Biological function of exosomes secreted by heat-stroke hepatocytes and induced liver damage / 解放军医学杂志

ABSTRACT

Objective To explore the biological function of exosomes secrected by heat-stroke hepatocytes and its effect on liver injury. Methods Exosomes were isolated from donor HepG2 hepatocytes and control hepatocytes by ultra-high-speed centrifugation. The morphology of exosomes was observed by transmission electron microscopy, the diameter of distribution was detected by nano-tracking technology and the expression of characteristic surface markers CD9, CD63 and CD81 were examined by Western blotting. The isobaric tags for relative and absolute quantification methods were applied to analyze the difference in protein composition between the control and HS hepatocyte derived exosomes. Biological information analysis on the differential protein set was performed by the Kyoto Encyclopedia of Genes and Genomes pathway analysis. Recipient hepatocytes were stimulated with sterilized PBS, control hepatocyte exosomes or HS exosomes (10 μg for 24 h), or directly exposed to HS or pretreated with exosome production inhibitor GW4869 2 h before HS. Supernatant alanine aminotransferase (ALT)/aspartate aminotransferase (AST)/lactic dehydrogenase (LDH) levels tested for evaluation of the hepatocyte damage. C57/BL6 mice were injected with sterilized PBS, control hepatocyte exosomes or HS exosomes (40 μg/per mice) through the tail vein, or treated with HS or intraperitoneally injected with GW4869 2 h before HS and sacrificed 9 h thereafter. Plasma ALT/AST/LDH level was examined to assess liver tissue damage. Results The microparticles secreted by hepatocytes are round or elliptical structures with a double-layer membrane coating, with a diameter ranging from 90–150 nm and highly expressed CD9, CD63 and CD81, suggesting the consistence to exosomes. The number of exosomes released by HS hepatocytes was significantly elevated than that in the control group [(8.46±1.38)×109/ml vs. (0.66±0.16)×109/ml, t=5.605, P=0.005]. HS significantly altered the protein expression profile of hepatocyte exosomes, and the enriched proteins were involved in necroptosis, PI3K-Akt signaling, antigen processing and presentation, apoptosis and NOD-like receptor signaling pathways. The supernatant level of ALT/AST/LDH of the recipients' hepatocytes in the HS exosomes-treated group was significantly increased, whereas that in the HS+GW4869 pretreatment group was significantly lower than that in the HS group. The serum ALT/AST/LDH level of the HS exosomes infusion group was significantly enhanced, and that of the pre-HS GW4869 treatment group was significantly reduced than that of the HS group. Conclusion HS may induce the release of exosomes from hepatocytes, which may lead to the changes in the protein profile of exosomes and affect the induction of acute liver injury.