Abstract
Hepatic ischemia/reperfusion (I/R) injury represents a major risk factor for liver transplantation and is related to graft dysfunction and acute/chronic rejection. However, a significant part of these processes remain poorly characterized. To reveal differences in the proteome during liver I/R injury, we collected human liver biopsy samples during hepatectomy before and after the Pringle maneuver and conducted a TMT-based proteomic analysis through quantitative high-throughput mass spectrometry. We used a fold-change threshold of 1.3 and a t-test p-value < 0.05 as the criteria to identify 5,257 total quantifiable proteins. The levels of 142 proteins were increased, while the levels of 103 proteins were decreased in response to hepatic I/R treatment. Bioinformatic analysis further revealed that these differentially expressed proteins are mainly involved in multiple biological functions and enzyme-regulated metabolic pathways. Most proteins whose expression was changed are related to the defense, immune and inflammatory responses as well as lipid and steroid metabolic processes. Based on this finding, we developed a panel for targeted proteomic analysis and used the parallel reaction monitoring (PRM) method, qPCR and western blotting experiments to validate alterations in the expression of some of the identified proteins. The upregulated proteins were found to be involved in immunity and inflammatory responses, and downregulated proteins were enriched in metabolic pathways. This study therefore may provide a research direction for the design of new therapeutic strategies for hepatic ischemia/reperfusion injury.
Highlights
Severe intraoperative bleeding represents a major risk during hepatectomy
Stepwise workflow based on Tandem mass tag (TMT)‐based LC–MS/MS to select and evaluate the protein signatures of the hepatic I/R and control groups For the purpose of this study, liver biopsy samples were collected from hepatic hemangioma patients who underwent hepatectomy prior to the Pringle maneuver (Control group) and 10 min after the restoration of blood supply (I/R group)
Tandem mass tag (TMT) proteomics has emerged as a new method of protein mass spectrometry and has been widely used for protein quantification and identification in the fields of cancer research [14, 15], neurobiology [16] and tissue analysis, such as biomarker research [17, 18]
Summary
Severe intraoperative bleeding represents a major risk during hepatectomy. To avoid massive blood loss, continuous or intermittent vascular clamping of the hepatic artery and portal vein, the so-called Pringle maneuver, is an efficient method to reduce hemorrhage. The Pringle maneuver can lead directly to reperfusion in the liver. Several factors and mechanisms implicated in the hepatic IRI process are anaerobic metabolism, mitochondria damage, oxidative stress and ROS, intracellular calcium overload, liver Kupffer cells and neutrophils, nitric oxide, cytokines and chemokines [3,4,5,6,7]. Liver IRI remains a major problem in clinical transplantation, causing more than 10% of early transplant failures and leading to a higher incidence of both acute and chronic rejection [8, 9]. Despite the obvious clinical importance of liver I/R, the mechanisms that account for I/R injury are only partially understood and remain one of the most understudied areas in clinical and experimental transplantation [3]
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