激动ALDH2减轻肠道缺血再灌注损伤的差异蛋白的筛选和功能验证

Intestinal ischemia/reperfusion (I/R) is a commonly seen critical condition in clinical practice, characterized by high mortality and high rates of complications. An effective treatment for the disease requires a thorough understanding of the pathogenesis in combination with specific diagnostic markers. Current academic views accept that intestinal I/R is a multifactorial disease involving many pathological aspects, in which oxidative stress is a crucial facet. Aldehyde dehydrogenase 2 (ALDH2) is the main enzyme in mitochondria to catalyze conversion of acetaldehyde (high toxicity) to carboxylic acid (low toxicity), thus detoxifying the deleterious effects related to aldehydes in oxidative stress. To this end, a study in the journal Science activated ALDH2 through its agonist Alda-1 and found that it could significantly reduce the infarct size by 60%. The study from Science has triggered a number of subsequent investigations published in many other journals. Under these circumstances, the current applicant evaluated the effects of ALDH2 activation in the organ of intestine, and found that enhancement of ALDH2 activity can exert protective effects against intestinal I/R injury, presenting the first account in the gut organ. These findings are encouraging and can cast light on the seeking of translational strategies for gut I/R. However, further investigations are still warranted, especially on the detailed mechanism of ALDH2 protection. It is of note that the first hint linking ALDH2 molecule with cardiac I/R arises from a proteomics high-throughput screening. Along with this indication and that from other publications, the current applicant hypothesized that the proteomics technique could provide invaluable clues for the illustration of mechanism of ALDH2. In our first-stage investigation, the applicant has utilized the iTRAQ technique and identified 488 differential proteins. Primary bioinformatics analyses suggest that the GO enrichment is mainly associated with oxidation-reduction process, extracellular exosome, and poly(A) RNA binding. Some of the results are consistent with previous reports, confirming the reliability of the experiment, while the other results have revealed a great many undocumented molecules or biological courses. Therefore, the current study aims to validate these primarily discovered candidate proteins from the perspectives of cell, animal and human samples. The applicant believes that this study is one further step closer to gaining an insight into the pathogenesis of intestinal I/R and the potential biomarkers for disease detection and prediction.

肠道缺血再灌注(I/R)损伤是临床常见的急症，氧化应激是其重要机制之一。Science曾报道，乙醛脱氢酶2(ALDH2)能够催化醛类代谢，进而抑制氧化应激，减轻心肌I/R。申请人既往也证实在肠道I/R中激活ALDH2具有保护作用(已发表,IF=5.016)，但具体机制仍需更多研究。在前期研究中，利用蛋白质组iTRAQ方法筛选出488个差异蛋白，生物信息学分析显示，一部分蛋白可以和以往机制对应，而另一部分则包含了从未报道过的分子(如Nme2、Gpd1l、Mtch1)。为进一步研究这些蛋白在肠道I/R的作用，本项目拟从细胞、动物、临床样本三个层面，用WB、ELISA和PCR等技术验证表达情况，并通过干预差异蛋白的表达，观测其对肠道I/R的影响，阐明差异蛋白在ALDH2激活后减轻肠道I/R损伤的作用机制。这些研究将为完善肠道I/R发病机制提供新视野，并为探索干预手段提供新的研究靶点。

肠道缺血再灌注（I/R）损伤是指肠道的血流中断一段时间后，再次恢复血流时，组织细胞功能损伤进一步加重的情况。肠道缺血再灌注损伤是临床常见的急症，氧化应激是其重要机制之一。既往研究发现乙醛脱氢酶2（ALDH2）能够减轻氧化应激，减轻心肌缺血损伤。本申请人前期发表文章也证实，激活ALDH2可减轻肠道I/R。本项目利用蛋白质组学技术，筛选出488个与ALDH2相关的差异蛋白，生物信息学发现，PTEN induced putative kinase 1（PINK1）蛋白是重要的候选蛋白。细胞实验提示，PINK1主要定位于线粒体，介导细胞线粒体自噬，在I/R状态下呈高表达趋势，ALDH2表达和PINK1表达呈显著负相关。下载KEGG线粒体自噬相关的所有基因后，经过热图和聚类分析发现，自噬相关基因在I/R组整体呈高表达状态，且聚类状态可正确区分对照组和I/R组。在Caco-2细胞中，使用ALDH2激活剂Alda-1（20 μmol/L）可显著下调PINK1表达，抑制PINK1与细胞色素c氧化酶IV（COX IV）共定位，减少细胞凋亡和降低线粒体电势，进而抑制线粒体自噬，以减轻I/R损伤。而使用自噬诱导剂（CCCP）能显著上调PINK1表达，部分逆转PINK1与COX IV共定位，增加细胞凋亡和升高线粒体电势，减弱ALDH2激活引起的保护作用。使用ALDH2抑制剂Daidzin（60 μmol/L）可显著上调PINK1表达，增加PINK1与COX IV共定位，增加细胞凋亡和升高线粒体电势，继而促进线粒体自噬，以增加I/R损伤。而使用自噬抑制剂氯喹（Chloroquine）能显著降低PINK1表达，部分抑制PINK1与COX IV共定位，减轻细胞凋亡和降低线粒体电势，进而减弱ALDH2抑制引起的损伤作用。通过以上研究，本项目发现ALDH2通过下调PINK1，减弱线粒体自噬，降低肠道缺血再灌注损伤。ALDH2通过PINK1/线粒体自噬途径减轻肠道缺血再灌注损伤的相关文章在国内较少有人报道，本项目内容具有一定的创新性，能为临床治疗肠道缺血再灌注提供新的选择，并对预防该疾病提供新的线索。