Equisetum arvense Inhibits Alveolar Bone Destruction in a Rat Model with Lipopolysaccharide (LPS)-Induced Periodontitis

Background and Aims Equisetum arvense extract (EA) exerts various biological effects, including anti-inflammatory activity. The effect of EA on alveolar bone destruction has not been reported; therefore, we aimed to determine whether EA could inhibit alveolar bone destruction associated with periodontitis in a rat model in which periodontitis was induced using lipopolysaccharide from Escherichia coli (E. coli-LPS). Methods Physiological saline or E. coli-LPS or E. coli-LPS/EA mixture was topically administered into the gingival sulcus of the upper molar region of the rats. After 3 days, periodontal tissues of the molar region were collected. Immunohistochemistry was performed for cathepsin K, receptor activator of NF-κB ligand (RANKL), and osteoprotegerin (OPG). The cathepsin K-positive osteoclasts along the alveolar bone margin were counted. EA effects on the expression of the factors regulating osteoclastogenesis in osteoblasts with E. coli-LPS-stimulation were also examined in vitro. Results Treatment with EA significantly reduced the number of osteoclasts by decreasing the RANKL-expression and increasing OPG-expression in the periodontal ligament in the treatment group compared to the E. coli-LPS group. The in vitro study showed that the upregulation of p-IκB kinase α and β (p-IKKα/β), p-NF-κB p65, TNF-α, interleukin-6, and RANKL and downregulation of semaphorin 3A (Sema3A), β-catenin, and OPG in the osteoblasts with E. coli-LPS-stimulation improved with EA-treatment. Conclusion These findings demonstrated that topical EA suppressed alveolar bone resorption in the rat model with E. coli-LPS-induced periodontitis by maintaining a balance in RANKL/OPG ratio via the pathways of NF-κB, Wnt/β-catenin, and Sema3A/Neuropilin-1. Therefore, EA possesses the potential to prevent bone destruction through inhibiting osteoclastogenesis attributed to cytokine burst under plaque accumulation.


Introduction
Periodontitis is an infammatory/infectious disease of the periodontal tissues caused by dental plaque at the interface between the teeth and the gingiva. Dental plaque contains bacterial components, such as lipopolysaccharide (LPS), which promotes osteoclast diferentiation through the production of proinfammatory factors, such as tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), and prostaglandin from the osteoblasts [1]. LPS promotes osteoclastogenesis not only indirectly through infammatory cytokine production from the osteoblasts but also directly through the production of the receptor activator of nuclear factor-κB ligand (RANKL), an osteoclastogenic factor. Moreover, LPS also suppresses the expression of osteoprotegerin (OPG), a decoy receptor for RANKL, thereby increasing the RANKL/ OPG ratio and fnally causing osteoclast formation and maturation, which consequently leads to bone resorption [2][3][4][5].
Hence, elimination of plaque accumulation, cytokine burst, overproduction of RANKL, and decreased production of OPG attributed to LPS are essential for preventing alveolar bone destruction associated with periodontitis.
Currently, plaque removal and the creation of an oral environment resistant to plaque adhesion form the basis of prevention and treatment of periodontitis [6]. Regular brushing is the most efective means of preventing periodontitis by maintaining good oral hygiene [7]. However, individuals who are disabled or bedridden tend to have some difculty in brushing satisfactorily and are prone to severe periodontitis. Terefore, the development of a supplementary oral care method to prevent the destruction of alveolar bone associated with periodontitis that can be easily applied by anyone daily in addition to brushing is desired.
Previously, by using a rat model of LPS-induced periodontitis, we confrmed that administration of EA into the gingival sulcus markedly reduced immunoexpression of TNF-α at the junctional epithelium [29]. TNF-α promotes the increase of osteoclast progenitor cells in vivo [30][31][32][33], and inhibition of human osteoclastogenesis in vitro by EA has also been reported [10,34]; therefore, we suggested that EA could inhibit alveolar bone destruction associated with periodontitis.
In this study, we aimed to clarify whether EA could inhibit alveolar bone destruction associated with periodontitis using a rat model in which periodontitis was induced using LPS from Escherichia coli (E. coli-LPS). Furthermore, we investigated whether EA is useful in the prevention and treatment of periodontal and other bone destructive diseases and also determined the mechanism of inhibitory efects of EA on bone destruction.

