Enhancing apoptotic cell clearance mitigates bacterial translocation and promotes tissue repair after gut ischemia-reperfusion injury

Abstract

A key aspect of intestinal ischemia/reperfusion (I/R) injury is the increased occurrence of apoptotic cell death in the gut. Insufficient clearance of apoptotic cells leads to increased inflammation and impaired tissue repair. Our recent studies have shown that administration of milk fat globule-epidermal growth factor-factor 8 (MFG-E8), a crucial molecule for apoptotic cell clearance, reduces apoptosis and inflammation under various disease conditions. The purpose of this study was to determine whether MFG-E8 reduces bacterial translocation and promotes tissue repair in a mouse model of gut I/R. Gut ischemia was induced by placing a microvascular clip across the superior mesenteric artery for 90 min in male adult mice. After removing the clip, recombinant murine MFG-E8 (rmMFG-E8) (0.4 µg/20 g BW) or normal saline (Vehicle) was intraperitoneally injected. At 4 h after reperfusion, apoptosis in the gut was measured by TUNEL staining. The mesenteric lymph node (MLN) complex was homogenized and plated on chocolate agar plates for bacterial culture. Neutrophil infiltration was assessed by examining myeloperoxidase (MPO) activity in the gut. Vascular endothelial growth factor (VEGF) levels in the gut, an indicator of tissue repair, were measured by western blotting. Out results showed that TUNEL-positive staining in the gut increased significantly in gut I/R vehicle-treated mice. Treatment with rmMFG-E8 markedly suppressed the number of apoptotic cells. Bacterial translocation to the MLN was minimal in sham mice, but was extensive in gut I/R vehicle-treated mice. rmMFG-E8 treatment significantly reduced bacterial translocation to the MLN. Similarly, gut I/R induced a significant increase in intestinal MPO activities in vehicle-treated mice. rmMFG-E8 treatment markedly reduced the increase in intestinal MPO activities after gut I/R. Intestinal levels of VEGF decreased significantly at 4 h after gut I/R. rmMFG-E8 treatment significantly increased intestinal VEGF levels. Thus, enhancing apoptotic cell clearance by rmMFG-E8 mitigates bacterial translocation, inhibits neutrophil infiltration and promotes tissue repair after gut I/R. Enhancing apoptotic cell clearance can be a novel concept in the treatment of gut I/R injury.

Figure 1.

Decrease in intestinal MFG-E8 protein levels after gut I/R. MFG-E8 protein levels were assessed by western blot analysis. A representative gel is presented. Data are expressed as means ± SE, ^*P<0.05 vs. Sham by Student’s t-test, n=5/group.

Figure 2.

Upregulation of Bcl-2 and downregulation of cleaved PARP-1 by rmMFG-E8 after gut I/R. Small intestinal levels of Bcl-2 and PARP-1 were measured by western blot analysis in sham-operated animals (Sham) and ischemia/reperfusion animals treated with normal saline (Vehicle) or rmMFG-E8 at 4 h after reperfusion. Representative blots were provided.

Figure 3.

Decreases in apoptosis by rmMFG-E8 after gut I/R. The small intestinal sections were stained with TUNEL (green fluorescence) and counterstained with propidium iodide (red). Photomicrographs of small intestinal sections from sham-operated animals (Sham) and ischemia/reperfusion animals treated with normal saline (Vehicle) or rmMFG-E8 at 4 h after reperfusion. Original magnification, x100.

Figure 4.

Attenuation of gut injury by rmMFG-E8 after gut I/R. Photo-micrographs of small intestinal sections from sham-operated animals (Sham) and ischemia/reperfusion animals treated with normal saline (Vehicle) or rmMFG-E8 at 4 h after reperfusion. Original magnification, x100.

Figure 5.

Inhibition of intestinal neutrophil infiltration by rmMFG-E8 after gut I/R. Neutrophil activity was assessed by MPO assay in sham-operated animals (Sham) and ischemia/reperfusion animals treated with normal saline (Vehicle) or rmMFG-E8 at 4 h after reperfusion. Data are expressed as means ± SE, ^*P<0.05 vs. Sham, ^#P<0.05 vs. Vehicle by one-way ANOVA and Student Newman-Keuls test, n=6/group.

Figure 6.

Suppression of bacterial translocation to mesenteric lymph nodes (MLN) by rmMFG-E8 after gut I/R. Bacterial translocation to mesenteric lymph nodes in sham-operated animals (Sham) and ischemia/reperfusion animals treated with normal saline (Vehicle) or rmMFG-E8 at 4 h after reperfusion. Data are presented as means ± SE (n=6), and compared by one-way ANOVA and Student Newman-Keuls test: ^*P<0.05 vs. Sham group; ^#P<0.05 vs. Vehicle group.

Figure 7.

Reduction of bacteremia by rmMFG-E8 after gut I/R. Bacteremia was determined by blood culture in sham-operated animals (Sham, n=6) and ischemia/reperfusion animals treated with normal saline (Vehicle, n=7) or rmMFG-E8 (n=6) at 4 h after reperfusion.

Figure 8.

Upregulation of VEGF by rmMFG-E8 after gut I/R. VEGF protein levels in the small intestine were measured by western blot analysis in sham-operated animals (Sham) and ischemia/reperfusion animals treated with normal saline (Vehicle) or rmMFG-E8 at 4 h after reperfusion. Data are presented as means ± SE (n=4), and compared by one-way ANOVA and Student Newman-Keuls test: ^*P<0.05 vs. Sham group; ^#P<0.05 vs. Vehicle group.