Abstract
Objectives
The mucoadhesive gingival patch is a topical drug delivery process it does not cause any irritation in the mucosa. EGCG (Epigallocatechin-3-gallate) it has potent antioxidant, antiangiogenic and antitumor effects. The present study investigates the ability of mucoadhesive gingival patch loaded with EGCG on periodontitis and its impact on IL-6 and IL-10 expression.
Method
Periodontitis model was developed in Wistar rat by induction of Porphyromonas gingivalis. Application of mucoadhesive gingival patch loaded with EGCG (GP-EGCG), mucoadhesive gingival patch loaded with doxycycline (GP-doxy) and blank patch, was done for treated periodontitis 1 h each day during 21 days. Indirect immunohistochemical analysis of IL-6 and IL-10 expressions were analyzed in the mandibular preparation of the anterior incisive region of animal.
Results
The GP-EGCG treatment for 3 days until 21 days, consistently increased the IL-10 expression in periodontitis (p < 0.05). In other hand, GP-EGCG treatment lowered the IL-6 expression after 7, 14 and 21 days (p < 0.05).
Conclusion
The GP-EGCG is promising for the periodontitis treatment by interfere the IL-6 and IL-10 expression.
Keywords: EGCG, Disease, Gingival patch, Medicine, Mucoadhesive patch, Periodontitis
Graphical abstract
1. Introduction
Mucoadhesive gingival patch used as collateral method to increase the standard mechanical treatment of periodontitis with scaling and root planning.1 This process is accepted as a good technique for its prolonged period and holding of the medicine at the area of the application. Permeability of the mucosa was more at that point which allowed fast absorption of medicine.2 It should have some properties such as strength and sustained drug release which provides the best chance to achieve the goals.3
Inflammatory disease of the periodontium called periodontitis is an advanced form that is categorized by the loss of periodontal ligament and devastation of the surrounding alveolar bone.4 The periodontitis caused by a particular microorganism or group of microorganisms which result in gradual devastation of periodontal ligament and alveolar bone, increasing the probing depth formation and gingival recession. Tooth attachment loss, periodontal pocket formation was clinical sign of periodontitis, it changes the alveolar bone density and height. This is one of the most quickly spreading chronic inflammatory disease in humans. It is considered as the leading cause of tooth loss and one of the major threats of oral health.5,6 Severe periodontitis outcomes can lead to pain, discomfort, impaired mastication, and teeth loss.7
In addition to pathogenicity a vital role is played by host immune system in periodontitis. Innate and acquired immunity primarily work together to fight diseases and save tissue damage.8 Innate immunity is a non-specific immunity that loses the ability to differentiate between the pathogenic antigens and the body tissues, resulting in tissue damage in periodontitis. The innate immunity activates the acquired immune response, so it starts to determine and fight the pathogenic antigens releasing the inflammatory cytokines that activate a cell-mediated and humoral immune response. Damage to periodontal tissue is done by the continuous release of cytokines and destructive enzymes, exaggerating the damage of periodontal tissues.9
Interleukin 6 (IL-6) and interleukin 10 (IL-10) is a significant cytokine present in the pathogenesis of periodontal disease. IL-6 It is secreted by T cells and macrophages responsible for activating formation of neutrophils in the bone marrow as well as protein synthesis in acute phase. It is abundant in the inflammatory lesions of periodontal tissues. Due to the presence of soluble IL-6 receptor it activates fibroblasts.10 It was first distinguished in the supernatant of T-lymphocyte cultures and found to induce final maturation of B cell into immunoglobulin manufacturing plasma cell.11 IL-10 which play important role in regulation of pro inflammatory cytokine. It is an anti-inflammatory cytokine produce by, monocytes, T-reg cells and B-cells. IL-10 increase the Osteoprotegerin (OPG) regulation and decrease the IL-6 regulation by the molecular mechanism of inhibition.12 Bone loss can be stopped by IL-10 inhibition and IL-6 stimulation.13
Plants and herbs used as medicine for hundreds of years for the treatment of many diseases. Natural sources for preventing immunological complications always attracted researchers. Using herbal medicine may decrease complications.14 The most popular beverage consumed worldwide green tea, which is gained from the dried leaves of the plant Camellia sinensis.15 Due to its scientifically proven advantages, it got considered attention. It is assessed that production of green tea leaves each year is about 2.5 million tons, in which approximately 20% of production was green tea.16 It is used for many years in India, China, Japan, and Thailand. Green tea has been demonstrated to have many functional properties, and at present, its consumption is widely recommended. Its polyphenol Epigallocatechin- 3- gallate (EGCC) are the most important catechins.17
As compared to other drug administration roots, the safe delivery system is a mucoadhesive drug delivery system. It has many benefits compared to other administrative roots; using this way, we can control the release of dosage at targeted sites with the extended retention time of drugs at target sites. The important benefit of these systems includes avoiding the first phase of metabolism.2 The present study investigates the ability of mucoadhesive gingival patch loaded with EGCG (GP-ECGC) on periodontitis model induced by Porphyromonas gingivalis and its impact on IL-6 and IL-10 expression.
