Study On Forensic Application Of Exosomes In Methamphetamine Toxic Injury And The Preliminary Study Of Its Role

Background:Methamphetamine(METH),also known as "ice",is the third most abused synthetic stimulant drug in the world,and the number one in China.The differernt feature of METH abuse is that it can cause damage to important organs of human while being addicted,especially the toxic damage to the central nervous system and cardiovascular system.In the practice of judicial identification,the determination of the state of injury and the cause of death caused by drug use has become a problem in forensic pratice.The lack of objective evaluation indicators of detoxification effect is also a problem that has been difficult to solve in the judicial field.Although a large number of studies have explored the mechanism of METH addiction and toxic injury,these results are still far from practical application.It is still a long way to go to find objective markers that can evaluate the degree of drug abuse injury,the recovery effect of detoxification and the cause of death.Exosomes have unique lipid bilayer,and contain rich contents.They not only participate in cell life activities as a medium of intercellular information transmission,but also play an important role in a variety of pathophysiological processes.They can be used as biomarkers for the diagnosis and prognosis of diseases.They are regarded as a treasure house for finding specific markers,and have shown important roles in many fields such as mechanism,diagnosis and treatment of diseases.But exosomes research in the field of drug started lately,especially in the field of METH research,there are only a few reports.The role and application of exosomes in METH toxic injury still have great exploration space.Objectives:In order to explore the role and application value of exosomes in METH toxicity injury,exosomes that secreted into blood and from cardiomyocytes respectively were studied.By constructing the single-dose and repeated-dose model of SD rats with METH,METH in blood and plasma exosomes were detected respectively to study its concentration-time variation rule and effect of plasma exosomes.The plasma exosomes were analyzed by miRNA microarray and verified,and the differentially expressed molecules were screened out to explore the application potential of plasma exosomes as biological markers of METH toxicity injury.In order to verify the mechanism of exosomes in METH toxic injury,the secretion,uptake and function of exosomes from cardiomyocytes stimulated by METH were explored through in vitro experiments,which enriched the connotation of METH toxic injury mechanism and provided new research directions and prevention strategies.Result:1.The concentration-time variation rule of METH in plasma exosomesAfter METH treatment,METH could be detected in the body fluid exosomes,suggesting that METH could be absorbed by the body fluid exosomes.Although plasma exosomes can not prolong the time window of METH detection for single METH administration,METH can accumulate in plasma exosomes for continuous METH treatment.Plasma exosomes can be used as a new candidate biological test material for METH detection and provide new evidence sources for METH detection.2.Verification of differentially expressed miRNAs in plasma exosomes after METH treatmentMETH leads to differences in the expression of plasma exosomes in rats,and many miRNA molecules with different expression levels are found.Bioinformatics analysis shows that most of the enriched biological functions of these miRNA target genes are involved in the regulation of nerve and cardiovascular system,and some of the differentially expressed genes are related to neurodegenerative diseases.They are enriched in calcium signaling pathway,mTOR signaling pathway,MAPK signaling pathway and VEGF signaling pathway.After further verification,the expression of miR-125b-5p,miR-187-5p,miR-206-3p and miR-200c-3p in plasma exosomes was up-regulated after METH treatment,which has the potential to be used as a molecular marker for METH abuse.Among them,the copy number of miR-200c-3p was correlated with the use duration of METH within a certain time range,and combined with the concentration of METH in the exosomes,which had the potential to be used as a marker for judging the duration of METH abuse and the degree of toxic injury.These miRNAs can provide new targets for the study of METH abuse biomarkers.3.Role of cardiomyocyte-derived exosomes in cardiovascular injury induced by METHMETH up-regulated the expression of Rab5 and Rab7,promoted the secretion of exosomes,and up-regulated the expression of miR-181,miR-1224,miR-25-3p,miR-29-3p and miR-200c-3p in exosomes.After these exosomes are uptaken by myocardial micro vascular endothelial cells,the phosphorylation levels of mTOR,ERK and eNOS in myocardial microvascular endothelial cells are up-regulated,and the migration,proliferation and angiogenesis of myocardial microvascular endothelial cells are enhanced.The mTOR signaling pathway may play a role.It provide a new direction for the role and application of exosomes in METH cardiovascular toxicity injury.Conclusions:The plasma exosomes induced by METH can not only carry METH,but also accumulate in the exosomes,which can be used as biological test materials for METH detection and may have the potential to judge the long-term abuse of METH.In addition to METH in plasma exosomes,there are differences in the expression of miRNA molecules in METH abuse.The copy number of miR-200c-3p is correlated with the duration of METH use in a certain time range,which may have the potential to judge the duration of METH abuse and the degree of toxic damage.METH stimulated the increase of secreted exosomes and the change of composition.These exosomes can enhance the migration,proliferation and tube formation of myocardial microvascular endothelial cells,thereby promoting the increase of angiogenesis and having the potential to repair damaged myocardial cells.This study not only provides a new direction for the study of biological materials for METH detection,but also provides a new direction and target for the study of METH toxic injury mechanism and the search for biomarkers.