Effect And Mechanism Of MiRNA On NSC And NSC-derived Exosomes In Rat Cerebral Ischemia Model

Objective Stroke is a serious disease which can result in physical disability and death.Ischemic stroke is the most common type of stroke.In the current study,the protective effect of mir-9a-5p up-regulated exosomes derived from neural stem cells on ischemic stroke and its molecular mechanism were investigated by in vitro cell experiments and in vivo animal experiments,aiming to reveal the therapeutic target of exosome for ischemic stroke and provide new ideas for the treatment of ischemic stroke.Methods(1)Rat hippocampal neural stem cells were isolated and cultured,and exosomes were extracted.The miRNAs differentially expressed in exosomes derived from neural stem cells at different differentiation periods were detected by miRNA high-throughput sequencing technology.The differentiallyexpressed miRNA,miR-9a-5p was selected as the research object.The effects of miR-9a-5p on proliferation and regulating the differentiation of neural stem cells were assessed By up-regulating miR-9a-5p.Furthermore,the activation of AMPK induced by miR-9a-5p was also detected by western blot.To investigate the protection effects of neural stem cell,an OGD/R model was built in vitro.The different effects between exosomes derived from normal and miR-9a-5p up-regulated neural stem cells on cell survival,proliferation,differentiation and AMPK activation were investigated.(2)MCAO/R model was established in SD rat.Exosomes derived from normal and miR-9a-5p up-regulated neural stem cells were used to treating ischemic stroke rat.TTC staining was performed to detect the area of cerebral infarction.Evans blue assay was performed to assess the blood-brain barrier(BBB)permeability.TUNEL staining was performed to detect the cell apoptosis after ischemic stroke.Immunofluorescence was performed to investigate the effects of miR-9a-5p on cell survival,and AMPK activation was detected by western blot.Results(1)MiRNA high-throughput sequencing analysis showed that miR-9a-5p was expressed in exosomes of neural stem cells at different stages of differentiation.After reviewing relevant literature broadly,miR-9a-5p was selected as the key research object.Up-regulation of miR-9a-5p expression in exosomes derived from hippocampal neural stem cells(miR-9a-5p exosomes)can promote cell proliferation and differentiation of neural stem cells,and AMPK protein activation was promoted when applied to hippocampal neural stem cells.For OGD/R model,the proliferation and differentiation were inhibited significantly.When treated by miR-9a-5p exosomes,the proliferation and differentiation of neural stem cells were restored,and the apoptosis was reduced significantly.Furthermore,the reduction of AMPK phosphorylation was also restored.(2)In vivo study revealed that miR-9a-5p exosomes decrease the area of cerebral infarction and BBB permeability.The results of immunofluorescence indicated that after treated with miR-9a-5p exosomes,the numbers of neuron cell,astrocytes and oligodendrocytes were maintained,which suggested that miR-9a-5p have the ability to regulate the differentiation of endogenic neural stem cells.The results of TUNEL staining indicated that miR-9a-5p could protect brain cells from death.AMPK activation was induced in MACO/R rat,and it was increased when treated by miR-9a-5p exosomes.All together,these results revealed that miR-9a-5p can protect the ischemic stroke disease progression by activating AMPK.Conclusion MiR-9a-5p exosomes can promote AMPK phosphorylation,so as to increase neural stem cell survival and promoter cell differentiation.Besides,miR-9a-5p exosomes have ability to inhibit ischemic stroke disease progression by maintaining brain cell number and promoting endogenic neural stem cell differentiation.