Molecular Mechanisms of Anti-Inflammatory Effects of Intravenous Anesthetic Agent Ketamine on Lipopolysaccharide-Activated Macrophages

Sepsis induced by gram-negative bacterial infection is a serious and life-threatening clinical syndrome occurred in intensive care unit patients. The prevalence of sepsis in hospitalized patients appears to significantly increase over the past decades, encountered by both surgeons and internists. Lipopolysaccharide (LPS), an endotoxin produced by gram-negative bacteria, is a main cause for endotoxic sepsis. Administration of rats with LPS can induce a sepsis-like syndrome. Immune cells in septic patients can be activated by LPS and increase the releases of inflammatory cytokines and oxidants into blood and tissues. These inflammatory cytokines and oxidants are important effectors to induce multiple-organs failure in sepsis. Ketamine is an intravenous anesthetic agent used for induction and maintenance of anesthesia during surgical procedures. Because ketamine has more-stable hemodynamics than barbiturates or inhaled anesthetic agents, this anesthetic agent is widely applied in critically ill patients as an inducer of anesthesia. Clinically, induction of anesthesia with ketamine may be associated with increases in cardiac output, arterial blood pressure, and heart rate. Previous studies using leukocytes and neutrophils demonstrate that ketamine has possible immunomodulating effects. Macrophages play critical roles in cellular host defense against infection and tissue injury. In response to stimuli, macrophages undergo a series of inflammatory processes, including chemotaxis, phagocytosis, intracellular killing, and release of inflammatory cytokines. In LPS-activated macrophages, ketamine was reported to inhibit TNF-α, IL-1β and IL-6 gene expressions. The molecular mechanisms of ketamine-induced inhibition of IL-1β and IL-6 gene expressions are known little. The mechanism of LPS-activated macrophages to induce the syntheses of inflammatory cytokines and NO is sequentially tranduced from the extracellular binding to intracellular signal transduction. First, LPS will bind to LPS-binding protein (LBP). Then, this LPS-LBP complex can bind to membrane protein CD-14 and activate Toll-like receptors (TLRs). TLRs are type I transmembrane proteins. The TLRs-involved signaling process responsible to LPS stimulation includes the adapter MyD88-dependent and –independent pathways. In the MyD88-dependent pathway, following activation of TLRs by LPS-LBP-CD-14, the intracellular signal can be tranduced from MyD88-TRAF6-MKK-MAKP to activate transcriptional factor AP-1. Another mechanism in the MyD88-dependent pathway is involved by the cascade of IκB signaling to activate another transcriptional factor NF-κB. AP-1 and NF-κB can translocate to nuclei, then bind to the promoters located in the 5’ end of inflammatory cytokine and iNOS genes as well as induce these gene expression. In the MyD88-independent pathway, following the binding of LPS to LBP, CD14 and TLRs, caspases will be activated and then metabolize the precursors of inflammatory cytokines into the mature cytokines. Our previous studies revealed that ketamine could inhibit the syntheses of nitric oxide, TNF-α, IL-1β and IL-6. This project is aimed to further investigate the molecular mechanism of ketamine -caused anti-inflammation in LPS-activated macrophages. This is a three-year project. The specific aims of this study are to evaluate the molecular mechanisms about the anti-inflammatory effects of ketamine on LPS-activated macrophages using murine macrophage-like Raw 264.7 cells as the experimental model. The goat of the first year will be specified to determine 1) if ketamine could interact with LPS to decrease the ability of this endotoxin in macrophage activation; 2) if ketamine could interrupt the LPS binding to LBP and CD-14; 3) if ketamine could modulate the syntheses of TLR4 mRNA and protein. The goat of the second year will be specified to determine 1) which TLR4 contributes the LPS-induced inflammatory responses in macrophages using RNAi and cloning methods; 2) the effects of ketamine on MyD88-dependent pathway, including MyD-88 -> TRAF-6 -> MKK -> MAKP -> AP-1. The goat of the third year will be specified to determine 1) the effects of ketamine on the other mechanism of MyD88-dependent pathway, including MyD-88 -> IκB-NFκB -> NF-κB; 2) the effects of ketamine on MyD-88-independent pathway, including the family of caspases. This project is expected to understand more about the drug interaction during the anesthesia of septic patients.