2. Material and Methods
Ethical approval
The approval of this was obtained by the Ethics Commission of the Faculty of Dental Medicine, Airlangga University, Surabaya Indonesia (Reference number 466/HRECC.FODM/X/2020).
2.1. Material
EGCG concentrate from green tea extract (Camellia sinensis) (Xi’An Rongsheng Biotechnology Co., Ltd., Shaanxi, China; polyethylene glycol (PEG) (Schuchardt OHG, Germany); PEG 4000 (SigmaAldrich, St. Louis, USA); sodium carboxymethyl cellulose (CMC-Na) (Teknis Indonesia); propylene glycol (PG) (Teknis Indonesia) and doxycycline (Kimia Pharma).
2.2. Experimental animal
This research was a laboratory experimental study using 45 healthy male Wistar rats (Rattus norvegicus) aged between five to six months having body weight of 250–300 g, characterized by energetic movement, and acclimatized for 1 week. The experimental animal was received by Animal Laboratory of the Faculty of Veterinary Medicine, Airlangga University. The sample size of experimental animal used in this study was determined by Lemeshow, 45 rats used in this study divided into three groups.18
2.3. EGCG preparation
The EGCG concentration <98% was prepared by combined with 80% of PEG 400 and 20% of PEG 4000.19
2.4. Preparation of blank patch
Mucoadhesive gingival patches (blank patch) preparation was done using the solvent casting technique. Primarily, patches were prepared using the CMC-Na. PG was used as a plasticizer to improve the performance and release characteristics of the patch.
The CMC-Na 0.6 g was dissolve in 30 ml of warm distilled water, then PG 1 g was added under continuous stirring to obtain a suitable viscosity dispersion. The mixture has poured into petri dishes, stored at 4 °C for 48 h to remove all the entrapped air bubbles, and finally oven-dried at 30 °C for 96 h. All patches were uniform, homogeneous and free from bubbles. Moreover, the patch's final thickness was 0.3 mm.
2.5. Preparation of GP-EGCG and GP-doxy
The GP-EGC and GP-doxy procedure similar with the blank patch preparation. The procedure begins with dissolved CMC-Na 0.6 g in 30 ml of warm distilled water with manual stirring. Then, EGCG (15 mg) or doxycycline (100 mg) was added into CMC-Na solution and continued stirring until it becomes a homogenous mixture. Then, PEG 1 g was added with continuous stirring until the researcher obtained a suitable viscosity dispersion. The mixtures were poured into petri dishes and dried in the oven at 50 °C for 168 h. All patches were uniform, homogeneous, and free from bubbles. Moreover, the patch's final thickness was 0.3 mm.
2.6. Porphyromonas gingivalis induce periodontitis
Porphyromonas gingivalis (ATCC 33 277) was produced in media containing tryptic soy broth (TSB) and then raised in anaerobic conditions for 18–24 h. Bacterial colonies were taken and transferred in 3 ml of BHI broth media then incubated at 37ᵒC for 18 h. The bacterial suspension was synchronized with a 0.5 McFarland standard (1010 CFU/ml) and taken with a micropipette and dropped on Mueller-Hinton agar media's surface and incubated for 24 h at 37ᵒC in under 5% CO2 anaerobic atmosphere.20,21
The periodontitis model achieved by injected Porphyromonas gingivalis (0.03 ml contain 1010 CFU) in PBS in the right and left mandibular gingival incisive sulcus of anterior teeth of animals once a day every two days during 14 days using a disposable syringe 0.5 cc.
