INTRODUCTION:

Periodontal abnormalities are abnormalities that occur in the periodontal tissue involving the structure of the tooth-supporting tissue around the circumference of the tooth and attaching the tooth to the jawbone so that the tooth cannot be separated from the socket. The main cause of periodontal disorders is oral commensal bacteria which are responsible for the initiation and spread of disease through a process of microbial imbalance¹. Periodontitis disorders can result in disease Periodontitis is a chronic, destructive, and irreversible inflammatory disease^2,3. There are 2 causes of periodontitis, namely direct and indirect causes, periodontitis can be directly caused by pathogenic bacteria, and indirectly it can be caused by a systemic reaction in the patient's body due to an immune reaction^4-7.

Other risk factors can be systemic or local such as medical conditions, obesity, stress, osteopenia, and inadequate consumption of calcium and vitamin D and diet⁸. Caries and periodontal disease are still a global burden of oral disease that is very important to address. Severe periodontitis, which can cause tooth loss, is found in 5-15% of most of the adult population aged 35-44 years⁹. The results of the 2018 Riskesdas data stated that the percentage of periodontitis cases in Indonesia was 74.1%, with the second highest prevalence after caries (88.8%). Judging from the type of work the prevalence was in PNS/TNI/Polri/BUMN/BUMD there were 68 cases. 1%. In the examination of Soldiers of the Dikjurtakes Abit Dikmata TNI AD with the Community Periodontal Index for Treatment Needs (CPITN) index, it was found that only 11.76% of soldiers had healthy periodontal while the rest were bleeding 17.65%, tartar 60.78% and shallow pockets 9.80%¹⁰. Several periodontal diseases and decreased effectiveness of periodontal disease treatment can be caused by psychological stress. Stress is a direct cause of periodontal problems which is connected through neuro-immunoendocrinological mechanisms¹¹.

Stress in humans can be caused by many things, the discrepancy between expectations and reality, physical pressure, psychological pressure, and others. This often involves many stress-causing factors that can have an impact on physical and psychological health. In previous research, there is a link where academic stress has a significant effect on periodontal health^11,12. Periodontitis-related stress is associated with increased expression of interleukin-1β (IL-1β)^13,14, where increased expression of IL-1β has a role that can damage periodontal tissue^8,15. In addition, stress has an effect on increasing the amount of interleukin-6 (IL-6), interleukin-10 (IL-10), and the hormone cortisol in the blood, as well as increased plaque formation and gingival inflammation^16-18.Polymorphonuclear (PMN) which is a component in leukocytes is one component that is believed to determine the severity of periodontitis. PMN or neutrophil cells which constitute 60% of the leukocyte component have a considerable role in the composition of the periodontal tissue¹⁹. Increased macrophage infiltration generally initiates periodontitis, macrophages phagocytize bacteria that infiltrate the gingival tissue^20,21. Immunoglobulin is a protein that plays a role in the immune system, among these proteins IgG and IgA have an important role in periodontitis because IgG is the main antibody that circulates in the blood and can move through the blood circulation to the tissues, while IgA has an important role in protecting the gingival mucosal surface²².

Similar studies have been carried out on psychological stress but none have had an impact on changes in periodontal tissue.

Periodontal disorders are the second highest cause of dental and oral disorders in the world, and have a fairly high prevalence and cannot be ignored from the total number of dental and oral diseases in the world. The resulting symptoms can cause discomfort. Stress is one of the triggers for periodontal disorders because stress can cause changes in interleukin expression, hormone expression, protein expression, and the number of PMNs and macrophages. This research uses the cellular method so experimental animals are needed as an initial stage of research.

The aim of this study was to analyze changes in the expression of IL-1β, IL-6, IL-10, cortisol, IgG, IgA, number of PMNs, number of macrophages, and widening of periodontal tissue, with the hypothesis that there is a change in the expression of biomolecular components as a marker of stress. periodontal abnormalities through increased expression (IL-1β, IL-6, cortisol, number of PMNs, number of macrophages, and widening of periodontal tissue); and decrease (IL-10, IgG, and IgA).

