INTRODUCTION:

Periodontitis is an inflammatory disease of the oral cavity that specifically occurs in periodontal tissue including the gingiva, periodontal ligament, cementum, and alveolar bone. Periodontitis has been linked to cardiovascular diseases, diabetes mellitus, obesity, etc.^1,2,3

The etiology of this disease is initiated by pathogenic bacterial invasions especially gram-negative types such as Porphyromonas gingivalis (P. gingivalis), Tannerella forsythia, and Prevotella intermedia.^4,5The condition is then exacerbated by the excessive inflammatory response, which is believed to be a major cause of periodontal tissue damage.^6,7

Pro-inflammatory mediators, such as cytokines, prostaglandins and metalloproteinase enzymes, are reported to have a role in the pathogenesis of periodontitis.^8,9,10 These mediators are used as biomarkers and therapeutic targets for periodontitis.^11,12 Synthetic antibiotic or anti-inflammatory drugs, combined with standard non-surgical therapies, are used to suppress those mediators. However, these drugs cause several side effects such as bacterial resistance, digestive disorders, kidney disorders, cardiovascular disorders, and tooth discoloration.^13,14,15

The Metalloproteinase-8 (MMP-8) enzyme is one of the pro-inflammatory mediators reported to have an important role in the development of periodontitis. In periodontitis, neutrophils and surrounding cells produce and release large amounts of the MMP-8 enzyme. This results in excessive destruction of type 1 collagen, which is the main structure of the extracellular matrix in periodontal tissue.¹⁶ Doxycycline is a tetracycline group that has an MMP-8 inhibitor effect. The use of low-dose doxycycline has the ability to prevent collagen breakdown and alveolar bone loss in animals under laboratory conditions. Doxycycline is also able to reduce both pocket depth and attachment loss in clinical trials. However, doxycycline has side effects on the development of bones and teeth in children as well as premature infants.^14,17

Various attempts have been made to find safer medications by examining natural phytochemicals isolated from plants that are easily found while having minimal side effects. Eleutherine americana Merr is one of the medicinal plants that is widely used by Kalimantan residents as a natural remedy for various diseases.¹⁸ This plant has many regional names in Indonesia including tiwai onion, mecca onion, sabrang onion, and dayak onion. Eleutherine bulbs are used as a diuretic, laxative, dysentery, astringent, antipyretic and antiemetic drug.¹⁹

Scientific findings indicate that ethanol extracted from Eleutherine americana Merr. (=EAM) bulb has an anti-oxidant effect that is stronger than vitamin C.²⁰. EAM inhibits the growth of P. gingivalis bacteria²¹ and also has an immunomodulatory and anti-inflammatory effect. ^22,23,24 A study on the effect of Eleutherine on the expression of metalloproteinase-8 and type 1 collagen enzymes in periodontitis had not yet been reported. The aim of this study, therefore, was to observe the effect of Eleutherine americana Merr bulb ethanol extract on MMP-8 and type 1 collagen expression in rat periodontitis models.

MATERIAL AND METHODS:

Material:

Eleutherine americana Merr. bulb, Na-Cmc, Doxycycline, Ligature wire (American orthodontic), Scalpel blade no. 15 (One Med), ATCC 33277 (Oxoid Ltd. ), Staining kit (Skytech Lab, USA), Primary antibodies MMP-8 (Rabbit Monoclonal 1: 100; Abcam, Cambridge, MA) and primary antibodies Collagen 1A1(Goat polyclonal antibody rabbit 1: 100; Santa Cruz Biotechnology, Inc.), Light Microscope (Optilab), Software Image Raster (Optilab).

Animal and Groups:

The Wistar rats (Rattus novergicus), with a weight of 200-300g, were obtained from the Department of Pharmacology, Faculty of Medicine, Universitas Mulawarman, Samarinda, Indonesia. They were kept under the standard condition with water and food ad libitum at room temperature. The animal treatment procedure was approved by the Ethical Committee at for the Faculty of Medicine, Universitas Mulawarman, Samarinda, Indonesia. A total of thirty-six rats were randomly arranged into six groups (six rats per group). Group 1 was periodontitis rats treated with Na-CMC (Negative control), Group 2 was periodontitis rats treated with EAM 100mg/Kg Body weight (BW) /day, Group 3 was Periodontitis rats treated with EAM 250mg/Kg BW/day), Group 4 was periodontitis rats with EAM 500mg/kg BW/day, Group 5 was periodontitis rats treated with EAM1000mg/KgBW/day, and Group 6 was periodontitis rats with Doxycycline 0,72mg/day.

