Expression of autophagy and apoptosis-related factors in the periodontal tissue of experimental diabetic rats: a histomorphometric, microtomographic and immunohistochemical study

Abstract

Objective: This study aimed to investigate the expression of autophagy-related factors microtubule-associated protein l light chain 3 (LC3) and the apoptosis-related factors BCL2-associated X protein (Bax) and B cell lymphoma-2 (Bcl-2) in the periodontal tissue of experimental diabetic rats. These data were used to explore the potential mechanism in diabetes-induced periodontal tissue lesions.

Methods: A total of 32 Sprague Dawley (SD) rats were randomly assigned into diabetes (group D, n = 16) and control groups (group N, n = 16). The diabetic group was induced by intraperitoneal injection of 1% streptozotocin (STZ, 60 mg/kg) and the control group was injected with citrate buffer (0.1mol/L). Rats were sacrificed after 4 and 8 weeks of feeding and collected as D1, N1 groups and D2, N2 groups, and the maxilla were retained for analysis. The changes in periodontal tissue structure were observed by hematoxylin-eosin (HE) staining. The expression and distribution of LC3, Bax and Bcl-2 in the periodontium of the rats was detected by immunohistochemical (SP) staining.

Results: Diabetic rats showed several changes compared to control animals including sparse alveolar bone trabecular structure, loss of the lamina dura and absorption of the local alveolar bone. The positive expression level of LC3 in the gingival epithelial, periodontal ligament and alveolar bone of group D1 was significantly higher than in the N1, N2 and D2 groups (P < 0.05). The level of Bax expression in the group D2 rats was significantly higher than those in the N1, N2 and D1 groups (P < 0.05), while the positive degree of Bcl-2 was significantly lower than those of other groups (P < 0.001). LC3 was negatively correlated with Bax and was irrelevant with Bcl-2; Bcl-2 was not correlated with Bax.

Conclusions: The expression of LC3, Bax and Bcl-2 changes in the periodontal tissue of diabetic rats may indicate that autophagy and apoptotic are involved in the process of periodontal tissue damage in diabetic rats. These changes may be one of the mechanisms of periodontal tissue lesions.

Keywords: Bax; Bcl-2; Diabetes; LC3; Periodontal tissue.

Figure 1. Schematic illustration of the distance measurements between the cement enamel junction (CEJ) and the alveolar bone crest (ABC).

The distance from the CEJ to the ABC was measured at six points (a–f) indicated on the third molar (M3) to the first molar (M1).

Figure 2. Alveolar bone loss was detected in M3 to M1 of each group.

(A–D) Alveolar bone loss was detected in M3 to M1 of each group. (E) Data represent the mean values ± SEM, (n = 6 for each group). *p < 0.05 using ANOVA, compared to the normal control group (group N). #p < 0.001 using ANOVA, compared to the D1 group.

Figure 3. Histological analysis of the maxillae from rats with DM (D1 and D2) and control groups (N1 and N2).

(A–H) Histological analysis of the maxillae from rats (N1 and N2) show the normal periodontium, the alveolar bone integrity and absence of or discrete cellular infiltration, and preserved alveolar bone. The periodontium from group D1 was mostly normal, the epithelial rete pegs proliferated. The infiltration of inflammatory cells was observed and the resorption of the alveolar bone was slight. The periodontium from DM rats (D2) showing alveolar bone resorption and accentuated inflammatory cell infiltration. Images are shown at 50×g and 100×g magnification. G (gingival); PDL (periodontal ligament); D (dentin); AB (alveolar bone); (I–T) Histological analysis of gingiva l (G), periodontal ligament (PDL) and alveolar bone (AB) respectively from rats with diabetes mellitus (D1 and D2) and control groups (N1 and N2). The images are shown at 200× magnification.

Figure 4. Photomicrographs of the periodontal tissue from DM rats (D1 and D2) and control groups (N1 and N2).

(A–L) Photomicrographs of the periodontal tissue from DM rats (D1 and D2) and control groups (N1 and N2) showing immunoreactivity to LC3, Bax and Bcl-2. Images are shown at 40×g magnification. Scale bar = 25 μm. The arrow indicates high or moderate labeling in the periodontal ligament or the alveolar bone. Small picture-Upper left (shown at 20×g magnification).