monoclonal, anti OPG (Lab Vision), and HRP kid for 24 hours at a temperature 4 0 C. Chromogenics used in this research were 3.3 -diaminobenzidine tetrahydrochloride, and then counterstained with HE. 9 Immunohistochemical staining with indirect method was carried out to show cells expressing BD-2 protein.The cells expressing BD-2 were shown with a brown cytoplasm, and then observed with a light microscope with a magnification of 400 x.Anova test, was performed to determine the differences in BD-2 expressions in the gingival epithelium of each group.HSD test was conducted to determine the significance of the differences between groups.

RESULTS
Immunohistochemical examination was performed to determine the distribution of cells expressing BD-2 in the gingival epithelium after induced with probiotics, L. reuteri bacteria, in each group (Table 1).
Based on Table 1, the results show that there was a significant difference of BD-2 expression in the gingival epithelium of Wistar rats among the treatment groups (p = 0.001).A decline in the mean of BD-2 expressions in the gingival epithelium was found in the positive control group (group induced by S. mutans), compared to the negative control group, namely from 15.80 into 4.80.An increase in the mean of BD-2 expressions in gingival epithelium was found in the treatment group 2 (induced by probiotic L. reuteri for 14 days and S. mutans for 14 days), compared to the treatment group 1 (induced with probiotic L. reuteri for 14 day and S. mutans for 7 days), namely from 24.00 into 27.00 The overall BD-2 expressions were significantly different among the groups.Based on the results of HSD test, it is known that there was a significant difference in BD-2 expressions between the negative control group and the positive control group.There were also significant differences in BD-2 expressions between the negative control group and the treatment group 1 as well as between the negative control group and the treatment group 2. Similarly, there were also significant differences in BD-2 expressions between the positive control group and the treatment group 1 as well as between the positive control group and the treatment group 2. However, there was no significant difference between the treatment group 1 and the treatment group 2.

DISCUSSION
The administration of L. reuteri as probiotics in this research could increase the expressions of BD-2 at gingival epithelial cells (Table 1 and Figure 1).Gingival epithelium is stratified squamous (flat stratified epithelium) which serves as a defense against pathogenic bacteria.Epithelial tissues in the oral cavity are continuously in contact with various kinds of bacteria, even so most individuals can maintain the balance of their health.Epithelial tissues of the oral cavity play a role in protecting the host not only with the physical defense, but also through the innate immune responses in the form of antimicrobial peptides.BDs are small antimicrobial peptides, which form cautions produced by epithelial cells playing an important role in mucosal defense and skin. 13Based on Figure 2, the results of immunohistochemical examination for BD-2 expressions in the gingival epithelium showed that BD-2 is expressed in stratum spinosum and stratum granulosum.The results is in line with the results of a research conducted by Weinberg et al., 14 stating that within the gingival tissue, mRNA from SW-1 and SW-2 is located in the suprabasal stratified epithelium, while peptide BD-2 is detected in the upper epithelial layers. 15ingiva, hard palate, and the dorsal part of the tongue is the keratinized epithelium of the oral cavity, while the floor of the mouth and the buccal area are not keratinized epithelium.The results of immunohistochemical examination conducted to observe BD-2 expressions in the keratinized epithelial in oral cavity (including gingiva) showed a weak positive result, while in the non-keratinized epithelial showed a negative result.This situation suggests that keratinization in the epithelium of the oral cavity plays an important role for the retention of peptides. 14he results found in the negative control group in this research can be associated to the results found in normal epithelium condition as stated by Abiko Y et al. since the group was not treated.BD-2 expressions in the gingival epithelium in the negative control group were 15.80, while BD-2 expressions in the treatment group 1were 24.00, and 127.00 in the treatment group 2 (Table 1).It indicates that BD-2 expressions increased after the induction of probiotic bacteria, namely L. reuteri and S. mutans bacteria.
The induction of probiotics in the treatment group 1 was assumed as a precaution.In this treatment group, L. reuteri bacteria were first induced from day 1 to day 14, whereas S. mutans bacteria causing caries were induced from day 8 to day 14.On the other hand, the induction of probiotics in the treatment group 2 was assumed as a therapeutic action.In the treatment group 2, L. reuteri and S. mutans were administered together from day 1 to day 14.
In Table 1, the number of gingival epithelial cells expressing BD-2 in the negative control group the group not induce L. reuteri and S. mutans bacteria was lower than in the treatment groups.This condition showed that BD-2 expressions were still expressed both at the gingival epithelium in normal circumstances and at the gingival epithelium in inflammation circumstances.In contrast, in most other epithelia, such as skin epithelium, tracheal epithelium, and intestinal epithelium, BD-2 is expressed only in inflammation circumstances. 16This statement is supported by the results of a research conducted by Whasun & Dale, stating that in most tissues, BD-2 is induced and expressed only in inflammation circumstances, but in epithelial tissues of the oral cavity, BD-2 is expressed only in normal circumstances (without inflammation) since the oral mucosa is continuously exposed to a variety of bacteria. 17he lowest number of BD-2 expressions was found in the positive control group (Table 1 17 Based on these results, it can be said that the administration of probiotics, L. reuteri bacteria, for 14 days with a concentration 4x10 8 CFU/ml could increase BD-2 expression significantly (p=0.001).This result can be seen in Table 1 and Figure 2 which show that the number of BD-2 expressions found in gingival epithelial cells in the treatment group 2 induced with probiotics, L. reuteri bacteria, for 14 days, was the highest one.The reason is that the active molecule on the cell wall of the probiotic bacteria, peptidoglycan, activates NOD-2 receptors in the cytoplasm.Activation of signaling through NOD-2 can trigger a recruitment of protein adaptor (RICK).RICK then will be activated, resulting in phosphorylating complex IKK.NF-kB is in the inactive form, which binds to IKB, inhibitor protein in the cytoplasm.This stimulus will result in phosphorylation, ubiquitination, and degradation of IKB proteins that will cause the translocation of NF-kB into the nucleus and the activation of NF-kB. 18,19It will activate target genes for synthesis of BD-2 protein.In conclusion, probiotics, L. reuteri, can improve BD-2 expressions in the epithelial cells of the gingiva.

Figure 1 .
Figure 1.Immunohistochemical examination results of BD-2 expressions in the gingival epithelium in each group (400x magnification); Black arrow (cells expressing BD-2); Red arrows (cells not expressing BD-2).A) BD-2 expressions in the gingival epithelium in the negative control group; B) BD-2 expressions in the gingival epithelium in the positive control group; C) BD-2 expressions in the gingival epithelium in the treatment group 1; D) BD-2 expressions in the gingival epithelium in the treatment group 2.
Open access under CC-BY-SA license.Available at http://e-journal.unair.ac.id/index.php/MKGDOI: 10.20473/j.djmkg.v49.i1.p49-53 ).The positive control group is a group only induced with S, mutans.Thus, it can be said that S. mutans is not able to induce BD-2.A research conducted by Wehkamp et al showed that E. coli Nissle 1917 strains can induce BD-2, while the other 40 isolates of E. coli cannot induce BD-2.The results of the isolation and purification of flagellant protein derived from E. coli Nissle 1917 are able to induce BD-2, while Flagellin deficient mutans derived from the bacteria are not able to induce defensins.LPS of E. coli Nissle 1917 even cannot stimulate the production of BD-2.Therefore, it indicates that flagellant protein is decisive MAMP in E. coli Nissle 1917 probiotics that can stimulate BD-2 excessively.

Table 1 .
The mean and standard deviation of BD-2 expressions in gingival epithelium after induced with Probiotic L. reuteri in each group.