M1型小胶质细胞源性外泌体携载miR-20a-5p对氧糖剥夺/复氧后神经元损伤的影响

Effect of M1 microglia-derived exosomal microRNA-20a-5p on neuronal injury after oxygen-glucose deprivation and restoration injury

目的探讨M1型小胶质细胞源性外泌体(M1-exo)对氧糖剥夺/复氧后神经元损伤的影响,并研究其作用机制。方法取对数期生长的小鼠小胶质细胞BV2,加入100μg/L脂多糖(LPS)和20μg/Lγ-干扰素(IFN-γ)诱导小胶质细胞极化为M1表型,通过蛋白质免疫印迹试验(Western blotting)、实时荧光定量聚合酶链反应(qPCR)、免疫荧光法鉴定M1型小胶质细胞。收取M1型小胶质细胞的上清液,用ExoQuick-TCTM试剂盒提取M1-exo,通过透射电镜及纳米颗粒粒径分析(NTA)观察外泌体形态,采用Western blotting法检测外泌体标记蛋白白细胞分化抗原(CD9、CD63)的表达。将生长良好的小鼠神经母细胞瘤细胞N2a分为6组:C组细胞常规培养;O组氧糖剥夺3 h后复糖复氧24 h制备N2a细胞氧糖剥夺/复氧损伤模型;E组的N2a细胞氧糖剥夺3 h后复糖复氧并与M1-exo共培养24 h;NC组、M组和I组分别构建阴性对照、过表达和敲低微小RNA-20a-5p(miR-20a-5p)的M1-exo,通过qPCR检测转染是否成功。NC组、M组和I组N2a细胞氧糖剥夺3 h后,与转染后的M1-exo共培养24 h后检测各项指标。采用细胞增殖检测试剂(CCK-8)法检测细胞活力,采用流式细胞术检测细胞凋亡,采用qPCR法检测miR-20a-5p表达。结果与M0型小胶质细胞相比,M1型小胶质细胞特异性标志物CD32和诱导型一氧化氮合酶(iNOS)荧光强度及其mRNA和蛋白表达均明显升高〔CD32(荧光强度):36.919±1.541比3.533±0.351,CD32 mRNA(2^(-ΔΔCt)):4.887±0.031比1.003±0.012,CD32/β-actin:2.663±0.219比1.000±0.028;iNOS(荧光强度):29.513±1.197比7.933±0.378,iNOS mRNA(2^(-ΔΔCt)):4.829±0.177比1.000±0.016,iNOS/β-actin:1.991±0.035比1.000±0.045;均P<0.01〕,证明M1型小胶质细胞被成功激活。电镜下可见M1-exo为圆形或卵圆形囊泡状小体,并有明显膜性结构,直径范围约100 nm。Western blotting结果显示,M1-exo表达特异性CD63和CD9蛋白。与C组比较,O组N2a细胞活力明显下降,细胞凋亡率和miR-20a-5p的表达明显升高〔细胞活力(A值):0.540±0.032比1.001±0.014,细胞凋亡率:(19.857±0.910)%比(13.508±0.460)%,miR-20a-5p(2^(-ΔΔCt)):5.508±0.291比1.033±0.101,均P<0.01〕。与O组比较,E组N2a细胞活力明显下降,细胞凋亡率和miR-20a-5p表达进一步升高〔细胞活力(A值):0.412±0.029比0.540±0.032,细胞凋亡率:(31.802±0.647)%比(19.857±0.910)%,miR-20a-5p(2^(-ΔΔCt)):8.912±0.183比5.508±0.291,均P<0.01〕,说明M1-exo进一步加重了氧糖剥夺/复氧后N2a细胞的损伤。与E组比较,M组N2a细胞活力明显下降,细胞凋亡率和miR-20a-5p表达明显升高〔细胞活力(A值):0.311±0.028比0.412±0.029,细胞凋亡率:(36.343±0.761)%比(31.802±0.647)%,miR-20a-5p(2^(-ΔΔCt)):32.348±0.348比8.912±0.183,均P<0.01〕;而I组N2a细胞活力明显升高,细胞凋亡率和miR-20a-5p表达明显下降〔细胞活力(A值):0.498±0.017比0.412±0.029,细胞凋亡率:(26.437±0.793)%比(31.802±0.647)%,miR-20a-5p(2^(-ΔΔCt)):6.875±0.219比8.912±0.183,均P<0.01〕。E组与NC组N2a细胞活力、细胞凋亡率和miR-20a-5p的表达比较差异均无统计学意义。结论M1-exo加重氧糖剥夺复氧后神经元的损伤,这一损伤作用可能与其携载的miR-20a-5p有关。

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 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,quantitative real-time polymerase chain reaction(qPCR)and immunofluorescence.The supernatant of M1 microglia was collected,and exosomes were extracted by ExoQuick-TCTM 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^(-ΔΔCt)):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^(-ΔΔCt)):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^(-ΔΔCt)):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^(-ΔΔCt)):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^(-ΔΔCt)):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^(-ΔΔCt)):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.