百草枯诱导Ⅱ型肺泡上皮细胞凋亡的调控机制研究

Paraquat (PQ), which is a herbicide, has been widely used throughout the world. But patients with PQ poisoning were associated with high mortality. Pulmonary fibrosis caused by PQ is the important reason for patients' death, and its pathogenesis hasn't been fully understood yet. Excessive apoptosis of type II alveolar epithelial cell (AT II) is considered as the critical event during the early phase of pulmonary fibrosis. Some supporting facts were found in our prophase work. The increasing expression of the proapoptotic proteins like Bad and Bax instead of Fas were noticed in AT II isolated from the lungs of PQ-treated mice, while the analysis of the activity of Caspases also indicated that the specific Caspases from endoplasmic reticulum stress (ER stress)and mitochondrial apoptotic pathway were activated. A hypothesis is developed by our team that the ER stress and mitochondrial apoptotic pathway would be the chief pathways in the PQ-induced apoptosis of AT II involving the reactive oxygen species (ROS) and intracellular calcium homeostasis. This research, through the cellular experiments, would attach the great importance to the regulation mechanism of PQ-induced apoptosis of AT II. The results will clarify the roles of ER stress mitochondrial apoptotic pathways,the trigger factors of those pathways, and the relationship between calcium homeostasis, ROS and two apoptotic pathways. This research will enhance the comprehension of the pathogenesis and role of apoptosis of AT II during PQ-induced pulmonary injury, provide the new insight and find a new therapeutic target point for the treatment.

百草枯(PQ)是目前世界范围内广泛使用的除草剂，中毒者病死率极高。PQ所致肺间质纤维化是患者死亡的重要原因，但其发病机制尚不十分清楚。II型肺泡上皮细胞（AT II）凋亡过度被认为是纤维化早期发生中的关键事件。我们在前期工作中发现，PQ致肺损伤过程中存在ATII凋亡增多，促凋亡蛋白Bad、Bax高表达，但Fas表达增高不明显。对Caspases激活的分析也发现PQ激活了内质网应激和线粒体途径特异性的Caspases。我们推测PQ致AT II凋亡调控机制可能以内质网应激和线粒体途径为主，涉及到活性氧和细胞内钙稳态。本研究将利用细胞实验，将PQ致AT II凋亡作用机制作为研究突破点。研究结果将阐明PQ诱导AT II凋亡过程中内质网应激及线粒体凋亡通路的作用及发生机制，以及Ca2+稳态和活性氧与内质网应激和线粒体凋亡通路的关系。本研究将深化对PQ致肺损伤发病机理的认识，为有效治疗本病提供新思路。

百草枯（PQ）是目前我国农业生产中广泛使用的除草剂。由于PQ具有强烈的毒性且无特异性解药，因此，研究PQ中毒的发病机理和救治方法具有迫切的现实意义。PQ致肺损伤的机制尚不十分清楚。本研究将PQ致肺泡Ⅱ型上皮细胞（AT II）凋亡调控机制作为研究突破点。.㈠.PQ引起内质网应激介导的AT II凋亡发生机制.我们检查PQ诱导人AT II细胞凋亡过程中ER应激相关信号分子mRNA和蛋白质水平的表达情况和变化规律。结果阐明了PQ介导AT II凋亡中内质网应激具有关键性作用。Salubrina（SAL）对PQ诱导人肺II型上皮样细胞A549发生凋亡具有保护作用，其机制与调节ERS反应中未折叠蛋白反应（UPR）的PERK-eIF2α通路相关。.㈡.PQ引起线粒体途径介导的AT II凋亡发生机制及上游信号P53蛋白的作用.我们检查PQ诱导人AT II细胞凋亡过程中，线粒体途径中相关通路蛋白和胞质中细胞色素C的表达、线粒体跨膜电位变化和P53蛋白表达。观察Pifithrin-α（PFT）抑制P53活性对PQ诱导的AT II细胞凋亡的影响。结果表明线粒体途径在PQ介导AT II中凋亡具有重要作用。上游信号P53蛋白可能以转录依赖方式介导了线粒体凋亡通路的激活。.㈢.PQ诱导AT II凋亡过程中细胞内Ca2+稳态的变化.使用流式细胞仪等方法分析PQ作用AT II细胞早期胞质内Ca2+随着作用时间的变化规律。结果表明PQ诱导AT II凋亡过程中，Ca2+存在细胞内重分布。Ca2+充当了第二信使的作用，使内质网应激与线粒体凋亡通路联系。.㈣.PQ诱导的活性氧产生与内质网应激和线粒体凋亡通路的关系研究.PQ能够引起ROS产生，而后者是引起凋亡的重要分子。利用抗氧化剂MnSOD的模拟物MnTMPyP预处理细胞，观察对PQ诱导的AT II细胞凋亡的是否具有保护作用。结果表明ROS是PQ诱导AT II凋亡的重要启动因素。MnTMPyP可以减轻活性氧的产生，可以减弱PQ引起的凋亡效应。.在本课题研究中，研究团队明确了PQ诱导AT II凋亡在PQ致肺损伤中的重要作用，提出了凋亡信号转导途径作为本病治疗靶点的可行性。本研究从细胞凋亡角度揭示PQ致肺损伤的发病机理，为有效治疗本病提供了新思路，为开发药物新靶点提供了依据。