INTRODUCTION:

One of society's most prevalent forms of periodontal disease is chronic periodontitis, with Porphyromonas gingivalis (Pg) bacteria as the primary causative agent. The main virulence factors of Pg bacteria are Lipopolysaccharide (LPS) and proteases, which can damage to the periodontal tissue, including gingiva, cementum, alveolar bone and periodontal ligament^1–3. Bacteria in the gingival sulcus in those with poor oral hygiene may potentially damage the junctional epithelium.

Junctional epithelium is part of the gingiva which attached to the tooth surface. It is considered as the first defence mechanism from bacterial attack and plays an important role in maintaining the health of the periodontal tissue by preventing the entry of microorganisms into the deeper periodontal tissues. Junctional epitheliumcomposed of epithelial cells. DAT cells are the epithelial cells that adhered to the surface of the tooth (directly attached to the tooth cells). DAT cells adhere to the tooth surface via an internal basal lamina in a hemidesmosome. Inflammation on the junctional epithelium can cause loss of attachment from DAT cells against the tooth surface, which can also cause the junctional epitheliummigration to the apical direction^4–6.

Matrix metalloproteinase (MMP) is a group of enzymes with zinc-dependent endopeptidase. MMP is an enzyme that plays a physiological and pathological role in the extracellular matrix. The most common type of MMP found in the junctional epithelium is MMP-7. MMP-7 is found among the supra basal cells of junctional epithelium, attached to the root surface of the tooth. Under normal conditions, the MMP-7 on junctional epithelium has several roles, including morphogenesis, cell migration, and repair. MMP-7 can degrade laminin, fibronectin, type IV collagen, gelatin, elastin, entactin, tenascin and proteoglycans⁷. MMP-7 also increases its activity after being triggered by the increase production of IL-17. In inflamed periodontal tissues, an increase in the production of IL-23 cytokines was found, which would trigger an increase in IL-17. This increase in IL-23 and IL-17 can potentially damage the periodontal tissue⁸.

Many methods have been used to treat periodontitis, including local and systemic antibiotics, periodontal curettage and periodontal flap surgery. Although the results are still unsatisfactory. Early prevention efforts are needed against the onset of periodontal disease. WHO also pays a special attention to the use of medicinal plants, which may have a lot of advantages, such as cheap, easy to obtain, and have low side effects.^9,10.

One of the most well-known medicinal plants is turmeric (Curcuma longa). Turmeric contains curcuminoids with curcumin as its main content and main active ingredient.¹¹. Curcumin has several main properties such, including anti-inflammatory, antioxidant, and anticancer.^12,13 Curcumin has the capacity to suppress NF-B, which can lower the expression of pro-inflammatory mediators involved in the migration of gingival junctional epithelium¹⁴. The purpose of this study was to observe changes in levels MMP-7 in the gingival junctional epithelium of periodontitis rats treated with 1% curcumin irrigation.

MATERIALS AND METHODS:

In this study, the experimental animals used were 20 weeks old, male Rattus norvegicus, with body weight of 325-350grams. Theses experimental rats were divided into a control group (Pg) consists of 24 rats, and a treatment group (Kur-Pg) consists of 24 rats. Each group was divided into 3 observation times (24hours, 7^th day and 14^th day), so that each group consisted of 8 periodontitis model rats.

Pg bacteria (ATCC 33277) was used in this study for periodontitis induction. Pg bacteria (ATCC 33277) were raised in media with tryptic soy broth (TSB), and cultivated for 24hours in an anaerobic environment, and then planted into a blood agar, which contains hemin and vitamin K. The blood agar was located in an anaerobic incubator for 24hours at 37⁰C. The biggest colonies were then placed into a liquid medium containing thioglycolate, where they were cultured for 24hours at 370C in an anaerobic environment. After that PBS was added, then with a wavelength spectrophotometry of 624nm, the concentration of bacteria was made as much as 1x10⁶ CFU. The Pg bacteria were given as much as 10⁶CFU in 30μl (0.03 ml). The PBS addition was carried out once every 3 days for 2 weeks with a micropipette.