Animal Experiments.
Te experimental protocol described as follows was approved by the Animal Care Committee of Hiroshima University (Permit Number: A20-32). A total of 11, 8-week-old male Wistar rats weighing 330 ± 11.9 g (range: 315-355 g) (Charles River Japan, Inc., Yokohama, Japan) were housed in a specifcpathogen-free facility in 12 hour light-dark cycles with access to water and food ad libitum and kept at a constant ambient temperature and humidity (22°C, 50 ± 5% relative humidity).
Rats were fxed on their back on an experimental stand. After the experiment, all the rats were sacrifced using CO 2 gas.

Each solution (physiological saline [PS], E. coli-LPS [5 mg/mL], and E. coli-LPS [5 mg/mL]/EA [15 μg/mL])
were topically applied into the right or left gingival sulcus of the maxillary molars every 10 minutes for 1 hour (six times, 2 μL each time). Periodontal tissue samples were subsequently obtained on day 3, following topical application, for immunohistochemistry. In this model, an increase in the osteoclasts on the surface of the alveolar bone toward the periodontal ligament was confrmed at 3 days following LPS-application [36]. Tissue preparation was performed as previously described by Yamano et al. [37]. To count the number of osteoclasts formed along the alveolar bone surface, 4.5 μm sections, including the root from the alveolar crestal area to the root apex area, were prepared from the periodontal tissue around the frst and second molars and stained with hematoxylin and eosin (H&E) for routine histological evaluation.

Histomorphometrical Analysis of Number of Osteoclasts.
For the histomorphometric evaluation of the osteoclasts, more than 12 representative sections containing the root apex of the disto-palatal roots of the right and left upper frst and second molars from each experimental group were selected. Tissue sections stained with cathepsin K were photographed at 40× and cathepsin K-positive osteoclasts formed along the alveolar bone margin contained within 1 mm from the alveolar crest were counted manually ( Figure 1(a)) to evaluate the LPS-induced osteoclast formation. Te mean value of the frst and second molars was used as the number of osteoclasts per sample.

Statistical Analysis.
Results are reported as the mean-± standard deviation. Intergroup diferences were compared using the Tukey-Kramer multiple comparisons test, which was conducted using the multcomp package in R [39]. Statistical signifcance was set at p < 0.05 or p < 0.01 or p < 0.001.

Topical Administration of EA Decreases Osteoclasts in the Alveolar Bone.
Te inhibitory efect of EA on osteoclastogenesis was analyzed using the rat model of LPS-induced periodontitis, in which the time changes in tissue destruction, including neutrophil migration, osteoclastogenesis, and cytokine expression, are well established [40].
Osteoclasts are multinucleated cells that are diferentiated from monocytic and macrophage progenitors and are the only cells capable of resorbing calcifed bone. Cathepsin K is a papain-like cysteine protease member of the cathepsin family and is only expressed at high levels in osteoclasts. Cathepsin K is reportedly present in lysosomes and intracytoplasmic vesicles along the osteoclast-bone resorption interface in osteoclasts [41]. Figure 1(b) shows the immunolocalization of cathepsin K in each experimental group. In the PS-applied control group, a few cathepsin K positive   Figure 1(c) shows the number of cathepsin K-positive osteoclasts that appeared along the alveolar bone margin in an area 1 mm from the alveolar bone crest (Figure 1(a)). Te number of cathepsin K-positive osteoclasts signifcantly increased in the LPS group compared to the control group (p < 0.05). Conversely, EA application signifcantly reduced the LPS-induced osteoclasts to the level of the control group (p < 0.05).  (Figures 2(b) and E′). Interestingly, in the E. coli-LPS/EA group, the expression of OPG recovered and tended to be more enhanced than that of the control group (Figures 2(b) F and F′). Moreover, in the control group, the RANKL expression was strong in the upper part of the alveolar bone; however, OPG was also expressed in this area, and osteoclastogenesis was not induced since the balance in RANKL/OPG was maintained (Figures 2(a) D and D′).

