Drp1介导的线粒体分裂在角膜碱烧伤中的作用及其机制研究

Corneal alkali burn is a kind of ocular trauma with very poor prognosis. It has been well documented that reactive oxygen species (ROS) play a crucial role in alkali burn-induced inflammatory responses and corneal neovascularization. There are two sources of ROS in cells. The main source of ROS is from mitochondria, and the other is from some enzymes, such as NADPH oxidases (NOXs). However, the role of Drp1-mediated mitochondrial fission in alkali burn-induced corneal injury and its underlying mechanism are still unclear. In the preliminary experiments, we found that mitochondrial fission, phosphorylation of Drp1 at ser616 and ROS increase in corneas ocurred at day1, and continued to day 7 after alkali burn. In contrast, upregulation of NADPH oxidases NOX2/4 ocurred at day 7 after alkali burn. Inhibition of Drp1 by Mdivi-1 efficiently attenuated alkali burn-induced ROS increase in corneas. Based on previous studies and our preliminary studies, we hypothesize that Drp1-mediated mitochondrial fission might upregulate the expression of NOXs via NF-κB signalling pathway, and involve in alkali burn-induced corneal injuries through synergistically producing ROS. To prove our hypothesis, we will further investigate the mechanism of Drp1 in mitochondrial fission, ROS production, and NOXs expression via NF-κB signalling pathway, and examine the therapeutic effect of Drp1 inhibitors on alkali burn-induced corneal injury. Therefore, this study will provide a potential therapeutic target for corneal alkali burn in the future.

角膜碱烧伤是预后极差的眼外伤。活性氧自由基ROS是角膜碱烧伤炎症反应和新生血管形成的关键致病因子。ROS主要来源于线粒体途径，其次是酶途径（如NADPH氧化酶NOXs），但有关Drp1介导的线粒体分裂在角膜碱烧伤中的作用及其机制均不清楚。本课题前期实验发现：小鼠角膜组织中线粒体分裂、Drp1-S616磷酸化和ROS水平升高出现在碱烧伤后第1天，并维持到第7天；而NOX2/4表达上调出现在碱烧伤后第7天；利用Mdivi-1抑制Drp1活性能有效减轻碱烧伤所致ROS升高。基于前期实验结果和文献报道，我们推测：Drp1介导的线粒体分裂可能经NF-κB信号通路上调NOXs表达，从而产生大量ROS参与到角膜碱烧伤中。为验证上述假说，将深入探讨Drp1介导的线粒体分裂在ROS产生、NF-κB通路激活及NOXs表达上调中的作用机制，并检测Drp1抑制剂对角膜碱烧伤的疗效。故本课题将为今后更好地治疗角膜碱烧伤寻找药物新靶点。

角膜碱烧伤是预后极差的眼外伤。活性氧自由基ROS是角膜碱烧伤炎症反应和新生血管形成的关键致病因子。ROS主要来源于线粒体途径，其次是酶途径（如NADPH氧化酶NOXs ），但有关Drp1介导的线粒体分裂在角膜碱烧伤中的作用及其机制均不清楚。本课题主要是研究Drp1介导的线粒体分裂是否可通过NFκB信号通路上调NOXs表达，从而产生大量ROS参与到角膜碱烧伤中。实验发现，角膜碱烧伤第三天线粒体分裂蛋白Drp1及其S616磷酸化水平均显著上调，而线粒体融合蛋白Mfn2表达下调。IκBα磷酸化和NOX2/4表达水平在角膜碱烧伤第七天上调。Drp1抑制剂Mdivi-1或利用Lenti-Drp1-shRNA敲低Drp1能有效阻止角膜碱烧伤后的ROS产生、NFκB通路激活、NOX2/4表达上调和炎症因子IL-6, IL-1β和TNF-α转录水平。在人角膜上皮细胞系HCE-2中，NaOH处理能刺激线粒体分裂和胞内ROS产生和NFκB p65的核移位，Drp1抑制剂能减轻NaOH所诱导的HCE-2细胞内线粒体分裂和ROS升高和p65核移位。整体动物实验中，通过药理实验我们发现Drp1抑制剂Mdivi-1和NOXS抑制剂Apocynin能有效减轻角膜碱烧伤所致炎症因子释放和新生血管形成，且联合应用Mdivi-1和Apocynin对角膜碱烧伤的保护具作用具有协同效应；结膜下注射线粒体抗氧化剂mito-TEMPO也能有效减轻碱烧伤后NOX2/4表达、炎症因子转录和角膜新生血管形成。这些实验结果证实了角膜碱烧伤可先刺激Drp1介导的线粒体分裂，然后经NFκB信号通路上调NOX2/4表达，从而促进胞内ROS产生，并以此参与角膜碱烧伤，也表明Mdivi-1和Apocynin有望成为治疗角膜碱烧伤的新型药物，故具有重要的科学价值和临床应用前景。