Abstract
BackgroundThe NF-κB signaling pathway plays a role in local and remote tissue damage following ischemia-reperfusion (I/R) injury to skeletal muscles. Evidence suggests that exosomes can act as intercellular communicators by transporting active proteins to remote cells and may play a role in regulating inflammatory processes. This study aimed to profile the exosomal protein expression in the serum of NF-κB knockout mice following skeletal muscle ischemia-reperfusion injury.ResultsTo investigate the potential changes in protein expression mediated by NF-κB in secreted exosomes in the serum following I/R injury, the levels of circulating exosomal proteomes in C57BL/6 and NF-κB−/− mice were compared using two dimensional differential in-gel electrophoresis (2-DE), liquid chromatography tandem mass spectrometry (LC-MS/MS), and proteomic analysis. In C57BL/6 mice, the levels of circulating exosomal proteins, including complement component C3 prepropeptide, PK-120 precursor, alpha-amylase one precursor, beta-enolase isoform 1, and adenylosuccinate synthetase isozyme 1, increased following I/R injury. However, in the NF-κB−/− mice, the expression of the following was upregulated in the exosomes: protease, serine 1; glyceraldehyde-3-phosphate dehydrogenase-like isoform 1; glyceraldehyde-3-phosphate dehydrogenase; and pregnancy zone protein. In contrast, the expression of apolipoprotein B, complement component C3 prepropeptide, and immunoglobulin kappa light chain variable region was downregulated in NF-κB−/− mice. Bioinformatic annotation using the Protein Analysis Through Evolutionary Relationships (PANTHER) database revealed that the expression of the exosomal proteins that participate in metabolic processes and in biological regulation was lower in NF-κB−/− mice than in C57BL/6 mice, whereas the expression of proteins that participate in the response to stimuli, in cellular processes, and in the immune system was higher.ConclusionsThe data presented in this study suggest that NF-κB might regulate exosomal protein expression at a remote site via circulation following I/R injury.
Highlights
The NF-κB signaling pathway plays a role in local and remote tissue damage following ischemia-reperfusion (I/R) injury to skeletal muscles
The electron microscopy experiments performed using the purified exosome samples obtained from the serum revealed the presence of vesicles that were derived from a C57BL/6 mouse, with an approximate diameter of 50–200 nm (Fig. 3)
A proteomics approach involving 2-DE was used to characterize the changes occurring in the serum exosomes following I/R injury and to compare such changes seen in C57BL/6 and NF-κB−/− mice
Summary
The NF-κB signaling pathway plays a role in local and remote tissue damage following ischemia-reperfusion (I/R) injury to skeletal muscles. Evidence suggests that exosomes can act as intercellular communicators by transporting active proteins to remote cells and may play a role in regulating inflammatory processes. This study aimed to profile the exosomal protein expression in the serum of NF-κB knockout mice following skeletal muscle ischemia-reperfusion injury. Ischemia-reperfusion (I/R) injury to the skeletal muscle leads to the production of oxygen free radicals, resulting in the occurrence of tissue lipid peroxidation upon re-oxygenation, and release of pro-inflammatory cytokines such as IL-6, IL-1, and TNF-α through the NF-κB signaling pathway. NF-κB plays an important role in the pathogenesis of I/R injury to the skeletal muscle. It has been reported that inhibition of NF-κB prevents local and remote organ injury following I/R injury [8]
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