| ObjectiveIschemic stroke(Ischemia,IS)is a common acute cerebrovascular disease,which is one of the main fatal diseases in middle-aged and elderly people.Astrocytes(Astrocytes)are the stromal cells of the central nervous system(CNS)and have a variety of regulatory functions,including the release of extracellular ions,removal of amino acid neurotransmitters,restriction of excitotoxicity and promotion of synapse development.Its mitochondrial dysfunction is the initiating event in ischemic stroke and plays an important role in neuronal survival and neural function protection processes.When IS occurs,astrocytes are rapidly induced as activated A1-type astrocytes(A1 astrocytes,or A1-AS).Therefore,under pathological stress,mitochondrial motility-related protein-1(Drp 1),as a key mitochondrial regulatory protein,mainly interacts with Fssion 1(Fis 1),leading to an exaggerated Fssion process manifested by excessive mitochondrial fragmentation,generation of reactive oxygen species(Reactive oxygen species,ROS)and loss of mitochondrial membrane potential(Membrane potential,JC-1).Thus,mitochondria produce significant fragmentation of mitochondrial pathology and mitochondrial function is significantly disrupted.Finally,damaged astrocyte mitochondria are further released into neighboring neurons to fuse with neuronal mitochondria to induce neuronal mitochondrial dysfunction,amplify neuronal damage and worsen neurological prognosis.In the study and treatment of ischemic stroke,most drugs are not easy to cross the blood-brain barrier(BBB)to play a therapeutic role,especially polypeptides and protein drugs.Exosomes have potential innate properties such as low immunogenicity,high biological permeability and high delivery capacity,which can be used for loading protein preparations and targeting ischemic regions.Macrophage exosomes(Macrophage Exosomes,EXO)is an excellent lipid bilayer carrier,which can cross the blood and brain barrier and reach the inflammatory site in the brain,and the preparation process is simple and easy to obtain in a large number,not easy to induce the immune response.Therefore,this topic will prepare EXO and investigate its loading capacity,and study the protection mechanism and therapeutic effect of EXO loading mitochondrial fission inhibitor heptapeptide(Heptapeptide,(Hep))on IS damage.MethodsIn this experiment,RAW 264.7 cells cultured in full growth medium were inoculated into culture plates(1×10~6cells).After 24 hours,the medium was collected and stirred.After centrifugation at 20000×g for 30 min,the supernatant was abandoned and the exosome microspheres were resuspended with phosphate saline(PBS).EXO was obtained by supercentrifugation at 100000×g for 150 min.Hep was loaded into EXO by incubation method,and the morphology,particle size and protein expression of EXO and EXO-Hep were characterized by transmission electron microscopy,particle size analysis and Western Blot.In uptake experiments,A1-astrocytes(A1-AS)were used as cell uptake experiment models to simulate cell uptake experiments.The uptake of EXO and EXO in brain tissue was observed by laser confocal microscopy and in vivo imaging of small animals,and the tissue distribution of EXO and EXO-Hep was detected.The astrocytes’activation by lipopolysaccharide(LPS)stimulation to A1-AS mimic the apoplexy-like astrocytes’activation state.The mitochondrial pathological fission mediated by Drp1/Fis1 in A1-AS was detected by Western Blot.The mitochondrial function of A1-AS was detected by ROS,ATP and membrane potential test,and the fragmentation of A1-AS external mitochondria was determined.The integrity of mitochondrial membrane outside A1-AS was examined by Western Blot.The mitochondria were extracted from the astrocyte culture medium by centrifugation,and red fluorescence labeling was performed.At the same time,mitochondria from neurons treated with oxygen-glucose deprivation(OGD)were given green fluorescence labeling,and the two were co-cultured.The fusion of mitochondria of astrocytes and neurons was investigated by laser confocal microscopy.Mitochondrial function in neuron cells was detected by ROS,ATP and membrane potential detection,and mitochondrial breakage was determined.The morphology of mitochondria in neuron cells was observed by laser confocal fluorescence microscopy to determine their integrity and morphological characteristics.The membrane integrity of neuron mitochondria was investigated by Western Blot assay.The apoptosis of neuronal cells was investigated by MTT colorimetry,and the damage to neuronal cells caused by inflammatory factors secreted by astrocytes and mitochondria was analyzed.The effect of astrocytes on neuronal apoptosis was detected by TUNEL.In the experiment to explore the neuroprotective effect,middle cerebral artery occlusion ischemia/reperfusion(t MACO)model,EXO and EXO-Hep drug administration models were made in SD rats,and neurological function was scored by Zea-Longa score and Ludmila Belayev score.Infarct size was detected by 2,3,5-triphenyltetrazolium chloride(TTC)test.Immunofluorescence staining experiment was conducted to investigate the effect of inhibitory neuron cell(Neu N)death and the activation of A1-AS in the ischemic area of t MACO group,EXO group and EXO-Hep group.ResultsIn this study,the extraction and purification of EXO by overspeed centrifugal method met the purity requirements.EXO particle size is about100nm,the morphology is relatively uniform,the structure is complete,and the exosome classic shape-cup,according to the requirements,to ensure that each extraction operation is the same,can extract the EXO particle size and shape of the same.Heps were loaded into EXO at 4°to obtain fully formed exosomes.In the cell uptake experiment,EXO and EXO-Hep could be taken up by polarized A1 astrocytes,and the contents of EXO and EXO-Hep were increased with the increase of time.In vivo uptake experiments,EXO and EXO-Hep can be ingested by brain tissue and reach target cells to play a major therapeutic role.These results indicate that EXO can carry drugs into cells and exert therapeutic effects both in vivo and in vitro.In Western Blot experiments,EXO-Hep significantly inhibited the activation and mitochondrial function of A1type astrocytes,and improved the damaged mitochondrial membrane potential and mitochondrial membrane integrity.By co-culture the pretreated mitochondria of astrocytes with neuronal cells,it was found that the mitochondria of astrocytes could be transferred into neuronal cells and fused with mitochondria in neuronal cells.Immunofluorescence and Western Blot experiments showed that the treatment of neuronal cells Astrocyte mitochondria can directly affect mitochondrial function and even death of neuron cells.In the neurological function experiment,TTC staining experiment,neurological function experiment and immunofluorescence experiment showed that EXO-Hep could significantly reduce the cerebral infarction area of IS rats.In order to better verify the role of astrocytes in vivo,the extracted mitochondrial lateral ventricle was injected into the ischemic site of rats,which significantly improved the stroke characteristics of rats.ConclusionsBecause astrocyte fragmentation and dysfunctional mitochondria can lead to neuronal damage during IS,we prepared drug-loaded macrophage-derived exosomes to reduce mitochondrial dysfunction in type A1 astrocytes and promote their transfer of healthy astrocyte mitochondria into neurons to mitigate IS-induced damage.These findings suggest that the application of EXO-Hep emphasizes the efficacy of mitochondrial transfer therapy and may represent a promising alternative therapeutic approach for the treatment of IS. |
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