Effect Of High Mobility Group Box-1and Toll Like Receptor4Pathway On Intestinal Ischemia/Reperfusion Injury

Background:Intestine is an important organ on immune, endocrine and barrier function. The intestinal ischemia reperfusion (I/R) injury, is believed to lead to ALI, SIRS, even MODS, but the specific mechanism remains in controversial. The traditional theory of bacterial translocation which is being questioned cannot explain a lot of questions and the theory of’gut-lymph’pathway gets more and more attention, and content of gut lymph is still unknown. Toll like receptor4(TLR4) has close relationship with intestine I/R injury, and the inhibition of TLR4signal transmission may have a protective effect on distant tissue injury and the local injury induced by intestine I/R.Objective:The aim of research is to study the effect of TLR4signal pathway and the contents of intestinal lymph on local damage and distant tissue injury induced by intestinal I/R with the model of mice of genetical defect and their wildtype.Methods:Free amino acid levels in plasma and serum endotoxin and HMGB1were precalculated in I/R rats.48TLR4genetically defected and48wildtype mice, were randomly divided into four groups respectively and were injected these through the tail vein in the following manners:A groups (A1and A2) were injected with intestinal lymph drained from Sham rats, B groups (B1and B2) with intestinal lymph drained from intestinal I/R injury, C groups (C1and C2) with endotoxin and D groups (D1and D2) with HMGB1protein. The animals were sacrificed after180min and the endotoxin, high mobility group box1(HMGBI), and inflammation cytokine in mice serum and the injected lymph were detected. We also observed the morphology injury in intestine. The expressions of HMGBI, NF-κB, MyD88and TRIF were examined with Western Blotting and the mRNAs were determined at the same time with real time PCR.Results:The levels of HMGB1and endotoxin in lymph drained in intestinal I/R rats were significantly increased, and the jejunal and ileal mucosa were damaged and free amino acid levels in plasma were changed a lot. The endotoxin levels in all mice was low, with no significant difference. In wildtype mice injected with the intestinal I/R lymph, tissue damage, TNF-a and IL-6were significantly increased, and the intestinal damage in TLR4gene deficient mice was improved a lot. In mice injected with normal lymph and endotoxin, it manifested minor tissue damage, and normal intestinal mucosal morphology. In wildtype mice injected with HMGB1, the inflammatory cytokines were moderately elevated, while in the TLR4-/-mice it showed normal intestinal morphology and slightly elevated inflammatory cytokines. In subgroup of mice injected with I/R lymph, compared with TLR4gene deficient mice, mice in the wild-type one indicated that both MyD88and TRIF mRNA and protein expression levels are relatively higher, and especially the latter one is comparatively predominant.Conclusions:Intestinal ischemia-reperfusion can cause intestinal mucosal injury and the changes of free amino acid levels in plasma and intestinal barrier damage may be related to the decline glutamine concentration and the increase of protein catabolism;’gut lymph’pathway plays a significant role in the gut I/R injury; TLR4is thought to mediate intestinal inflammation and intestinal damage induced by I/R, and MyD88and TRIF pathways are both likely to get involved in the injury, especially the latter one; endotoxin in mesenteric lymph maybe is not involved in mediating intestinal I/R-induced systemic inflammation and mucosal damage; Probably HMGB1is involved in mediating I/R-induced intestinal inflammation and damage.