Introduction
Periodontal disease, characterized by alveolar bone resorption and bacterial pathogen-induced inflammation, is a chronic inflammatory disease. It has a significant impact on oral health [1]. Bacteria in subgingival dental plaque (biofilm) are the major causative agent of periodontal disease. Porphyromonas gingivalis, is one of the key pathogens in periodontal destruction and is highly associated with periodontal disease development [2]. P. gingivalis induced the secretion of inflammation-inducing molecules, including interleukin (IL)-6, IL-8, prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, and matrix metalloproteinases (MMPs) and causes osteoclast formation in periodontal tissue [3]. Zhang et al. [4] observed that P. gingivalis invaded alveolar osteoblasts in a periodontitis mouse model. So, it is necessary to study de- velopment of materials inhibiting expression of these inflammatory factors and inhibitory effect of alveolar bone resorption. Development of the therapy has evolved from use of natural materials. Among these, Garcinia mangostana L. (mangosteen) and propolis have been shown to have anti-inflammatory and osteogenic effects in the wound healing process [5-7].
Propolis is a natural product produced by the honey bee. It possesses several biological properties, such as antioxidant and anti-inflammatory activities [6,8]. Previous research has shown that ethanol extract of propolis on mouse paw edema inhibited production of nitric oxide (NO) [9]. Additionally, bone mineral density was higher in rats that received oral propolis treatment [8]. Mangosteen extract contains abundant xanthones such as α, β, and γ-mangosteens [10]. Many biological properties such as anti-inflammatory, anti-oxidative damage and antioxidant activities from xanthones have been reported [11,12]. Previous studies have shown that α-mangosteen significantly inhibits NO, PGE2, tumor necrosis factor (TNF)-α, and iNOS production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells [3,13].
Animal models have been used to study pathogenesis of periodontal disease and to develop new treatment and prevention agents. Ligature-induced periodontitis model in rat has been frequently used in periodontitis-related research [14]. Ligature-induced periodontitis in rats is reliable and extensively used to reproduce periodontitis because periodontal anatomy in the molar is similar to that of humans [15]. The purpose of this study was to evaluate the effect of mangosteen extract complex (MEC; G. mangostana L. and propolis extract) on inhibition of inflammation and alveolar bone loss in ligatureinduced periodontitis model in rat.
Materials and Methods
1. Bacteria culture and extraction of lipopolysaccharide
P. gingivalis KCOM 2804 was used in this study, and was obtained from the Korean Collection for Oral Microbiology (KCOM; Gwangju, Korea). The strain was cultured in tryptic soy broth (TSA; BD Difco Laboratories, Franklin Lakes, NJ, USA) supplemented with 0.5% yeast extract, 0.05% cysteine HCl- H2O, 0.5 mg/mL hemin, and 2 μg/mL vitamin K1 at 37℃ in an anaerobic chamber (Bactron I; Sheldon Manufacturing Inc., Cornelius, OR, USA) with 10% H2, 5% CO2, 85% N2 gas condition. LPS of P. gingivalis KCOM 2804 was extracted, according to enclosed protocol using the LPS Extraction Kit (iNtRon, Seongnam, Korea). Extracted LPS was dissolved in sterilized water filtered with 0.22 μm membrane. Extracted LPS was lyophilized and weighed (P. gingivalis KCOM 2804 strain LPS extracted, was called pgLPS, and pgLPS is denoted by L).
2. Experimental animals
A total of 40 male Wistar rats (weighing 250–400 g) were used in the experiment. In this study, selection and management of the animals, surgical protocol, and preparation procedures, were approved by the Chosun University Institutional Animal Care and Use Committee (CIACUC2017-S0020).
The animals were randomly divided into four groups. The mangosteen complex extracts powder corresponding to each group, were suspended in water and taken into rats. Experimental groups were as follows:
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1) Non-ligation (Lig) group (negative control, n = 10): nonligation + distilled water (DW)
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2) Lig + L + DW group (positive control, n = 10): silk-ligation + pgLPS injection + DW
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3) Lig + L + MEC 0.5:34 group (n = 10): silk-ligation + pgLPS injection + 8 μg mangosteen extract powder and 544 μg propolis extract powder (0.5:34)
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4) Lig + L + MEC 1:34 group (n = 10): silk-ligation + pgLPS injection + 16 μg mangosteen extract powder and 544 μg propolis extract powder (1:34)
3. Silk-ligature-induced periodontitis rat model
The experiment was conducted for three weeks and all procedures were performed under anesthesia with intramuscular injection of Zoletil® (20 mg/kg; Virbac, Carros, France) and Rompun® (7.5 mg/kg; Bayer, Ansan, Korea). A 5-0 silk-ligature (0.5–0.6 mm in diameter) was fixed in the cervical part of the maxillary second molar and all ligatures were positioned subgingivally. After ligation, 1 μg/mL pgLPS diluted by phosphatebuffered saline (PBS) was injected into buccal and palatal gingiva of the ligated teeth to induce periodontitis. The pgLPS was reinjected under anesthesia after one and two weeks (Fig. 1).
In experimental groups, mangosteen extract powder and propolis extract powder were suspended in 0.1 mL of sterilized water and administered orally using a feeding needle. In the control groups, the same amount of DW was administered. At three weeks, rats were sacrificed by carbon dioxide inhalation. After sacrifice, gingival tissue around the ligated maxillary sec ond molar was obtained. It was stored at –80℃, for analysis of expression of specific inflammatory mediators. Remaining tissues were fixed with 10% formaldehyde for taking microcomputed tomography (micro-CT).
