2-芳基呋喃香豆素对埃及伊蚊的作用机理研究

Furocoumarins show important pharmacological and biological activities. Early, we designed and synthesized a series of 2-substituted furocoumarin derivatives, which showed significantly photosensitive activities. In order to clarify the mode of action of insecticide, the aedes aegypti from the tropical area is selected as target insect, 2-aryl furocoumarins are as the tested insecticides in this project. Based on the preliminary symptoms and fluorescence localization, the midgut of aedes aegypti is the target organs. Then, utilization of cytology, enzymology and molecular biology, the midgut of aedes aegypti is further studied. At first, the ultrastructure and ultrastructure of the midgut treated by 2-aryl furocoumarin will be observed. Then, the intestinal digestive enzymes and material metabolism, energy metabolism will be tested; the activity of oxidative stress will be detected. At last, the apoptosis and DNA damage of intestinal cells will be researched. According to above experiments, the mode of action of 2-aryl furocoumarins will be formed, which will help for the further structural improvement of highly active pesticide.

呋喃香豆素是一类具有重要药理和生物活性的物质。课题组前期设计合成了一系列2-取代呋喃香豆素衍生物，显示出很强的光敏活性。为明确其杀虫机理，本项目以热带地区埃及伊蚊为靶标试虫，以高活性2-芳基呋喃香豆素为供试药剂，基于前期症状学和荧光定位确定的中肠靶器官，通过细胞学，酶学和分子生物学手段，以中肠靶器官为切入点，观察2-芳基呋喃香豆素对埃及伊蚊中肠的超微形态和超微结构，测试试虫肠道消化酶系和中肠的物质代谢、能量代谢，检测药剂的氧化应激活性，明确试虫中肠细胞凋亡及DNA损伤，从而揭示化合物的作用机理，为优化高活性的农药潜药奠定基础。

呋喃香豆素是一类来源于植物的光活性化合物，具有抗氧化、抗癌、杀虫、杀菌等多种生物活性,具有重要研究价值。为此通过缩合、酯化、溴化、Wittig反应合成2-芳基呋喃并香豆素衍生物，发现2-噻吩呋喃并香豆素（Ⅰy）具有较好的光敏活性。因此，本研究以 Ⅰy三个不同浓度（LC25、LC50、LC75）为处理，埃及伊蚊四龄幼虫为研究对象，从亚致死剂量、线粒体功能障碍、氧化应激水平、肠道屏障功能障碍、细胞凋亡等方面进行研究。结果表明，在紫外光照射下，Ⅰy能够诱导中肠细胞产活性氧（ROS），3h时出现ROS迸发，引起线粒体复合酶活性受到抑制，尤其对复合酶Ⅲ抑制效果最显著。超微结构分析显示，线粒体嵴模糊和出现空泡化，三羧酸循环酶活性受到抑制，ATP含量和线粒体膜电位降低。因此线粒体功能受到破坏，进而诱导ROS过度产生，随后包括SOD、CAT、GPx、GR在内的抗氧化酶系受到抑制，使得ROS消除受阻，进一步使中肠细胞处于高氧化应激状态，从而加剧机体氧化损伤程度。观察到中肠组织学改变，从而导致肠道屏障功能障碍。当ROS消除受阻，在细胞内不断蓄积，诱导激活AeDronc、AeCaspase7、AeCaspase8在内的凋亡相关基因， 凋亡染色证实凋亡细胞存在，并且观察到凋亡典型特征核固缩。此外，Ⅰy在亚致死浓度下，能对埃及伊蚊生长发育造成影响，且存在明显的后致死效应。综合表明，Ⅰy具有很好的光敏活性，能引起埃及伊蚊幼虫中肠损伤和细胞凋亡，具有重要的应用前景。