Effect of M1 microglia-derived exosomal microRNA-20a-5p on neuronal injury after oxygen-glucose deprivation and restoration injury / 中华危重病急救医学

ABSTRACT

Objective:To investigate the effect of M1 microglia-derived exosomes (M1-exo) on neuronal injury after oxygen-glucose deprivation and restoration, and to explore its mechanism.Methods:The mouse microglia BV2 cells grown in logarithmic growth phase were added with 100 μg/L liposolysaccharide (LPS) and 20 μg/L interferon-γ (IFN-γ) to induce the polarization of microglia into M1 phenotype. M1 microglia were identified by Western blotting, quantitative real-time polymerase chain reaction (qPCR) and immunofluorescence. The supernatant of M1 microglia was collected, and exosomes were extracted by ExoQuick-TC TM kit. The morphology of exosomes were observed by transmission electron microscope and nanoparticle tracking analysis (NTA), and the expression of characteristic proteins CD9 and CD63 of exosomes were detected by Western blotting. The well-growing mouse neuroblastoma N2a cells were divided into six groups the cells in group C were conventionally-cultured; and the cells in group O were subjected to oxygen-glucose deprivation for 3 hours followed by restoration of oxygen-glucose supply 24 hours to establish the model of oxygen-glucose deprivation and restoration injury; and the N2a cells in group E were co-cultured with M1-exo 24 hours after oxygen-glucose deprivation 3 hours; NC group, M group and I group constructed negative control, overexpression and knockdown of microRNA-20a-5p (miR-20a-5p) M1-exo, respectively. The succession of transfection was detected by qPCR and N2a cells in group NC, group M and group I were co-cultured with such transfected M1-exo for 24 hours after oxygen-glucose deprivation 3 hours. Cell viability were detected by cell counting kit-8 (CCK-8) assay, cell apoptosis were detected by flow cytometry, and the expression of miR-20a-5p were detected by qPCR. Results:Compared with M0 microglia, the fluorescence intensity and mRNA and protein expressions of CD32 and inducible nitric oxide synthase (iNOS), specific markers of M1 microglia, were increased [CD32 (fluorescence intensity) 36.919±1.541 vs. 3.533±0.351, CD32 mRNA (2 -&Delta;&Delta;Ct) 4.887±0.031 vs. 1.003±0.012, CD32/β-actin 2.663±0.219 vs. 1.000±0.028; iNOS (fluorescence intensity) 29.513±1.197 vs. 7.933±0.378, iNOS mRNA (2 -&Delta;&Delta;Ct) 4.829±0.177 vs. 1.000±0.016, iNOS/β-actin 1.991±0.035 vs. 1.000±0.045; all P < 0.01], indicating M1 microglia were successfully activated. Under electron microscopy, M1-exo had round or oval vesicular bodies with obvious membranous structures, with diameters ranging from 100 nm. Western blotting showed that the exosomes expressed specific CD63 and CD9 proteins. Compared with group C, the cell viability was decreased, the apoptosis rate and the expression of miR-20a-5p were significantly increased in group O [cell viability ( A value) 0.540±0.032 vs. 1.001±0.014, apoptosis rate (19.857±0.910)% vs. (13.508±0.460)%, miR-20a-5p (2 -&Delta;&Delta;Ct) 5.508±0.291 vs. 1.033±0.101, all P < 0.01]. Compared with O group, cell viability was decreased, apoptosis rate and the expression of miR-20a-5p were increased in group E [cell viability ( A value) 0.412±0.029 vs. 0.540±0.032, apoptosis rate (31.802±0.647)% vs. (19.857±0.910)%, miR-20a-5p (2 -&Delta;&Delta;Ct) 8.912±0.183 vs. 5.508±0.291, all P < 0.01], indicating that M1 microglia-derived exosomes further aggravated the damage of N2a cells after oxygen-glucose deprivation and restoration. Compared with group E, cell viability was decreased, apoptosis rate and the expression of miR-20a-5p were increased in group M [cell viability ( A value) 0.311±0.028 vs. 0.412±0.029, apoptosis rate (36.343±0.761)% vs. (31.802±0.647)%, miR-20a-5p (2 -&Delta;&Delta;Ct) 32.348±0.348 vs. 8.912±0.183, all P < 0.01]; and the cell viability was increased, apoptosis rate and the expression of miR-20a-5p were decreased in group I [cell viability ( A value) 0.498±0.017 vs. 0.412±0.029, apoptosis rate (26.437±0.793)% vs. (31.802±0.647)%, miR-20a-5p (2 -&Delta;&Delta;Ct) 6.875±0.219 vs. 8.912±0.183, all P < 0.01]. There was no significant difference in cell viability, apoptosis rate and the expression of miR-20a-5p between group E and group NC. Conclusion:M1 microglia-derived exosomes aggravate the injury of neurons after oxygen and glucose deprivation and reoxygenation, which may be related to miR-20a-5p carried by M1-exo.