Next, 1% curcumin is obtained by standardizing curcumin, which is available in powder form, taken and then weighed with a digital scale to obtain a weight of 1 gram, then put in 100ml corn oil (FFA < 0.08; SG = 0.915). The administration of 1% curcumin every 3 days for 2 weeks was given to the experimental groups using a micropipette in the gingival sulcus, proximal to the lower incisors of the treatment group, while the control group did not receive any curcumin administration. Observations for the control and treatment groups were carried out at 24hours, 7^th and 14^th days. Observations were made based on samples from GCF, using periopaper which was placed in the gingival sulcus. The gingival tissue in the proximal part of the lower incisors were then examined by the ELISA method. Statistical analysis was performed by T-test (α = 0.05).

RESULTS:

The results of the Games-Howell test for changes in MMP-7 activity from 24hours to day 7 gave almost the same pattern in the control and treatment groups, which is a significant increase (p <0.05). This result is different from the observation on day 7 to day 14. MMP-7 activity from day 7 to day 14 in the control group experienced a significant increase (p<0.05), whereas in the treatment group there was no significant change (p>0, 05). The mean difference in MMP-7 activity between the control and treatment groups was greater on the 14^th day of observation compared to the 7^th day (Figure 1). The inhibitory properties given by the curcumin to inhibit the increase of MMP-7 activity was greater in the 14-day period. The MMP-7 activity in the treatment group on day 14 was not significantly different from the control group of 1 day of administration, which means that curcumin administration for 14 days gave the best results in inhibiting the increase of MMP-7 activity in gingiva exposed to Pg bacteria.The results of assessing MMP-7 activity on the junctional epithelium in the control group and the treatment group are shown in Table 1 and Figure 1.

DISCUSSION:

Porphyromonas gingivalis (Pg) bacteria is the main cause of chronic periodontitis. Pg bacteria accumulate and multiply in the gingival sulcus. The presence of bacteria in the gingival sulcus is potentially damaging the junctional epithelium. Bacterial products that are closely related to the initiation and progression of periodontal disease are LPS and proteases. LPS triggers an inflammatory response through activation of the TLR2 membrane receptor which generates a signal for the activation of the transcription factor NF-κB, which is the main component of the inflammatory process. In the nucleus, NF-κB cells produce pro-inflammatory cytokines by co-stimulating transcription and translation processes of these cytokines, including: IL-1, IL-6 and TNF-α. If these cytokines increase in periodontal tissue, they will cause damage to cells and the extracellular matrix^15–17.

The junctional epithelium proliferates and migrates to the apical in periodontitis, causing damage to the connective tissue. MMP-7 is secreted by osteoclasts, macrophages and endothelial cells. It plays an important role in angiogenesis, cell invasion and inflammation. Excessive MMP production leads to accelerated matrix degradation in periodontitis and other pathological processes. As a result, inflammatory mediators like cytokines, prostaglandins, and reactive oxygen species are released, along with proteolytic enzymes such matrix metalloproteinases (MMPs). Additionally, MMPs activate proinflammatory mediators that are dormant, like membrane-bound TNF-α, IL-1b, and several other MMPs. Matrilysins predominantly affect laminin, fibrinogen, fibronectin, type IV collagen, and gelatin. MMP-7 has a wider range of collagen types I, III, V, IX, X, XI. The extracellular matrix of the periodontal tissue consists of collagen, non-collagenous proteins, and proteoglycans, which most of the constituents of this matrix are degraded by MMP-7. MMP-7 causes extracellular matrix remodeling and contributes to the etiology of periodontitis by destroying periodontal tissue.^18–20.

Curcumin is an extract derived from the turmeric plant. The main chemical content of turmeric is curcumin, which is an active ingredient that is widely used as a basic ingredient for making traditional medicines. Curcumin has anti-inflammatory properties and it has been shown to exhibit anti-inflammatory biological activity^13,21–27. In the previous study, 20 patients were reported with gingival enlargement accompanied by bleeding, which was found on 200 tooth surfaces. After being given 1% curcumin by gargling for 6 weeks, an improvement can be observed in the patient’s gingival condition. The degree of inflammation (redness), bleeding and pocket depth was significantly reduced. After 5 days of curcumin administration, there was a reduced gingival bleeding by up to 100%, while redness is reduced by 96%. Researchers also compared the effect of curcumin with a standard mouthwash for periodontal disease, namely 0.2% chlorhexidine. The results showed that curcumin reduced the degree of gingival inflammation more quickly than chlorhexidine. On the 3^rd day of administration, curcumin was shown to give a real improvement effect²⁸.