2.7. Mucoadhesive gingival patch treatment
After the periodontitis develop, each group of animals received treatment after anaesthetized with 10% ketamine (dose 0.1 ml/100 g body weight) delivered intramuscular injection. Each group received GP-EGCG, GP-doxy or blank patch was placed in periodontitis for 1 h in a day. During this period, animals were sacrificed consecutive after received treatment for 3, 5, 7, 14 and 21. The 10% ketamine (dose 0.1 ml/100 g body weight) with intramuscular injection was used as anaesthesia agent. The mandibular bone and lower anterior incisive was biopsied.
2.8. IL-6 and IL-10 expression
The IL-6 expression was analyzed in the neutrophils using indirect immunohistochemical method with anti-IL-6 monoclonal antibodies (Biogear Indonesia) with light microscope with 400x magnification.
The IL-10 expression was analyzed in the lymphocyte using indirect immunohistochemical method with anti-IL-10 monoclonal antibodies (Biogear Indonesia) with light microscope with 400x magnification.
2.9. Statistical analysis
Data of IL-6 and IL-10 is shown in the form of mean ± standard deviation (X ± SD). SPSS (IBM, Chicago, IL, USA) version 25 was used for statistical analysis. Normality test was conducted using Kolmogorov Smirnov test, while the homogeneity test was using the Levane test. Two-way Analysis of Variance (two-way ANOVA) was conducted to find differences in the number of IL-6 and IL-10 showing statistically significant results. Two-way ANOVA test results which state there are differences, will then be done Tukey High Significant Difference (Tukey-HSD) test (p < 0.05).
3. Results
3.1. IL-6 expression
The immunohistochemistry of IL-6 was showed in the Fig. 1 (A, B, C). The GP-EGCG showed the lowest IL-6 expression, then followed by GP-doxy. The duration of treatment showed different IL-6 expression. The duration of treatment for 3 days showed the lower IL-6 expression in GP-EGCG compared GP-doxy (p = 0.039) but the treatment for 5 days showed no differences of IL-6 expression compared GP-doxy and blank patch (p = 0.183; p = 0.05) (Fig. 2). The duration of treatment for 7, 14 and 21 showed the lower IL-6 expression in GP-EGCG compared GP-doxy and blank patch ((p = 0.043; p = 0.004; p = 0.002; p = 0.001; 0.011; p = 0.001; respectively) (Fig. 2).
Fig. 1.
The immunohistochemistry of periodontal tissue with 400x magnification. IL-6 expression (A, B, C) and IL-10 (D, E, F).
Fig. 2.
The IL-6 expression.
3.2. IL-10 expression
The immunohistochemistry of IL-10 was showed in the Fig. 1 (D, E, F). The GP-EGCG showed the highest IL-10 expression, then followed by GP-doxy. The duration of treatment showed different IL-10 expression.
The duration of treatment for 3, 5, 7, 14 and 21 days showed the higher IL-10 expression in GP-EGCG compared GP-doxy and blank patch (p = 0.001; p = 0.001; p = 0.002; p = 0.001; p = 0.005; p = 0.001; p = 0.006; p = 0.001; p = 0.015 and p = 0.001) (Fig. 3).
Fig. 3.
The IL-10 expression.