Preparation of Eleutherine americana Merr bulb extract:

The Eleutherine americana Merr bulbs used had a size of 0.5 - 2cm. The bulbs were dried and mashed. Extracts of 500g of Eleutherine americana Merr bulbs were macerated with ethanol solvent and the filtrate was filtered after 24 hours. The pulp was macerated again with ethanol solvent, with the extraction process being carried out for a maximum of 3 times. The filtrate obtained is combined and evaporated until a concentrated extract is obtained and dried with a water bath at 40⁰C.¹⁸

Preparation for P. gingivalis Suspension:

The ATCC 33277 (Oxoid TM) bacteria were cultured and made into a suspension, according to the Mc Farland 0.5 standard which was equivalent to 1.5 x 10⁸ CFU/ml. Furthermore, the suspension was injected into the subgingival labial area of the rat Incisors.^21,25

Periodontitis Rat Model:

The incisors were ligated with orthodontic wire ligature (American Orthodontic) in cervical incisors of rats that had been anesthetized with 0.1ml/200g ketamine. Furthermore, P. gingivalis suspension injection was performed on the subgingival of labial incisors of the rats²⁷ on days 1 and 4. Ligation was left for 14 days until periodontitis occurred. On the 15th day, the ligation was removed and treated with a drug preparation that was dissolved in 0.5% Natrium carboxymethylcellulosa (Na-CMC) once a day for 10 days. At the end of treatment, the animal overdosed with ketamine and the gingiva was remove carefully from animal jaw. The procedure given to utilize on the animals for the experiment was approved by the ethics committee of medical faculty of Mulawarman University.

Gingival Tissue Preparation:

The gingival tissue of labial incisor region was collected by surgical excision with a carbon steel scalpel blade no. 15 (One Med). Gingival tissue was then fixed with 10% formalin buffer for 48 hours. Immunohistochemical examination was then performed to observe the expression of MMP-8 and type 1 collagen.

Immunohistochemical assay:

Immunohistochemical staining for observing MMP-8 and type 1 collagen expressions follow the procedure as describe previously.²⁶ Paraffin-embedded 3-µm tissue sections were deparaffinized with xylene and then rehydrated with ethanol. Immunostaining was then carried out using a staining kit, in alignment with the manufacturer's instructions. Primary antibodies used for staining include MMP-8 and type 1 Collagen (Col 1A1). Slides were visualized using diaminobenzidine (DAB) and counterstained with hematoxylin. Cells stained in brown are observed under a light microscope with 400x magnification. They are then counted and measured in 20 fields of view using the Image Raster software.

Statistical analysis:

All data are presented as mean±SD. Differences between groups were determined by one way ANOVA test, followed by post hoc test using Tukey HSD. All technical data processing results were analyzed by computerization using IBM SPSS Statistics 21, with a significance level of 0,05.

RESULTS:

MMP-8 expression in Gingiva:

The results showed that there were no significant differences in expression of MMP-8 in the gingiva between the Na-cmc groups (13.67±2.94), EAM100 groups (13.83±2.99) and EAM250 groups (13.00±3.68). MMP-8 expression was seen to decrease in the EAM500 group (8.33±1.63), EAM1000 group (7.5±1.87) and Doxy group (7.17±2.23).

Figure 1A. Expression of MMP-8 in each treatment group.

Results are expressed as the mean ± SD, a and b denote the difference annotations that imply significant difference (p<0,05). Abbreviations: Na-cmc, Natrium carboxymethylcellulosa ; EAM, Eleutherine americana Merr.; Doxycycline

MMP-8 expression in groups EAM500, EAM1000 and Doxy did not show significant differences. However, significant differences were seen in the Na-cmc group when compared to the EAM500, EAM1000 and Doxy groups with p-value <0.05. The MMP-8 expression EAM100 and EAM250 groups are also appeared to be significantly higher than EAM500, EAM1000 and Doxy (Figure 1A).

