Warm Ischemia-induced Alterations in Oxidative and Inflammatory Proteins in Hepatic Kupffer Cells in Rats

Abstract

The aim of the study was to investigate the impact of ischemia/reperfusion injury on the proteome of Kupffer cells. Lean Zucker rats (n ‫؍‬ 6 each group) were randomized to 75 min of warm ischemia or sham operation. After reperfusion for 8 h, Kupffer cells were isolated by enzymatic perfusion and density gradient centrifugation. Proteins were tryptically digested into peptides and differentially labeled with iTRAQ (isobaric tags for relative and absolute quantitation) reagent. After fractionation by cation exchange chromatography, peptides were identified by mass spectrometry (ESI-LC-MS/MS). Spectra were interrogated against the Swiss-Prot database and quantified using ProteinProspectorா. The results for heat shock protein 70 and myeloperoxidase were validated by ELISA. There is substantial evidence that indicates that Kupffer cells play a central role in the pathogenesis of liver parenchymal cell damage as seen during ischemia/reperfusion injury (1). The activation of Kupffer cells that occurs during ischemia results in the production of oxygen radicals and the modulation of proinflammatory and anti-inflammatory cytokines (2). These mediators are implicated in hepatic cell injury and sinusoidal function. It has been demonstrated that modulation of Kupffer cells can protect the liver (3, 4). The mechanisms that have been postulated include a decreased production of oxygen radicals and neutrophil infiltration. However, little is known about the differential effects of protein expression and changes in post-translational modifications in Kupffer cells that are exposed to warm ischemia. One objective of proteome research is to identify and describe the complex responses of an organism to different stimuli (5, 6). Rapid progress in both protein separation and identification techniques has made mass spectrometry a powerful tool for characterizing the protein content of a biological system (7-13).Isotope ratio mass spectrometry with isobaric tags for relative and absolute quantitation (iTRAQ) 1 allows for the quantification and identification of the protein content of up to four samples in one experiment by labeling all peptides in a given sample (14). Peptides of the samples are modified by incorporating the four different reagents. The derivatized peptides are indistinguishable in MS but exhibit intense low mass MS/MS signature ions that support quantitation.To our knowledge there have been no studies to date that have used a proteomic approach to study the effects of warm ischemia on protein expression in Kupffer cells. We hypothe- The abbreviations used are: iTRAQ, isobaric tags for relative and absolute quantitation; AIF-1, allograft inflammatory factor-1; C3, complement factor 3; C3a, activated C3; FDR, false discovery rate; IL, interleukin. Research

L-arginine in the ischemic phase protects against liver ischemia-reperfusion injury

“…IRI was established by clamping the vessels to the left lateral and median hepatic lobes, which account for 70% of the rabbit liver mass 25 . In other animal models, it has been reported that hepatic insult is similar to the clinical situation in which the liver is rendered ischemic during total vascular exclusion for liver resection [25][26][27][28] . Indeed, in the present study, 60 minutes of hepatic warm ischemia followed by 120 minutes of reperfusion caused severe liver injury in rabbits, as demonstrated by the structural damage to the liver and the increased serum levels of AST, ALT, and LDH.…”

A toxicoproteomic study on cardioprotective effects of pre-administration of docetaxel in a mouse model of adriamycin-induced cardiotoxicity

“…T he f luorogenic r eagent ( DAABD-Cl) i s ex cessivly added to the sample; therefore, all the proteins with one or more cystein residues are labeled and are able to be detected by fluorescence detector. The cystein-free proteins are excluded from the analytical targets of FD-LC-MS/MS; however, most of proteins contain one or more cystein r esidues [30] and ot her pr oteomic m ethods with la beling technologies, s uch a s isotope-coated a ffinity t ag (ICAT) targeting cystein thiol, ha ve be en widely applied t o proteomic studies [31,32]. For the sensitivity of FD-LC-MS/MS, the detection limit o f an 14 actin s tandard ( MW 43000, i ncluding 6 c ystein r esidues) w as 440 f emtomol pe r H PLC injection [ 24].…”

Daintain/AIF-1 accelerates the activation of insulin-like growth factor-1 receptor signaling pathway in HepG2 cells

“…It has been demonstrated that inflammatory factors such as TNF-α, IL-6 and IL-1α contribute to the progression of HCC (19,20). Daintain/AIF-1, as a novel inflammatory factor, has been found to be expressed in activated Kupffer Daintain/AIF-1 accelerates the activation of insulin-like growth factor-1 receptor signaling pathway in HepG2 cells cells lining the walls of liver sinusoids (21,22). Therefore, we hypothesized that daintain/AIF-1 may be involved in the development of HCC.…”