Increases In MiR-150-3p In Neural Stem Cells Derived Exosomes Confer Neuroprotective Effects By Targeting Caspase 2 Following Hypoxic-ischemic Brain Injury

The brain is one of the most sophisticated and crucial organs for human beings,and brains are more vulnerable to ischemic/hypoxic damage.Hypoxia and ischemia cause extensive damage to the brain,and result in irreversible damage even brain death.At present,the therapeutic strategies for cerebral ischemia and hypoxia are very limited,and all the therapeutic methods cannot improve the neurological defects.Therefore,it is urgent to develop new therapeutic methods against cerebral ischemia and hypoxia.Neural stem cells(NSCs)display a capacity of self-renewal and are able to differentiate into a variety of neural lineages.By secreting exosomes and communicating in formation with other tissue cells,stem cells play a therapeutic role.Stem cell derived exosomes have shown encouraging therapeutic effects in several different types of diseases,including kidney injury,heart injury,brain injury,and liver and lung injury.However,there are few studies on neural stem cells and exosomes from which they are derived,and their biological functions and mechanisms of action are still unclear.In-depth study on neural stem cells will provide new ideas and theoretical basis for the treatment of hypoxic-ischemic brain diseases.Methods:Neural stem cells were isolated from neonatal rat cerebral cortex and cultured in vitro.Immunofluorescence was applied to examine the expression of stem cell-labeled Nestin in primary rat neural stem cells.The supernatant of neural stem cells was collected and exosomes were extracted through supercentrifugation.Exosomes morphology was detected by transmission electron microscope,the expression levels of exosomes markers CD63 and CD81 were detected by Western blot,and the particle size of neural stem cell exosomes was analyzed by Nano particle tracking analyzer.Flow cytometry was applied to examine death rate of SH-SY5Y cells cultured under normoxic and oxyglucose deprivation.MTT assay was applied to examine the effects of neural stem cell exosomes treatment on the proliferation of SH-SY5Y cells cultured under normoxic and oxyglucose deprivation conditions.HE staining was used to observe the brain structure,Nissl staining was used to detect the effect on nissrosia,and TUNEL staining was used to detect the effect on nerve cell apoptosis.Immunofluorescence technique was used to detect the effects on the number of MBP,NeuN and NG2 positive cells in MCAO model rat brain.Neural stem cells and exosomes of neural stem cells were collected for microRNA chip sequencing detection to explore the changes of miRNA spectrum between the two.Sh-sy5y cells were treated with Mir-150-3p mimics and Mir-150-3p inhibitor in vitro,and their apoptosis and proliferation were detected by flow cytometry and CCK-8 respectively.HE staining was used to obsenve the brain structure.Nissl staining was used to observe the effects on Nissl.TUNEL staining was used to detect the effects on neuronal apoptosis.The effects on the number of MBP,NeuN and NG2 positive cells in MCAO model rats were detected by immunofluorescence technique.Use the miRNA target genes to predict site miRDB(http://mirdb.org/miRDB/)and TargetScan(http://www.targetscan.org)to predict the potential target genes of miR-150-3p.The dual luciferase reporter gene was used to detect the targeted regulatory relationship between miR-150-3p and the predicted target gene caspase 2.qPCR was used to detect the effect of regulated miR-150-3p expression on its target gene caspase 2.The effect of miR-150-3p mimics and miR-150-3p inhibitor treatment on the protein expression of caspase 2 in MCAO mice brain was detected by immunofluorescence.Western blot was used to detect the effects of miR-150-3p mimic and miR-150-3p inhibitor on the expression levels of Bax,Cleaved Caspase3,Caspase2 and Bcl-2 in SH-SY5Y cells.Western blot analysis of the effects of neurostem cell exosome treatment on the expression levels of Bax,Cleaved Caspase3,Caspase2 and Bcl-2 in SH-SY5Y cells under normoxic and glycoxic deprivation conditions.The effect of neural stem cell exosomes on caspase 2 protein expression was detected by immunofluorescence.Results:The primary neural stem cell culture