H2O2 activates matrix metalloproteinases through the nuclear factor kappa B pathway and Ca2+ signals in human periodontal fibroblasts

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Abstract

Background:

The mechanisms involved in reactive oxygen species and matrix metalloproteinase (MMP)-mediated periodontal tissue breakdown are unknown.

Objective:

To determine the effect of H2O2 in MMP-2 and MMP-9 activity, and the involvement of nuclear factor kappa B (NFκB) and Ca2+-mediated signals in human periodontal ligament fibroblasts.

Material and Methods:

Primary cultures were characterized for their phenotype and exposed for 24 h to sublethal doses (2.5–10 μm) of H2O2 or control media. NFκB involvement was evaluated through immunofluorescence of p65 subunit, using the NFκB blocking peptide SN50 and catalase. Ca2+ signals were analyzed by loading the cells with Fluo4-AM and recording the fluorescence changes in a confocal microscope before and after the addition of H2O2. 1,2-bis(o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl was used to chelate intracellular Ca2+. The activity and levels of MMP-2 and MMP-9 were analyzed by gelatin zymogram and densitometric scanning, and enzyme-linked immunosorbent assay, respectively. Statistical analysis was performed with stata V11.1 software using the ANOVA test.

Results:

H2O2 at concentrations of 2.5–5 μm induced Ca2+ signaling and NFκB subunit p65 nuclear translocation, whereas catalase, SN50 and BAPTA-AM prevented p65 nuclear translocation. H2O2 at 2.5–5 μm significantly increased MMP-9 and MMP-2 activity, while SN50 resulted in lower MMP-2 and MMP-9 activity rates compared with controls.

Conclusion:

Sublethal H2O2 induces Ca2+-dependent NFκB signaling with an increase in MMP gelatinolytic activity in human periodontal ligament.

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