MATERIAL AND METHODS:

Experimental design:

This study used Wistar rats with two treatment groups and one control group for each treatment and control period (one day and five days). In one group of Wistar rats, there were 6 in each treatment and control group which were used with the following conditions: male type, weight 300 grams, age 5 months. Each treatment group was exhausted (treadmill) for 15 minutes and rested for 45 minutes and the treatment group was afraid (cats as predators of mice). Experimental animal tests in this study were carried out using CBCT, SEM, Elisa, IHK, and HE.

Ethical approval:

This study has been evaluated and approved by the animal ethics committee of IPB University with Ethical Clearance Number: 019/KEH/SKE/XI/2020.

RESULT AND DISCUSSION:

Expression of IL-1β, IL-6, IL-10, IgG, IgA, PMN, and Macrophage on the 1st and 5th-day treatment gave a brownish reaction to anti-monoclonal anti-Il-1β, IL-6, IL-10, IgG, IgA, PMN, dan Macrophage using a Nikon Eclipse Ci camera, Camera DS-Ri 16.25 megapixels, magnification 100x, 400x, 1000x. As shown in Figures 1 and 2, the statistical results of IL-1β expression showed that the L1 treatment group was higher than K1 on the first day and compared to K2, L2, and P2 on the fifth day. IL-1β expression decreased significantly in the L2 treatment group compared to the L1 treatment group on day 1, while on day 5 the P2 treatment group was higher than the L2 treatment group.

Figure 1. Expression of IL-1β

Figure 2. Line graph of statistical analysis of IL-1β expression between groups

The decrease in the L2 treatment group was above the K1 and K2 control groups. The P2 treatment was also still above the K2 control group on the fifth day.Statistical results on the expression IL-6 (Figures 3 and 4) showed that the L2 treatment group on day 5 had an increase compared to the L1 group on day 1, while between treatment groups on day 1, it showed that the P1 treatment group was higher than the L1 treatment group. On day 5, the L2 treatment group was higher than the P2 treatment, although it was still below the control group.

Figure 3. Expression of IL-6

Figure 4. Line graph of statistical analysis of IL-6 expression between gropus

The statistical results of the CPI examination on IL-10 expression (Figures 5 and 6) showed that the L2 treatment group on day 5 experienced a significant increase compared to the L1 treatment group on day 1 which can be seen in the line graph of the statistical results for IL-10 expression. treatment group on both days.In the treatment group on day 1, it was significant in the P1 treatment group compared to the P2 treatment group and it was still below the K1 control group. In the 5th-day treatment group, it was also significant in the L2 treatment group compared to the P2 treatment group and it was still below the K2 control group.

Figure 5. Expression of IL-10

Figure 6. Line chard statistical analysis of II-10 expression between groups

Statistical analysis (Figures 7 and 8) indicated that IgG was increased in L2 on day 5 compared to L1 although it was still below the control group K1 and K2. L1 and P1 on day 1 showed a significant difference compared to K1 and K2 where p = 0.000 and there was a significant difference in P2 and L2 on the fifth day compared to K2 and K1 p = 0.000 (Figure 8).

Figure 7. Expression of Ig G

Figure 8. line graph of statistical analysis of Ig G expression between groups

The results of statistical calculations showed that IgG and IgA expression (Figures 9 and 10) gave an increase on day 5 in the L2 treatment group compared to the L1 treatment on day 1 and an increase in the P2 treatment group on day 1 compared to the P1 treatment group on day 1. The L1 treatment gave significant results compared to the control group K1 and K2.The results of statistical analysis on PMN (Figures 11 and 12) showed that the P1 treatment group had a significant value of p = 0.37 with the K1 control group and the fifth day of the P2 treatment group had a significant significant value of p = 0.11 with the K2 control group on day 5. There was an increase in the L2 treatment group on the first day compared to the L1 day and also an increase in L2 day 5 compared to L1 day 1. All treatment groups were above the control group either K1 or K2.

