基于外泌体蛋白质组学探讨叶酸缺乏对N2a-APP细胞β-淀粉样蛋白的影响

Effect of folic acid deficiency on amyloid-βin N2a-APP cells by exosomes proteomic analysis

目的观察叶酸缺乏对N2a-APP细胞β-淀粉样蛋白(amyloid-β,Aβ)、外泌体数量的影响,利用外泌体蛋白质组学探讨叶酸缺乏影响神经细胞外泌体分泌的可能分子机制。方法体外培养N2a-APP细胞,根据有无叶酸添加,分成叶酸缺乏(folic acid deficiency,FD)组(0μmol/L)和叶酸正常(folic acid normal,FN)组(10μmol/L)。采用酶联免疫吸附法测定检测细胞内外Aβ_(1-40)和Aβ_(1-42)水平;试剂盒提取细胞培养液外泌体;电镜观察、粒径分析鉴定外泌体;Western blot检测外泌体标志蛋白及细胞内自噬相关蛋白;非标记定量技术检测外泌体蛋白质组变化,进行生物信息学分析。结果FD组N2a-APP细胞内外Aβ_(1-40)和Aβ_(1-42)水平高于FN组(P<0.05)。两组N2a-APP细胞分泌的物质均呈现外泌体典型结构,表达外泌体标志蛋白CD36和Flotillin-1。FD组外泌体蛋白浓度高于FN组[(637.56±47.31)μg/mL vs.(365.76±76.21)μg/mL,P<0.001]。蛋白质组学结果显示,FD组与FN组间有50种蛋白差异表达,其中FD组细胞分泌的外泌体内14种蛋白表达下降,36种蛋白表达增加。FD组细胞内自噬相关蛋白Beclin-1(1.33±0.11 vs.0.88±0.13)、LC3Ⅱ/Ⅰ(0.55±0.08 vs.0.40±0.04)表达高于FN组,p62(0.88±0.07 vs.1.27±0.16)低于FN组,差异均有统计学意义(P<0.05)。结论叶酸缺乏可影响N2a-APP细胞自噬水平、外泌体的分泌数量及内容物,增加细胞内外Aβ水平。

Objective To investigate the effect of folic acid deficiency on the levels of amyloid-β(Aβ)in N2a-APP cells,and the number of exosomes secreted by N2a-APP cells.To explore the possible molecular mechanisms by which folate deficiency affects exosome secretion based on exosome proteomics.Methods N2a-APP cells were cultured in vitro,according to whether folic acid was added,and then divided into folic acid deficiency group(FD,0μmol/L)and folic acid normal group(FN,10μmol/L).The expression levels of Aβ_(1-40) and Aβ_(1-42) in brain tissues were measured with enzyme-linked immunosorbent assay.Exosomes were isolated from cells culture medium by using exosome extraction kit,and identified with electron microscopic observation and Nanoparticle Tracking Analysis.Exosome and autophagy related marker proteins were detected with Western blot.The study combined proteomics analysis and biochemical approach to identify proteins and pathways affected by folic acid deficiency in exosomes.Results The intra-or extracellular levels of Aβ_(1-40) and Aβ_(1-42) are significantly higher in the FD group than in the FN group(P<0.05).The substances secreted by both groups of N2a-APP cells showed the typical structure of exosomes.The exosomes marker proteins CD36 and Flotillin-1 were expressed in both groups.The exosomes protein concentration was significant higher in the FD group than in the FN group[(637.56±47.31)μg/mL vs.(365.76±76.21)μg/mL,P<0.001].Proteomics analysis showed that there were 50 differential proteins in the exosomes of the two groups compared,with 14 proteins down-regulated and 36 proteins up-regulated in the FD group.Compared to FN group,the autophagy related makers Beclin-1(1.33±0.11 vs.0.88±0.13)and LC3Ⅱ/Ⅰ(0.55±0.08 vs.0.40±0.04)expression levels are higher in the FD group(P<0.05),p62 levels lower in the FD group(0.88±0.07 vs.1.27±0.16,P<0.05).Conclusions Folic acid deficiency can affect N2a-APP cell autophagy levels,the number of exosomes secreted and their contents,and increases intra-and extracellular Aβlevels.