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

Abstract

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 quantitative 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 "gut-lymph" pathway, improve intestinal barrier function, and attenuate distant organ injury incurred by intestinal I/R.

Keywords: Cytokines; Distant organ injury; Endotoxin; High mobility group protein box 1; Intestinal ischemia/reperfusion; Lymph drainage.

Figure 1

Immunohistochemistry staining of high mobility group protein box 1 in the jejunum (A), ileum (B), liver (C), kidney (D) and lung (E) (n = 8). I/R + D: Ischemia/reperfusion and drainage; I/R: Ischemia/reperfusion. Images shown at 100× magnification (A and B) and 200× magnification (C, D and E).

Figure 2

Expression levels of high mobility group protein box 1 protein in the jejunum and ileum (n = 8). Top: Western blotting analysis of high mobility group protein box 1 (HMGB1) in the jejunum (left) and ileum (right). Lane 1: Blank; Lane 2: Sham; Lane 3: I/R + D; Lane 4: I/R; Bottom: Quantification of HMGB1 protein levels in the jejunum (left) and ileum (right). HMGB1 levels were normalized to the levels of the β-actin internal control. ^aP < 0.05 vs the blank group; ^cP < 0.05 vs the sham group; ^eP < 0.05 vs the I/R group. I/R + D: Ischemia/reperfusion and drainage; I/R: Ischemia/reperfusion.

Figure 3

Expression levels of high mobility group protein box 1 in the liver, lung, and kidney (n = 8). Top: Western blotting analysis of high mobility group protein box 1 (HMGB1) in the liver (left), lung (middle), and kidney (right). Lane 1: Blank; Lane 2: Sham; Lane 3: I/R + D; Lane 4: I/R; Bottom: Quantification of HMGB1 protein levels in the liver (left), lung (middle), and kidney (right). HMGB1 levels were normalized to the levels of the β-actin internal control. ^aP < 0.05 vs the blank group; ^cP < 0.05 vs the sham group; ^eP < 0.05 vs the I/R group. I/R + D: Ischemia/reperfusion and drainage; I/R: Ischemia/reperfusion.