Effects of metformin on inflammation, oxidative stress, and bone loss in a rat model of periodontitis

Abstract

Aim: To evaluate the effects of metformin (Met) on inflammation, oxidative stress, and bone loss in a rat model of ligature-induced periodontitis.

Materials & methods: Male albino Wistar rats were divided randomly into five groups of twenty-one rats each, and given the following treatments for 10 days: (1) no ligature + water, (2) ligature + water, (3) ligature + 50 mg/kg Met, (4) ligature + 100 mg/kg Met, and (5) ligature + 200 mg/kg Met. Water or Met was administered orally. Maxillae were fixed and scanned using Micro-computed Tomography (μCT) to quantitate linear and bone volume/tissue volume (BV/TV) volumetric bone loss. Histopathological characteristics were assessed through immunohistochemical staining for MMP-9, COX-2, the RANKL/RANK/OPG pathway, SOD-1, and GPx-1. Additionally, confocal microscopy was used to analyze osteocalcin fluorescence. UV-VIS analysis was used to examine the levels of malondialdehyde, glutathione, IL-1β and TNF-α from gingival tissues. Quantitative RT-PCR reaction was used to gene expression of AMPK, NF-κB (p65), and Hmgb1 from gingival tissues. Significance among groups were analysed using a one-way ANOVA. A p-value of p<0.05 indicated a significant difference.

Results: Treatment with 50 mg/kg Met significantly reduced concentrations of malondialdehyde, IL-1β, and TNF-α (p < 0.05). Additionally, weak staining was observed for COX-2, MMP-9, RANK, RANKL, SOD-1, and GPx-1 after 50 mg/kg Met. OPG and Osteocalcin showed strong staining in the same group. Radiographically, linear measurements showed a statistically significant reduction in bone loss after 50 mg/kg Met compared to the ligature and Met 200 mg/kg groups. The same pattern was observed volumetrically in BV/TV and decreased osteoclast number (p<0.05). RT-PCR showed increased AMPK expression and decreased expression of NF-κB (p65) and HMGB1 after 50 mg/kg Met.

Conclusions: Metformin, at a concentration of 50 mg/kg, decreases the inflammatory response, oxidative stress and bone loss in ligature-induced periodontitis in rats.

Fig 1. Radiographic evaluation after Met treatment and experimental periodontitis.

(A) Representative sagittal 2D and 3D microCT images of no ligature (NL), ligature (L), Met 50, Met 100, and Met 200 groups. (B) Graph representing linear bone loss in the area of the mesial second molar. Data are mean ± standard area of the mean. *p<0.05, *p<0.01, ***p<0.001, (n≥3 for all groups/time points). (C) Graph representing the % BV/TV in the area under the second molar bifurcation. Data are mean ± standard area of the mean. *p<0.05, *p<0.01, ***p<0.001, (n≥3 for all groups/time points).

Fig 2. Microscopic analyses.

A, D and G No ligature. B, E, H: Ligature, Periodontium from a rat with periodontitis (treated with saline) showing alveolar bone and cementum resorption (discontinuous cementum) and inflammatory cell infiltration. F: Treatment with Met (100 mg/kg) and I: Treatment with Met (200 mg/kg) showing no reduced inflammation and increased alveolar bone loss. F: Periodontium from a rat with periodontitis (treated with Met, 50 mg/kg) showing reduced inflammation and decreased alveolar bone loss. Sections were stained with H&E. Original magnification 40×. Scale bars = 100 μm. PL, periodontal ligament; D, dentin; AB, alveolar bone; C, cementum; a, bone loss; b, resorption of cementum; c, inflammatory process; e, decreased inflammation process.

Fig 3. Quantitation of osteoclast numbers after MET treatment and experimental periodontitis.

