The decreasing of NF κ B level in gingival junctional epithelium of rat exposed to Porphyromonas gingivalis with application of % curcumin on gingival sulcus

Background: Periodontal disease is a chronic, multi-factorial disease. Chronic periodontitis is one of the main causes of tooth loss. Chronic periodontitis is usually caused by Porphyromonas gingivalis (P. gingivalis). P. gingivalis can induce NF κ B activation resulting in the increasing of periodontal extracellular matrix degradation. Curcumin can inhibit NF κ B activation and reduce the severity of periodontal degradation. Purpose: This research was aimed to observe level of NF κ B in gingival junctional epithelium of rat exposed to Porphyromonas gingivalis with local administration of curcumin. Methods: Sixteen Wistar rat were divided into two groups. Group 1 (treatment) consisted of eight rat given 2 x 10 6 CFU/ml P. gingivalis and 1% curcumin. Meanwhile, group 2 (control) consisted of eight rat given 2 x 10 6 CFU/ml P. gingivalis only. GCF samples were collected from gingival sulcus. The samples were biochemically analyzed with ELISA method. Data were then analyzed statistically by using independent t-test ( α =0.05). results: The examination of NF κ B level showed that there was significant difference between treatment group and control group (p<0.05). The level of NF κ B in the treatment group was significantly lower than the control group. Conclusion: It can be concluded that 1% curcumin application can reduce NF κ B level in gingival junctional epithelium of rat exposed to P. gingivalis.

Periodontal disease is irreversible so that if there is no optimal treatment, most patients will potentially have lifelong disorder.However, this disease is likely painly, so people are not aware, as a result, it is generally found in the advanced level. 1 Chronic periodontitis is one of the types of periodontal disease widely spread in community.Porphyromonas gingivalis (Pg) bacteria are a major cause of this disease. 2In person with poor oral hygiene, the presence of Pg bacteria in the gingival sulcus can potentially damage junctional epithelium, causing periodontitis characterized by loss of attachment.
Loss of attachment to the junctional epithelium may be caused by the invasion of Pg bacteria.Pg bacteria causing chronic periodontitis can produce certain endotoxin, namely LPS and proteases, that can activate neutrophils and macrophages, which then will activate the transcription factor of nuclear factor-kappa B (NFκB).The activation of NFκB can trigger transcription and translation processes producing mediators that play a role in inflammation process. 3A research in rat showed that LPS in 10 days can cause inflammation in periodontal tissue. 4Inflammatory mediators in the optimal number actually can serve to neutralize antigen.If the number of inflammatory mediators produced are excessive, the extracellular matrix

Dental Journal (Majalah Kedokteran Gigi)
205 March; 48(): 5-8 will be damaged, so the periodontal tissues then can be damaged. 5arious attempts have been made to treat chronic periodontitis, such as by scaling and root planning as well as by giving antibiotics and anti-inflammatory either locally or systemically.Anti-inflammatory is required in the treatment of chronic periodontitis since it can neutralize the effects caused by the increasing of pro-inflammatory mediators.Anti-inflammatory commonly used in the treatment of chronic periodontitis is administered systemically.Systemic administration has few adverse side effects.Therefore, it needs proper efforts that are effective, efficient and safe for treating this disease.One of those is by using herbal ingredients, which have medicinal properties and can be administered locally, such as turmeric (Curcuma longa). 6he main active ingredient contained in turmeric is curcuminoid which main content is curcumin.Curcumin has useful properties, such as anti-inflammatory, antioxidant, antibacterial, antitumor and antihepatotoxic.Curcumin is also considered as a potent inhibitor of transcription factor NFκB. 6,7 Nevertheless, the role of curcumin against NFκB associated with periodontal disease, especially chronic periodontitis with indicators taken from GCF has never been proven.
Based on those facts, curcumin is expected to reduce the degree of inflammation in the gingival junctional epithelium caused by bacterial Pg invasion.Curcumin is also expected to inhibit the increasing of NFκB level so that pro-inflammatory mediators produced by NFκB that cause periodontitis can be decreased.The purpose of this research is to investigate NFκB level in gingival junctional epithelium of rat exposed to Porphyromonas gingivalis with local administration of curcumin.

