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

Abstract

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.

Figure 1

Effect of Equisetum arvense extract (EA) on lipopolysaccharide (LPS)-induced osteoclast formation. Effects of EA (15 μg/mL) on osteoclast formation by Escherichia coli (E. coli)-LPS (5 mg/mL). Periodontal tissue was collected 3 days following treatment, and immunohistochemical (IHC) staining was performed. (a) Illustration of the osteoclast counting area. (b) Immunoexpression of cathepsin K, an osteoclast marker after 3 days following (A, A′) physiological saline (control), (B, B′) E. coli-LPS, and (C, C′) E. coli-LPS/EA application. Scale bars = 100 μm. (c) The number of cathepsin K-positive osteoclasts formed along the alveolar bone margin within 1 mm of the alveolar crest is counted. Data are presented as means ± standard deviation (n = 7 for each group). Tukey–Kramer multiple comparison test, ^∗p < 0.05.

Figure 2

Immunohistochemical examination of periodontal tissue. (a) Hematoxylin and eosin (H&E) staining of tissue obtained from animals 3 days after Escherichia coli (E. coli)-lipopolysaccharide (LPS) topical administration. (A) Control (PS-applied control), (B) E. coli-LPS, (C) E. coli-LPS/Equisetum arvense extract (EA) application. Immunoexpression of the receptor activator of NF-κB ligand (RANKL) in the upper part of the alveolar bone area, (D, D′) control, (E, E′) E. coli-LPS, and (F, F′) E. coli-LPS/EA application. Scale bars = 100 μm. (b) H&E staining of tissue obtained from animals 3 days after E. coli-LPS topical administration. (A) Control, (B) E. coli-LPS, and (C) E. coli-LPS/EA application. Immunoexpression of osteoprotegerin (OPG) in the upper part of the alveolar bone area, (D, D′) control, (E, E′) E. coli-LPS, and (F, F′) E. coli-LPS/EA application. Scale bars = 100 μm.

Figure 3

Effect of Equisetum arvense extract (EA) on osteoclast formation-related factors. (a) It has been reported that stimulation of osteoblasts with lipopolysaccharide (LPS) alters the expression of osteoclast formation-related factors. Therefore, we examined the effect of EA on osteoclast formation-related factors. Tumor necrosis factor-α, interleukin (IL)-6, and the receptor activator of NF-κB ligand (RANKL) mRNA expression of ST2 stimulated with Escherichia coli (E. coli)-LPS (1 μg/mL) with or without EA (3 μg/mL) for 2 hours, and total RNAs are extracted (A, B, C). 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.

Figure 4

Effects of EA on the LPS induced NF-κB and MAPKs' signaling pathway in ST2. (a) E. coli-LPS (1 μg/mL) is applied to ST2 cells and collected at several time points. NF-κB p65 and MAPKs (JNK and p38) in ST2 cells are analyzed by Western blotting. β-actin is used as a loading control. (b) EA: Equisetum arvense extract (3 μg/mL) is pretreated in ST2 cells for 30 minutes, and then E. coli-LPS (1 μg/mL) is additionally applied to ST2 cells. IKKα/β, NF-κB p65, and MAPKs (JNK and p38) in ST2 cells are assessed at 30 minutes by Western blotting. β-actin is used as the loading control.

Figure 5

Effects of EA on the LPS induced downregulation of Wnt/β-catenin signaling in ST2. (a) E. coli-LPS (1 μg/mL) is applied to ST2 cells and collected at several time points. β-Catenin in ST2 cells is analyzed by Western blotting. β-actin is used as a loading control. (b) E. coli-LPS (1 μg/mL) and/or EA; Equisetum arvense extract (3 μg/mL) are treated in ST2 cells for 10 hours on ST2 cells. β-Catenin in ST2 cells is analyzed by Western blotting. β-actin is used as the loading control.

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 inflammatory 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. This suppresses osteoclast formation and excessive bone resorption.