Mechanisms linking periodontitis and diabetes

Background + Aims

• Diabetes mellitus (DM) is a significant risk factor for periodontal disease, characterised by hyperglycaemia, chronic inflammation, and compromised immune defence. Poorly controlled blood glucose levels increase the prevalence and severity of periodontal disease, impacting the quality of life for affected individuals. Recent research has introduced the concept of ‘inflamm-ageing’- a pro-inflammatory state linked to diabetes that exacerbates tissue damage through macrophage activation.
• SETDB1 is an epigenetic enzyme that plays a crucial role in regulating gene expression by modifying chromatin structure. It is involved in the trimethylation of histone H3 at lysine 9 (H3K9me3), which is associated with gene silencing and maintaining genomic stability. In the context of inflammation and disease, SETDB1 has been shown to influence cellular processes such as senescence and immune responses, particularly in macrophages. Its dysregulation can contribute to various conditions, including chronic inflammation and age-related diseases.
• LINE-1 de-repression refers to the activation of Long Interspersed Nuclear Element-1 (LINE-1) retrotransposons, which are repetitive DNA sequences in the genome. Under normal conditions, these elements are kept in check and do not actively transpose or replicate. However, certain stressors, such as hyperglycaemia, can lead to the loss of this regulatory control, resulting in increased expression and activity of LINE-1.
• This animal model aims to investigate the effects of hyperglycaemia on macrophage behaviour and the subsequent impact on periodontal health in diabetic mice.

Materials + Methods

• Four-week-old male C57BL/6 mice were obtained from Nanjing University and bred at Sichuan University.
• Gingival tissue samples were collected from diabetic mice and healthy controls, with clinical characteristics reported in a previous study.
• Mice were housed in standard conditions with a 12-hour light-dark cycle. Five-week-old mice were made hyperglycemic with a single injection of streptozotocin (STZ) or given saline as a control. Blood glucose levels were measured biweekly after fasting, categorising mice into normal, moderate, and severe hyperglycemia groups based on their glucose readings. After 8 weeks, the mice were euthanised for further analysis.
• Four-week-old male C57BL/6 mice were divided into three groups: control (C), diabetic (D), and diabetic treated with metformin (DM). Diabetic groups were placed on a high-glucose, high-fat diet for 4 weeks before STZ injections to induce diabetes. Mice in the DM group received daily metformin treatment starting at 13 weeks of age, while control groups received water. The diabetic evaluation was conducted in a single-blind manner, and all groups were euthanised at the study’s conclusion.
• RAW 264.7 macrophage cells were cultured in DMEM with 10% fetal bovine serum at 37°C in a CO2 incubator. Cells were exposed to varying glucose concentrations and other compounds (mannitol, 3TC, metformin) for 24 hours to assess the effects of high glucose levels.
• Statistical Analyses involved one-way ANOVA and Tukey’s test for multiple comparisons, with a significance threshold set at p < .05.

Results

• Mice in moderate and severe hyperglycaemia groups exhibited greater alveolar bone loss and decreased bone volume fraction compared to normal controls.
• Elevated levels of senescence markers (p16INK4A, p21CIS1) and increased LINE-1 transcript abundance were observed in the gingival tissues of hyperglycemic mice.
• Hyperglycemia promoted senescence-like changes in macrophages, evidenced by increased SA-β-Gal staining and upregulation of inflammatory markers (p21, p16, ORF1p).
• High glucose levels impaired macrophage migration and phagocytic capacity.
• SETDB1 expression varied with glycemic levels; higher in moderate but reduced in severe hyperglycemia.
• Knockdown of SETDB1 in macrophages led to enhanced senescence and inflammatory responses, while its overexpression reduced these effects.
• Metformin improved periodontal health by enhancing SETDB1 activity and reducing inflammation markers like p16 and ORF1p.

Limitations