materials and methods
This research can be considered as an in vivo experiment using male rat (Rattus norvegicus) Wistar aged 5-6 months old and weighed 250-300 grams.The choosing of the age was based on the fact that the size of the jaw and gingival of the rat in that age is big enough to be applied with the medicines.This research was conducted at Laboratory of Biology in Faculty of Natural Science and Mathematics, Laboratory of Microbiology, and Laboratory of Biochemistry in Universitas Brawijaya.The samples of this research were selected randomly with the sample size determined through trial.Based on a sample size formula the representative number of samples must be eight. 7urcumin powder was prepared in solution by using corn oil solvent to achieve a concentration of 1%.The concentration of 1% was determined based on a previous research conducted by Suhag. 9 Meanwhile, to stimulate periodontitis disease in those rat, Pg bacteria (ATCC 33 277) were given and grown in a medium containing tryptic soy broth (TSB).They were incubated in an anaerobic atmosphere for 24 hours.They were grown in blood agar containing 10% sheep blood, 0.4 mL/ml vitamin K1 and 5 mL/ml hemin and then put into an anaerobic incubator with a composition of 80% N 2 , 10% H 2, and 10% CO 2 for 24 hours at a temperature of 37 0 C. The largest colonies were transferred into liquid medium containing thioglicolat, and then incubated for 24 hours at 37 0 C in an anaerobic atmosphere.After PBS was given, spectrophotometry with 624 nm wavelength was used to make bacterial concentration as many as 1 x 10 6 CFU. 10 Rat used as experimental animals were adapted for 1 week before the research, by conditioning them in a cage with feeding (certain concentrate) and beverages (distilled water) according to standards of animal care in Biological Laboratory, Faculty of Natural Science and Mathematics, Universitas Brawijaya.Those rat were then locally given 0.03 ml of live Pg ATCC 33 277 with the concentration of 2 x 10 6 CFU/ ml in the gingival sulcus bottom of their left and right mesial incisor teeth.The same procedure was then repeated every three days for two weeks.This procedure was conducted to make the animals get periodontitis.In the treatment group, 1% curcumin in corn oil as much as 0.03 ml was administered locally in the same area as the provision of bacteria every day for two weeks to provide a therapeutic effect.Meanwhile, in the control group, only corn oil was administered every day for two weeks in the same area as the provision of bacteria.
NFκB level was measured on day 14 by using periopaper put into the gingival sulcus for 30 seconds to get gingival crevicular fluid (GCF). 11The examination was conducted two weeks after the treatment.Periodontitis can be occurred in the past 10 days. 4Samples derived from GCF (in periopaper) put into eppendorf tubes and stored at -80 0 C until all the samples were collected and ready for examination.After all the samples were collected from the GCF, then the preparation of eLISA examination was conducted.each eppendorf tube containing periopaper was added with 0.5ml, 0.02M PBS with pH 7.0 to 7.2 as the solvent, and then centrifugation was conducted at 3000 rpm for 20 minutes.Finally, the supernatant of all of those samples was taken for examination in accordance with eLISA procedure using specific rat monoclonal antibody for NFκB.results Table 1 and Figure 1 show that NFκB levels in both the control group (bacteria and corn oil) and the treatment group (bacteria and curcumin in corn oil) on day 14.The results of Kolmogorov-Smirnov Goodness of Fit Test showed the value of p was 0.954 (p>0.05).It indicates that the survey data were normally distributed.
Based on the analysis of independent t-test results between the treatment group and the control group, it is known that there were significant differences between the control group and the treatment group with p=0.000 (p<0.05).It is also showed that NFκB level in the treatment group given 1% curcumin was lower than that in the control group.

