Down-regulation of toll-like receptor 4 alleviates intestinal ischemia reperfusion injury and acute lung injury in mice

Abstract

Intestinal ischemia reperfusion (IR) injury is a critical problem, which can cause intestinal injury locally and acute lung injury (ALI) distally by inflammatory responses and oxidative stress. Toll-like receptor 4 (TLR4) is involved in innate immune and inflammatory responses. This study was to determine whether TLR4 mutant can attenuate intestinal and lung injuries after intestinal IR. Wild type (WT) and TLR4 mutant mice were submitted to intestinal IR by occluding the superior mesenteric artery. Histological assessment of the intestine and the lung were conducted by HE staining. The levels of proinflammatory cytokines, oxidative stress markers, apoptotic index and other mediators were measured. In addition, a 24-hour survival study was performed. Histological assessment showed that intestinal IR caused serious injuries in the intestine and the lung, corroborated by increased proinflammatory cytokines in the circulation. TLR4 mutant suppressed the histological injuries as demonstrated by significantly decreased pathological scores. Consistent with the morphological results, the TLR4 mutant mice exhibited remarkably lowered cytokine expressions in the intestine (TNF-α, IL-6, IL-1β, and NF-κB) and the lung (NO, iNOS, MCP-1, MIP-2, NF-κB, and Caspase-3). ALT and creatinine were also significantly dampened in the liver and kidney, respectively. Furthermore, the survival rate over the course of 24 hours was significantly improved. Collectively, the findings reveal that TLR4 mutant significantly abated the intestinal IR injury and ALI at least in part by alleviating the inflammatory response and oxidative stress.

Keywords: Toll-like receptor 4 (TLR4); acute lung injury (ALI); inflammatory response; intestinal ischemia reperfusion (IR); oxidative stress.

Figure 1. Intestine tissue damage in mice submitted to intestinal IR or sham surgery in each groups

TLR4 mutant mice and wild-type (WT) mice were submitted to 60 min of ischemia by blocking the superior mesenteric artery (SMA) and then 120 min of reperfusion. Sham surgery operated the same procedure except for clamping the artery. Representative images of HE staining sections from the WT mice and TLR4 mutant mice are shown (original magnification: X 100). HE staining from the intestine unveiled widespread mucosal damage, loss of villi, and infiltration of inflammatory cells in the WT IR group, while TLR mutant showed beneficial effects. The severity of the intestinal injury was scored by Chiu's grading criteria and each column represents the means ± SD (n = 7/group). Data were compared by Kruskal-Wallis test and Mann-Whitney U test. #P < 0.05 vs. WT mice with sham surgery, and *P < 0.05 vs. WT mice with IR.

Figure 2. Local cytokine expressions in the intestine after IR

WT and TLR4 mutant mice were subjected to intestinal IR or sham surgery. The SMA was clamped for 60 min, followed by 120 min of reperfusion. Intestinal tissue expression of (A) TNF-α, (B) IL-1β, (C) IL-6, (D) NF-κB, and (E) IκBα were determined 120 min after reperfusion by ELISA. Measurements were in triplicate. Results are given as mean ± SD (n = 7/group). #P < 0.05 vs. WT sham and *P < 0.05 vs. WT IR by one-way ANOVA and LSD test.

Figure 3. Effects of TLR4 mutant on the circulation and distant organs after intestinal IR

WT mice and TLR4 mutant mice were sham-operated or subjected to 60 min of IR followed by 120 min reperfusion. Serum levels of (A) TNF-α, (B) IL-1β, and (C) IL-6 were measured by ELISA. Renal levels of (E) creatinine were measured by commercial kits. Hepatic expressions of (D) alanine aminotransferase (ALT) were determined by Western blot 120 min after reperfusion. Measurements were in triplicate. Bars denote the mean ± SD (n = 7/group). Data were compared by one-way ANOVA and LSD test. #P < 0.05 vs. WT sham and *P < 0.05 vs. WT IR.

Figure 4. Alteration in the lung morphology after intestinal IR

Representative micrographs at original 100 X magnification are shown. The SMA was occluded for 60 min, followed by 120 min reperfusion, to achieve intestinal IR. The lung tissues from the WT and TLR4 mutant mice submitted to IR or sham surgery were fixed and stained with HE. The lung injury was graded observing the presence of exudates, hyperemia/congestion, neutrophil infiltration, intra-alveolar hemorrhage/debris and cellular hyperplasia. Data were presented as mean ± SD (n = 7/group) and compared by one-way ANOVA and LSD test (#P < 0.05 vs. WT sham and *P < 0.05 vs. WT IR).

Figure 5. Lung cytokine expressions after intestinal IR

WT mice or TLR4 mutant mice were submitted to 60 min IR followed by 120 min reperfusion (n = 7). After harvesting the tissues, cytokine expressions in the lungs were measure: (A) iNOS, and (B) NO by commercial kits; (C) MCP-1, (D) MIP-2, (E) IκBα and (F) NF-κB by ELISA; (G) Caspase-3 by Western blot with β-actin used as internal control for loading; (H) Wet-to-dry ratios; (I) Bronchoalveolar lavage (BAL) to serum protein levels; (J) Hydrogen peroxide (H2O2); (K) MDA. Results are representative of triplicate experiments from each group. Data were expressed as mean ± SD and compared by one-way ANOVA and LSD test. #P < 0.05 vs. WT sham and *P < 0.05 vs. WT IR.

Figure 6. Survival benefit in TLR4 mutant mice after intestinal IR injury

WT mice and TLR4 mutant mice were subjected to intestinal IR and then observed for 24 hours. Each point in the figure shows the mean survival rate at each time point. The survival rate was estimated by Kaplan-Meier method and compared by the log-rank test (n = 16/group). #P < 0.05 vs. WT mice with IR.