二氢丹参酮I调控RIP3介导的易损斑块形成的药效及机制

Vulnerable plaque is an important cause of acute cardiovascular events that threatens human health seriously. At present, there are no ideal drugs for stabilizing plaque. Chinese medicine theory holds that atheromatous plaque belongs to the syndrome of phlegm and blood stasis. Qi-deficiency and blood-stasis syndrome are the main pathological basis of geriatric disease. Danshen has the pharmacological action of activating circulation to remove blood stasis and is widely used in clinical for cardiovascular diseases. Dihydrotanshinone I (DHT) is an active ingredient of Danshen. Our previous study showed that DHT significantly reduced the area of necrotic core in vulnerable plaque of ApoE-/- mice. Meanwhile, DHT reversed necroptosis of vascular smooth muscle cells which is one important component of vulnerable plaque, decreased RIP3 expression and ROS production. RIP3 is a key mediator of necroptosis and involved in the process of atherosclerosis. Above study suggests that RIP3 may mediate the formation of vulnerable plaque and DHT has potential effects on its stability. However, related mechanisms remain to be studied. Therefore, this proposal will further investigate the intervention effects and molecular mechanisms of DHT on ameliorating plaque development by focusing on the activation of RIP3 both in vivo and in vitro. Methods such as siRNA technique and western blot analysis will be adopted in trials. This study is conductive to provide new ideas and new ways for Traditional Chinese Medicine in treatment of acute cardiovascular diseases.

易损斑块是造成急性心血管事件的重要因素，严重威胁人类健康。目前尚无理想的稳定斑块的药物。中医药理论认为粥样斑块多属痰、瘀之范畴，气虚血瘀证是老年病的主要病理基础。中药丹参具有活血化瘀功能，临床上广泛应用于治疗心血管疾病。二氢丹参酮I(dihydrotanshinone I，DHT)是丹参活性成分。我们前期研究发现，DHT明显减少ApoE-/-小鼠易损斑块坏死中心的面积，逆转血管平滑肌细胞（易损斑块重要组成细胞）程序性坏死(necroptosis)，减少RIP3表达和ROS释放。RIP3是necroptosis关键蛋白且被证实参与粥样硬化病理过程，这提示RIP3可能介导易损斑块形成且DHT有潜在的稳定作用，但相关机理尚待研究。本项目拟以RIP3作为切入点，通过基因沉默，蛋白免疫印迹等手段从体内、体外探讨DHT对易损斑块的干预作用及相关机制。本课题开展为传统中药治疗心血管疾病提供新思路和方法。

相比癌症、慢性呼吸系统疾病和糖尿病，心血管疾病依旧位居全球各种死因之首，形势非常严峻。易损斑块是严重心脑血管事件的重要发病机制，斑块一旦破裂会引起心肌梗死、猝死、中风等急性心脑血管事件，对患者产生严重的后果。因此稳定易破损斑块对预防心脑血管事件有十分重要的意义。目前尚无理想的稳定斑块的药物。近几年中药以其“治疗效果好，不良反应少”受到了国际社会越来越多的认可。 丹参是我国历史上被研究最多的中草药之一，广泛用于心脑血管疾病的防护。二氢丹参酮I（dihydrotanshinone I, DHT）是丹参的重要活性成分之一。我们的前期的预试验发现DHT对高糖损伤的血管平滑肌细胞有明显的保护作用，为DHT改善动脉粥样硬化提供了立题依据。本项目主要从两个方面进行课题研究：1）体内实验，高糖高脂饲料喂养ApoE-/-的C57BL/6J小鼠建立易损斑块模型，检测DHT及临床药物辛伐他汀对易损斑块相关病理指标的干预作用；体外实验，LPS/zVAD诱导血管平滑肌细胞构建Necroptosis模型，在此基础上探讨DHT对坏死平滑肌细胞的干预及调控机制。团队通过深入研究及齐心合作，获得了关键数据：1）体内实验数据显示：DHT明显改善动脉粥样硬化小鼠的脂质代谢紊乱及氧化应激，更为重要的是DHT减少模型组小鼠的胸主动脉中易损粥样斑块的大小及坏死中心的面积，同时恢复了斑块中平滑肌细胞的数量，降低了坏死marker蛋白RIP3在斑块中的表达；体外实验结果显示：DHT通过靶向RIP3阻断LPS/zVAD诱导的程序性坏死通道RIP3/MLKL,从而保护血管平滑肌细胞。这两点为后期DHT的临床应用提供了理论支持。本研究的科学意义如下:1)丰富了“动脉粥样硬化斑块引起心血管疾病”的发病机制，进一步增加了人们对易损斑块的关注; 2)以DHT为切入点，揭示以丹参为代表的中药治疗心血管疾病的优势，揭示其分子机制，为其临床应用提供科学数据支持。