4. Discussion
Interleukin-10 vanquish both macrophage and dendritic cell function, with antigen-show presenting cell function and the formation of proinflammatory cytokines. it is a broad-spectrum cytokine having anti-inflammatory properties. This can happen consequently in feedback regulation of T-helper 1 (Th1)-type and Th2-type responses. IL-10 antagonist plays major role in inhibitory or valuable vaccines against chronic infections or cancer. The ultimate leading role for IL-10 appears to be as an immunosuppressive cytokine with extensive anti-inflammatory properties, in particular by its inhibition of macrophage and DC function, with the development of pro-inflammatory cytokines. IL-6 is a pleiotropic cytokine that has important physiological effects on extensive range of functions such as endorsing B cell differentiation, T cell activation and inducing acute phase proteins.12
Herbal medicine has no side-effects therefore it is widely used as an alternative approach for treatment. It gained allot of importance as subsequent for chemical drugs. It has been demonstrated that green tea extract has antibacterial properties against Porphyromonas gingivalis bacteria it is most common pathogenic bacteria which causes periodontitis. The most popular beverage consumed worldwide green tea, obtained from the dried leaves of the plant Camellia sinensis.15 Due to its scientifically proven advantages, it got considered attention.16 Green tea has been demonstrated to have many functional properties, and at present, its consumption is widely recommended. Its polyphenol Epigallocatechin- 3- gallate are the most important catechins.17
The objective of the present study was to measure the number of inflammatory mediators' cytokines IL- 6, and anti-inflammatory mediators cytokines IL-10 in Wistar rats with periodontitis that was treated locally with GP-EGCG, GP-doxy and blank patch. Additionally, it was investigated whether there is significant variability in the levels of these mediators. As well as the significant difference between the treatment group, control positive and negative control groups.
IL-6 observed on all groups it can revealed that significant differences is among control negative group and positive control group, treatment group and control negative group, positive control group and treatment group, In days there were significant differences between days 3 and day 5,7,14 and 21. Day 5 and day 7,14, and 21. But no significant differences between day 7 and day 14, day 7 and day 21, day 14 and day 21.
IL-10 observed on the whole observation it can revealed that there are significant differences between control negative group and positive control group, treatment group and control negative group, treatment group and control negative group, positive control group and treatment group. It is observed in the results of this study that number for IL-10 increased the number of cytokines counted on day 3, 5, 7, 14 and 21. They increased in all groups getting treatment. But on the other side, the number of IL-6 start to decreasing in all groups with the passage of time from day to day 3 to day 5, 7, 14 and 21.
IL-1 β, IL-6, IL-8, prostaglandin-E2 (PGE2), TNF- α, and many others that modify innate and adaptive immune responses were detected immediately at infected periodontal sites.22 Studies done Previously tell us about the application of green tea extract gel used topically help to decrease the infection of periodontium, that give idea that the formulation could be used for periodontal treatment as an adjuvant.
In a silico study we use EGCG compounds as a ligand the formulas obtained from Pub Chem database. Targeted compounds of 3D structures undergoes energy minimization at PyRx the purpose to stable the structure of molecules and get the data bank.23 Proteins marked in this study involved Sclerostin, TRAP, Rank-rank, Osteocalcin, NFATC1, Osterix, and RUNX2, these proteins were obtained from the RCSB PDB database. The 3D structure was downloaded in the pdb protein format and then sterilized the water molecules and native ligand through the PyMol software.24,25 When EGCG binding occurs in NFACT with Sclerostin TRAP, Rank-rank Molecular it has inhibitory affects.25
After providing EGCG, the biomolecular mechanism of periodontitis tissue regeneration exposed to Porphyromonas gingivalis bacteria was retained. The mucoadhesive gingival patch has been introduced as loaded with EGCG, and it has been discovered to be a viable technique for periodontitis therapy. Many references discuss the use of EGCG. However, the mucoadhesive model for periodontitis has yet to be tested. As a result, we used this model to demonstrate, and this point is the study's originality.
4.1. Limitation of the study
The most significant limitation to this study is the sample size and dose, both of which could be expanded in future investigations. As our study conforms to 3,5,7,14, and 21 days for trails, the frequency of different days can be seen.
5. Conclusion
The GP-EGCG treatment for 3 days until 21 days, consistently increased the IL-10 expression in periodontitis. In other hand, GP-EGCG treatment lowered the Il-6 expression after 7, 14 and 21 days. The GP-EGCG is promising for the periodontitis treatment by interfere the IL-6 and IL-10 expression.
Source of funding
The department of dentistry at the University of Airlangga in Surabaya, Indonesia, scrutinized and provided both ethical approval and finance. The Directorate General of Higher Education, Ministry of Education, Culture, Research and Technology, Indonesia, funded this research under the grant number 275/NU3/2021.
Declaration of competing interest
All author declares no conflict of interest.
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