Mechanisms linking periodontitis and diabetes

Background + Aims

• Diabetic periodontitis (DP) is a term used to describe periodontitis influenced by diabetes.
• Diabetes is thought to exacerbate periodontitis through various pathogenic mechanisms, including mitochondrial dysfunction.
• Previous studies identified mitochondrial dysfunction as a key factor in DP and highlighted hydroxytyrosol’s potential to prevent periodontitis by modulating mitochondrial function. These findings suggest mitochondrial protective strategies as promising pharmacological approaches for managing DP.
• Impaired mitochondrial biogenesis is driven by reduced peroxisome proliferator-activated receptor gamma-coactivator 1-alpha(PGC-1α) activity.
• Silibinin, a bioactive compound in silymarin, has shown promise in restoring mitochondrial function and mitigating bone loss in diabetes-related conditions.
• This study explored silibinin’s therapeutic effects in DP, demonstrating its ability to enhance PGC-1α activity, promote mitochondrial biogenesis, and alleviate DP.

Materials + Methods

• The study explored the therapeutic effects of silibinin on DP using both in vivo and in vitro methods.
• In vivo experiments involved:
  • Inducing diabetes in Wistar rats using streptozocin and creating periodontitis through silk ligation.
  • Rats were divided into six groups:
    • Control
    • Diabetic
    • DP
    • DP treated with different doses of silibinin (100-, 150, and 200-mg/kg/day).
  • Silibinin was administered via gastric lavage
  • Parameters assessed involved:
    • Microcomputed tomography (micro-CT) was used to assess alveolar bone loss
    • Mitochondrial dysfunction and cellular apoptosis were assessed using micro-CT, TUNEL staining, and mitochondrial assays
• In vitro experiments involved:
  • Human periodontal ligament cells (hPDLCs) were harvested from healthy donors and cultured under conditions mimicking diabetic periodontitis using hydrogen peroxide and silibinin at concentrations of 0.75 mM and 75 µM, respectively.
  • RNA interference was employed to knock down PGC-1α using siRNA, facilitating the assessment of its role in mitochondrial biogenesis.
  • Cell viability was determined through colorimetric 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays.
  • Apoptosis (cell death) was evaluated using flow cytometry and TUNEL staining.
  • Mitochondrial function was analszed through MitoSOX staining for mitochondrial reactive oxygen species (mtROS) and tetramethylrhodamine methyl ester (TMRM) staining for mitochondrial membrane potential (MMP).
  • Real-time PCR and mitochondrial DNA (mtDNA) copy number analyses were performed to quantify gene expression and mitochondrial content.
  • Adenosine triphosphate (ATP) levels were assessed using a luminescence assay, while mitochondrial complex I activity was measured by the oxidation of NADH. Alkaline phosphatase (ALP) activity and osteogenic differentiation were evaluated through ALP activity assays and Alizarin Red S staining, respectively.
  • Western blotting was used to quantify PGC-1α and other related proteins.
  • A drug affinity responsive target stability assay identified silibinin’s interaction with PGC-1α.
• Statistical analyses were performed using ANOVA and Tukey’s post hoc test, with significance set at p<0.05.

Results

• In vivo:
  • Silibinin at 150 mg/kg/day reduced alveolar bone loss and improved bone volume/total volume (BV/TV) and bone mineral density (BMD) in rats with DP. It also decreased gingival and periodontal ligament cell apoptosis while enhancing ALP activity, crucial for bone regeneration.
  • Mitochondrial assessments showed that silibinin reduced mitochondrial reactive oxygen species (mtROS) levels and preserved ATP production in DP rats. It also restored mitochondrial biogenesis by increasing mitochondrial DNA (mtDNA) copy numbers and upregulating critical regulatory genes, including PGC-1α, TFAM, and NRF2.
  • Immunohistochemical analysis confirmed increased PGC-1α expression in silibinin-treated DP rats.
• In vitro:
  • Hydrogen peroxide-treated hPDLCs mimicked oxidative stress conditions.
  • Silibinin improved cell viability, reduced apoptosis, and enhanced ALP activity and mineralization capacity. It also mitigated mitochondrial dysfunction by decreasing mtROS levels, restoring mitochondrial membrane potential, increasing ATP production, and enhancing mitochondrial complex I activity.
  • The protective effects of silibinin were linked to PGC-1α regulation. PGC-1α activation by silibinin reversed mitochondrial dysfunction and oxidative damage. A PGC-1α activator (ZLN005) showed similar effects, supporting its critical role.
  • PGC-1α knockdown abolished silibinin’s protective benefits, highlighting its dependence on this mitochondrial biogenesis pathway.

Limitations