The anti-inflammatory potential of curcumin is mainly achieved through inhibition of the transcription factors NF-κB and AP-1²⁹. NFκB activation also determines MMP-7 activity. This means that when curcumin is administered, NFκB activation decreases. Curcumin then inhibits the phosphorylation of inhibitory factor I-B kinases that the translocation of NFκB into the cell nucleus is inhibited, thus, the transcriptional activity carried out by NFκB to express the MMP-7 gene is also inhibited. Inhibition to its expression will surely decrease its activity as well, compared to no inhibition at all.

In this study, the MMP-7 activity in the control group showed a significant increase at 24 hours, 7 days and 14 days of observation. This data shows that MMP-7 plays a role in the mucosal immune response. MMP-7 expression increases rapidly in injured epithelium where exposure to Pg bacteria in junctional epithelium gingiva stimulates and activates NFκB which in turn will express MMP-7³⁰.

MMP-7 plays a role in the innate immunity and its activity increase was seen in the early phase of bacterial invasion. The results of the Kivela-Rajamaki study comparing MMP-7 levels in healthy periodontal tissues and those in peri-implantitis concluded that in inflamed periodontal tissues (peri-implantitis), MMP-7 levels which were taken from GCF samples showed higher levels (Kivela-Rajamaki et al., 2003a). In adult periodontitis and localized juvenile periodontitis patients, there is a slight increase in MMP-7 expression compared to healthy individuals. A significant increase was also seen in MMP-8. It was also stated that MMP-7 can function as an activator of pro-MMP-8 to become active MMP-8 (Tervahartiala et al., 2000). This condition can be interpreted that the damage to the periodontal tissue in periodontitis patients is caused more by the role of MMP-8, although indirectly it is also inseparable from the influence of MMP-7. Lundmark et al stated that the RNA on the gingival tissue biopsy of periodontitis patients showed a higher level of MMP-7 in the GCF compared to healthy patients³¹. In Eka Fitria Augustina's study, application of LPS resulted in inflammation of the junctional epithelium, resulting in a high expression of MMP-7. This result indicates that when there is inflammation, MMP-7 expression increases and plays a part in the mucosal immune response³².

In this study, curcumin administration can reduce MMP-7 activity in a 7-day period and curcumin's effect to inhibit the increase of MMP-7 activity was seen to be greater in the 14-day period of curcumin administration. The decrease in MMP-7 activity in the treatment group was related to the activity of curcumin which is known to inhibit histone acetyltransferase (HAT). Curcumin is a HAT p300/CBP inhibitor³³. If p300/CBP HAT activity on the specific site of the NFκB subunit p65 is inhibited, acetylation does not occur at p65 so that the gene expressed by NFκB is also inhibited. When the expression is inhibited, its activity will decrease as well.

The decrease in MMP-7 activity is also related to JNK and AP-1 activity. Pg bacterial invasion can cause JNK phosphorylation and activate transcription factor AP-1³⁴which can increase MMP-7 gene expression, while the transcriptional activity of genes regulated by AP-1 can be inhibited by curcumin. Therefore, MMP-7 activity significantly differed between the control and treatment groups since the observation at 7 days, as well as a decrease in MMP-7 activity for the treatment group at 14 days of observation due to inhibition by curcumin on the AP-1 transcription factor.

CONCLUSION:

In this study, there was an increase in MMP-7 levels in rats exposed to Pg bacteria. This suggests that MMP-7 acts as an inflammatory response in the gingiva exposed to Pg bacteria. Administration of 1% curcumin topically on the Junctional epithelium exposed to Pg bacteria can reduce the proteolytic activity of MMP-7 in periodontitis cases, which can inhibit the migration progression of the Junctional epithelium gingiva apically. This suggests that curcumin irrigation can reduce MMP-7 activity.

Table 1: MMP-7 Activities (u/L) on Gingiva for each Group and Time

Group	Time			t-test
Group	24 hours	7^th day	14^th day	t-test
Control	15.38± 2.125^a,b	22.76± 0.980^c	29.12± 1.669^d	p=0.001
Treatment	14.04± 1.185^a	18.32± 2.158^b	15.43± 3.739^a,b	p=0.001
Brown-Forsythe	p=0.001

Note: The same superscript shows no significant difference in each group (Games-Howell)

Figure 1: MMP-7 Activities (u/L) on Gingiva for each Group and Time

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Received on 30.01.2023 Modified on 13.05.2023

Research J. Pharm. and Tech 2024; 17(1):309-313.

DOI: 10.52711/0974-360X.2024.00048