Role Of Exosome MiR-503-3P In The Pathogenesis Of Depression And The Potential Mechanism

Depression is a common mental illness that seriously affects human health.Its high incidence and social hazards,including suicide,make the prevention and treatment of depression a major public health problem worldwide.Exosomes are carriers of material transfer and information exchange between cells,and are nano-scale vesicles secreted by living cells to the outside.Exosomes carry proteins,m RNA and mi RNA,and can realize the information transfer and exchange between donor cells and recipient cells through various methods such as target cell membrane fusion.Thus,exosomes and the contents,have been taken as important mediums for the interaction between different cells.Increasing evidences have demonstrated the important role of exosomes and their important mi RNAs in the pathogenesis of neuropsychiatric diseases.In this study,both an animal model and a cell model of depression were established in the present study,and a series of behavioral tests were adopted,combined with a variety of experimental methods such as electron microscopy,serum exosome isolation,and mi RNA sequencing analysis techniques.The aim is to investigate the potential role of exosomes and the contained mi RNAs in the pathogenesis of depression,especially the role of mi R-503-3p in regulating the expression of BDNF/Trk B pathway-related proteins.This study is helpful to clarify the pathogenesis of depression and develop new therapeutic targets with detailed experimental evidence.Objective:To investigate the potential role of exosomes and the contained mi RNAs in the pathogenesis of depression,especially the role of mi R-503-3p in regulating the expression of BDNF/Trk B pathway-related proteins.Methods:1. Whole animal experiment:Establish rat depression model by chronic unpredictable mild stress?CUMS?,observe neuropsychological behavior changes in rats,extraction and isolation of serum exosomes for mi RNA sequencing analysis.Forty-five male Sprague-Dawley?SD?rats were divided into a control group and a stress-treated group according to the weight.One week after the adaptive feeding,the rats in the stress treatment group began to receive chronic unpredictable mild stress for4 weeks,and the rats in the control group did not receive stress stimulation on a free diet.After 2 weeks,the rats in the stress treatment group were divided into a CUMS group and a fluoxetine group?5 mg/kg?and continued to receive stress stimulation.Rats in the fluoxetine group were administered orally once a day for 2 weeks.Rats in the control group and CUMS group were orally administered with an equal volume of vehicle.Observe weight changes weekly.The open field test,sucrose preference test and forced swimming test were used to observe the neuropsychological and behavioral changes in each group of rats.Serum exosomes were extracted and isolated.Electron microscopy and surface markers were verified to sequence and analyze the differential expression of mi RNA in rat serum.GO analysis and KEGG enrichment analysis were performed on mi RNA sequencing results of different groups.Western Blot and q RT-PCR were used to detect the expression of BDNF,Trk B,TREM1,TREM2 and Synaptotagmin I in the hippocampus,prefrontal cortex and serum exosomes of rats.2.Cell experiment:mi R-503-3p regulates BDNF/Trk B pathway in SH-SY5Y cells stimulated by corticosteroneHuman neuroblastoma SH-SY5Y cells were cultured in vitro in DMEM-F12medium?1:1?containing 10%fetal bovine serum?FBS?,penicillin and streptomycin,in a 37?,5%CO₂ incubator.Cells in logarithmic growth phase were seeded in 6-well plates at a density of 1×10⁴,and corticosterone?25?mol/L?was used to stimulate the model.After 24 hours,transfection was performed using GP-si RNA-Mate Plus.Cells were divided into control group,corticosterone group,corticosterone plus NC?negative control,100?mol/L?group and corticosterone plus mi R-503-3p mimic?100?mol/L?group.Seven hours after transfection,the medium was changed,and a fluorescent inverted microscope was used to observe the cells.q RT-PCR was used to detect mi R-503-3p in SH-SY5Y cells.Western Blot and q RT-PCR were used to detect the protein and m RNA expression of BDNF,Trk B,TREM1,TREM2 and Synaptotagmin I.Results:1.Compared with the control group,the weight gain rate of rats in the CUMS group was significantly reduced,and the sucrose water preference index decreased significantly in the sucrose preference test.In the open field test,the total movement distance,center movement distance,and number of rearing of the CUMS group significantly decreased and the number of defecations increased.In the forced swimming test,the CUMS group of rats had a longer immobility time and shorter swimming time.Compared with the CUMS group,the rats in the fluoxetine-treated group had more activities and exploring behaviors,and both lack of pleasure and despair behavior were improved.Compared with the control group,the protein expressions of BDNF,Trk B,TREM1 and Synaptotagmin I in the hippocampus,prefrontal cortex,and serum exosomes of the CUMS group were reduced,while TREM2 protein expression was significantly up-regulated;serum exosomal mi RNA sequencing The results showed that the mi RNAs differentially expressed and targeted to the BDNF gene in rat serum exosomes were mi R-503-3p,mi R-7a-1-3p,mi R-16-5p and mi R-382-5p;KEGG enrichment analysis showed that CUMS-induced differentially expressed mi RNAs are involved in the regulation of multiple signal transduction pathways including the MAPK pathway,Wnt pathway,and m TOR pathway.2. Compared with the control group,q RT-PCR experiments showed that the levels ofserum exosomes mi R-503-3p in the CUMS group decreased significantly while that in the fluoxetine group increased significantly.Western blot results in cell experiments showed that corticosterone stimulation the protein expression of BDNF,Trk B,TREM1and Synaptotagmin I in the model group of SH-SY5Y cells was significantly reduced,while the expression of TREM2 was significantly increased.After mi R-503-3p was transfected into SH-SY5Y cells,the m RNA and protein expression of BDNF,Trk B,TREM1,and Synaptotagmin I were significantly increased,while the expression of TREM2 was decreased,with or without corticosterone stimulation.Conclusion:Chronic unpredictable mild stress can not only induce depression-related behaviors and imbalanced expression of proteins related to the BDNF/Trk B signaling pathway in the hippocampus and prefrontal cortex,but also cause differential expression of serum exosomes and mi RNAs in rats.Exosomal mi RNA,especially mi R-503-3p,may be involved in the occurrence of depression by regulating the expression of BDNF/Trk B signaling pathway-related proteins,and is expected to become a potential diagnostic biomarker and therapeutic target for stress-related depression.