Cui Yiran, Liu Xin, Li Xianyu, Yang Hongjun
Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, 100700 Beijing, China.
Beijing Key Laboratory of Traditional Chinese Medicine Basic Research on Prevention and Treatment for Major Diseases, Experimental Research Center, China Academy of Chinese Medical Sciences, 100700 Beijing, China.
Int J Mol Sci. 2017 Jun 24;18(7):1355. doi: 10.3390/ijms18071355.
Stroke is the second most common cause of death worldwide. A systematic description and characterization of the strokes and the effects induced in the hippocampus have not been performed so far. Here, we analysed the protein expression in the hippocampus 24 h after cerebral ischaemic injury and repair. Drug intervention using Danhong injection (DHI), which has been reported to have good therapeutic effects in a clinical setting, was selected for our study of cerebral ischaemia repair in rat models. A larger proteome dataset and total 4091 unique proteins were confidently identified in three biological replicates by combining tissue extraction for rat hippocampus and LC-MS/MS analysis. A label-free approach was then used to quantify the differences among the four experimental groups (Naive, Sham, middle cerebral artery occlusion (MCAO) and MCAO + DHI groups) and showed that about 2500 proteins on average were quantified in each of the experiment group. Bioinformatics analysis revealed that in total 280 unique proteins identified above were differentially expressed ( < 0.05). By combining the subcellular localization, hierarchical clustering and pathway information with the results from injury and repair phase, 12 significant expressed proteins were chosen and verified with respect to their potential as candidates for cerebral ischaemic injury by Western blot. The primary three signalling pathways of the candidates related may be involved in molecular mechanisms related to cerebral ischaemic injury. In addition, a glycogen synthase kinase-3β (Gsk-3β) inhibitor of the candidates with the best corresponding expression trends between western blotting (WB) and label-free quantitative results were chosen for further validation. The results of Western blot analysis of protein expression and 2,3,5- chloride three phenyl tetrazole (TTC) staining of rat brains showed that DHI treatment and Gsk-3β inhibitor are both able to confer protection against ischaemic injury in rat MCAO model. The observations of the present study provide a novel understanding regarding the regulatory mechanism of cerebral ischaemic injury.
中风是全球第二大常见死因。迄今为止，尚未对中风以及海马体中引发的影响进行系统的描述和特征分析。在此，我们分析了脑缺血损伤和修复24小时后海马体中的蛋白质表达情况。本研究选用了据报道在临床环境中具有良好治疗效果的丹红注射液（DHI）进行药物干预，以研究大鼠模型中的脑缺血修复。通过结合大鼠海马体组织提取和液相色谱-串联质谱（LC-MS/MS）分析，在三个生物学重复中可靠地鉴定出了一个更大的蛋白质组数据集以及总共4091种独特蛋白质。然后采用无标记方法对四个实验组（未处理组、假手术组、大脑中动脉闭塞（MCAO）组和MCAO + DHI组）之间的差异进行定量分析，结果显示每个实验组平均约有2500种蛋白质得到定量。生物信息学分析表明，上述总共鉴定出的280种独特蛋白质存在差异表达（<0.05）。通过将亚细胞定位、层次聚类和通路信息与损伤和修复阶段的结果相结合，挑选出12种显著表达的蛋白质，并通过蛋白质印迹法验证它们作为脑缺血损伤候选物的潜力。候选物相关的主要三条信号通路可能参与了与脑缺血损伤相关的分子机制。此外，选择了在蛋白质印迹（WB）和无标记定量结果之间具有最佳相应表达趋势的候选物糖原合酶激酶-3β（Gsk-3β）抑制剂进行进一步验证。大鼠脑蛋白质表达的蛋白质印迹分析结果和2,3,5-氯化三苯基四氮唑（TTC）染色结果表明，DHI处理和Gsk-3β抑制剂均能够对大鼠MCAO模型中的缺血性损伤起到保护作用。本研究的观察结果为脑缺血损伤的调节机制提供了新的认识。
