Abstract
Aim
To investigate the influence of auxiliary chemical substances (ACS) and calcium hydroxide [Ca(OH)2] dressings on LPS/lipid A detection and its functional ability in activating Toll-like receptor 4 (TLR4).
Methodology
Fusobacterium nucleatum pellets were exposed to antimicrobial agents as following: 1) ACS: 5.25%, 2.5% and 1% sodium hypochlorite solutions (NaOCl), 2% chlorhexidine (CHX) (gel and solution) and 17% ethylenediaminetetraacetic acid (EDTA); 2) Intracanal medicament: Ca(OH)2 paste for various periods (1h, 24h, 7 days, 14 days and 30 days); 3) Combination of substances: a) 2.5% NaOCl (1h), followed by 17% EDTA (3 min) and Ca(OH)2 (7 days); b) 2% CHX (1h), afterwards, 17% EDTA (3 min) followed by Ca(OH)2 (7 days). Saline solution was the control. Samples were submitted to LPS isolation and lipid A purification. Lipid A peaks were assessed by MALDI-TOF MS while LPS bands by SDS-PAGE separation and silver staining. TLR4 activation determined LPS function activities. Statistical comparisons were carried out using one-way ANOVA with Tukey-Kramer post hoc tests at the 5% significance level.
Results
MALDI-TOF MS analysis of control lipid A demonstrated the ion cluster at mass/charge (m/z) 1882 and an intense band in SDS-PAGE followed by silver staining of control LPS. In parallel, LPS control induced a robust TLR4 activation when compared to ACS (p≤.001). 5.25% NaOCl treatment led to absence of lipid A peaks and LPS bands, while no changes occurred to lipid A/LPS after treatment with others ACS. Concomitantly, 5.25% NaOCl treated LPS did not activate TLR4 (p<.0001). As for Ca(OH)2, lipid A was not detected by MALDI-TOF nor by gel electrophoresis within 24h. LPS treated with Ca(OH)2 was a weak TLR4 activator (p<.0001). From 24h onwards, no significant differences were found among the time periods tested (p > 0.05). The addition of Ca(OH)2 for 7 days to cells treated either with 2.5% NaOCl or 2% CHX led to absence of lipid A and LPS bands, leading to a lower activation of TLR4.
Conclusion
5.25% NaOCl and Ca(OH)2 dressings from 24h onwards are able to induce loss of lipid A peaks and LPS detection, rendering a diminished immunostimulatory activity through TLR4.
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