摘要目的:探讨在蛛网膜下腔出血(SAH)早期阶段运用p38抑制剂后神经细胞线粒体蛋白表达的差异。方法:采用氧合血红蛋白(OxyHb)刺激Neuro-2a细胞(小鼠来源神经瘤母细胞，购买自中国科学院上海细胞库)，模拟SAH后血液中氧合血红蛋白对于神经细胞的刺激状态，建立体外SAH细胞模型，设立对照组(Con)、蛛网膜下腔出血组(SAH)及p38抑制剂组治疗组(p38+SAH组)，提取线粒体蛋白进行质谱分析，Maxquant软件和Perseus软件进行查库和非标记定量分析，两组间蛋白差异比较应用 t检验。对差异蛋白(p38+SAH/SAH组)进行基因本体论(GO)和京都基因与基因组百科全书(KEGG)通路的富集分析；应用Multiple Experiment Viewer软件进行分层聚类热图分析；STRING在线工具构建出蛋白与蛋白互作网络(PPI)；通过蛋白质印迹法(Western blot)和实时定量反转录聚合酶链反应(RT-qPCR)验证差异蛋白。 结果:3组中共筛出239个差异蛋白，上调差异蛋白105个，下调差异蛋白134个(符合差异倍数(FC)>1.5或<0.667， P<0.05)。GO富集分析表明，差异蛋白富集在调节丝裂原活化蛋白激酶(MAPK)的级联正调控、内吞作用、凋亡过程负调控等细胞生物学功能。在KEGG中：差异蛋白主要在p53信号通路、磷脂酰肌醇3激酶(PI3K)-蛋白激酶B(Akt)信号通路、轴突导向信号等通路富集。通过PPI分析：以Rab7a、Cox5a、Cacybp等蛋白为核心的9个蛋白互作网络SAH组Rab7a的表达低于CON组(蛋白：0.345±0.014比0.226±0.045， t＝13.920， P<0.05；mRNA：1.00比0.76±0.04， t＝9.813， P<0.05)；P38+SAH组中Rab7a的表达高于SAH组(蛋白：0.226±0.045比0.282±0.023， t＝-4.768， P<0.05；mRNA：0.76±0.04比0.95±0.02， t＝-6.802， P<0.05)，与蛋白组学结果一致。 结论:p38抑制剂并不是通过单一途径改善SAH后神经细胞线粒体功能障碍，而与多种蛋白及信号通路的调控有关。

abstractsObjective:Objective To investigate the difference of mitochondrial protein expression in the early stage of subarachnoid hemorrhage (SAH) treated with p38 inhibitor.Methods:The mouse neuroblastoma N2a cells were used to establish SAH model in vitro. Control (CON) group, SAH group and p38 inhibitor+ SAH group were set up. Mitochondrial protein was extracted and analyzed by mass spectrometry. Maxquant software and Perseus software were used for database searching and unlabeled quantification t test was used to compare the protein difference between the two groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed on the differential proteins (p38+ SAH/SAH group). The multiple experiment viewer (MeV) software was used for hierarchical clustering analysis. The protein-protein interaction network (PPI) was constructed by online tool string. Western blotting and RT-qPCR were used to verify the differential proteins. Results:A total of 239 differential proteins were screened out in the three groups, including 105 up-regulated proteins and 134 down regulated proteins (FC>1.5 or <0.667, P<0.05). GO enrichment analysis showed that different proteins were enriched in the regulation of mitogen-activated protein kinase (MAPK) cascade positive regulation, endocytosis, apoptosis process negative regulation and other cellular biological functions. In KEGG, the differential proteins were mainly enriched in p53 signaling pathway, phosphoinositide-3-kinase (PI3K)-protein kinase B (Akt) signaling pathway and axon guidance signaling pathway. PPI analysis showed that there were 9 protein interaction networks with rab7a, cox5a and CacyBP as the core. Compared with CON group, the expression of rab7a decreased in SAH group (protein: 0.345±0.014 vs. 0.226±0.045, t=13.920, P<0.05; mRNA: 1 vs. 0.76±0.04, t=9.813, P<0.05). Compared with SAH group, the expression of rab7a increased in p38 inhibitor+ SAH group (protein: 0.226±0.045 vs. 0.282±0.023, t=-4.768, P<0.05; mRNA: 0.76±0.04 vs. 0.95±0.02, t=-6.802, P<0.05). The results were consistent with those of proteomics. Conclusion:The p38 inhibitors do not improve the mitochondrial dysfunction of neurons after SAH through a single pathway, but are related to the regulation of a variety of proteins and signaling pathways.