Exosome Shuttled MiR-92b-3p From Astrocyte Is A Paracrine Mediator Of Ischemic Preconditioning In The Brain

Background and Objective:Ischemic stroke is a central nervous system disease with high incidence,high mortality and high morbidity.The treatment of ischemic stroke has always been the emphasis and difficulty to medical researchers.Ischemic preconditioning(IPC)is a viable strategy to treat ischemic cerebral injury.However,the mechanisms underlying the IPC-induced neuroprotection remain unclear.MicroRNA(miRNA)is a class of endogenous single-stranded non-coding RNA(containing about 18-25 nucleotides).MiRNAs participate in a variety of biological processes by binding to the 3'UTR of their target mRNA to interfere with their translation.In recent years,studies have suggested that miRNA is involved in the development,plasticity and differentiation of neurons and plays an important role in the development and progression of ischemic stroke.Exosomes are membrane vesicles with a diameter of 30-100 nm secreted by many cell types.Exosomes can transport and deliver various miRNA to recipient cells and can modify cell function.Numerous studies have been shown that exosomes played an important role in intercellular communication.Accordingly,we try to explore whether the neuroprotective effect of IPC is mediated by exosomes.This study will help to underpin the theory of ischemic preconditioning and may provide novel potential approach for the management of ischemic stroke.Methods:1.Primary cultured astrocytes,neurons and BV2 cell line.Cells were subjected to oxygen-glucose deprivation(OGD,astrocytes for 3 h,neurons for 30 min and BV2 for 2 h)to build the cell ischemic preconditioning model.Neurons were cultured with or without the conditioned medium for 24 h and subj ected to 2.5 h of OGD.Cell counting kits,LIVE/DEAD BacLight Viability kits and TUNEL staining to assess the effect of conditioned medium on neurons.2.Extraction of exosomes in conditioned medium by ultracentrifuge and exosome isolation kits.Transmission electron microscope and Western Blot were used to confirm the presence of exosomes in the conditioned medium.Immunofluorescence staining was used for detecting whether exosomes from astrocytes were taken up by neurons.Neurons were cultured with or without exosome for 24 h and subjected to 2.5 h of OGD.Cell counting kits,LIVE/DEAD BacLight Viability kits and TUNEL staining to assess the effect of exosomes from astrocyte conditioned medium on neurons.3.MiRNA sequence was carried out in exosomes before and after astrocytes ischemic preconditioning.Significantly differentially expressed miRNAs were identified by real time PCR.4.Neurons were subjected to 2.5 h OGD after down-regulated or up-regulated the expression of miR-92b-3p by miR-92b-3p inhibitor or miR-92b-3p mimic.Cell counting kits,LIVE/DEAD BacLight Viability kits and TUNEL staining to assess whether exosome was required and sufficient to confer neuroprotection in cultured neurons.5.Dual luciferase reporter assays and Western Blot were used to verify the target mRNA of miR-92b-3p.We used 15 min middle cerebral artery occlusion(MCAO)surgery to induce IPC model in mice.Inhibited or overexpressed the expression of miR-92b-3p by intraventricular injection of miR-92b-3p inhibitor or miR-92b-3p mimic.Modified Neurological Severity Scores(mNSS)and adhesive removal test were used to evaluate the neurological function of mice after MCAO surgery.TTC staining and TUNEL staining were used to assess the infraction volume and apoptosis after MCAO surgery.Western Blot was used to detect the effect of miR-92b-3p on PTEN/Akt/mTOR signaling pathway.Results:1.Culture medium from ischemic preconditioned astrocytes but not neurons and BV2 ameliorated OGD-induced neuron injury.2.Conditioned medium from astrocytes were enriched with exosomes.Exosomes from astroglial conditioned medium were taken up by neurons and protected neurons from OGD-induced injury.3.The expression of miR-92b-3p in exosomes released by ischemic preconditioned astrocytes was significantly increased.4.Downregulated the expression of miR-92b-3p in neurons resisted the neuroprotective effect of astrocyte conditioned medium.However,overexpression of miR-92b-3p with miR-92b-3p mimic exerted similar protection effects on the cell survival compared with astrocyte conditioned medium.5.PTEN was a target mRNA of miR-92b-3p.Deletion of miR-92b-3p aggravated the neurological impairment and increasd the infraction volume and TUNEL-positive cells after MCAO surgery.Overexpression of miR-92b-3p exert similar protection effects on the mice.The neuroprotection of ischemic preconditioning was regulated by miR-92b-3p/PTEN/Akt/mTOR pathway.Conclusion:Exosomes released by ischemic preconditioning astrocytes exert neuroprotection in vitro and in vivo.The mechanism of the neuroprotection of exosome maybe associated with miR-92b-3p mediated PTEN/Akt/mTOR pathway.