Abstract
Intestinal ischemia and reperfusion injury is a model system of possible consequences of severe trauma and surgery, which might result into tissue dysfunction and organ failure. Neutrophils contribute to the injuries preceded by ischemia and reperfusion. However, the mechanisms by which intestinal ischemia and reperfusion stimulate and activate circulating neutrophils is still not clear. In this work, we used proteomics approach to explore the underlying regulated mechanisms in Wistar rat neutrophils after ischemia and reperfusion. We isolated neutrophils from three different biological groups; control, sham laparotomy, and intestinal ischemia/reperfusion. In the workflow, we included iTRAQ-labeling quantification and peptide fractionation using HILIC prior to LC-MS/MS analysis. From proteomic analysis, we identified 2,045 proteins in total that were grouped into five different clusters based on their regulation trend between the experimental groups. A total of 417 proteins were found as significantly regulated in at least one of the analyzed conditions. Interestingly, the enzyme prediction analysis revealed that ischemia/reperfusion significantly reduced the relative abundance of most of the antioxidant and pro-survival molecules to cause more tissue damage and ROS production whereas some of the significantly up regulated enzymes were involved in cytoskeletal rearrangement, adhesion and migration. Clusters based KEGG pathways analysis revealed high motility, phagocytosis, directional migration, and activation of the cytoskeletal machinery in neutrophils after ischemia and reperfusion. Increased ROS production and decreased phagocytosis were experimentally validated by microscopy assays. Taken together, our findings provide a characterization of the rat neutrophil response to intestinal ischemia and reperfusion and the possible mechanisms involved in the tissue injury by neutrophils after intestinal ischemia and reperfusion.
Highlights
The intestine is the most sensitive organ to ischemia and reperfusion (IR) injury
To assign the proteins according to their abundance in the best number of clusters, two validation indices, Xie-Beni index (Xie and Beni, 1991) and minimal centroid distance (Schwammle and Jensen, 2010), were applied and proteins were assigned to five different clusters based on their regulation trend (Figure 2, Supplementary Table S2)
Our proteomic approach revealed that intestinal ischemia/reperfusion causes the down regulation of important antioxidants together with the up regulation of enzymes involved in reactive oxygen species (ROS) production
Summary
The intestine is the most sensitive organ to ischemia and reperfusion (IR) injury. This injury can result from various clinical situations, such as intestinal obstruction, acute mesenteric ischemia, incarcerated hernia, small intestine transplantation, neonatal necrotizing enterocolitis, trauma, and shock, taking the patient to relentless clinical syndromes, and even death (Mojzis et al, 2001; Mallick et al, 2004; Guneli et al, 2007). It is clear that the reperfusion following ischemia leads to significantly greater mucosal intestinal injury as compared to the ischemia alone (Crissinger and Granger, 1989) whereas development of the systemic inflammatory response syndrome (SIRS) and multiple organ failure (MOF) can be the final consequences of IR (Ceppa et al, 2003). Among the polymorphonuclear leukocytes (PMNs), neutrophils are the first line of defense against bacterial and fungal infections (Kaufmann, 2008). A multistep process of neutrophil recruitment to the site of infection requires three types of adhesion receptors, like integrins, selectins, and adhesion receptors of the immunoglobulin superfamily (Rao et al, 2007)
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