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.


METHODS
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.


METHODS
University research laboratory.


METHODS
Male BALB/c mice.


METHODS
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.


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.
1Department of Emergency, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. 2Research Institute of Digestive Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. 3Department of Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
He
H
Meimei
Min
M
Dejin
Chen
C
Erzhen
Shi
S
Minmin
Jiang
J
Songyao
Li
L
Jianfang
Chen
C
Hao
D000818:Animals; D008099:Liver / Q000098:blood supply*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D017472:Receptors, Interleukin-1 / Q000037:antagonists & inhibitors*; D015427:Reperfusion Injury / Q000517:prevention & control*; D015398:Signal Transduction; D051197:Toll-Like Receptor 4 / Q000037:antagonists & inhibitors*; D052096:Warm Ischemia
D017472:Receptors, Interleukin-1; D051197:Toll-Like Receptor 4