红花黄色素黄酮成分激活自噬负调控NLRP3活化抗MIRI的分子机制及构效关系研究

The activation of the NLRP3 inflammasome and its downstream signal pathways during myocardial ischemia/reperfusion injury (MIRI) amplify the inflammatory response and mediate further damage. Activating autophagy and inhibiting NLRP3 inflammasome may provide a pivotal strategy for prevention and treatment of MIRI. Carthamus tinctorius L., which belongs to Compositae family, has the effects of promoting blood circulation and removing blood stasis. Safflow yellow (SY), is the main bioactive ingredients of Carthamus tinctorius L., has significant effects against coronary heart disease. Our previous studies found that SY and HSYA could inhibit IL-1β、IL-6 production and decrease NF-κB expression levels, which indicates that the anti-inflammatory effects are the main mechanisms against MIRI. Further research showed that SY could up-regulate autophagy related protein expressions, diminish NLRP3 inflammasome activation, suggesting that the anti-inflammatory effects may be related tothe regulation of autophagy and then inhibition of the NLRP3 inflammasome. In this respect, 4 papers have been published and we have awarded 2 prizes. In this study, we aim to study on the structure activity relationship of the protective effects of main active safflow yellow components against the MIRI, and to investigate whether the underlying mechanisms are closely associated with the up-regulation of autophagy that leads to inhibiting NLRP3 inflammasome activation, and subsequently reducing the expression levels of inflammatory factors and cell apoptosis, thereby exploring the molecular mechanisms and targets of the cardioprotective effects of safflow yellow components.

NLRP3炎症小体及其下游信号分子的激活是诱发心肌缺血后炎症反应的主要途径。通过诱导自噬，负反馈调控NLRP3炎症小体活化，进而减轻炎症反应是治疗心肌缺血再灌注损伤的关键策略。红花为菊科植物，具有活血祛瘀之功效。SY是红花的主要效应物质。临床用于冠心病等疾病的治疗，效果显著。我们前期研究发现SY及HSYA显著抑制IL-1β、IL-6升高，抑制NF-κB的表达，提示其抗炎作用是其抗MIRI的重要分子机制。进一步研究发现SY可上调线粒体自噬的相关蛋白的表达，抑制NLRP3炎症小体活化，推测其减少IL-1β分泌，减轻炎症反应可能与调控自噬，负反馈调控NLRP3活化有关。发表了4篇论文，获奖二项，本课题拟对SY主要活性成分进行构效关系研究，并探讨其对自噬及NLRP3的影响。论证其心肌保护作用可能与直接上调自噬进而抑制NLRP3的激活，抑制炎症因子表达，减少细胞凋亡有关，确证其作用信号通路与靶分子。

通过诱导自噬，负反馈调控NLRP3炎症小体活化，进而减轻炎症反应是治疗心肌缺血再灌注损伤的关键策略。红花黄色素（SY）是红花的主要效应物质，临床用于冠心病等疾病的治疗，效果显著。本项目主要研究内容为：①红花黄色素不同类型黄酮类成分活性筛选：利用缺氧复氧诱导H9C2心肌细胞损伤模型检测不同类型黄酮类成分对心肌细胞存活率的影响。发现羟基红花黄色素A（HSYA）、脱水红花黄色素B（AHSYB）等在一定浓度对缺氧复氧损伤H9C2细胞具有显著保护作用。②SY对急性心肌缺血再灌注损伤大鼠的心肌保护作用研究：SY对心肌缺血再灌注损伤的急性期炎症反应有明显抑制作用，其机制之一可能是通过抑制Toll样受体启动，抑制NF-κB产生免疫应答，减少炎性因子的产生从而抑制炎症。另一机制可能是通过阻断或减弱mTOR/Akt/PI3K通路，上调自噬相关蛋白表达促进细胞自噬，减少线NLRP3炎症小体活化，从而减轻炎症反应保护心肌。③HSYA、AHSYB对缺氧/复氧诱导HUVECs内皮细胞损伤的保护作用及调控自噬体抑制炎症小体NLRP3的机制研究：HSYA、AHSYB在一定浓度范围能显著保护HUVECs细胞活性，并能激活缺氧/复氧损伤的HUVECs细胞自噬，负反馈调控炎症小体NLRP3活化，降低细胞炎症反应，减轻细胞损伤。氯喹抑制剂组自噬溶酶体大量增多，LC3-II表达也增多，炎症小体NLRP3激活，炎症因子分泌增加，细胞炎症反应加重，细胞活性降低。④HSYA对心肌缺血再灌注损伤的保护作用及激活自噬负调控NLRP3的机制研究：HSYA能明显减轻大鼠心肌缺血/再灌注（I/R）损伤及心肌细胞缺氧/复氧（H/R）损伤，可通过调节AMPK信号通路促进心肌自噬发生，抑制NLRP3炎症小体的激活，进而减少下游炎症因子的表达达到保护缺血心肌的目的。⑤山奈酚（Kae）可通过调控AMPK信号通路，抑制NLRP3炎症小体活化，减轻炎症反应，从而减少缺氧/复氧造成的心肌细胞损伤。本研究可为红花的临床应用和开发提供实验依据，为心肌缺血再灌注损伤的防治提供新的靶点及有效药物，具有良好的科学价值。本项目已发表相关学术论文4篇，其中包括SCI论文2篇；培养研究生3名。