DISCUSSION:

The paradigm of conventional periodontitis therapy has shifted towards the development of host modulation therapy. The therapeutic goal is to prevent overproduction of pro-inflammatory mediators. Matrix metalloproteinase (MMP) -8 is a proteinase enzyme that is targeted due to its role in the progressive destruction of periodontal tissue in periodontitis. MMP-8 was made a biomarker by several researchers to assess its therapeutic effect on periodontitis.^27,28,29 Other researchers also developed a chairside MMP-8 test to diagnose and evaluate periodontitis therap.³⁰

MMP-8 (Collagenase-2) has a very low level under normal circumstances but increases significantly in chronic pathological processes that damage tissue. These enzymes are present as a family of zinc-dependent endopeptidases that are widely involved in various physiological and pathological processes. They play a role in bone growth and remodeling, wound healing, cancer and inflammatory diseases. The main source of MMP-8 comes from neutrophils but it is also produced by other inflammatory cells and resident cells in periodontal tissue. In periodontitis, the collagenase (MMP-1, MMP-8, and MMP-13) which is most dominantly induced is MMP-8 (collagenase 2). MMP-8 is stored in neutrophil specific granules. Neutrophils will release large amounts of MMP-8 when recruited to the inflammatory area.³¹

From the results of the study, an increase in gingival MMP-8 expression of periodontitis rat models was seen after 14 days. MMP-8 increased significantly in the administration of Na-CMC as negative control which showed no inhibitory effect on the material. However, the administration of doxycycline decreased MMP-8 expression (Figure 1A). Scientific evidence states that doxycycline is a metalloproteinase enzyme inhibitor.³² Doxycycline is able to down-regulate these enzymes through various mechanisms including inhibiting active MMPs by binding to Ca2+ and Zn2+, inhibiting activation of latent MMPs, inhibiting MMP and Reactive Oxygen Species. This serves to protect proteinase inhibitors, and thereby indirectly reducing the activity of tissue proteinase.^33,34 Administration of EAM at doses of 500mg/kg and 1000mg/kg showed a decrease in MMP-8 expression, which was not significantly different from the administration of doxycycline in periodontitis rats. This proves the effect of Eleutherine inhibitors on MMP-8. This is probably because EAM has the ability to inhibit the enzymes lipase and protease. The presence of secondary metabolites of polyphenols in Eleutherine, such as naphthoquinones, will form soluble protein complexes with high molecular weight so that they will react with enzymes in the cytoplasm and cell walls.³⁵

Collagen is the main protein constituent of the extracellular matrix which plays an important role in shaping the periodontal tissue architecture. Type 1 collagen is the main type found in periodontal tissue and is a marker of progression of periodontal disease when this collagen becomes degraded.³⁶ In this study, type 1 collagen showed decreased expression in periodontitis rats that were given Na-CMC. The results of the study are in accordance with the report of Eijel ³⁷, which stated that areas experiencing severe inflammation will undergo a collagen reduction of about 20%.

EAM treatment showed an increase in the expression of type 1 collagen, which increased according to the dose given (Figure 2A). Increased expression of type 1 collagen in the administration of EAM is probably due to the presence of Isoeleutherine secondary metabolites which can increase fibroblast proliferation and collagen synthesis through TGFβ-smad signaling pathway stimulation.³⁸Besides the decrease in MMP-8 expression in EAM, the administration can also affect the increase in expression of type 1 collagen. MMP-8 is a collagenase that has a threefold effect compared to collagenase 1 and collagenase 3 in initiating the outbreak of type 1 collagen so that the collagen fibrillar fragments will be easily destroyed by various other types of MMPs.³⁹ This is the first study to observe the expression of MMP-8 and type 1 collagen in periodontitis rats. This was done by administering an ethanol extract from the Eleutherine americana Merr bulb. Although it appears that EAM increases type 1 collagen production and inhibits MMP-8, further research is needed by measuring clinical parameters and the use of secondary metabolites that are thought to play a role in MMP-8 inhibition thereby strengthening the potential of EAM for use in the management of periodontitis.

CONCLUSION:

Eleutherine americana Merr. extract reduces MMP-8 expression and increases expression of type 1 collagen in periodontitis rats induced by ligature and P. gingivalis. The results indicate the potential of this plant for use in periodontitis therapy.

ACKNOWLEDGMENTS:

The authors would like to thank the Faculty of Medicine Universitas Mulawarman and Faculty of Medicine Universitas Brawijaya, for providing the laboratory facilities.

CONFLICT OF INTEREST:

The authors report no conflicts of interest in this study.

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Received on 24.09.2019 Modified on 10.11.2019

Research J. Pharm. and Tech 2020; 13(5):2407-2412.

DOI: 10.5958/0974-360X.2020.00432.1