AimTo evaluate the effects of metformin (Met) on inflammation, oxidative stress, and bone loss in a rat model of ligature-induced periodontitis.Materials & methodsMale 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.ResultsTreatment 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.ConclusionsMetformin, at a concentration of 50 mg/kg, decreases the inflammatory response, oxidative stress and bone loss in ligature-induced periodontitis in rats.

Evidence shows that metformin is an antidiabetic drug, which can exert favorable anti-inflammatory effects and decreased bone loss. The development of nanoparticles for metformin might be useful for increased therapeutic efficacy. The aim of this study was to evaluate the effect of metformin hydrochloride-loaded Poly (d,l-Lactide-co-glycolide) (PLGA)/(MET-loaded PLGA) on a ligature-induced periodontitis model in diabetic rats. MET-loaded PLGA were characterized by mean diameter, particle size, polydispensity index, and entrapment efficiency. Maxillae were scanned using Microcomputed Tomography (µCT) and histopathological and immunohistochemical analysis. IL-1β and TNF-α levels were analyzed by ELISA immunoassay. Quantitative RT-PCR was used (AMPK, NF-κB p65, HMGB1, and TAK-1). The mean diameter of MET-loaded PLGA nanoparticles was in a range of 457.1 ± 48.9 nm (p < 0.05) with a polydispersity index of 0.285 (p < 0.05), Z potential of 8.16 ± 1.1 mV (p < 0.01), and entrapment efficiency (EE) of 66.7 ± 3.73. Treatment with MET-loaded PLGA 10 mg/kg showed low inflammatory cells, weak staining by RANKL, cathepsin K, OPG, and osteocalcin, and levels of IL-1β and TNF-α (p < 0.05), increased AMPK expression gene (p < 0.05) and decreased NF-κB p65, HMGB1, and TAK-1 (p < 0.05). It is concluded that MET-loaded PLGA decreased inflammation and bone loss in periodontitis in diabetic rats.

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The aim of this study was synthesize and evaluate the effects of Poly (D, L-Lactide-co-29 glycolide) (PLGA) Nanoparticles (NPs) of metformin (PLGA+ Met) on inflammation, and bone loss 30 in a ligature-induced periodontitis rat model. The prepared NPs were characterized by mean 31 diameter, size particle, polydispensity index and encapsulation efficiency by Atomic force 32 microscopy (AFM). Male albino Wistar rats were randomly divided into four groups of 20 rats in 33 each group, and given the following treatments for 10 days to evaluate in vivo activity: (1) Sham: no 34 ligature + water; (2) Positive control: ligature + water (with Periodontal disease and Diabetes); (3) 35 ligature + PLGA+ 10 mg/kg Met (With Periodontal disease and Diabetes); and (4) ligature + PLGA+ 36 100 mg/kg Met (with Periodontal disease and Diabetes). Water or PLGA + Met was administered 37 orally by gavage. Maxillae were fixed and scanned using Micro-computed Tomography (μCT) to 38 quantify linear of bone loss. Histopathological characteristics were assessed through 39 immunohistochemical staining for Osteocalcin, Cathepsyn K, RANKL/RANK/OPG pathway. IL-1β 40 and TNF-α from gingival tissues were analysed by Elisa immunoassay. Quantitative RT-PCR 41 reaction was used to evaluate gene expression of AMPK, NF-κB p-65, Hmgb1 and TAK-1 from 42 gingival tissues. Statistical analysis was performed using one-way ANOVA at 5% significance. The 43 mean diameter of MET-loaded PLGA nanoparticles was in a range of 457.1 ± 48.9 nm with a 44 polydispersity index of 0.285, zeta potential: 8.16 ± 1.1 mV and entrapment efficiency (EE) was 70%. 45The results suggest that the addition of MET in the core slightly affected the particle sizes. Treatment 46 with PLGA+ 10 mg/kg Met showed low inflammatory cells, decreased bone loss and integrity Preprints (www.preprints.org) | NOT PEER-REVIEWED | staining was shown by RANKL, Cathepsyn K, OPG, and osteocalcin. Radiographically, linear 49 measurements showed a statistically significant reduction in bone loss after treatment with PLGA+ 10 mg/kg 50 Met compared to the positive control (p < 0.05). RT-PCR showed increased AMPK expression (p < 0.05) 51 and decreased expression of NF-κB P65, HMGB1 and TAK-1 after PLGA+ 10 mg/kg Met (p < 0.05). 52The PLGA nanoparticle + 10 mg/kg Met decreased glucose levels and also decreased the inflammatory 53 response, and bone loss in ligature-induced periodontitis in rats. 54