Hyperglycemia induces ROS production in human gingival fibroblasts (HGFs)

Abstract

Objectives: Periodontopathogens initiate inflammatory reactions in human periodontium leading to tissue destruction. Diabetic patients with chronic hyperglycemia causing tissue accumulation of reactive oxygen species (ROS) could be one reason underpinning the increased periodontitis susceptibility of the former. This study investigated the effects of hyperglycemic with or without Porphyromonas gingivalis or Escherichia coli lipopolysaccharide (LPS) on ROS production in human gingival fibroblasts (hGF) or human periodontal ligament cells (hPDLCS). Methods: hGF (donors 35-60 years, n=10) hPDLCS (donors <25 years, n=15) were challenged by P. gingivalis or E. coli LPS(Invivogen)(0.5-2.0 μmol/L) for 24h, and at various glucose concentrations (10-100mM/L) over 72 hours. ROS production was measured using ROS assay kit(Abcam) and ROX GREEN staining (CellROX™ Green Flow Cytometry Assay Kit, Invitrogen). Inflammatory cytokines signal transduction were detected by qPCR. Results: Under normal glucose concentration, nil increase in ROS production from hGFs (3.52±0.31unit) or hPDLCs (13.8±0.3unit) could be observed upon various LPS stimulation (0.5, 1.0 or 2.0 μmol/L) while at high glucose concentrations (10-100 mmol/L), increased production/accumulation of ROS in hGFs could be observed at 60mmol/L(8.92±0.1unit p<0.0001). mRNA levels of IL1b, TNF-a, and IL-6 under 80 mmol/L glucose showed 12.75 fold increase in hGFs(One-way ANOVA with Bonferroni correction, 12.75±0.65 P < 0:0001). No increase of mRNA level for IL1b, TNF-a, or IL-6 in hGF/hPDLC could be observed upon LPS stimulation at normal glucose condition while hPDLCs signal of the tested cytokines were not upregulated upon high glucose with or without LPS（OD≈14）. Conclusions: In contrast to earlier studies(Wang, P.-L. and Ohura, K. 2002), this investigation indicated that P. gingivalis or E. coli LPS at the test concentrations did not stimulate ROS production in hGFs. Rather, under hyperglycemia, significant increase in ROS production was observed in hGF. Further investigation is on the way to explore the effects of LPS under hyperglycemia on hGF.