Mechanisms linking periodontitis and diabetes

Background + Aims

• Periodontitis, characterised by inflammatory-driven alveolar bone destruction, is exacerbated by diabetes mellitus (DM), which increases inflammatory and pro-osteoclastogenic (formation of bone-resorbing cells) factors in the periodontium.
• Conventional therapies for those with diabetes and periodontitis are limited, necessitating innovative treatments.
• Gut microbiota plays a critical role in regulating inflammation and metabolism, with positive modulation showing potential in alleviating disease.
• Probiotic intervention effectively regulates gut microbiota, influencing host immune function, intestinal barrier integrity, and metabolite production. Intestinal metabolites, acting as messengers between microbiota and the host, impact inflammatory and metabolic diseases. Raised intestinal metabolites mediated by gut microbiota may contribute to conditions such as colitis and cardiac disease.
• Lactobacillus casei (L. casei), a widely used probiotic, was employed in this study to modulate gut microbiota.
• The study hypothesizes that L. casei modulates gut microbiota, reducing alveolar bone loss in diabetic periodontitis (DP) by regulating the production and absorption of these metabolites.

Materials + Methods

• The study employed both in vivo and in vitro approaches to investigate the effects of probiotic casei and metabolite α-tocopherol acetate (α-TA) on diabetic periodontitis.
• In vivo study:
  • Six-week-old male db/db mice were used and monitored daily for a week in a specific pathogen-free space.
  • DP was induced by ligating the maxillary second molars with silk sutures and smearing Porphyromonas gingivalis on the sutures.
  • Mice were randomized into groups for 3 experimental setups:
    • Experimental setup 1: DP mice received intragastric gavage of either L. casei (test group) or phosphate buffer saline (PBS) (control group) for 4 weeks.
    • Experimental setup 2: DP mice received similar treatments but were housed on their own (singly-housed mice (Sh_mice)) or co-housed (Ch_mice).
    • Experimental setup 3: DP mice were injected intraperitoneally with α-TA (test group) or saline (control group) for 3 weeks.
  • Mice were euthanised at the end of the trials
  • Samples collected including blood, faeces, gingival tissues, maxillary bones, and ileum.
• In vitro study:
  • Human periodontal ligament cells (hPDLCs) and primary human gingival fibroblasts (HGFs) were extracted and cultured to assess whether increased serum metabolites directly affect human periodontal tissues.

Results

• In vivo:
  • The study demonstrated that positive regulation of gut microbiota using L. casei reduced alveolar bone loss in DP mice.
  • Microbiota modulation improved gut composition and increased α-tocopherol acetate (α-TA) absorption, reducing alveolar bone resorption and osteoclast activity. Increased osteoprotegerin (OPG) and decreased receptor activator of NF-ҡB ligand (RANKL) expression supported these effects.
  • Positive regulation of gut microbiota using casei altered metabolic profiles and increased serum α-TA levels in DP mice. Metabolomics identified significant changes in metabolites, with α-TA showing a 9.91-fold increase, likely due to enhanced intestinal absorption. Improved intestinal morphology, including increased villus height and decreased paracellular permeability, supported this absorption. These findings suggest α-TA, an antioxidant, as a key mediator linking gut microbiota to reduced alveolar bone loss.
  • Co-housing L. casei-treated DP mice with untreated mice increased serum α-T levels in untreated mice, likely through microbiota transfer. Principal coordinate analysis confirmed a closer gut microbiota composition between co-housed groups. ELISA analysis revealed significantly higher serum α-T levels in co-housed untreated mice compared to singly housed untreated mice, demonstrating the connection between gut microbiota and α-T levels.
  • Co-housing experiments confirmed the link between gut microbiota and α-TA levels, and α-TA administration reduced bone resorption, enhanced OPG, and suppressed RANKL expression in DP mice.
• In vitro:
  • α-T (the hydrolysed form of α-TA) promoted osteogenesis in hPDLCs and reduced inflammation in HGFs under high glucose and lipopolysaccharide conditions.
  • α-T upregulated osteogenic markers (ALP, RUNX2) and suppressed inflammatory cytokines (IL-1β, IL-6) and matrix metalloproteinase (MMP-9) expression.
  • Transcriptomic analysis linked these effects to the suppression of the STAT3 signaling pathway.
    • α-TA and α-T decreased p-STAT3 levels, reduced nuclear translocation of STAT3, and reduced the inflammatory responses.

Limitations