EA Maintains a Balance in the RANKL/OPG Ratio in the
In the control group, the expression of RANKL is suppressed to a low level, and OPG is expressed (Figures 2(a) D and 2(b) D). In the LPS-administered group, RANKL levels in the periodontal ligament fbroblasts and osteoblasts have increased, but the expression of OPG has suppressed (Figures 2(a) E and 2(b) E). In contrast, in the E. coli-LPS/EA-administered group, RANKL is suppressed, while the OPG expression is restored, and the staining intensity is higher than that of the control expression (Figures 2(a) F and 2 Te results demonstrated that EA signifcantly inhibited TNF-α mRNA expression attributed to LPS stimulation (Figure 3(a) A) and revealed the inhibitory tendency of RANKL, IL-6, and IL-1β mRNA expression (Figures 3(a) B-D). Subsequently, we examined the efects of EA on E. coli-LPS induced RANKL protein secretion in ST2 cells by the enzyme-linked immunoassay (ELISA) system. Te results showed that EA signifcantly suppressed the RANKL protein secretion (Figure 3(b) A). Moreover, EA signifcantly increased the OPG secretion reduced by LPS stimulation (Figure 3(b) B).

EA Inhibited LPS-Induced Phosphorylation of IKK, NF-κB in ST2.
Nuclear factor-κB (NF-κB) and MAPKs play important roles in infammatory cytokine production caused by LPS-TLR4 signaling [42,43]. Since our results showed that EA reduced LPS-stimulated expression of infammatory cytokines, we suggest that EA may also inhibit phosphorylation of NF-κB and MAPKs; c-JunN-terminal kinase (JNK) and p38, which are upstream of infammatory cytokines. We investigated the efects of EA on these signaling pathways caused by E. coli-LPS (1 μg/mL) by Western blotting. Te time course analysis showed that E. coli-LPS  (Figure 4(a)). Subsequently, the efects of EA on the phosphorylation of IkB kinase alpha, IkB kinase beta (IKKα/β), NF-κB p65, JNK, and p38 are examined at 30 minutes after LPS stimulation. EA strongly downregulated p-IKKα/β and p-NF-κB p65 activated by E. coli-LPS but did not downregulate pJNK or p-p38 (Figure 4(b)).

EA Inhibited LPS-Induced Degradation of β-Catenin in ST2.
Since OPG expression is regulated by Wnt/β-catenin signaling pathways in osteoblasts [44], it was suggested that EA increased the OPG reduced by LPS stimulation via the Wnt/β-catenin signaling pathway. Terefore, we investigated the efects of EA on the Wnt/β-catenin signaling pathways caused by E. coli-LPS (1 μg/mL) by Western blotting. Te time course analysis shows that E. coli-LPS induced degradation of β-catenin after 30 min upon stimulation with E. coli-LPS ( Figure 5(a)). Subsequently, we examined the efect of EA on the degradation of β-catenin by E. coli-LPS. Te results demonstrates that EA inhibited β-catenin degradation in ST2 stimulated cells with E. coli-LPS ( Figure 5(b)).