(A) Representative H&E stained sagittal images of no ligature (NL), ligature (L), Met 50, Met 100, and Met 200 groups. Black arrows highlight osteoclasts, multinucleated cells lining the bone areas. Sections were stained with H&E. Original magnification 20×. Noted the increased bone destruction and porosity in the ligature and Met 200 groups. (B) Graph representing osteoclast numbers quantitated (avg:average). Data are mean ± standard error of the mean. *p<0.05, ***p<0.001, (n≥2 for all groups/time points). (ANOVA test followed by Bonferrone).

Fig 4. Photomicrographs of periodontal tissue of PD rats treated with Met and No ligature and Ligature groups, showing immunoreactivity to RANK, RANK-L, OPG, and Cathapsyn.

Rats subjected to saline (A, D, G, J); rats subjected to ligation (B, E, H, K); rats subjected to ligation and treated with Met (50 mg/kg) (C, F, I, L). Images are shown at 40× magnification. Bar = 100 μm. Arrow indicates high or moderate labeling in the periodontal ligament or the alveolar bone. Asterisk indicates mild labeling in the periodontal ligament or the alveolar bone. Triangle and asterisk indicate mild labeling of OPG in osteoclasts. Triangle and arrow indicate high labeling of osteoclasts. Triangle indicates intense labeling of Cathepsin on alveolar bone. Asterisk and triangle indicate mild labeling of cathepsin on alveolar bone. Small picture- Upper right (non-immune or IgG controls).

Fig 5. Photomicrographs of periodontal tissue of PD rats treated with Met and No ligature and Ligature groups showing immunoreactivity to MMP-9, COX-2, SOD-1 and GPX-1.

Rats subjected to saline (A, D, G, J); rats subjected to ligation (B, E, H, K); rats subjected to ligation and treated with Met (50 mg/kg) (C, F, I, L). Images are shown at 40× magnification. Bar = 100 μm. Arrow indicates high or moderate labeling in the periodontal ligament or the alveolar bone. Asterisk indicates mild labeling in the periodontal ligament or the alveolar bone. Small picture- Upper right (non-immune or IgG controls)

Fig 6. Representative confocal photomicrographs of fluorescently-tagged osteocalcin (green) in the periodontal tissue.

Samples were counterstained with DAPI nuclear counterstained (blue). (A) No ligature group: The rat periodontium presents osteocalcin labelling, showing presence of osteoblasts. (B) Weak osteocalcin labelling (red arrows) was seen in the ligature group. (C) Osteocalcin labelling (white narrow) was diffuse and strong in the Met50 group. Scale bar, 100 mm, 10x. (G,H). (D) Densitometric analysis confirmed significant increases in osteocalcin immunoreactivity in the Met (50mg/kg) that were blocked in the positive control group. Five immunofluorescence sections from each animal in each group were analyzed (N = 3 animals per group) (***p < 0.001, Kruskal-Wallis test followed by Dunn’s test). Small picture- Upper right (non-immune or IgG controls)

Fig 7. MDA and GSH in NL, L, and groups treated with 50 mg/kg, 100 mg/kg, and 200 mg/kg Met (*p <0.05, **p < 0.01).

(ANOVA test followed by Bonferrone).

Fig 8

Levels of A) IL-1β, B) TNF- α, in NL, L, and Met-treated animals (50 mg/kg, 100 mg/kg, and 200 mg/kg) (*p <0.05). (ANOVA test followed by Bonferrone).

Fig 9. Real-Time RT-PCR analysis of gene expression.

Met effects on AMPK, NF-KB p65, and Hmgb1 mRNA expression in rats with Periodontal disease. The expression of AMPK mRNA was increased in Met 50 mg/kg group (*p< 0.01, Fig 8) and decreased in L and NL group. The expression of NF-Kb p65 mRNA was decreased in All Met groups and NL group compared to the L group (*p< 0.05)Hmgb1 mRNA levels appeared to be lower in the Met 50 mg/kg group and NL group compared to the L group (*p>0.05, Fig 8). (N = 5 animals per group; ANOVA test followed by Bonferrone).