Ischemia-like Hypoxic Preconditioning Of Astrocyte Exosomes Regulates Neural Damage After Ischemic Stroke Through CircSHOC2

Background Stroke is a common clinical disease with high mortality and high disability,which seriously endangers human health.Among them,ischemic stroke accounts for 60%-70% of the total number of stroke cases.Cerebral neuron death due to hypoxia after ischemia is the most important damage mechanism.Therefore,finding effective methods to reduce neuronal death is the current research hotspot in the treatment of ischemic stroke.In recent years,many studies have shown that after ischemic stroke,astrocytes around neurons will undergo reactive migration and proliferation.As an important part of the nervous system microenvironment,astrocytes play an important role in regulating the survival of neuronal cells after cerebral ischemia and hypoxia.Current studies have confirmed that in the process of ischemic stroke,astrocytes release signal molecules through a variety of ways and interact with other central nervous system cells.For example,astrocytes can promote the proliferation of vascular endothelial cells by releasing HMGB1 after cerebral ischemia.However,there are few studies on whether astrocytes directly regulate neuronal cell survival after cerebral ischemia.Hypoxia caused by ischemia is a huge stimulus to human cells or tissues.This stimulus makes many kinds of cells secrete a large number of exosomes in a short time.Exosomes are vesicles with a diameter of 30-150 nm.They are derived from multivesicular bodies formed by intracellular lysosomal particles and can be actively secreted by cells.The outer membrane of exosomes is in contact with the cell membrane and fuse,and then is released into the extracellular matrix.Due to the lipid bilayer membrane structure of exosomes,they can well protect the material covered by them,which contains proteins,lipids,and genetic material.The main function of exosomes is to transfer protein and RNA between cells and play the role of molecular signal communication between cells.Previous studies have found that exosomes secreted by human umbilical vein endothelial cells pretreated with hypoxia can reduce the neuroinflammatory response caused by traumatic brain injury,reduce the area of cortical damage,and improve the motor function of mice after brain injury.Autophagy is an evolutionary conserved basic life activity of cells.It maintains cell homeostasis mainly by isolating non-essential cellular components and degrading in lysosomes.However,after the occurrence of ischemic stroke,the neuronal cells in the ischemic penumbra will undergo excessive autophagy,which leads to the death of a large number of neurons in this area,which further worsens the patient's condition.circ RNA is an endogenous non-coding RNA with a unique covalently closed circular singlestranded RNA.It can resist the destruction of RNA exonuclease,so it has higher stability than mi RNA and Lnc RNA,and can be transmitted as a signal factor between cells to perform corresponding functions.Studies have shown that exosomes derived from neural stem cells can repair ischemic muscle damage through the circ RNA they carry.However,whether circ RNA in hypoxic preconditioning astrocyte exosomes can play a neuroprotective effect on neurons suffering from hypoxic damage is still unknown.There are many mi RNAs binding sites on Circ RNA,which competitively bind to mi RNA through base complementation,inhibit the ability of mi RNA to bind to m RNA,and thereby weaken the regulation of mi RNA on target gene expression.This method of binding mi RNA through sequence complementarity competition is called "mi RNA sponge".Circ RNA acts as a mi RNA sponge to act as the most widely explored mechanism in many circ RNArelated studies,and it is also the main mode for some circ RNA to regulate the function of downstream target genes.Circ RNA plays an important role in the biological processes of cell growth,cell proliferation,cell differentiation and cell apoptosis.The latest research results show that after a transient ischemic stroke,the level of E3 ubiquitin protein ligase 1(circ RNAHECTD1)in the Circ RNA HECT domain in the brain tissue of patients increases,and circ RNAHECTD1 can act as a sponge for mi R-142 and regulate itself.Phagocytosis affects the