Figure 9. Expression of IgA

Figure 10. Line chart of statistical analysis of IgA expression between groups

Figutr 11. Differences in the number of PMN cells (black arrows)

Figure 12. Line chart of Statistical analysis of PMN expression between groups

The results of statistical analysis on macrophages (Figures 13 and 14) showed that the P1 treatment group was significant compared to the K1 treatment group with a p-value = 0.46, while the P2 treatment group was significant compared to the K2 control group with a p-value = 0.007. In the L2 treatment group, it was significant compared to the K2 treatment group with a value of p = 0.018.In the L2 treatment group there was a decrease in the L1 treatment group and in the P2 treatment group there was an increase in the P1 treatment group and the K2 and K1 control groups (Figure 15). Periodontal disorders can be identified by complex signs by looking at the level of proinflammatory IL-1β.

Figure 13. Differences in the number of macrophage (red arrows)

Figure 14. Line chart of statistical analysis of Marcophage expression between group

Figure 15. Line chart of statistical analysis of Macrophage expression between groups

Proinflammatory IL-1β turns active and shows an increase in gingival tissue that has periodontitis²³. IL-1β is a group of first-order cytokines that play a role in the immune response which is an important mediator of the inflammatory response to strengthen the activation of Th lymphocytes by antigen-presenting cells (Antigen Presenting Cells = APCs). The amount of IL-1β will increase in various events of the immune response, including antibody secretion, fibroblast proliferation, bone resorption, and inflammatory responses such as gingivitis and periodontitis.In this study, it was found that the expression of IL-1β in all treatment groups on day 1 and day 5 was above the control group, indicating an increase in IL-1β. Even though there was a decrease in IL-β expression in the treatment group, it was still above the control group. This indicates that the pro-inflammatory factor is still ongoing and still high.On the first day of treatment, there was a difference between the control group and the running treatment with significant results, namely an increase in IL-1β which indicated that there were markers of periodontal abnormalities due to stress from running. The predator on day 5 also showed significant results compared to the control on the fifth day.

This shows that the IL-1β factor was most dominant in the running group on the first day and the predator group on the fifth day. The presence of IL-1β expression as a pro-inflammatory in the treatment group can result in the widening of the periodontal tissue which directly affects a periodontal disorder. According to Martinez's research, 2021 The highest value of alveolar bone loss occurred in the periodontitis and chronic mild stress group showing the expression of IL-1β as one of the highest pro-inflammatory mediators²⁴. Likewise, in Julie T's research, in 2020, it was stated that there was an increase in IL-1β expression in periodontitis gingival tissue. The cytokine response is complex and a variety of proinflammatory cytokines, one of which is IL-6, is produced. These cytokines act in a coordinated network to precisely regulate the host response²⁵.

The role of IL-6 is very important in the dysbiotic response of the host in the development of periodontitis²⁶, it also acts as an indicator of periodontal disease²⁷. In the proinflammatory factor IL-6, an increase occurred in the running group but a decrease occurred in the predator group. It can be explained in this case that the experimental animals were trying to adapt to the psychological stress obtained from predators. However, all treatment groups, both running and predators, are still under the control group, which means that inflammation is still ongoing and has the potential for periodontal widening which will result in periodontitis. IL-6 expression was below the control group. It could be explained that IL-6 is pro-inflammatory, but this cytokine also has regenerative and anti-inflammatory activity. Biofilm formation also expresses several proteins that have the ability to block complement activation so that the production of pro-inflammatory proteins also decreases. This anti-inflammatory activity may occur depending on the level of concentration and the concomitant presence of other inflammatory cytokines^28,29. According to Hedge's study, 2018, individuals with periodontitis experienced increased systemic acute phase protein levels, plasma antibody levels, coagulation factors, total white blood cell counts, neutrophils, reactive protein C, and cytokines such as IL-6³⁰. IL-10 is an anti-inflammatory cytokine that is a response to M2. Expression of IL-10 as an anti-inflammatory will result in an increase in macrophages³¹. Both running and predator treatment groups on the first and fifth days were under the control group. The occurrence of a decrease in IL-10 was significantly associated with an increased risk of periodontitis^32-34. There was a significant decrease in IL-10 in the moderate predator treatment group in the running group on the first day under the control group then an increase occurred.

