Calcitriol-enhanced autophagy in gingival epithelium attenuates periodontal inflammation in rats with type 2 diabetes mellitus

Abstract

Type 2 diabetes mellitus (T2DM)-associated periodontitis is a common disease with high prevalence, associated with persistent infection and complicated manifestations. Calcitriol (1 alpha, 25-dihydroxyvitamin D3, 1,25D) is the active form of vitamin D that plays a protective role in immune regulation, bone metabolism, and inflammatory response. In this study, we constructed a T2DM model in rats by combining a high-fat diet with low-dose streptozotocin. The periodontitis model in rats was developed by ligation and Porphyromonas gingivalis (ATCC 33277) inoculation. Rats were randomly divided into five groups: non-diabetic blank, diabetic blank, diabetes with calcitriol treatment, diabetes with 3-methyladenine (3-MA) treatment, or diabetes with calcitriol and 3-MA treatment. The diabetic rats exhibited an intense inflammatory response and decreased autophagy compared with the non-diabetic rats. Intraperitoneal injection of calcitriol and autophagy inhibitor (3-MA) allowed us to explore the effect of calcitriol on inflammation in the gingival epithelium and the role of autophagy in this process. Treatment with calcitriol resulted in the decreased expression of NFκB-p65, p62/SQSTM1 and inflammatory response and increased expression of LC3-II/LC3-I. Application of 3-MA significantly suppressed autophagy, which was apparently retrieved by calcitriol. Antibacterial peptide (LL-37) is the only antimicrobial peptide in the cathelicidin family that is found in the human body, and it exhibits a broad spectrum of antibacterial activity and regulates the immune system. In the present study, our findings indicated that calcitriol-enhanced autophagy may attenuated periodontitis and the decrease of LL-37 was rescued by calcitriol treatment in the gingival epithelial cells of T2DM rats. Our study provides evidence for the application of calcitriol as an adjunctive treatment for T2DM-associated periodontitis.

Keywords: LL-37; Porphyromonas gingivalis; autophagy; calcitriol; rats model; type 2 diabetes mellitus.

Figure 1

Establishment of the experimental type 2 diabetic model in rats. (A) The flow chart of the experimental design. HFD, High-Fat Diet; STZ, streptozotocin (35 mg/kg); HE, hematoxylin and eosin; IHC, immunohistochemical. (B) The random blood glucose (RBG) of the rats were measured. (C) Body weight of the diabetic rats were measured. The data were presented as the mean ± SD (n=4/group, **P <0.01, compared with 7 week).

Figure 2

Establishment of the experimental periodontitis model in rats. (A, B) The figures presented the gross specimens and sections obtained individually from the control group and the periodontitis group. (a, e): As shown in the gross specimen, the distance from the cemento-enamel junction (CEJ) to the alveolar bone crest in the periodontitis group was remarkably longer than that in the control group, that is alveolar bone resorption occurred. (b, c): As shown in the section from the control group with hematoxylin and eosin (HE) staining, the junctional epithelium is located at the CEJ site. (f, g): The section from the periodontitis group shows that the junctional epithelium was located on the root, that is attachment loss (AL) occurred. (d, h): Compared with that from the control group, more inflammatory cells and more severe vasodilatation was observed in the gingival tissue from the periodontitis group. The black arrows in (d, h) indicates the inflammatory cell (especially neutrophile) infiltration. The red arrows in (h) indicates vasodilatation. M1, the maxillary first molar; M2, the maxillary second molar.

Figure 3

Diabetic rats with periodontitis showed more severe alveolar bone loss and intense gingival inflammatory response than non-diabetic rats. The figures present the micro-CT images and sections obtained individually from the non-DM control group, non-DM periodontitis group, DM control group and the DM periodontitis group. Non-DM control group, health control without type 2 diabetes mellitus (T2DM) and periodontitis; non-DM periodontitis group, ligation/P. gingivalis-induced periodontitis but without T2DM; DM control group, high-fat diet and low dose of streptozotocin-induced T2DM but without periodontitis; DM periodontitis group, combination periodontitis and T2DM. (A) Micro-computed tomography images of the maxillary first molars from (a-d). The distance between two red lines, cemento-enamel junction (CEJ) and the alveolar bone crest, represents the alveolar bone resorption (e-h). (B) Statistical analysis of the alveolar bone loss. Data are presented as mean ± SD (n=4) and are shown in the bar graphs, repeated 3 times (**P <0.01, compared with the control group or the non- DM group). (C) Hematoxylin and eosin staining of sections (i-p).

Figure 4

Autophagy in gingival epithelium was attenuated in diabetic rats. The expression of autophagy-related protein of gingival epithelium in non-DM control group, non-DM periodontitis group, DM control group and the DM periodontitis group were detected. Non-DM control group, health control without type 2 diabetes mellitus (T2DM) and periodontitis; non-DM periodontitis group, ligation/P. gingivalis-induced periodontitis but without T2DM; DM control group, high-fat diet and low dose of streptozotocin-induced T2DM but without periodontitis; DM periodontitis group, combination periodontitis and T2DM. (A) Immunohistochemical (IHC) staining and semi-quantitative analysis of the expression of LC3 in the gingival epithelium of the four groups (a-e). (B) Western Blot reveals the expression of LC3 and p62 in four groups (f-h). All data are presented as mean ± SD (n = 3) relative to non- DM control are shown in bar graphs (*P <0.05, **P <0.01).

Figure 5

Calcitriol attenuated the gingival epithelium inflammatory response in diabetic rats by promoting autophagy. The diabetic rats with periodontitis were randomly divided into blank group (propylene glycol: water: ethyl glycol=60:30:10), calcitriol group (2 µg/kg), 3-MA group (15 mg/kg) and calcitriol + 3-MA group. They received intraperitoneal injections for 1 week. (A) The expression of LC3 (a-e) and NFκB-p65 (f-j) in the gingival epithelium of the four groups were detected by IHC staining. (B) HE staining showed the inflammatory response in the subepithelial fiber of the four groups (k-n). (C) The expression of NFκB-p65, LC3-II/LC3-I and p62 in the gingival epithelium of the four groups were detected by western-blot (o-r). Semi-quantitative analysis of IHC and western-blot were performed by gray density analysis. All data are presented as mean ± SD (n = 3) relative to blank group are shown in bar graphs (*P <0.05, **P <0.01).

Figure 6

Decrease of LL-37 in the gingival epithelial cells of T2DM rats, which was rescued by calcitriol treatment. (A) IHC staining of the gingival epithelium obtained individually from the non-DM control group, non-DM periodontitis group, DM control group and the DM periodontitis group (a-h, r). Non-DM control group, health control without type 2 diabetes mellitus (T2DM) and periodontitis; non-DM periodontitis group, ligation/P. gingivalis-induced periodontitis but without T2DM; DM control group, high-fat diet and low dose of streptozotocin-induced T2DM but without periodontitis; DM periodontitis group, combination periodontitis and T2DM. (B) IHC staining of the LL-37 expression in the gingival epithelium of different groups (i-p, s). The diabetic rats with periodontitis were randomly divided into blank group (propylene glycol: water: ethyl glycol=60:30:10), calcitriol group (2 µg/kg), 3-MA group (15 mg/kg) and calcitriol + 3-MA group. (C) Western Blot reveals the expression of LL-37 in four groups (q) analysis of the LL-37 expression in the gingival epithelium (t). All data are presented as mean ± SD (n = 3) relative to non-DM and blank group are shown in bar graphs (*P <0.05, **P <0.01, compared with the blank group).

Figure 7

Mechanism schema diagram.