survival of astrocytes.Therefore,this research will focus on studying the possible regulatory mechanism of hypoxia preconditioning astrocyte exosomes-derived circ RNA in the pathological process of ischemic stroke and provide new clinical treatment for ischemic stroke in the future.Research direction.PART I Therapeutic effect of exosomes extracted from astrocytes of ischemic preconditioning on hypoxic neural injuryPurpose This study will mainly focus on the effect of exosomes secreted by hypoxic preconditioning astrocytes on neuron survival after hypoxic injury.Method We constructed oxygen-sugar deprivation/reperfusion astrocytes(Ischemia-like Hypoxic Preconditioning Astrocytes,IPAS)in vitro and used ultra-high-speed centrifugation to extract exosomes from the IPAS culture supernatant.Western Blot analysis and identification of the expression of the surface marker protein of exosomes;transmission electron microscopy to observe the morphology of exosomes;Zetasizer-Nano-ZS method was used to measure the diameter of exosomes.Use the red fluorescent dye Dil to label exosomes and observe the uptake of exosomes by neuronal cells.TUNEL staining experiment and LDH detection experiment were used to determine the effect of IPAS exosomes on neuron survival after hypoxic injury.Furthermore,the mechanism of IPAS exosomes regulating neuronal hypoxia tolerance was analyzed by Western Blot.Finally,a mouse brain ischemia-reperfusion injury model was established,and hypoxic preconditioning astrocyte exosomes were injected into the brain of mice.In vivo experiments verified that IPAS exosomes can alleviate ischemic stroke.The brain tissue damage caused and can accelerate the recovery of brain nerve function in mice.Result The exosomes identification results showed that the exosomes extracted by high-speed centrifugation are homogeneous spheres with nanometer size,and their average sizes are 91nm±0.3nm respectively;Western-blot results show that the surface of the extracted exosomes is rich in CD9,ALIX,TSG101 and CD63.We also confirmed that the exosomes can be taken up by neurons.Furthermore,we found that IPAS exosomes can attenuate neuronal apoptosis caused by hypoxia injury,and western-blot results suggest that exosomes can exert neuroprotective effects by inhibiting excessive autophagy of neurons.Finally,in vivo experimental results show that injection of IPAS exosomes can significantly reduce the volume of cerebral infarction in mice after 3 days of constructing a mouse transient cerebral ischemia model and improve the recovery of behavioral function dominated by the brain of mice.Conclusion This study preliminarily proves that exosomes secreted by hypoxic preconditioning of astrocytes can exert neuroprotection on neurons and brain tissues that suffer from hypoxia damage.PART II circ SHOC2 derived from IPAS-EXOs regulates ischemic InjuryPurpose To explore the effect of hypoxic preconditioning of the highly expressed circ RNA in astrocyte exosomes on neuronal cells damaged by hypoxia.Method First,we used the circ RNA microarray chip to screen the differences in the expression of circ RNA in hypoxic preconditioned astrocyte exosomes(IPAS-EXOs)and normal oxygen supply astrocyte exosomes,which were quantified by real-time fluorescence PCR technology verifies the expression level of circ RNA screened by the chip in IPAS-EXOs,and further screens the target circ RNA based on the results of PCR verification.Secondly,we used in situ hybridization to determine the main distribution sites of circ SHOC2 in neuronal cells.Furthermore,we co-cultured mouse cerebral cortex neuronal cells treated with oxygen glucose deprivation/reperfusion with circ SHOC2 mimics or circ SHOC2 inhibitors(circ SHOC2 inhibitor)and evaluated neurons by LDH detection and TUNEL staining.Cell viability,Western blot detects changes in protein expression related to apoptosis and autophagy.Finally,use plasmids to transplant circ SHOC2 mimics or circ SHOC2 inhibitor into the brain of middle cerebral artery occlusion(MCAO)model mice.According to the research results of modified neurological deficit score(m Nss),2,3,5-triphenyltetrazolium chloride(TTC)staining and TUNEL staining,explore the effect of circ SHOC2 on the repair of hypoxic cerebral infarction and neurological deficits.Result The expression of circular