Proteomic Analysis Of Exosomes Released From Heat Stress Hepatocyte Reveals Promotion Of Programmed Cell Death Pathway

BackgroundHeatstroke(HS)is a life-threatening disease,defined as a core temperature rise of ≥40℃,and more complicated Multiple Organ Dysfunction Syndrome(MODS).In recent years,due to the greenhouse effect,the incidence of heat stroke has also increased year by year.Different degrees of liver damage can occur in the early stage of heatstroke,and the degree of damage is closely related to the development and prognosis of heat stroke.Epidemiology shows that the mortality of patients with severe heat stroke and liver failure will also increase significantly,even as high as 50%.At present,the causes of liver damage caused by heat stroke are complicated,and the mechanism has not yet been elucidated.The exosomes are vesicular structures surrounded by bilayer phospholipids with a diameter of 30-200 nm.Exosomes contain thousands of biologically active molecules,such as proteins,lipids,and nucleic acids,that deliver these molecules to recipient cells,causing their function to change.Hepatogenic exosomes play an important role in various liver pathophysiological mechanisms.Exosomal proteins or miRNAs can serve as novel biomarkers for liver disease.People have explored the exocrine contents secreted in liver diseases.Different sources or different stimuli,the formation,assembly,secretion and function of the internal components of the exosomes are quite different.In addition,the effects of exosome from cell origin on different target cells are also different.However,little is known about the role of hepatocyte-derived exosomes in heatstroke liver damage and protein expression profiles.Therefore,studying the protein profile changes of hepatocyte-derived exosomes in heat stroke and their functional role in liver injury can improve our understanding of the pathogenesis of heat stroke and may provide potential clinical value.ObjectionThe research of this subject is mainly to carry out proteomic analysis of exosomes released from heat-stressed hepatocytes,and to study the possible molecular mechanisms and signaling pathways of differential protein-mediated hepatocyte injury,and provide new mechanisms for expanding the mechanism of heat-induced liver injury.The idea provides new targets and strategies for clinical intervention in heatstroke liver injury.Methods1.Heat stress stimulates the liver cells,collects the supernatant,and extracts the exosomes by ultra-high speed gradient centrifugation.The general characteristics of exosomes were identified by transmission electron microscopy,nanoparticle tracer analysis(NTA),and Western blot.2.Differences in protein composition between the two exosomes of the control and heat-stressed hepatocytes were analyzed by the isotope multi-label relative quantitative proteomics(iTRAQ)method.3.Bioinformatics analysis of differential proteins:GO function annotation,KEGG analysis.4.Through in vitro direct absorption experiments and in vivo tracer experiments to verify that exosomes can be taken up by liver cells and liver.5.Perform the following grouping:CTRL,CTRL-EXO,HS-EXO,HS,HS+GW4869.The ratio of apoptosis and necrotic apoptosis of hepatocytes was detected by flow cytometry.The liver damage of each group was evaluated by supermatant/serum ALT,AST and LDH levels.The activated Western blot was used to detect activated Caspase-3/8 and RIPKI/REPK3/phosphorylated MLKL expression,verification of hepatocyte apoptosis and necrotic apoptosis;liver injury by HE staining,TUNEL staining in mice.6.In addition,the most differential protein TRAIL was selected for further validation.Western blot analysis confirmed whether exosome TRAIL levels were associated with liver damage and overall disease severity.Results1.The microparticles secreted by hepatocytes have the characteristics of exosomes:the particle size is about 100 nm,a single distribution,and the morphology is a circular or cup-shaped classic bilayer membrane structure.2.iTRAQ method analysis There were 74 differential proteins in the exosome of the control group and heat-stressed hepatocytes.The GO ftunctional annotation analysis found that the differential proteins of the two exosomes played different roles in the body.Functionally,it is mainly divided into binding function,molecular function,catalytic activity,and transportation activity.The differential proteins above the cellular components are mainly localized to cells,organelle parts,and cell junctions.We then found that the proportion of programmed cell death was higher in the GO functional enrichment experiment.3.Through KEGG analysis,we found that the main enrichment pathways include necrotic apoptosis,PI3K-Akt,antigen processing and presentation,and apoptosis.4.Heat-stressed liver cell-derived exosomes can be absorbed by liver cells and liver.5.After heat stress,hepatocytes can release a large number of exosomes,causing liver damage:the number of apoptosis and necrotic apoptosis of hepatocytes in HS-EXO group is significantly increased.The CRTL-EXO group did not increase the above phenomenon,and the HSH-GW4869 group significantly reduced the occurrence of hepatocyte apoptosis and necrotic apoptosis.6.Among the positively regulated proteins involved in programmed cell death,TNFSF 10(TRAIL)has the highest fold change.At the same time,in the protein-protein interaction network map,we found that TRAIL plays a central role.7.Western blotting demonstrates the enrichment of TRAIL in HS-exosomes.Conclusion1.Heat-stressed hepatocytes can release a large amount of exosomes,causing liver damage.2.There are significant differences in the protein profiles carried by the two groups of exosomes,and the enrichment of programmed cell death signaling pathway is closely related to liver injury.3.Serum exosomes TRAIL may become a therapeutic target for liver damage.