The effect of sword bean extract on the relationship between rheumatoid arthritis and periodontitis in mice

Objectives Several studies in humans and experimental animals have reported an interaction between rheumatoid arthritis (RA) and periodontitis (PD). We previously showed that extracts of Canavalia gladiata (sword bean extract, SBE) can treat PD in rats. Here, we investigated the relationship between RA and PD and the effects of SBE in an experimental mouse model. Methods Female SKG mice were assigned to eight groups (n=6/group): (1) Untreated controls, (2) RA (induced at 6 weeks of age), (3) PD (induced at 10 weeks of age), (4) RA + PD, (5) SBE (2 mg/ml in drinking water starting at 5 weeks of age), (6) PD + SBE, (7) RA + SBE, and (8) RA + PD + SBE. Mice were sacrificed at 13 weeks of age, and alveolar bone resorption, periodontal tissue inflammation, and paw joint inflammation were assessed by histology and immunohistochemistry. Results Mice in the RA + PD group exhibited significantly higher inflammation scores in the joint tissues as well as more abundant IL-17-positive cells and cathepsin K-positive osteoclasts in the radial bone compared with the RA mice. Alveolar bone resorption was also significantly more severe in the RA + PD mice than in the PD mice. SBE treatment significantly improved all bone resorption and tissue inflammation scores in mice with RA + PD. Conclusion Concomitant RA and PD exacerbates the tissue destruction symptomatic of each condition. SBE suppresses all parameters evaluated, suggesting that it is has anti-inflammatory activities in both RA and PD.


1 anti-inflammatory effects of SBE.
2 In the present study, we employed the SKG mouse model of RA to examine 3 the association between RA and ligature-induced PD, and investigated the 4 anti-inflammatory effect of SBE in mice with RA and/or PD.  2 RA and PD were induced at 6 and 10 weeks of age, respectively (See Methods for 3 details). Mice were provided free access to drinking water with or without SBE (2 4 mg/ml). All animals were sacrificed at 13 weeks of age.

6 Preparation of SBE
7 Sword beans were cultivated in Kagoshima, Japan and SBE was prepared by 8 Yoshitome Co., LTD. (Chiba, Japan). In brief, ground sword beans were mixed with 9 50% ethanol and filtered to remove the insoluble fraction. The extract was lyophilized 10 to a powder, which was reconstituted in water for use in experiments [17].

11
12 RA induction 13 Mannan was purchased from Sigma-Aldrich (Tokyo, Japan) and dissolved in 14 phosphate-buffered saline at 100 mg/ml. RA was induced when mice were 6 weeks old 15 by intraperitoneal injection of 200 μl of the mannan solution [18]. 16 17 PD induction 18 At 10 weeks of age, mice were anesthetized with pentobarbital sodium (50 19 mg/kg) and PD was induced by tying a 5-0 silk ligature (Alfresa Pharma, Osaka, Japan) 20 around the maxillary second molar on both sides (Kyoritsu Seiyaku, Tokyo, Japan). The 7 1 ligature was left in place until sacrifice 3 weeks later [19]. Paw swelling was assessed and scored by three examiners who were blinded 5 to the treatments at every weeks until the end of the experiment. Each paw was given a 6 visual score (0, no joint swelling; 0.1, swelling of one digit or toe joint; 0.5, mild 7 swelling of the wrist or ankle; and 1.0, severe swelling of the wrist or ankle) and the 8 four scores were summed to give the total score for each mouse [20]. 9 10 Morphometric analysis of alveolar bone loss 11 Using a digital camera system, the buccal surface of the maxillary posterior 12 dentition was recorded in a standardized manner using fixed reference points on the      Mice in the RA and RA + PD groups had more severe paw redness and 3 swelling than mice in the other groups (Fig 2A). By the end of the experiment (13 4 weeks of age), both symptoms were more severe in the RA + PD mice than in the RA 5 mice. However, SBE treatment reduced the paw redness and swelling in the RA + PD 6 mice at 13 weeks ( Fig 2B).  14 Inflammation scores were significantly higher for the RA + PD mice than 15 the RA mice, and significantly lower for the RA + PD + SBE mice than for the RA + 16 PD mice (Fig 4B). There was no significant difference between the inflammation scores 17 for the RA mice and the RA + SBE mice, although the scores tended to be lower for the 18 SBE-treated group (Fig 4B).    18 magnification (a, b, c, d, e, f, g, h) and ×40 magnification (a1, b1, c1, d1, e1, f1, g1, h1).
19 RA, rheumatoid arthritis; PD, periodontal disease; SBE, sword bean extract. T, teeth; 20 AB, alveolar bone, 13 1 2 IHC analysis of IL-17 3 Although very few IL-17-positive cells were detected around the radial bone 4 of mice in the control, PD, SBE, and PD + SBE groups (Figs 6a, b, e and f), they were 5 present in the RA, RA + SBE, and RA + PD + SBE groups (Figs 6c, g and h).
6 IL-17-positive cells were particularly abundant in mice in the RA + PD group (Fig 6d).     7 Among the various reports on the association between RA and PD, several have 8 implicated periodontal pathogenic bacteria such as P. gingivalis, Treponema denticola, 9 and Tannerella forsythia, the major PD-related species [23]. Since P. gingivalis can 10 produce peptidyl arginine deiminase, which citrullinates proteins, it has been suggested 11 to play an important role in the development of RA [24]. For example, P. gingivalis 12 infection exacerbates arthritis in a collagen antibody-induced mouse model [11] and in 13 another SKG mouse model of RA [25]. However, PD, but not P. gingivalis 14 colonization, was associated with arthritis activity in treatment-naïve arthritis patients 15 [13]. We believe that P. gingivalis is unlikely to be involved in the alveolar bone loss 16 reported in the current study because we induced PD by ligation. Indeed, our finding 17 that alveolar bone resorption and joint inflammation scores were significantly higher in 18 mice with RA and PD than in mice with either disease alone suggests that they interact 19 to induce inflammation without bacterial infection. In the present study, SBE 20 suppressed bone resorption, periodontal tissue inflammation, and joint inflammation in 1 and activation may have contributed to the bone resorption observed in the present 2 study. Moreover, SBE-mediated suppression of the periodontal inflammatory response 3 may also have dampened the RA symptoms. Although we do not know the mechanism 4 by which SBE inhibits IL-17 production or osteoclast differentiation, our pilot study 5 strongly suggests that SBE inhibits proinflammatory cytokine production. 9 In the present study, we found that serum CRP levels were higher in mice with RA and 10 PD compared with mice with RA or PD alone. One possibility is that CRP is induced by 11 inflammatory cytokines, such as IL-6, that are produced locally in the periodontal tissue 9 demonstrated that epigallocatechin, which is contained in green tea, has synergistic 10 anti-cancer effects in combination with gallate and (-)-epicatechin Thus, synergism 11 between various components of SBE may be responsible for its anti-inflammatory 12 effects. In the future, we plan to investigate possible applications for SBE to prevent or 13 treat inflammatory diseases such as RA and PD. It will also be important to identify the 14 constituents of SBE to determine the mechanism of its anti-inflammatory effects.

17
In this study, we found that concomitant RA and PD exacerbates the 18 destruction of periodontal and joint tissue in a mouse model, suggesting an association 19 between the two diseases. Alveolar bone resorption and joint inflammation were both 20 significantly suppressed by treatment with SBE. Our preliminary data suggest that SBE 1 inhibits inflammatory cytokine production, providing a possible mechanism by which it 2 may suppress the interaction between RA and PD in vivo.

4 Acknowledgments 5
There is none.