Role of the nucleotide-binding oligomerization domain-containing protein 1 pathway in the development of periodontitis.

OBJECTIVES: During innate immune defense, host pattern recognition receptors, including toll-like receptors and nucleotide-binding oligomerization domain-like receptors (NLRs), can activate downstream pathways by recognizing pathogen-associated molecular patterns produced by microorganisms, triggering immune responses. NOD1, an important cell membrane protein in the NLR-like receptor protein family, exerts anti-infective effects through γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP) recognition. Oral epithelial cells resist bacterial invasion through iE-DAP-induced interleukin (IL)-8 production, recruiting neutrophils to sites of inflammation in response to bacterial threats to periodontal tissues. To date, the regulatory mechanisms of iE-DAP in gingival epithelial cells (GECs) are poorly understood. This study was conducted to investigate the role of the NOD1 pathway in the development of periodontitis by examining the effect of iE-DAP on IL-8 production in Ca9-22 cells. METHODS: IL-8 production by iE-DAP-stimulated-Ca9-22 cells was assessed using an enzyme-linked immunosorbent assay. Phosphorylation levels of intracellular signaling molecules were evaluated using western blot analyses. RESULTS: iE-DAP induced NOD1 receptor expression in Ca9-22 cells. Additionally, iE-DAP induced expression of pro-IL-1ß protein without extracellular secretion. Our results suggest that iE-DAP regulates IL-8 production by activating p38 mitogen-activated protein kinase (MAPK) and ERK1/2 signaling pathways. iE-DAP also promoted nuclear factor kappa-B p65 phosphorylation, facilitating its nuclear translocation. Notably, p38 MAPK and ERK1/2 inhibitors suppressed iE-DAP-stimulated IL-8 production, suggesting that JNK is not involved in this mechanism. CONCLUSIONS: Our results indicate that p38 MAPK and ERK1/2, but not JNK, are involved in innate immune responses in GECs.

P38α contributes to TNF-α-induced IL-8 production in human gingival cells.

Tumor necrosis factor-alpha (TNF-α) is a major inflammatory cytokine that induces interleukin (IL)-8 production. Although some studies have reported the involvement of the p38 MAPK signaling pathway in TNF-α-induced IL-8 production, its specific regulatory mechanisms in gingival epithelial cells (GECs) are still poorly understood. In the present study, Ca9-22 cells were used as representative GECs to investigate the effect of p38 signaling on TNF-α-induced IL-8 production. We found that TNF-α enhanced IL-8 production in Ca9-22 cells by activating the p38 signaling pathway, and one of its isoforms, p38α, played a key role. P38α deletion markedly inhibited TNF-α-induced IL-8 expression in Ca9-22 cells, while p38α gene rescue could reverse this effect. Further studies revealed that TNF-α-induced IL-8 production was markedly reduced when the threonine 180 and tyrosine 182 p38α phosphorylation sites were targeted for mutagenesis to alanine and phenylalanine, respectively, suggesting their critical role in the process. In conclusion, p38α plays an important role in TNF-α-induced IL-8 production, providing a potential therapeutic target to prevent and treat periodontal disease.

Effects of S-PRG filler eluate on MMP-1 and MMP-3 secretion by human gingival fibroblasts.

The aim of this study was to investigate the effects of surface reaction-type pre-reacted glass-ionomer (S-PRG) filler eluate on Matrix metalloproteinase (MMP)-1 and MMP-3 secretion by human gingival fibroblasts (HGF). The S-PRG filler eluate contains 6 ions (F, Na, Al, B, Sr and Si) released from the S-PRG filler. The S-PRG filler eluate stimulation induced a slight secretion of MMP-1 and MMP-3 by HGF. It also enhanced the phosphorylation of p38 and ERK. The increase in MMP-1 and MMP-3 secretion by the inflammatory cytokine TNF-α was suppressed by the S-PRG filler eluate. TNF-α-induced increases in the phosphorylation of ERK were slightly enhanced by S-PRG filler eluate. These findings may prompt the development of new therapeutic agents for oral inflammation with materials composed of S-PRG filler eluate.

Molecular depth profiling with cluster secondary ion mass spectrometry and wedges.

Secondary ion mass spectrometry and atomic force microscopy are employed to characterize a wedge-shaped crater eroded by 40 keV C(60)(+) bombardment of a 395 nm thin film of Irganox 1010 doped with four delta layers of Irganox 3114. The wedge structure creates a laterally magnified cross section of the film. From an examination of the resulting surface, information about depth resolution, topography, and erosion rate can be obtained as a function of crater depth in a single experiment. This protocol provides a straightforward way to determine the parameters necessary to characterize molecular depth profiles and to obtain an accurate depth scale for erosion experiments.

Construction of electrochemical aptasensor of carcinoembryonic antigen based on toehold-aided DNA recycling signal amplification.

Carcinoembryonic antigen (CEA), serves as a broad-spectrum tumor marker, and plays an important role in reflecting the existence, therapeutic evaluation, development, monitoring and prognosis of many types of cancer. An electrochemical aptasensor was designed for CEA detection based on toehold-aided DNA recycling. A partially hybridized Probe-4 (i.e. P2/P3/P4) was self-assembled on the surface of a gold electrode serving as the sensing platform. For CEA detection, CEA can bind with aptamer and free probe-1 (P1) can hybridize with P4, triggering toehold-aided DNA recycling. This enables the hybridization of more probe-5 (P5) (labeled with methylene blue (MB)) with P4, causing more methylene blue (MB) to be brought close to the electrode surface. An amplified current signal was thus generated due to more MB in the electrode surface. The proposed design showed good linearity between current response and log CEA concentration ranging from 0.1 to 50 ng·mL-1, with a detection limit of 20 pg mL-1. This aptasensor also showed high specificity for CEA detection, and was successfully used in spiked biological samples.