Discussion
In this study, we confrmed that EA inhibited alveolar bone destruction through the control of osteoblast-mediated osteoclastogenesis by reducing phosphorylated NF-κB, TNF-α, and RANKL and upregulating Sema3A, β-catenin, . IL-1β and Sema3A mRNA expression of ST2 stimulated with E. coli-LPS with or without EA for 12 hours, and total RNAs are extracted (D, F). Osteoprotegerin (OPG) mRNA expression of ST2 stimulated with E. coli-LPS with or without EA for 24 hours, and total RNAs are extracted (E). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is used as an internal control. Data are presented as means ± standard error (n � 7 for each group). Tukey-Kramer multiple comparison test, * * * p < 0.001, * * p < 0.01, * p < 0.05. (b) RANKL and OPG protein expression of ST2 stimulated with Escherichia coli (E. coli)-lipopolysaccharide (LPS) (1 μg/mL). Cell culture supernatants were collected 3 hours (for RANKL) or 48 hours (for OPG) after treatment and analyzed using an ELISA assay. Data are presented as means ± standard error (n � 6 for each group). Tukey-Kramer multiple comparison test, * * * p < 0.001. and OPG with LPS stimulation. Notably, EA only suppressed the increase in osteoclasts induced by LPS application to the control level, suggesting that EA may not inhibit physiological bone remodeling. Tis is the frst report demonstrating the inhibitory efect of EA on osteoclastogenesis in vivo. Periodontitis is caused by the repeated challenge of harmful irritants, such as LPS, on the periodontal tissues owing to persistent infection by periodontal bacteria resulting in chronic host immune and infammatory responses and tissue destruction, including alveolar bone resorption. Bone homeostasis is maintained via concerted communication between bone-building osteoblasts, bonedegrading osteoclasts, and osteocytes as the mechanosensory cells of the bone.
EA has osteogenic properties and has been reported to induce human osteoblasts in vitro [45]. EA can reportedly promote bone healing following bone surgery and fractures. However, considering the inhibitory efect of EA on osteoclastogenesis, it is reported that EA suppressed osteoclast progenitor cell diferentiation caused by the macrophage colony-stimulating factor and RANKL stimulation in vitro [34]. Besides, there is no report verifying the efect of EA on bone resorption associated with infammation in vivo We are the frst to demonstrate the inhibitory efect of EA on LPS-induced osteoclastogenesis using an animal model.
Osteoclast diferentiation and activity are regulated by RANKL and OPG, mainly produced by osteoblast lineage cells. In this study, we found that EA suppressed the production of RANKL, which was enhanced by LPS, and restored the production of OPG downregulated by LPS, using the rat model of periodontitis. Particularly, OPG expression in the periodontal ligament tissue of the E. coli-LPS/EA group was markedly upregulated in comparison with that of the control group. OPG is a soluble molecule that regulates bone destruction by potently inhibiting osteoclasts.
Since OPG expression is regulated by Wnt/β-catenin signaling pathways in osteoblasts [44], we performed Western blotting using osteoblast lineage cells (ST2) to investigate the efect of EA on the upstream signaling of RANKL and OPG induced by LPS. Figure 6 shows a schematic representation of the proposed mechanism by which EA suppresses osteoclastogenesis by LPS: LPS induces activation of IKKα/β and its downstream NF-κB via TLR4, upregulates TNF-α and RANKL expression, and inactivates the Wnt/β-catenin signaling pathway, leading to downregulation of OPG expression. Furthermore, the production of the secreted protein Sema3A is reduced, inhibiting neuropilin-1-mediated β-catenin degradation and nuclear translocation and suppressing OPG expression as well (Figure 6(a)). In contrast, EA strongly suppresses IKKα/β and NF-κB activation caused by LPS stimulation, resulting in reduction of RANKL.
Several proinfammatory cytokines enhance osteoclast diferentiation and function. For example, TNF-α promotes the increase of osteoclast progenitor cells in vivo [30][31][32][33]. Furthermore, IL-1α and β directly act on the osteoclasts without RANKL to prolong the osteoclast life and activate bone resorption [46]. Infammatory cytokines can also afect bone formation. For instance, TNF-α acts in an inhibitory manner on osteoblast diferentiation and survival; TNF-α reportedly suppresses the production of insulin-like growth factor 1, which acts to increase the number of osteoblast progenitor cells [47], and suppresses the transcription of runt-related transcription factor 2, a master transcription factor for osteoblast diferentiation [48]. Furthermore, TNF-α and IL-1β have been reported to act on osteoblasts to increase Fas expression and induce apoptosis [49]. Previously, by using the rat model of LPSinduced periodontitis, we confrmed that EA treatment markedly reduced immunoexpression of TNF-α at the junctional epithelium [29]. In the present study, evaluation using osteoblast lineage cells demonstrated the strong inhibitory efect of EA on E. coli-LPS-induced production of infammatory cytokines (TNF-α, IL-1β, and IL-6). Tese fndings suggest that EA may strongly inhibit osteoclastogenesis by restoring OPG production and suppressing the production of infammatory cytokines and RANKL induced by LPS.
Tese efects of EA could be attributed to the compounds contained in E. arvense. For instance, quercetin in E. arvense has been shown to inhibit the formation, proliferation, and maturation of osteoclasts [50,51]. Osteoblast-like cells with quercetin treatment reportedly show an increased tendency in OPG production and a signifcant decrease in RANKL [52,53]. Moreover, quercetin has an efect of promoting mature osteoclast apoptosis [50,54].
In the present study, we focused on the inhibitory efect of EA on bone destruction; however, positive efects are also expected in osteogenesis. Te current in vitro study demonstrated that EA restored the LPS-induced downregulation of Sema3A and β-catenin, which is homeostatically produced by osteoblast lineage cells. Sema3A inhibits osteoclast diferentiation via neuropilin-1 on osteoclast progenitor cells, which also acts on osteoblasts themselves, promoting osteoblast diferentiation through the Wnt/β-catenin signaling pathway [55]. E. arvense contains several compounds that reportedly promote osteoblast formation. Ursolic acid contained in E. arvense has been shown to promote osteoblast diferentiation and mineralization in vitro [26]. Furthermore, silica, rich in E. arvense, promotes the deposition of calcium and other minerals, decreases the number of osteoclasts, stimulates the activity of osteoblasts, promotes the synthesis of collagen, and facilitates the synthesis of glycosaminoglycans and collagen, resulting in the formation of bone and connective tissue [28,56]. Tis evidence indicates that the compounds in EA promote osteoblast formation; therefore, EA is expected to have the efect of not only inhibiting osteoclast formation but also promoting bone formation by International Journal of Dentistry osteoblasts in vivo. Te osteogenic activity of EA should be verifed in future studies.
Antiresorptive and anabolic agents are efective treatments for osteoclastic lesions; however, some side efects have been reported. For instance, antiresorptive osteonecrosis of the jaw caused by anti-RANKL antibodies and bisphosphonates has become a major problem [57]. Hormone-replacement therapy can attribute to an increased risk of venous thromboembolism and cardiovascular events [58,59]. A prolonged treatment period and high administration dose of recombinant human parathyroid hormone (teriparatide) increases the incidence of bone neoplasms [60]. Terefore, natural compounds such as EA with potent osteoprotective properties and few side efects could be an alternative strategy to overcome the shortcomings of existing treatments for osteoclastic lesions as well as to develop oral care methods to prevent the destruction of alveolar bone associated with periodontitis.