This can be explained by the psychological stress generated by the running group had begun to adapt, at certain times IL-10 showed a response towards the end of the inflammatory phase with increased IL-10. The predator group has the potential for widening the periodontal tissue because the expression of IL-10 as an anti-inflammatory continues to decrease. As in the Elyased study, 2020 inflammatory mediators in periodontitis cases can cause marked damage to the entire periodontium. The inflammatory process is accompanied by regulatory cytokines and also increased production of IL-10 cytokines³⁵. In accordance with Lu Sun's research, 2020 found that periodontal tissue with low IL-10 showed significantly more severe clinical conditions³⁶. IgG expression in this study in the predator group and the running group were under the control group. This decrease in the secretion of IgG expression is a decrease in the immune system. In the predator treatment, the decline continued on the fifth day, while in the running treatment, there was an increase, but it was still under control. This indicated that in the predator treatment, the immune system was severely compromised and there was the potential for periodontitis to occur, as well as in the fatigue treatment, even though improvements had been seen. adaptation to stress.

A decrease in antibodies occurs associated with the degree of severity of a disease. Tissue defense in the oral cavity occurs by involving the local production of IgG in inflamed tissues as a local defense in the gingiva. Antibody capacity has a destructive capacity through hypersensitivity reactions until gingivitis occurs which can progress to periodontitis.Any type of stress, physical or neurogenic causes an increase in cortisol secretion and then decreases migration of white blood cells to the inflamed area and phagocytosis of damaged cells. It suppresses the immune system by decreasing the secretory expression of IgA and IgG and causes lymphocyte reproduction to be greatly decreased³⁷. Inhibition of secretory IgA, IgG, and neutrophil function, can damage the defense against infection and produce uncontrolled bacterial proliferation that causes periodontal disease³⁸. In accordance with the study of Jt Marchesan, 2021 which stated that low levels of IgG antibodies indicate failure to increase a sustainable immune response against periodontal pathogens³⁹. The first line of defense for the oral cavity is tissue starting from IgA. The secretory component IgA in saliva has a role as protection against microbes in the oral cavity. Decreased immunity coupled with the presence of microbes will exacerbate the periodontal condition with a sharp decrease in ribs in the predator treatment, while in the running treatment, it appears that an adaptive response has occurred resulting in an increase in IgA although it is still below the control group.This shows that when stress causes a decrease in IgA. Psychological stress is carried out continuously which results in a decrease in immunity with an impact on the widening of the periodontal tissue which can manifest as periodontitis.

The relationship between the response of IgA and stressors is not linear, because IgA increases in response to relaxation and stressors. Acute stress has the potential to increase the immune system as an adaptive response, while chronic stress results in decreased immune function, associated with high susceptibility⁴⁰. As A Piasetka's study, 2023 stated a decrease in the IgA and IgG concentration index values with a deepening of the stress reaction tends to be more pronounced in patients with chronic general periodontitis⁴¹. The increase in PMN was evident in the predator group (fear), although the running group (fatigue) also experienced an increase above that of the control group. The release of PMNs by these proinflammatory cytokines results in the widening of the periodontal tissue into periodontitis.Release of inflammatory mediators namely macrophages and PMN, PMN neutrophils are at the site of infection and use three main ways to fight microbes including phagocytosis, degranulation, and neutrophil extracellular trapping⁴². During periodontitis, host cells release proinflammatory cytokines against pathogens in the gingival sulcus that stimulate infiltration of polymorphonuclear leucocytes (PMNs), this is the first line of host cell defense. The exacerbated inflammatory response to bacterial plaque leads to the release of reactive oxygen species (ROS) such as hydrogen peroxide and superoxide from leukocytes which, together with an imbalance of antioxidant defenses, results in oxidative stress and apoptosis of the periodontal connective tissue⁴³. According to Elena, 2018 PMN numbers are higher, more apoptotic, and have an increased level of degranulation markers in periodontitis⁴⁴. The running group and the predator treatment group experienced a decrease in the number of macrophages. The decrease in macrophages is a good thing, because the running group and the predator group adapted to the treatment they received, thereby minimizing the occurrence of periodontitis. Because macrophages apart from functioning as phagocytosis can play a role in conveying antigens to lymphocytes to work together in the immune system.