RNA SHOC2(circ SHOC2)in hypoxic preconditioning astrocyte exosomes was significantly increased In vitro.Overexpression of circ SHOC2 in neuronal cells can reverse the decrease in neuronal cell viability induced by hypoxic injury and reduce the expression of apoptotic proteins.In addition,Western blot results showed that overexpression of circ SHOC2 can inhibit the expression of apoptosis-related protein Caspase-3,autophagy-related protein LC3-II and p62.Therefore,we believe that hypoxic preconditioning of circ SHOC2 overexpressed in astrocyte exosomes mainly exerts a neuroprotective effect by inhibiting excessive autophagy of neurons.In vivo experiments further confirmed that after transfection of circ SHOC2 mimics,the cerebral infarction volume of MCAO model mice was significantly reduced,and the neurological recovery of MCAO model mice was significantly improved;on the contrary,specific inhibition of the expression of circ SHOC2 in neurons can aggravate neurological function of MCAO model mice was damaged,and the cerebral infarction volume of mice was also significantly increased compared with the control group.Conclusion Circ SHOC2 overexpressed in astrocyte exosomes inhibited neuronal apoptosis induced by ischemic injury.And this exosome could reduce the infarct volume of brain tissue after ischemia injury and enhances the subsequent recovery of neural function by inhibiting Neuron autophagy.PART III Circ SHOC2 regulates SIRT1 by targeting mi R-7670-3p to affect neuron survival after ischemic strokePurpose This study initially explored the neuroprotective mechanism of circ SHOC2 overexpressed in astrocyte exosomes pretreated by hypoxia and its specific regulatory signal pathways.Method Firstly,circ RNA-mi RNA prediction databases such as star Base was used to screen mi RNAs that may bind to circ SHOC2.Subsequently,we initially verified the target mi RNAs we screened through experiments such as q RT-PCR analysis and pull-down analysis;secondly,through molecular cloning technology,we constructed a luciferase reporter gene plasmid of circ SHOC2 and target mi RNA,using dual luciferase Reporter gene detection experiments further confirmed that circ SHOC2 binds to the target mi RNA.Furthermore,the ability of circ SHOC2 to bind to the target mi RNA can be visually observed through fluorescence in situ hybridization through mutated circ SHOC2 or target mi RNA.Next,by using the results of multiple bioinformatics analysis software(starbase,Targetscan,and mi Rnada),we predict the downstream neuroprotection-related target genes regulated by the target mi RNA.By Using dual luciferase reporter gene detection and q RTPCR research the mutual binding ability of target mi RNA and target gene.Finally,through in vivo experiments in mice,we specifically overexpress or inhibit the expression level of target mi RNAs in the mouse brain and evaluate the effects of target mi RNAs on the neurological function recovery and downstream target gene expression levels in MCAO model mice;Finally,Western blots were used verifies the influence of the expression level of circ SHOC2 in regulated neurons on the target gene expression level.Result Bioinformatics analysis revealed that circ SHOC2 has mi R-7670-3p binding sites.Gene prediction software and dual luciferase reporter gene test results confirmed that circ SHOC2 acts as a "molecular sponge" in hypoxia-damaged neurons,down-regulating the expression level of mi R-7670-3p.A series of experiments such as dual luciferase reporter gene analysis,q RT-PCR analysis and Western blot confirmed that circ SHOC2 can be used as an endogenous mi R-7670-3p sponge to inhibit the activity of mi R-7670-3p and affect the downstream of mi R-7670-3p The expression of SIRT1 inhibits the lethal autophagy of neurons caused by hypoxia.Therefore,we found that the circ SHOC2/mi R-7670-3p/SIRT1 signaling pathway inhibits excessive neuronal autophagy and reduces neuronal damage caused by ischemic stroke,thereby enhancing the recovery of neurological function after stroke.Conclusion Circ SHOC2 can be used as a molecular sponge of mi R-7670-3p in neurons to positively regulate the expression of SIRT1 and regulate the circ SHOC2/mi R-7670-3p/SIRT1 signaling pathway to affect the ischemic penumbra after ischemic stroke.Neuronal function plays a protective role.