Study On TLR4, HMGB1 And Downstream Signaling Pathway In Distal Tissue Injury Following Intestinal Ischemia Reperfusion

Objective:To characterize HMGB1-TLR4 and downstream signalling pathways in intestinal ischemia/reperfusion (I/R) injury.Methods:40 Specific-Pathogen-Free male C57BL/6 mice were randomly divided into five groups (n=8 per group):sham, control, anti-HMGB1, anti-MyD88, and anti-TRIF antibody groups. The control IgG antibody, or anti-HMGB1, or anti-MyD88, or anti-TRIF antibodies (all,1 mg/kg,0.025%) were injected via the caudal vein 30 min prior to ischemia. After anesthetization, the abdominal wall was opened and the superior mesenteric artery was exposed, followed by 60 min mesenteric ischemia and then 60 min reperfusion. For the sham group, the abdominal wall was opened for 120 min without I/R. We measured levels of serum NF-κBp65, IL-6, and TNF-a, and MPO activity in the lung and liver, as well as, morphological changes that occurred in the lung and intestinal tissues. Levels of mRNA transcripts encoding HMGB1 and NF-κB were measured by real-time quantitative PCR, and levels of HMGB1 and NF-κB protein were measured by western blot. Results were analysed using one-way analysis of variance.Results:Blocking HMGB1, MyD88, and TRIF expression by injecting anti-HMGB1, anti-MyD88, or anti-TRIF antibodies prior to ischemia can significantly reduce the levels of inflammatory cytokines in serum, including TNF-a, IL-6, and NF-κB, and can also alleviate tissue injury in the lung and small intestine compared with the control group in the mouse intestinal I/R model. The administration of anti-HMGB1, anti-MyD88, and anti-TRIF antibodies can significantly reduce damage caused by I/R, which might have therapeutic implications and the role of anti-HMGB1 antibody was the most obvious.Conclusions:HMGB1 and its downstream signalling pathways play important roles in the mouse intestinal I/R injury and the effect of the TRIF-dependent pathway are slightly larger.