Inhibitory Effect of Periodontitis through C/EBP and 11β-Hydroxysteroid Dehydrogenase Type 1 Regulation of Betulin Isolated from the Bark of Betula platyphylla

Abstract

Periodontitis is an infectious inflammatory disease of the tissues around the tooth that destroys connective tissue and is characterized by loss of periodontal ligaments and alveolar bone. Currently, surgical methods for the treatment of periodontitis have limitations and new treatment strategies are needed. Therefore, this study evaluated the efficacy of the compound betulin isolated from bark of Betula platyphylla on the inhibition of periodontitis in vitro and in vivo periodontitis induction models. In the study, betulin inhibited pro-inflammatory mediators, such as tumor necrosis factor, interleukin-6, inducible nitric oxide synthase, and cyclooxygenase-2, in human periodontal ligament cells stimulated with Porphyromonas gingivalis lipopolysaccharide (PG-LPS). In addition, it showed an anti-inflammatory effect by down-regulating 11β-hydroxysteroid dehydrogenase type 1 and transcription factor C/EBP β produced by PG-LPS. Moreover, PG-LPS inhibited the osteogenic induction of human periodontal ligament cells. The protein and mRNA levels of osteogenic markers, such as inhibited osteopontin (OPN) and runt-related transcription factor 2 (RUNX2), were regulated by betulin. In addition, the efficacy of betulin was demonstrated in a typical in vivo model of periodontitis induced by PG-LPS, and the results showed through hematoxylin & eosin staining and micro-computed tomography that the administration of betulin alleviated alveolar bone loss and periodontal inflammation caused by PG-LPS. Therefore, this study proved the efficacy of the compound betulin isolated from B. platyphylla in the inhibition of periodontitis and alveolar bone loss, two important strategies for the treatment of periodontitis, suggesting the potential as a new treatment for periodontitis.

Keywords: 11β-hydroxysteroid dehydrogenase type 1; betulin; human periodontal ligament cells; inflammation; osteogenic induction; periodontitis.

Figure 1

Quantitative analysis of Betulin in bark of B. platyphylla extract. And analysis of bark of B. platyphylla extract and betulin using HPLC DAD (A). Diode array detector wavelength (203 nm). Confirmation of linearity of isolated betulin and analysis of betulin content in bark of B. platyphylla extract (B).

Figure 2

Effects of betulin on HPDL cells cytotoxicity and confluency. HPDL cells were seeded 1 × 10⁴ cell/mL for 24 h, then after treatment with each indicated concentration (5–40 μM) of betulin for 24 h, cell viability was analyzed through MTT assay (A). The cell confluents were analyzed using the Incucyte^® Live-Cell assay system to marked the cell confluency of normal cells (B).

Figure 3

Effect of Betulin on C/EBP and 11β-HSD1 Activity Regulation in PG-LPS Stimulated HPDL Cells. The cells (1 × 10⁶ cells/mL) were pre-treated with 10 and 40 μM for the 6 h then after, PG-LPS (1 μg/mL) was treated for the 24 h. The expression of each proteins (A) and gene level (B) were analyzed by Western blot and real time pcr. The cell confluents were analyzed using the Incucyte^® Live-Cell assay system to marked the cell confluency of normal cells (C). * p < 0.05 was considered significant differences between each treated groups.

Figure 4

Inhibitory Effect of Betulin on Inflammatory Mediators by Regulating 11β-HSD1 and C/EBP Activities. The cells (1 × 10⁶ cells/mL) were pre-treated with 10 and 40 μM for the 6 h then after, PG-LPS (1 μg/mL) was treated for the 24 h. The expression of each proteins (A) and pro-inflamatory cytokine gene level (B) were analyzed by Western blot and real time pcr. * p < 0.05 was considered significant differences between each treated groups.

Figure 5

Inhibitory Effect of Betulin on Inflammatory Mediators by Regulating 11β-HSD1 and C/EBP Activities. The HPDL cells (5 × 10³ cells/mL) were pretreated with or without the indicated concentration of betulin, after treatment with PG-LPS, it was cultured for 14 days. The result of mineralization was measured by alizarin red s (ARS) staining (A). The level of osteogenic induction specific genes (alp, opn, runx2) and proteins were analyzed by real-time PCR (B) and Western blot (C). The results were normalized to gapdh and β-actin expression. * p < 0.05 was considered significant differences between only PG-LPS treated groups.

Figure 6

Protective effect of betulin on alveolar bone and fibrous tissue lost with PG-LPS in a periodontitis-induced in vivo in vivo model. Methods of inducing periodontitis in vivo in vivo models are described in materials and methods Section 2.9. The images of the alveolar bone newly formed by betulin were taken through micro-CT (3 mm) (A). Confirmation of periodontal fibrous tissue protective effect through Goldner’s Masson Trichrome staining (B). The CEJ-ABC distance and BMD were quantified through VGStudio MAX 1.2.1 software by setting the red part as the quantitative area (C). Measurement of the thickness of periodontal fibrous tissue by setting the red part as the quantitative area (D). * p < 0.05 was considered significant differences between only PG-LPS treated groups. Each group (n = 3).

Figure 7

Inhibitory effect of betulin on periodontal tissue infiltration in PG-LPS-induced periodontitis in vivo model. Periodontal tissue infiltration due to periodontitis was analyzed by hematoxylin and eosin (H&E) (A) staining and is described in materials and methods Section 2.11. The quantification of infiltration was expressed as a percentage (%) of the infiltrating area from the total area of the marking (red area) (B). Measurement of pro-inflammatory cytokines using ELISA kit from serum of rats induced with periodontitis by PG-LPS (C). * p < 0.05 was considered significant differences between only PG-LPS treated groups. Each group (n = 3).