Proteomic Analysis of Exosomes Released from Heatstressed Hepatocytes Reveals Promotion of Programmed Cell Death Pathways

Background: Liver injury is a common feature of heat stroke (HS). Exosomes, which are recognized as a novel cell-cell communication pathway that harbour functional cargo, may be involved in the pathogenesis underlying HS. However, little is known about the role of hepatocyte-derived exosomes in HS liver injury and their protein expression patterns under HS. Methods: In this study, isobaric tags for relative and absolute quantitation (iTRAQ) technology with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis were applied to explore changes in the proteomic profiles of HS-hepatocyte exosomes. Findings: In total, 53 and 21 proteins were identified as up- and downregulated in the HS-exosomes. Based on bioinformatics analysis using gene ontology (GO) and the Kyoto encyclopedia of genes and genomes (KEGG) pathway database, we found that regulation of programmed cell death was the most significantly altered pathway. Next, a candidate protein (TRAIL, TNF-related apoptosis-inducing ligand) that played a central role in the target pathway was chosen for validation. Western blotting clearly demonstrated enrichment of TRAIL in the HS-exosomes. We also confirmed that serum exosomal TRAIL could be used as a clinical marker for disease severity assessment and prognostic prediction of HS liver injury. Interpretation: Taken together, this study presented the first comprehensive analysis of the HS-hepatocyte exosomal proteome, confirming the induction of HS liver injury by exosomes and revealing novel potential pathways and proteins awaiting further investigation for clinical application. Funding Statement: This work was supported by the National Natural Science Foundation of China (grant No. 81471839, No. 81671896 and No. 81873583). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: All animal experiments were conducted in compliance with the criteria outlined in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication 86-23, 1985 revision) and were approved by the Animal Care and Use Committee of the General Hospital of Guangzhou Military Command.