Tumor necrosis factor-α antagonist diminishes osteocytic RANKL and sclerostin expression in diabetes rats with periodontitis

Abstract

Type 1 diabetes with periodontitis shows elevated TNF-α expression. Tumor necrosis factor (TNF)-α stimulates the expression of receptor activator of nuclear factor-κB ligand (RANKL) and sclerostin. The objective of this study was to determine the effect of TNF-α expression of osteocytic RANKL and sclerostin in type 1 diabetes rats with periodontitis using infliximab (IFX), a TNF-α antagonist. Rats were divided into two timepoint groups: day 3 and day 20. Each timepoint group was then divided into four subgroups: 1) control (C, n = 6 for each time point); 2) periodontitis (P, n = 6 for each time point); 3) diabetes with periodontitis (DP, n = 8 for each time point); and 4) diabetes with periodontitis treated with IFX (DP+IFX, n = 8 for each time point). To induce type 1 diabetes, rats were injected with streptozotocin (50 mg/kg dissolved in 0.1 M citrate buffer). Periodontitis was then induced by ligature of the mandibular first molars at day 7 after STZ injection (day 0). IFX was administered once for the 3 day group (on day 0) and twice for the 20 day group (on days 7 and 14). The DP group showed greater alveolar bone loss than the P group on day 20 (P = 0.020). On day 3, higher osteoclast formation and RANKL-positive osteocytes in P group (P = 0.000 and P = 0.011, respectively) and DP group (P = 0.006 and P = 0.017, respectively) than those in C group were observed. However, there was no significant difference in osteoclast formation or RANKL-positive osteocytes between P and DP groups. The DP+IFX group exhibited lower alveolar bone loss (P = 0.041), osteoclast formation (P = 0.019), and RANKL-positive osteocytes (P = 0.009) than that of the DP group. On day 20, DP group showed a lower osteoid area (P = 0.001) and more sclerostin-positive osteocytes (P = 0.000) than P group. On days 3 and 20, the DP+IFX group showed more osteoid area (P = 0.048 and 0.040, respectively) but lower sclerostin-positive osteocytes (both P = 0.000) than DP group. Taken together, these results suggest that TNF-α antagonist can diminish osteocytic RANKL/sclerostin expression and osteoclast formation, eventually recovering osteoid formation. Therefore, TNF-α might mediate alveolar bone loss via inducing expression of osteocytic RANKL and sclerostin in type 1 diabetes rats with periodontitis.

Fig 1. Alveolar bone loss.

Representative images of the distance from CEJ to ABC in each group. Black arrows and dotted lines represent the CEJ and ABC, respectively (upper panels, H&E stain, scale bar = 100 μm). Measurements of the distance from CEJ to ABC in each group (lower panel, C, n = 6; P, n = 6; DP, n = 8; DP+IFX, n = 8 at each time point). Data are presented as mean ± SE. Data were evaluated by one-way analysis of variance (ANOVA) followed by Scheffe’s test. A P value of less than 0.05 was considered statistically significant. * P < 0.05 compared to C group; # P < 0.05; C: control group; P: periodontitis group; DP: diabetes with periodontitis group; DP+IFX: diabetes with periodontitis treated with infliximab group.

Fig 2. Cathepsin K-positive osteoclasts and RANKL-positive osteocytes in alveolar bone and IL-1β mRNA expression levels in gingival tissues.

(A) Representative images of the number of cathepsin K-positive multinucleated cells in each group. Red arrowheads represent cathepsin K-positive multinucleated cells (upper panels, scale bar = 100 μm). Quantification of the number of cathepsin K-positive multinucleated cells in each group (lower panel, C, n = 6; P, n = 6; DP, n = 8; DP+IFX, n = 8 at each time point). (B) Percentage of RANKL-positive osteocytes in each group (C, n = 6; P, n = 6; DP, n = 8; DP+IFX, n = 8 at each time point). (C) IL-1β mRNA expression levels in gingival tissues of each group (C, n = 3; P, n = 3; DP, n = 4; DP+IFX, n = 4 at each time point). Data are presented as mean ± SE. Data were evaluated by one-way analysis of variance (ANOVA) followed by Scheffe’s test. A P value of less than 0.05 was considered statistically significant. * P < 0.05 compared to C group; # P < 0.05; n = number.

Fig 3. Osteoid area in alveolar bone.

Representative images of osteoid area in each group. Black dotted lines indicate the osteoid area (upper panels, H&E stain, scale bar = 100 μm). Quantification of osteoid area in each group (lower panel, C, n = 6; P, n = 6; DP, n = 8; DP+IFX, n = 8 at each time point). Data are presented as mean ± SE. Data were evaluated by one-way analysis of variance (ANOVA) followed by Scheffe’s test. A P value of less than 0.05 was considered statistically significant. * P < 0.05 compared to C group; # P < 0.05.

Fig 4. Sclerostin expression in osteocytes of alveolar bone.

(A) Representative images of sclerostin-positive osteocytes in each group. White and red arrowheads indicate representative sclerostin-negative and -positive osteocytes, respectively (upper panels, immunohistochemistry stain, scale bar = 100 μm). Percentage of sclerostin-positive osteocytes in each group (lower panel, n = 6; P, n = 6; DP, n = 8; DP+IFX, n = 8 at each time point). (B) Sclerostin mRNA expression in each group (C, n = 3; P, n = 3; DP, n = 4; DP+IFX, n = 4 at each time point). Data are presented as mean ± SE. Data were evaluated by one-way analysis of variance (ANOVA) followed by Scheffe’s test. A P value of less than 0.05 was considered statistically significant. * P < 0.05 compared to C group; # P < 0.05.