system was successfully established in vitro.After 14 days of culture,the suspended nerve appeared fusion.The positive rate of Nestin protein in neural stem cells was over 90%.The isolated exosomes showed typical circular double-layer dish-shaped vesicles.The isolated exosomes highly expressed specific markers CD63 and CD81,but did not express endoplasmic reticulum specific markers calnexin.The collected exosomes had an average size of 146.5 nm and a concentration of 1.79E+11.The treatment of NSCS exosomes could inhibit the apoptosis of SH-SY5Y cells and promote the proliferation of SH-SY5Y cells in normoxic and oxygen-glucose deprived culture conditions.A total of 198 miRNAs with different expressions were identified between neural stem cell exosomes and neural stem cells,of which 146 miRNAs were up-regulated and 52 miRNAs were down-regulated.The top five increased miRNAs were miR-150-3p,miR-1248,miR-5089-5p,miR-3916 and miR-6803-3p.The top five decreased miRNAs were miR-551b-5p,miR-548az-5p,miR-548,miR-3175 and miR-6763-5p.miR-150-3p was the most significantly up-regulated miRNAs.miR-150-3p mimics inhibited apoptosis of SH-SY5Y cells in normoxic and oxygen-glucose deprived cultures,while miR-150-3p inhibitor significantly promoted apoptosis of SH-SY5Y cells.miR-150-3p mimics significantly enhanced the proliferation of SH-SY5Y cells,while miR-150-3p inhibitor significantly inhibited the proliferation of SH-SY5Y cells.Nissl staining showed orderly arrangement of neurons in mir-150-3p mimics group,showing Nissl staining and a significant increase in the number of Nissl,while miR-150-3p inhibitor intensified neuronal damage and reduced the loss of Nissl.TUNEL staining showed that apoptotic neurons in mir-150-3p mimics group were significantly reduced,while apoptotic cells in miR-150-3p inhibitor group were significantly increased.Immunofluorescence staining showed that miR-150-3p mimics increased the number of MBP positive oligodendrocytes,NeuN positive nerve cells,and NG2 positive cells,while miR-150-3p inhibit or treatment decreased the number of MBP positive oligodendrocytes,NeuN positive nerve cells and NG2 positive cells.Analysis of miRNA target gene prediction website showed that Caspase-2(caspase 2)was a potential target gene of miR-150-3p.Two luciferase reporter genes confirmed the existence of targeted binding sites between miR-150-3p and caspase 2.miR-150-3p mimic significantly reduced the mRNA level of caspase 2 in SH-SY5Y cells under normoxia and oxyglucose deprivation,while miR-150-3p inhibitor significantly induced the mRNA level of caspase 2 in SH-SY5Y cells.Immunofluorescence assay showed that miR-150-3p mimic inhibited the protein level of caspase 2,while miR-150-3p inhibitor promoted the protein expression level of caspase 2.miR-150-3p mimic significantly reduced the expression of Bax,cleaved caspase3 and Caspase2 in SH-SY5Y cells,and induced the expression of Bcl-2,while miR-150-3p inhibitor significantly induced the expression of Bax,Cleaved caspase3 and Caspase2 in SH-SY5Y cells,and inhibited the expression of Bcl-2.In addition,immunofluorescence assay showed that neural stem cell exosome treatment inhibited caspase 2 protein expression in MCAO model brain tissue.Conclusion:Neural stem cell-derived exosomes promote the proliferation of neural cells and inhibit their apoptosis both in vitro and in vivo,showing neuroprotective effects.The miRNA expression profiles is different between neural stem cell exosomes and neural stem cells.miR-150-3p is identified as the most obviously up-regulated miRNAs.miR-150-3p promotes the proliferation while inhibits apoptosis of nerve cells.In addition,miR-150-3p reverses the phenomenon ofNissl loss in MCAO model.miR-150-3p increases the number of MBP positive,NeuN positive and NG2 positive cells.Silencing of miR-150-3p inhibits the proliferation while promotes apoptosis of nerve cells.The loss of nestenite in MCAO model increased and the number of MBP positive,NeuN positive and NG2 positive cells decreased after miR-150-3p silencing.miR-150-3p is the upstream miRNA of caspase 2.miR-150-3p inhibits the expression of apoptosis-related genes Bax,Cleaved-caspase3,and Caspase2,induces the expression of anti-apoptotic gene Bcl-2,and promotes the proliferation of nerve cells via targeting caspase 2.