TLR4-HMGB1-, MyD88- and TRIF-dependent signaling in mouse intestinal ischemia/reperfusion injury

AIM: To characterize high-mobility group protein 1-toll-like receptor 4(HMGB1-TLR4) and downstream signaling pathways in intestinal ischemia/reperfusion(I/R) injury.METHODS: Forty specific-pathogen-free male C57BL/6 mice were randomly divided into five groups(n = 8 per group): sham, control, anti-HMGB1, anti-myeloid differentiation gene 88(My D88), and anti-translocatingchain-associating membrane protein(TRIF) antibody groups. Vehicle with the control Ig G antibody, antiHMGB1, anti-My D88, 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. Levels of serum nuclear factor(NF)-κB p65, interleukin(IL)-6, and tumor necrosis factor(TNF)-α were measured, along with myeloperoxidase activity in the lung and liver. Inaddition,morphologic changes that occurred in the lung and intestinal tissues were evaluated. Levels of m RNA 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 analyzed using one-way analysis of variance.RESULTS: Blocking HMGB 1, MyD 8 8, and TRIF expression by injecting anti-HMGB1, anti-My D88, or anti-TRIF antibodies prior to ischemia reduced the levels of inflammatory cytokines in serum; NF-κB p65: 104.64 ± 11.89, 228.53 ± 24.85, 145.00 ± 33.63, 191.12 ± 13.22, and 183.73 ± 10.81(P < 0.05); IL-6: 50.02 ± 6.33, 104.91 ± 31.18, 62.28 ± 6.73, 85.90 ± 17.37, and 78.14 ± 7.38(P < 0.05); TNF-α, 43.79 ± 4.18, 70.81 ± 6.97, 52.76 ± 5.71, 63.19 ± 5.47, and 59.70 ± 4.63(P < 0.05) for the sham, control, anti-HMGB1, anti-My D88, and anti-TRIF groups, respectively(all in pg/m L).Antibodies also alleviated tissue injury in the lung and small intestine compared with the control group in the mouse intestinal I/R model. The administration of antiHMGB1, anti-My D88, and anti-TRIF antibodies markedly reduced damage caused by I/R, for which anti-HMGB1 antibody had the most obvious effect.CONCLUSION: HMGB1 and its downstream signaling pathway play important roles in the mouse intestinal I/R injury, and the effect of the TRIF-dependent pathway is slightly greater.

Impact of intestinal ischemia/reperfusion and lymph drainage on distant organs in rats

AIM:To investigate the impact of intestinal ischemia/reperfusion(I/R) injury and lymph drainage on distant organs in rats.METHODS:Thirty-two Sprague-Dawley male rats,weighing 280-320 g,were randomly divided into blank,sham,I/R,and ischemia/reperfusion and drainage(I/R + D) groups(n = 8).All rats were subjected to 60 min ischemia by clamping the superior mesenteric artery,followed by 120 min reperfusion.The rats in the I/R + D group received intestinal lymph drainage for 180 min.In the sham group,the abdominal cavity was opened for 180 min,but the rats received no treatment.The blank group served as a normal and untreated control.A chromogenic limulus assay kit was used for quantita-tive detection of serum endotoxin.The serum concentrations of tumor necrosis factor-α(TNF-α),interleukin(IL)-6,IL-1β,soluble cell adhesion molecules(sICAM-1),and high mobility group protein box 1(HMGB1) were determined with an enzyme-linked immunosorbent assay kit.Histological evaluations of the intestine,liver,kidney,and lung were performed by hematoxylin and eosin staining and immunohistochemistry.HMGB1 protein expression was assayed by western blot analysis.RESULTS:The serum levels of endotoxin and HMGB1 in the I/R and I/R + D groups were significantly higher than those in the sham group(endotoxin,I/R and I/R + D vs sham:0.033 ± 0.004 EU/mL,0.024 ± 0.003 EU/mL vs 0.017 ± 0.009 EU/mL,respectively,P < 0.05;HMGB1,I/R and I/R + D vs sham:5.473 ± 0.963 EU/mL,4.906 ± 0.552 EU/mL vs 0.476 ± 0.406 EU/mL,respectively,P < 0.05).In addition,endotoxin and HMGB1 were significantly lower in the I/R + D group compared to the I/R group(P < 0.05).The serum inflammatory factors IL-6,IL-1β,and sICAM-1 in the I/R and I/R + D groups were significantly higher than those in the sham group(IL-6,I/R and I/R + D vs sham:41.773 ± 9.753 pg/mL,19.204 ± 4.136 pg/mL vs 11.566 ± 2.973 pg/mL,respectively,P < 0.05;IL-1β,I/R and I/R + D vs sham:144.646 ± 29.378 pg/mL,65.829 ± 10.888 pg/mL vs 38.178 ± 7.157 pg/mL,respectively,P < 0.05;sICAM-1,I/R and I/R + D vs sham:97.360 ± 12.714 ng/mL,48.401 ± 6.547 ng/mL vs 33.073 ± 5.957 ng/mL,respectively;P < 0.05).The serum TNF-α in the I/R group were significantly higher than in the sham group(45.863 ± 11.553 pg/mL vs 18.863 ± 6.679 pg/mL,respectively,P < 0.05).These factors were significantly lower in the I/R + D group compared to the I/R group(P < 0.05).The HMGB1 immunohistochemical staining results showed no staining or apparent injury in the blank group,and slight staining at the top of the microvillus was detected in the sham group.In the I/R group,both the top of villi and the basement membrane were stained for HMGB1 in most areas,and injury in the I/R + D group was less than that in the I/R group.HMGB1 expression in the liver,kidney,and lung of rats in the I/R + D group was significantly lower than the rats in the I/R group(P < 0.05).CONCLUSION:Lymph drainage could block the "gutlymph" pathway,improve intestinal barrier function,and attenuate distant organ injury incurred by intestinal I/R.