Conclusion
Te main limitation of this study was the use of E. coli-LPS for the animal experiment. Although we should have used LPS from periodontal pathogen, we chose this model because of the established time course of histological changes in periodontitis caused by E. coli-LPS administration. E. coli-LPS is known to be a type of Aggregatibacter actinomycetemcomitans-LPS, which is one of the main periodontal  Figure 6: Proposed mechanism of EA inhibition of osteoclast formation caused by LPS. (a) In LPS-induced periodontitis, TLR4-mediated phosphorylation of IκB kinase α and β (IKKα/β) and NF-κB induces the expression of infammatory cytokines such as tumor necrosis factor (TNF)-α in periodontal tissue cells and the generation of the osteoclastogenic receptor activator of NF-κB ligand (RANKL), an osteoclastogenic factor. However, it inactivates the Wnt/β-catenin signaling pathway and promotes β-catenin degradation. Furthermore, production of the secreted protein semaphorin 3A (Sema3A) is decreased; Sema3A/neuropilin-1 (Nrp1)-mediated β-catenin degradation and nuclear translocation are suppressed, as is transcription and expression of osteoprotegerin (OPG), the decoy receptor for RANKL. Finally, the RANKL/OPG ratio is increased, leading to osteoclast formation and maturation and subsequent bone resorption. (b) Proposed mechanism of EA-induced suppression of osteoclastogenesis by LPS: EA strongly suppresses the activation of IKKα/β and NF-κB, indicating that the Wnt/β-catenin signaling pathway is activated and β-catenin degradation is suppressed. Furthermore, Sema3A production is elevated, Sema3A/Nrp1-mediated β-catenin degradation is suppressed, and nuclear migration is promoted. Tis suppresses osteoclast formation and excessive bone resorption. pathogens; however, the efects of EA on LPS from periodontal pathogen-induced periodontal changes in vivo should be clarifed in future studies.
Tis study suggests that EA could be useful in preventing or treating periodontal disease and other bone-destroying lesions. Considering the clinical application, the daily use of toothpaste and mouthwash containing EA could inhibit alveolar bone resorption associated with plaque accumulation in periodontal pockets and suppress tooth mobility and tooth loss attributed to periodontitis, eventually resulting in the maintenance of healthy teeth for a lifetime.

Data Availability
All data are available from the corresponding author upon reasonable request.

Conflicts of Interest
Te authors declare that there are no conficts of interest regarding the publication of this article.