discussion
This research was conducted to find an alternative solution for healing chronic periodontitis.The data from previous researches indicate that oral hygiene is the main cause of pathogen bacteria accumulation.This accumulation of bacteria causes periodontal tissue damage characterized by loss of attachment and migration of gingival junctional epithelium to apical.The results of this research showed that the group of rat not given curcumin had higher NFκB level than the group given curcumin.It means that curcumin has an effect in inhibiting NFκB activity in gingival junctional epithelium cells of those rat.
Curcumin can inhibit the phosphorylation of IκB kinase, so the translocation of NFκB into the nucleus of the cell is inhibited, resulting the decreasing of NFκB activity so that the expression of pro-inflammatory cytokines is also decrased. 7The inhibition of the phosphorylation of the inhibitor factor of IκB kinase in the cytoplasm can occur because curcumin is lipophilic 13 so it could penetrate cell membranes and be located in either the cytoplasm or nucleus. 14urcumin can inhibit NFκB activation prior to its translocation into the cell nucleus.NFκB activation in patients with periodontitis has increased about 90%, while that in healthy tissue is only 30%.The expression of IκB as an NFκB inhibitor in patients with periodontitis is only 5%. 15urcumin as an anti-inflammatory is a potential inhibitor of NFκB.NFκB determines the expression of genes encoding cytokines, chemokine, growth factors, cell adhesion molecules, some acute protein phases, such as iNOS and COX-2, and receptors on the cell membrane, such as TLR. 16The activation of NFκB can be caused by a wide variety of triggers, such as bacteria, viruses, cytokines, free radicals and toxic environments.The activation of NFκB is associated with inflammatory process found in diseases, such as periodontitis, arthritis, atherosclerosis, and other degenerative diseases. 17On the other hand, the complex and persistent inhibition of NFκB is also connected directly with apoptosis, impaired immune cell development, and cell growth inhibition. 16hronic periodontitis disease is generally caused by Pg bacteria. 2The increasing of NFκB is generally caused by products produced by Pg bacteria, such as protease, LPS, and fimbriae. 18Protease produced by Pg bacteria can trigger a series of immune response.Arg-gingipain (RGP), protease produced by Pg bacteria, is a potential activator for protease-activated receptor-2 (PAR-2). 19It means that protease is capable of breaking peptide in N-terminal of PAR-2 molecule, resulting in a new amino acid (neo-ligand) binding to the trans-membrane domain triggering PAR-2 activation.PAR-2 will further stimulate signals to NFκB, so NFκB is activated and trans-located into the nucleus. 20n addition to proteases, LPS produced by Pg bacteria is also a potent stimulator to produce pro-inflammatory cytokines.It means that in periodontal tissue exposed to LPS, monocytes will be activated, and pro-inflammatory cytokines will be secreted.LPS may also activate receptors on the cell membrane, such as TLR-2, TLR-4 and TLR-7.The activation of TLR causes intracellular signals passing through MyD88 pathway that induces p38 MAPK and subsequently activates NF-κB. 21imbriae contained on live Pg can also be considered as an effective inducer that can activate NFκB due to signals from TLR-2.TLR-2 can enhance the activity of NFκB due to fimbriae of Pg bacteria. 21The fimbriae of Pg bacteria can stimulate TLR-2 which signals will further activate NFκB. 22n other words, the activation of NFκB is stimulated by TLR-2 signals more dominantly activating cytokines induced by TNF in acute responses of osteoclastogenesis process. 23The increasing of osteoclasts then will cause damage to the periodontal tissues.
In addition to extracellular signals, the increasing of NFκB level may also be caused by intracellular signals derived from virulence factor of the structure of Pg bacteria, ie peptidoglycan.Peptidoglycan, a bacterial cell wall, can be recognized by TLR-2 located on the cell membrane or by NOD1 and NOD2 located in the cytoplasm.This synergism between TLR and NOD-2 then can activate NFκB. 24 The focus of this research is to analyze the activity of the transcription factor NFκB used as a strategy for the prevention and treatment of periodontal disease, especially chronic periodontitis.From the results of this research, it is known that the administration of 1% curcumin can make NFκB level in the treatment group lower than that in the control group.This means that inflammation is the problem of tissue damage in chronic periodontitis disease, but its severity can be reduced by administration of curcumin.It can be concluded that 1% curcumin locally given in gingival sulcus can reduce the degree of inflammation in the periodontal tissues, especially gingival junctional epithelium, by decreasing NFκB level.
figure1.NFκB levels in the control group and the treatment group.

table 1 .
The mean and standard deviation levels of NFκB Figure1.NFκB level in the control group and the treatment group