Human endothelial cells were incubated with or without HMGB1 (1 μg/mL) in the presence or absence of ketamine, an nuclear factor (NF)-κB inhibitor (PDTC), anti–toll-like receptor (TLR)2/4 antibody, or small interfering RNA (siRNA). The anti-inflammatory activities of ketamine were determined by measuring solute flux, leukocyte adhesion and migration, and activation of proinflammatory proteins in HMGB1-activated endothelial cells. The effect of ketamine on TLR-2/4 and NF-κB activation was evaluated using enzyme-linked immunosorbent assays and immunofluorescence confocal microscopy assay.
We found that ketamine inhibited the HMGB1-mediated barrier disruption, neutrophil adhesion and migration, and expression of cell adhesion molecules in a dose-dependent manner. Furthermore, ketamine downregulated the TLR-2 and -4, expression in HMGB1-activated endothelial cells. Treatment with ketamine also significantly inhibited the activation of TLR2/4 and the nuclear translocation of NF-κB p50/p65. Furthermore, our study shows that the HMGB1-induced release of inflammatory mediators was suppressed by PDTC, anti-TLR2/4 antibody, and siRNA.
Our study has demonstrated that ketamine exerts anti-inflammatory effects in HMGB1-mediated proinflammatory responses in a dose-dependent manner. The mechanism responsible for these effects involves the TLR2/4 and NF-κB signaling pathway.
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