Signal-transducing mechanisms of ketamine-caused inhibition of interleukin-1 beta gene expression in lipopolysaccharide-stimulated murine macrophage-like Raw 264.7 cells.
Ketamine may affect the host immunity. Interleukin-1 beta (IL-1 beta), IL-6, and tumor necrosis factor-alpha (TNF-alpha) are pivotal cytokines produced by macrophages. This study aimed to evaluate the effects of ketamine on the regulation of inflammatory cytokine gene expression, especially IL-1 beta, in lipopolysaccharide (LPS)-activated murine macrophage-like Raw 264.7 cells and its possible signal-transducing mechanisms. Administration of Raw 264.7 cells with a therapeutic concentration of ketamine (100 microM), LPS, or a combination of ketamine and LPS for 1, 6, and 24 h was not cytotoxic to macrophages. Exposure to 100 microM ketamine decreased the binding affinity of LPS and LPS-binding protein but did not affect LPS-induced RNA and protein synthesis of TLR4. Treatment with LPS significantly increased IL-1 beta, IL-6, and TNF-alpha gene expressions in Raw 264.7 cells. Ketamine at a clinically relevant concentration did not affect the synthesis of these inflammatory cytokines, but significantly decreased LPS-caused increases in these cytokines. Immunoblot analyses, an electrophoretic mobility shift assay, and a reporter luciferase activity assay revealed that ketamine significantly decreased LPS-induced translocation and DNA binding activity of nuclear factor-kappa B (NF kappaB). Administration of LPS sequentially increased the phosphorylations of Ras, Raf, MEK1/2, ERK1/2, and IKK. However, a therapeutic concentration of ketamine alleviated such augmentations. Application of toll-like receptor 4 (TLR4) small interfering (si)RNA reduced cellular TLR4 amounts and ameliorated LPS-induced RAS activation and IL-1 beta synthesis. Co-treatment with ketamine and TLR4 siRNA synergistically ameliorated LPS-caused enhancement of IL-1 beta production. Results of this study show that a therapeutic concentration of ketamine can inhibit gene expression of IL-1 beta possibly through suppressing TLR4-mediated signal-transducing phosphorylations of Ras, Raf, MEK1/2, ERK1/2, and IKK and subsequent translocation and transactivation of NF kappaB.
Chen
Ta-Liang
TL
Department of Anesthesiology, Taipei Medical University, Taipei, Taiwan.
Chang
Chia-Chen
CC
Lin
Yi-Ling
YL
Ueng
Yune-Fang
YF
Chen
Ruei-Ming
RM
D000818:Animals; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics*; D053583:Interleukin-1beta / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics; D007649:Ketamine / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000502:physiology*; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D051197:Toll-Like Receptor 4 / Q000037:antagonists & inhibitors / Q000378:metabolism
D053583:Interleukin-1beta; D008070:Lipopolysaccharides; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; D007649:Ketamine