Effect And Mechanism Of M1-EXO-miR-20a-5p On Neuronal Injury After Oxygen-Glucose Deprivation/Glucose-reoxygenation

ObjectiveTo investigate the biological effect of M1 microglia-derived exosomes（M1-EXO）on neuronal injury after oxygen-glucose deprivation/glucose-reoxygenation（OGD/R）,and to explore the effect of MFN2 on neuronal injury after OGD/R,and to elucidated the specific molecular mechanism of M1microglia-derived exosomal miR-20a-5p targeting MFN2 aggravated neuron damage after OGD/R.MethodsThe mouse microglia BV2fcells grown in logarithmic growth phase were added with 100μg/L liposolysaccharide（LPS）and 20μg/L interferon-γ（IFN-γ）to induce the polarization of microglia into M1 phenotype.M1 microglia were identified by Western blotting（WB）,quantitative real-time polymerase chain reaction（q PCR）and immunofluorescence.The supernatant of M1 microglia were collected,and exosomes were extracted by Exo Quick-TC^TMkit.The morphology of exosomes were observed by transmission electron microscope and nanoparticle tracking analysis（NTA）,and the expression of characteristic proteins CD9,CD63 and HSP70 of exosomes were detected by Western blotting.PKH26 fluorescently labeled exosomes were used to trace exosomes and analyze whether exosomes could be ingested by N2a cells.Bioinformatics and dual-luciferase analysis showed a targeting relationship between miR-20a-5p and MFN2.After transfection of miR-20a-5p mimic,inhibitor and NC,q PCR was used to verify the expression of miR-20a-5p in M1-EXO.The activity of N2a cells in different groups after OGD/R was analyzed by CCK8.q PCR was used to detect MFN2mRNA expression in N2a cells after OGD/R.WB was used to detect the expression of MFN2,Cleaved-caspase3,Bcl-2 and Bax in N2a cells after OGD/R.The oncosis of N2a cells in different groups after OGD/R were determined by flow cytometry.The ultrastructure of mitochondria was observed by transmission electron microscope.Results1.Compared with M0 microglia,the fluorescence intensity and mRNA and protein expressions of CD32 and inducible nitric oxide synthase（i NOS）,specific markers of M1 microglia,were increased（P<0.01）,indicating M1 microglia were successfully activated.Compared with group C,the cell viability was decreased（P<0.01）,the apoptosis rate was significantly increased in group OGD/R（P<0.01）;Compared with group OGD/R,the cell viability was decreased（P<0.01）,the apoptosis rate was significantly increased in group OGD/R+M1-CM（P<0.01）;Compared with group OGD/R+M1-CM,the cell viability was increased（P<0.01）,the apoptosis rate was significantly decreased in group OGD/R+M1-CM+GW4869（P<0.01）,indicating M1-CM aggravated N2a cells damage after OGD/R.Under electron microscopy and NTA,M1-EXO had round or oval vesicular bodies with obvious membranous structures,with diameters ranging from 100 nm.WB showed that the exosomes expressed specific proteins CD63,CD9 and HSP70.3D confocal images showed that PKH26-labeled exosomes（red）were localized in the cytoplasm of MAP2⁺N2a cells（green）,suggesting exosomes were taken up by N2a cells.Compared with group C,the cell viability was decreased（P<0.01）,the apoptosis rate was significantly increased in group OGD/R（P<0.01）;Compared with group OGD/R,the cell viability was decreased（P<0.01）,the apoptosis rate was significantly increased in group OGD/R+M1-EXO（P<0.01）,indicating M1-EXO aggravated N2a cells damage after OGD/R.2.The transduction efficiency of lentivirus plasmid was measured by inverted fluorescence microscope.Lentivirus plasmid transduction rate is about 90%.Compared with group C,the expression of MFN2 mRNA and its protein and cell viability were decreased（P<0.01）,the apoptosis rate was significantly increased（P<0.01）,the expression of anti-apoptosis-related proteins Bcl-2 was decreased（P<0.01）,and the expression level of apoptosis-related proteins Bax and Cleaved-caspase3were increased in group OGD/R（P<0.01）;Compared with group OGD/R,the expression of MFN2mRNA and its protein and cell viability were decreased（P<0.01）,the apoptosis rate was significantly increased（P<0.01）,the expression of anti-apoptosis-related proteins Bcl-2 was decreased（P<0.01）,and the expression level of apoptosis-related proteins Bax and Cleaved-caspase3 were increased in group OGD+si-MFN2（P<0.01）;We further observed mitochondrial morphology by TEM.The results showed that after OGD/R treatment,the outer membrane of mitochondria ruptured with mitochondrial fragmentation increases,and the mitochondrial cristae partical disappearance.Compared with group OGD/R,the outer membrane of mitochondria ruptured massively with mitochondrial fragmentation increases,and the mitochondrial cristae disappearance,even severe vacuolization.3.Bioinformatics analysis and dual luciferase reporting assay demonstrated that MFN2 was a direct target gene of miR-20a-5p.q PCR assay showed that compared with EXO-CN,the expression of miR-20a-5p in M1-EXO were increased（P<0.01）;compared with M1-EXO,the expression of miR-20a-5p in M1-EXO-miR-20a-5p-mimic were increased（P<0.01）,the expression of miR-20a-5p in M1-EXO-miR-20a-5p-mimic were decreased（P<0.01）.4.Compared with group C,the apoptosis rate and the expression of miR-20a-5p,Cleaced-caspase3and Bax proteins were significantly increased（P<0.01）,the expression of MFN2 mRNA and its proteins,Bcl-2 and cell viability were decreased in group OGD/R（P<0.01）;Compared with group OGD/R,the apoptosis rate and the expression of miR-20a-5p,Cleaced-caspase3 and Bax proteins were significantly increased（P<0.01）,the expression of MFN2 mRNA and its proteins,Bcl-2 and cell viability were decreased in group OGD/R+M1-EXO（P<0.01）;Compared with group OGD/R+M1-EXO,the apoptosis rate and the expression of miR-20a-5p,Cleaced-caspase3 and Bax proteins were significantly increased（P<0.01）,the expression of MFN2 mRNA and its proteins,Bcl-2 and cell viability were decreased in group OGD/R+M1-EXO miR-20a-5p mimic（P<0.01）,the apoptosis rate and the expression of miR-20a-5p,Cleaced-caspase3 and Bax proteins were significantly decreased（P<0.01）,the expression of MFN2 mRNA and its proteins,Bcl-2 and cell viability were increased in group OGD/R+M1-EXO miR-20a-5p IN（P<0.01）.ConclusionM1 microglia-derived exosomal miR-20a-5p may inhibit the expression of MFN2 in N2a cells after OGD/R and aggravate cell damage.