Inhibiting the toll-like receptor 4 Toll/interleukin-1 receptor domain protects against hepatic warm ischemia and reperfusion injury in mice.

OBJECTIVE: During Toll-like receptor 4 signaling, the Toll/interleukin-1 receptor domain is essential for interactions with downstream Toll/interleukin-1 receptor domain-containing adaptor proteins. The aim of this study is to investigate the role of the Toll/interleukin-1 receptor domain in the Toll-like receptor 4 signaling pathway during hepatic ischemia and reperfusion injury.

DESIGN: We genetically blocked the function of Toll/interleukin-1 receptor domain in mice and examined the effect on Toll-like receptor 4 signaling and the response to hepatic ischemia and reperfusion.

SETTING: University research laboratory.

SUBJECTS: Male BALB/c mice.

INTERVENTIONS: Male BALB/c mice were hydrodynamically administrated the target gene plasmid pTIR-IRES2-EGFP, empty vector containing enhanced green fluorescent protein, or normal saline. Animals underwent 90 minutes of partial hepatic ischemia, followed by 6 or 24 hours of reperfusion. Hepatic injury was assessed by measuring serum alanine transaminase, hepatic histology, and malondialdehyde. The expression of inflammatory cytokines and nuclear factor-κB phosphorylation was examined in liver tissues. Hepatic apoptosis was evaluated by caspase-3 assays, terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick-end labeling staining, and the activation of Jun N-terminal kinase and p38 mitogen-activated protein kinase.

MEASUREMENTS AND MAIN RESULTS: Blocking the Toll/interleukin-1 receptor domain resulted in markedly lower serum alanine transaminase levels, reduced histologic injury, and lower malondialdehyde levels following 6 or 24 hours of hepatic reperfusion than mice receiving the vector alone or normal saline. Anti-Toll/interleukin-1 receptor treatment also reduced Toll-like receptor 4 expression and disrupted Toll/interleukin-1 receptor-Toll/interleukin-1 receptor interactions in Toll-like receptor 4 signaling pathways. Blocking the Toll/interleukin-1 receptor domain also prevented Toll-like receptor 4-mediated mitogen-activated protein kinase activation (via Jun N-terminal kinase and p38 mitogen-activated protein kinase), an activation that mediated liver ischemia and reperfusion injury via caspase-3 activation, resulting in increased hepatocellular apoptosis. Lastly, blocking the Toll/interleukin-1 receptor domain decreased inflammatory cytokine production by inhibiting nuclear factor-κB activation.

CONCLUSIONS: Toll/interleukin-1 receptor domain inhibition disrupts the interaction of Toll-like receptor 4 with its adaptor proteins, which abrogates downstream signaling pathways and prevents the activation of nuclear factor-κB and Jun N-terminal kinase/p38. This reduction in signaling consequently reduces hepatic inflammation, cell apoptosis, and hepatic damage. Toll/interleukin-1 receptor domain-targeted therapy thus represents a new approach to inhibit the intracellular Toll-like receptor 4 signaling pathway and reveals novel therapeutic target sites, which will facilitate the development of specific therapeutic agents.