Investigation of brain damage mechanism in middle cerebral artery occlusion/reperfusion rats based on i-TRAQ quantitative proteomics
Investigation of brain damage mechanism in middle cerebral artery occlusion/reperfusion rats based on i-TRAQ quantitative proteomics
复制标题
基于i-TRAQ定量蛋白质组学研究大脑中动脉闭塞/再灌注大鼠脑损伤机制
DOI：
10.1007/s00221-021-06054-3
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发表时间：
2021-02-18
影响因子：
2
通讯作者：
Zhao, Baosheng
中科院分区：
文献类型：
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作者：
Ma, Quantao;Wang, Chunguo;Zhao, Baosheng
本研究旨在分析大鼠大脑中动脉缺血/再灌注（MCAO/R）后大脑皮质蛋白质的差异表达谱，从蛋白质水平探讨MCAO/R的脑损伤机制，为寻找MCAO/R的特异性标志蛋白提供实验基础。采用改良线栓法建立大鼠MCAO/R模型，通过脑组织TTC染色和HE染色评价模型的建立。取假手术组（Sham）和MCAO/R组大鼠大脑皮质，进行fasp酶解、i-TRAQ定量标记和反相液相色谱纯化分离。使用Orbitrap Q Exactive质谱法对总差异蛋白表达谱进行定性和定量分析。MCAO/R大鼠脑梗死灶明显，脑梗死相对表面积与假手术组相比有显著差异，提示MCAO/R大鼠模型制备成功。有199个显著差异蛋白（MCAO/R vs Sham，p <0.05，|倍数变化|> 1.2），其中上调蛋白104个，下调蛋白95个。基因本体（GO）富集分析显示，上调蛋白主要集中在NF-κ B转录的正向调节和I-κ B激酶-NF-κ B等生物学过程中，下调蛋白主要集中在长时程突触增强、细胞对DNA损伤刺激的应答、KEGG途径分析表明，差异蛋白的表达途径包括氧化磷酸化途径、代谢途径和Ras信号途径。差异蛋白的网络分析显示Alb、ndufb5、ndufs7、ApoB、Cdc42、Ndufa3、Igf1r、P4hb、Mbp、Gc、Nme1、Akt2等蛋白可能在MCAO/R的氧化应激、凋亡和炎症反应中起重要调节作用。基于i-TRAQ标记的定量蛋白质组学研究揭示了炎症和凋亡在MCAO/R脑损伤机制中的作用。本研究为寻找和确定MCAO/R的潜在治疗靶点提供了一定的实验基础。
The objective of this study is to analyze the differential protein expression profile in cerebral cortex of rats with middle cerebral ischemia/reperfusion (MCAO/R), explore the brain damage mechanism of MCAO/R at protein level, and provide experimental foundation for searching specific marker proteins of MCAO/R. Rat model of MCAO/R was established by modified suture-occluded method, and the model was evaluated by the results of brain 2,3,5-triphenyltetrazolium chloride (TTC) and hematoxylin-eosin (HE) staining. Cerebral cortex of rats from sham-operated group (Sham) and MCAO/R groups was used for FASP enzymatic hydrolysis, i-TRAQ quantitative labeling, and reverse-phase liquid chromatography purification and separation. Orbitrap Q Exactive mass spectrometry was used for qualitative and quantitative analyses of total differential protein expression profiles. MCAO/R rats had obvious cerebral infarction lesions, and the relative surface area of cerebral infarction was significantly different compared with sham rats, suggesting that MCAO/R rat model was successfully prepared. There were 199 significant difference proteins (MCAO/R vs Sham, p < 0.05, |fold change|> 1.2), including 104 up-regulated proteins and 95 down-regulated proteins. Gene ontology (GO) enrichment analysis showed that the up-regulated proteins were mainly concentrated in the biological processes of positive regulation of NF-kappa B transcription and I-kappa B kinase-NF-kappa B, etc. Down-regulated proteins were mainly concentrated in long-term synaptic potentiation, cellular response to DNA damage stimulus, etc. KEGG pathway analysis showed that the pathway involved in differential proteins includes oxidative phosphorylation, metabolic pathway, and Ras signaling pathway. Network analysis of differential proteins showed that Alb, ndufb5, ndufs7, ApoB, Cdc42, Ndufa3, Igf1r, P4hb, Mbp, Gc, Nme1, Akt2, and other proteins may play an important role in regulating oxidative stress, apoptosis, and inflammatory response in MCAO/R. Quantitative proteomics based on i-TRAQ labeling reveals the effect of inflammation and apoptosis in brain damage mechanism of MCAO/R. Besides, this research provide some experimental foundation for search and determination of potential therapeutic targets of MCAO/R.