To investigate if high glucose (HG) exacerbates Porphyromonas gingivalis (P.g) lipopolysaccharide (LPS)-induced inflammatory response in human gingival fibroblasts (HGFs) and to explore the underlying mechanisms. To provide a basis for the mechanism of diabetes aggravating periodontitis.

HGFs were divided into four groups: the control group (basal medium), the LPS group (treated with 5 μg/mL P.g-LPS for 24 h), the HG group (treated with 25 mmol/L glucose for 24 h), and the HG+LPS group (treated with 25 mmol/L glucose + 5 μg/mL P.g-LPS for 24 h). After culturing for 24 h in the respective media, the cells were harvested for experiments. Intracellular reactive oxygen species (ROS) and mitochondrial reactive oxygen species (mtROS) were detected using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and MitoSOX Red staining, respectively. Fluorescence intensity was analyzed by confocal fluorescence microscopy and directly measured in cell suspension. Immunofluorescence was used to detect changes in mitochondrial DNA (mtDNA) content of HGFs. Real-time fluorescence quantitative PCR was used to detect the content of mtDNA in cytoplasm and cell supernatant. Protein expression of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway was assessed by western blot, while mRNA expression levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) were detected by PCR.

Compared to the control group, both the LPS group and the HG group exhibited a significant increase in ROS and mtROS, with a more pronounced elevation in the HG+LPS group, demonstrating a synergistic effect (ROS: F = 396.5, P < 0.001; mtROS: F = 29.38, P < 0.001, CI < 1). The cytoplasmic mtDNA content was significantly elevated in the LPS group, with a more marked increase in the HG+LPS group (F = 27.85, P < 0.001). The supernatant mtDNA levels were significantly higher in both the LPS and HG groups, with a more pronounced elevation in the HG+LPS group (F = 15.26, P < 0.001). The phosphorylated proteins p-STING, p-TBK1, and p-P65 in the cGAS-STING pathway showed varying degrees of activation in the LPS and HG groups, reaching the highest levels in the HG+LPS group (p-STING: F = 52.67, P < 0.001; p-TBK1: F = 15.67, P = 0.001; p-P65: F = 9.83, P = 0.005), while p-IRF3 showed no significant differences among the groups (P = 0.072). Pro-inflammatory cytokine TNF-α was significantly higher in the HG+LPS group compared to the control group (F = 15.05, P < 0.001), and IL-1β increased in both the LPS and HG groups, with a more pronounced rise in the HG+LPS group (F = 30.98, P < 0.001). IL-6 showed no significant differences among the groups (P = 0.847).

High glucose and LPS act synergistically to enhance oxidative stress, accompanied by increased mtDNA release, which activates the cGAS-STING pathway, thereby amplifying the inflammatory response in HGFs.

To investigate the impact of metabolic labeling on Porphyromonas gingivalis (Pg) and compare the imaging effects of two fluorescent probes.

This study was reviewed by the unit Ethics Committee and was approved by the Experimental Animal Welfare Ethics Branch of the Unit Experimental Biomedical Ethics Committee. Pg integrated N-azidoacetylgalactosamine (Ac₄GalNAz) via a bioorthogonal reaction and was labeled with Cy5-DBCO or Cy7-DBCO via a click chemistry reaction. The bacteria were divided into Pg group (control, not fluorescently labeled), Cy5-Pg group (tagged by Cy5-DBCO), and Cy7-Pg group (tagged by Cy7-DBCO). A live/dead staining kit was applied to test the viability of Pg, Cy5-Pg, and Cy7-Pg. The mRNA levels of interleukin-6 (IL-6) and IL-8 and cell proliferation were examined in human gingival fibroblasts (HGFs) after the challenge of Cy5-Pg, Cy7-Pg, or Pg. To investigate the stability of metabolic labeling, Cy5-Pg or Cy7-Pg was cocultured with Escherichia coli (E. coli). Cy5-Pg and Cy7-Pg signal intensity with serial dilutions were examined using an in vivo imaging system (IVIS). Finally, C57BL/6J mice were orally administered Cy5-Pg or Cy7-Pg for IVIS detection, and the signal-to-background ratios were calculated.

Metabolic labeling could be applied to label live Pg in vitro. The optimal labeling concentrations for Cy5 and Cy7 were 20 μmol/L and 30 μmol/L, respectively. The area ratios of live to dead bacteria were approximately 2.0 in the three groups (F = 0.318, P＞0.05). After a 6-h challenge with Cy5-Pg, Cy7-Pg, or Pg, the mRNA levels of HGFs increased by 7.86-, 7.46-, and 6.56-fold for IL-6, respectively (F = 40.886, P＜0.001) and 12.43-, 13.03-, and 13.71-fold for IL-8 (F = 18.781, P＜0.01), were spectively, compared to that of the Ctrl group, which was not challenged by bacteria, where no significant differences were observed among the three groups (P＞0.05). HGFs were further challenged by Cy5-Pg, Cy7-Pg, or Pg at different MOIs, and cell proliferation was significantly inhibited (MOI = 10⁴∶1, F = 153.52, P＜0.001; MOI = 10⁵∶1, F = 331.21, P＜0.001; MOI = 10⁶∶1, F = 533.65, P＜0.001), with no significant differences among the three groups (P＞0.05). Within 24 h of co-culturing Cy5-Pg or Cy7-Pg with E. coli, minimal E. coli was detected. The intensities of Cy5 and Cy7 remained stable for 3 h. Additionally, the fluorescence signal intensities of Cy5 and Cy7 were linearly correlated with the concentration (R² = 0.97). After oral gavage of Cy5-Pg or Cy7-Pg in mice for the abdominal region at 1 h and 3 h, the signal-to-background ratios of Cy7-Pg were approximately 4.24-fold (t = 6.893, P＜0.01) and 3.77-fold (t = 4.407, P＜0.05) higher, respectively, than those of Cy5-Pg. For the isolated gastrointestinal tracts at 3 h, the signal-to-background ratio of Cy7-Pg was 5.19-fold higher than that of Cy5-Pg (t = 4.418, P＜0.05).

Metabolic labeling did not significantly affect viability, immunomodulatory ability, and toxicity. The imaging effect of Cy7 on IVIS was better than that of Cy5. Our study provided experimental evidence for the correlation between periodontitis and overall health.