蒺藜皂苷基于线粒体调节NLRP3干预缺血性脑损伤的物质基础及作用的分子机制研究

It has been found that NLRP3 in neurons and glia plays an important role in cellular injury and inflammation during cerebral ischemia. Mitochondria regulate NLRP3-mediated inflammatory response via ROS. So it has become a new strategy for drug therapy to regulate the inflammasome NLRP3 to inhibit the inflammation in cerebral ischemia. Caltrop,belonging to zygophyllaceae, has the founction of relieving heat and promoting the blood circulation. We started the research on caltrop from the 1990s. We have developed total saponins of the caltrop fruit as a new drug of traditional Chinese medicine for cerebral infarction, which has received clinical permission and authorized patent. Our research in caltrop has been published in 27 papers. Recently, we found that saponins of the caltrop fruit can reduce the area of cerebral infarction areas and decrease the level of IL-1β and IL-18 in focal cerebral ischemia of rat. Given our previous research, we will analyze the effects and molecular mechanisms of active ingredient of saponins on mitochondria-regulated inflammasome NLRP3. Ultimately, we will demonstrated that NLRP3 pathway （NLRP3, ASC,pro-caspase-1, and pro-IL-1β）may be involved in the protective effects of monomers of saponins in cerebral ischemia. In addition, the protective mechanismis may be mediated by mitochondria regulating NLRP3 through ROS. It is aimed to confirm molecular target of caltrop saponins and explore the molecular mechanism of their protection in cerebral ischemia. Our research will provide theoretical basis for development and clinical application of caltrop and find new target and effective drugs for the treatment of cerebral ischemia.

神经元和胶质细胞的NLRP3 在细胞损伤和介导炎症发挥重要作用。线粒体通过ROS调控NLRP3炎性小体进而抑制脑缺血介导的炎症反应成为药物干预脑缺血损伤的新途径。蒺藜系蒺藜科植物，具有散风，行血的功效。我们从90年代开始了对蒺藜的研究，将蒺藜总皂苷开发为治疗脑梗塞的新药，已获临床批件(具授权专利)，发表27篇论文。近期我们发现蒺藜总皂苷可缩小大鼠局灶性脑缺血脑梗死面积，降低缺血大鼠IL-1β和IL-18的水平。本课题拟在此基础上对蒺藜总皂苷及其主要成分进行基于线粒体调节炎性小NLRP3-ASC-(pro- caspase-1)分子平台干预脑缺血炎性反应的分子机制研究，论证蒺藜单体成分脑细胞保护作用可能与直接干预NLRP3炎性小体途径有关，且其分子机制是由线粒体通过ROS调控NLRP3介导的，以确证蒺藜皂苷类成分抗脑缺血作用的靶分子，探索其保护脑缺血损伤的物质基础及分子机制。

本项目通过对蒺藜总皂苷及其主要活性成分基于NLRP3干预脑缺血炎性反应的分子机制展开研究，发现NLRP3干预脑缺血炎性反应直接影响脑缺血部位NLRP3炎性小体（NLRP3, ASC, pro-caspase-1及pro-IL-1β），蒺藜皂苷及其两个单体皂苷成分保护脑缺血损伤的分子机制与线粒体通过ROS调控的NLRP3表达有关。进一步对蒺藜甾体皂苷中抗MCAO的活性成分进行筛选，发现两种具有明显活性的单体成分，同时对其基于NLRP3干预脑缺血炎性反应的分子机制进行了深入研究，表明它们能够有效降低脑缺血引起的神经性功能损伤，改善炎性细胞浸润，降低炎症细胞因子表达，提示可能通过干预炎症反应减轻局灶性脑缺血损伤。进一步研究发现两个化合物在大鼠脑缺血3 h和6 h，主要影响NLRP3、IL-1β mRNA水平的表达，而在缺血12 h后可降低NLRP3蛋白表达，至缺血24 h后则进一步抑制IL-1β的蛋白表达，改善脑缺血损伤所致炎症反应。基于线粒体调节的NLRP3信号途径介导炎症反应机制发现，两种单体皂苷可抑制OGD引起的NLRP3炎性小体激活，但不能逆转ROS激动剂引起的的NLRP3炎性小体的激活。同时，结果显示，两种单体皂苷可通过调控AMPK通路、细胞自噬过程和AIM2炎性小体活化途径，抑制OGD诱导的IL-1β和casepase-1过表达，但其对NLRC4炎性小体活化无明显抑制作用。本项目明确了蒺藜皂苷成分发挥抗脑缺血作用的抗炎分子靶标，为蒺藜的临床应用及进一步开发提供可靠的实验依据。本项目已发表相关学术论文5篇，其中包括SCI论文2篇，核心期刊论文2篇；获省科技进步一等奖1项；培养毕业硕士研究生2名，在读研究生1名。