| Research Background:There are three main types of extracellular vesicles(EVs):exosomes(30-150nm),microvesicles(100-1000nm)and apoptotic bodies(1000nm-),of which exosomes are the most studied object,Exosomes are small vesicles that are actively secreted outside the cell after the fusion of intracellular multivesicular bodies(MVB)with the cytoplasmic membrane.The diameter of exosomes is between 30-150nm,surrounded by lipid bilayers,which contain proteins,lipids,mRNA,miRNA and other substances,and exist in blood,saliva,urine,cerebrospinal fluid and breast milk.in body fluids.The process is that first the cell membrane invaginates to form an early endosome,and then the early endosome membrane buds inward and encapsulates proteins,RNAs and miRNAs in the cytoplasm to form intraluminal vesicles(ILVs).With the gradual accumulation of intraluminal vesicles(ILVs),early endosomes grow and mature to form late endosomes,also known as multivesicular bodies(MVBs).There are two fates of late endosomes:1.The vesicle contents are degraded after fusion with lysosomes;2.After fusion with the cell membrane,these vesicles are released outside the cell,that is,exosomes.For a long time after it was discovered,exosomes were only regarded as garbage carriers for cells to excrete waste,but as the contents of exosomes gradually became known,especially functional proteins and mRNAs,miRNAs The discovery of exosomes has made exosomes one of the research hotspots in the past decade.Exercise can prolong life and is a non pharmacological method that can reduce the risk of aging related diseases and improve human health.Short term high-intensity exercise is a very effective method that can improve metabolic function and aerobic capacity.It is characterized by short-term and intense exercise.It has been proved to be more effective than moderate endurance exercise.It is generally believed that exosomes pass through at least two mechanisms,i.e.dependent or independent transport essential endosomal sorting complex(eccrt)mechanism,and the independent ESCRT mechanism may be related to ceramide.Therefore,we propose a hypothesis that high-intensity exercise mediates the formation and increase of exosomes through the secretion of ceramide in mice.After consulting the literature,we tried to take C57BL/6 mice as experimental animals to explore the possibility of the above hypothesis.Given that the current definition of extracellular vesicles is not fully defined,and due to the limitations of current technology,many studies to date have analyzed crude extracellular vesicle preparations,guidelines issued by the International Society for Extracellular Vesicles(ISEVs)propose Use the term extracellular vesicles instead of exosomes and(exfoliated)microvesicles.Therefore,although we use exosomes as the research object,we still use extracellular vesicles to express more in line with the actual situation,which is also a common saying in many literatures.Research purposes:1.EVs are released after inactivity and high-intensity exercise.2.Release of EVs in the peripheral and central nervous system.3.Possible mechanisms to promote the release of EVs.Research methods:In this project,C57BL/6 mice were used as the research objects and were fed to 4-5 months old.They were divided into exercise group and non-exercise group,and exercise group was divided into different observation time points.After high-intensity exercise,exosomes released from brain tissue and exosomes in peripheral blood were extracted and analyzed by immunofluorescence.Detection of exosome marker protein expression,western-blot detection of protein quantification,Nanosight LM10 to detect the particle size distribution and number of exosomes,detection of ceramides,comparison of the difference in expression between exercise and non-exercise groups,and brain tissue The release of exosomes in different time periods after high-intensity exercise was statistically analyzed.To observe the secretion of exosomes in the brain tissue and the periphery of mice under high-intensity exercise,and explore whether short-term high-intensity exercise promotes the release of exosomes in brain tissue and plasma through a ceramide mechanism.Statistical analysis:Quantitative data were expressed as mean ± standard deviation,t-test was used for comparison between two groups,and one-way analysis of variance was used for comparison between multiple groups,and a P value less than 0.05 was considered statistically significant.Statistical analysis was performed using SPSS 22.0.Research result:1.To study the relationship between the release of EVs in serum and high-intensity exercise,mice in the exercise group performed incremental treadmill exercise for 30 minutes.Serum EVs were purified from peripheral blood,and the expression of EVs markers TSG101,CD63,and HSC70 were analyzed by western blot.The protein spectrum showed that the level of HSC70 increased rapidly(P<0.01)after high-intensity exercise(0 min),and returned to the initial level rapidly(P>0.05)after exercise(90 min).There was no significant difference in the levels of TSG101 and CD63 before and after exercise(P>0.05).2.The effect of short-term exercise on hippocampal EVS release and colocalization with Tuj 1+neurons was investigated by immunofluorescence staining of brain tissue.This study examined the expression of selective exosomal markers HSC70 and TSG101 in neuronal exosomes.Among them,the proportions of TSG101+Tuj1+cells and HSC70+Tuj1+cells in the total Tuj1+cells in the hippocampus of the non-exercise group and the immediate post-exercise assessment group were significantly different(TSG101 P<0.05;HSC70 P<0.05).These results suggest that EVs in hippocampal neurons are increased after exercise compared to before exercise.3.To investigate the relationship between high-intensity exercise and the release of EVs in the brain,mice in the exercise group performed 30-min incremental treadmill exercise.We collected mouse brain tissue before exercise,immediately after exercise(0 min),and 90 min after exercise(90 min).Western blot analysis of EVs protein confirmed that the amount of EVs released at 0 min immediately after exercise was significantly greater than that before exercise.In addition,short-term and high-intensity exercise had more significant effects on HSC70 and TSG101-positive vesicles(P<0.05),while CD63-positive vesicles had no significant change after exercise(P>0.05).Western blot analysis also showed that TSG101 and HSC70 levels almost returned to baseline levels 90 minutes after exercise(P>0.05).In addition,the secretion of extracellular vesicles in mice before exercise,after exercise(0 min)and 90 minutes after exercise(90 min)was further confirmed by NTA.The concentration of extracellular vesicles(×108 particles/ml)was higher after exercise than before exercise(P<0.01),and returned to the baseline level 90 min after exercise(P>0.05),while there was no significant change(P>0.05)of the average diameter of EVs before and after exercise.4.To determine whether EVs secretion is dependent on ceramide synthesis,we measured ceramide secretion in brain tissue by thin-layer chromatography(TLC).The results showed that the release of ceramide at 0 min after short-term high-intensity exercise was significantly higher than that before exercise(P<0.05),and the release of ceramide basically returned to the baseline level at 90 minutes after exercise(P>0.05).In conclusion:Short-term high-intensity exercise altered the level of EVs in the brain and increased the release of EVs in the brain tissue and serum of C57BL/6 mice.It was confirmed by western blot,immunofluorescence and NTA detection.At the same time,short-term high-intensity exercise significantly affected the release of ceramide in the brain tissue of C57BL/6 mice,and the release of ceramide may be one of the mechanisms that promote the increased release of exosomes. |
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