4. Expression of inflammatory cytokines in gingival tissues
To measure expression changes of inflammatory mediators (PGE2, IL-8, iNOS, MMP-8, COX-1, and COX-2) in gingival tissue, the ELISA kit of the Mybiosource Company (San Diego, CA, USA) was used, according to manufacturer’s instructions. Stored gingival tissue was homogenized with PBS and centrifuged to separate the supernatant. The supernatant was used and analyzed according to manufacturer’s instructions. Results are expressed as the mass (ng/mL, pg/mL) or unit (U/L) of each protein present in gingival tissue according to manufacturer’s instructions.
5. Micro-computed tomography imaging
Collected tissues were scanned with a Quantum GX micro- CT Imaging System (PerkinElmer, Hopkinton, MA, USA) for two minutes at an acceleration potential of 90 kVp, and a beam current of 88 μA. Micro-CT data were reconstructed and formatted using OnDemand3DTM (Cybermed, Seoul, Korea) software. Through this process, three-dimensional structures around the teeth and around the alveolar bone were evaluated in all directions and all widths. Distance between the cementoenamel junction (CEJ) and coronal plane of alveolar bone crest (ABC) was measured at six sites. It was measured at mesiobuccal, disto-buccal, mid-buccal, mesio-palatal, disto-palatal, and mid-palatal sites of the maxillary left and right second molars.
6. Statistical analysis
Data are presented, as average mean ± standard deviation. Statistical analysis was performed, using SPSS (version 12; SPSS Inc., Chicago, IL, USA). To compare the control and experimental groups, the Mann–Whitney U test was used. Statistical significance was determined by p ＜ 0.05 (95% confidence level).
Results
1. Expression of inflammatory cytokines in gingival tissuess
Expression level of inflammatory cytokines, such as PGE2, IL-8, iNOS, MMP-8, COX-1, and COX-2 in gingiva tissues had a tendency to increase in the group Lig + L + DW than that of group Non-Lig. In the group Lig + L + MEC 1:34, expression levels of PGE2, IL-8, iNOS, MMP-8, COX-1, and COX-2 were significantly decreased compared to the groups Non-Lig, Lig + L + DW, and Lig + L + MEC 0.5:34 (Fig. 2). Compared with the group Lig + L + DW, expression levels of PGE2, IL-8, iNOS, MMP-8, COX-1, and COX-2 in the group Lig + L + MEC 1:34 were significantly reduced and in particular, expression of iNOS was downregulated by 91.1% (p ＜ 0.05) (Fig. 2).
Considering these results, the group Lig + L + MEC 1:34 was most effective in inhibiting expression levels of PGE2, IL-8, iNOS, MMP-8, COX-1, and COX-2 in gingival tissues of ligature- induced periodontitis rats model. The inhibition of inflammatory cytokines in gingival tissues of rats model suggested that Lig + L + MEC 1:34 may be associated with decreased progression of periodontitis and inhibition of alveolar bone resorption.
2. Alveolar bone loss in ligature-induced periodontitis rats model
Alveolar bone loss was significantly increased in the group Lig + L + DW compared to the group Non-Lig. In contrast, it was reduced in the groups Lig + L + MEC 0.5:34 and Lig + L + MEC 1:34 compare to the group Lig + L + DW (Fig. 3). To evaluate degree of periodontal destruction, the distance between the CEJ and ABC was expressed as the amount of alveolar bone destruction. Distance from the CEJ to ABC in the groups Lig + L + MEC 0.5:34 and Lig + L + MEC 1:34 was significantly reduced by 4.2% and 13%, respectively, compared to the group Lig + L + DW (p ＜ 0.05) (Fig. 3). These data showed that MEC exhibited an inhibitory effect on alveolar bone loss by marked decrease of CEJ-ABC distances in rats by ligatureinduced periodontitis.
Discussion
Periodontitis, one of the most common diseases in adults, is an inflammatory disease caused by periodontal pathogens in plaque on the surface of teeth. These pathogens promote production of inflammatory cytokines such as PGE2, and cytokines IL-1, IL-6, IL-8, TNF-α, and MMPs resulting in bone destruction [16,17]. Results of this study showed that MEC was significantly inhibited expression levels of inflammatory mediators (PGE2, iNOS, MMP-8, IL-8, COX-1, and COX-2) in gingival tissues induced periodontitis by silk-ligature and injection of pgLPS. Especially, the expression PGE2 in the group Lig + L + MEC 1:34 markedly decreased than that in the group Non-Lig. In previous study, MEC 1:34 showed anti-inflammatory and in vitro bone formation effects in human gingival fibroblasts and human osteosarcoma cell lines (in submission). This result suggests that MEC 1:34 may alleviate the inflammatory state of rat by reducing the expression of PGE2 at the basic level. PGE2 is known to be an important inflammatory mediator causing alveolar bone destruction and osteoclast formation [18].
Degree of alveolar bone loss is one of the indicators of severity of periodontal disease [19]. Previous studies have shown that 500 μg/mL mangosteen extract did significantly inhibition, in LPS-induced bone destruction by osteoclast [20]. It has been reported that the group treated with 100 mg/kg propolis in rats with expanded premaxillary suture reduced alveolar bone loss and a number of osteoclast compared to the control group [21]. Micro-CT study showed significant reduction of alveolar bone loss (13%) in the group Lig + L + MEC 1:34. This study validates for the first time that MEC (mixture of mangosteen and propolis) reduced alveolar bone loss through inhibition of inflammatory responses in rats with ligature- and pgLPS-induced periodontitis. These results suggest that MEC could be useful in preventing and treating periodontitis.