The classic phagocyte-activating cytokines are recent evidence showing that T-helper 17/interleukin-17, alone or in conjunction with proinflammatory cytokines and T-helper 1, mobilizes macrophages and neutrophils, which may also contribute to exacerbating the host response in the periodontal tissues⁴³. The ratio of M1 and M2 provides information related to the health of the periodontal tissues, an increased ratio of M1 and M2 can be a cause of alveolar bone resorption, while a decreased ratio of M1 and M2 can inhibit bone resorption^45,46. The macrophage infiltration process is generally seen on the 5th day after treatment, this is because it takes a 48 to 72-hour delay after monocytes move or migrate to the tissue, to differentiate into macrophages in the injured area. stated that macrophage activation has important consequences in inflammatory periodontitis⁴⁷. The field of psycho-neuroimmunology has demonstrated that stress-related disorders have increased the expression of systemic inflammatory burdens. The increased inflammatory burden and its co-factors remain relevant measures in the risk assessment of periodontal disease⁴⁸. Cortisol responsiveness is an important determinant of stress although the response of cortisol to stress or to adrenocorticotropin varies widely. The results of Cortisol on the first day showed an increase, especially in the Running treatment as well as in the predator treatment, this resulted in the severity of periodontal abnormalities. In the running treatment on the fifth day, reactive coping was obtained resulting in a decrease in the hormone cortisol. This difference in susceptibility occurs in the stress treatment by predators where there is still an increase in the hormone cortisol so the chance of exacerbating periodontal disease is still very large. In accordance with Joae's study, in 2018 aggressive periodontitis had higher salivary cortisol levels than healthy or patients with chronic periodontitis⁴⁹.

Overall examination showed the description of the effect of psychological stress on periodontal disorders in Wistar rats. The highest expression of IL-1β was on the first day of running (L1). The expression of IL-1β in the predator treatment (P1) on the first day also shows a high value, the role of IL-1β as the main biomarker of the effects of chronic stress is by increasing IL-1β converting this inflammatory signal into a nerve signal and with the endocrine system (melatonin and cortisol) to compensate. This increase in lL-1β occurs mainly when individuals succumb to stressful events ²¹. This shows that the rats on the first day experienced changes in activity patterns that caused stress effects both from running and predators and then showed a decrease on the fifth day due to the adaptation process. Although it is known that the stress response is different for each individual.

CONCLUSION:

In conclusion, there was an increase in IL-1β, IL-6, PMN, Macrophage, and cortisol while a decrease in IL-10, IgG, and Ig A, as well as widening of the periodontal tissue under stress conditions as a marker of periodontal abnormalities in Wistar rats which showed a major role in IL-1β, IL-6, IL-10, Ig G, Ig A, PMN, Macrophage, and Cortisol on psychological stress mechanisms in periodontal disorders.It is necessary to carry out further research with primate animals to clinical trials, using gene transcription techniques (qPCR) that support immunohistochemical techniques, more in-depth research by looking at time series, and to look at the biomolecular components of stress and changes in the pathophysiology of periodontal disorders.

ACKNOWLEDGMENTS:

This study has been supported and discussed with the teaching staff of the military dentistry doctoral program at Padjadjaran University, Bandung, Indonesia. We would like to thank and appreciate the University of Padjadjaran, Bandung, Indonesia, and the Defense Ministry of the Republic of Indonesia for supporting the Ph.D. program at Padjadjaran University in Bandung.

CONFLICT OF INTEREST:

The authors confirm that this article's content has no conflicts.

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Received on 14.06.2023 Revised on 03.04.2024

Accepted on 17.09.2024 Published on 24.12.2024

Available online from December 27, 2024

Research J. Pharmacy and Technology. 2024;17(12):5729-5737.

DOI: 10.52711/0974-360X.2024.00872