基于NLRP3炎性小体信号通路抑制泡沫细胞焦亡探讨清热解毒法治疗动脉粥样硬化的机制

Atherosclerosis (AS), which is a severe threat to human health, is a chronic inflammatory process characterized by accumulating of foam cells on endarterium . Recently, it was found that pyroptosis of foam cells, which is activated by NLRP3 inflammasome mediate pathway, is the main culprit for the expansion of lipid cord in AS plague and the deteriorating of plague inflammation. In Traditional Chinese Medicine, “Breeding of heat toxin within human body" was regarded as the important mechanism of AS development therefore "Heat-scavenging and Detoxicating Remedy" were used to treat it. Our preliminary data showed that one of the formula fit in this kind, ”Huang Lian Jie Du Decoction” although not working as an antihyperlipidemics ，can decrease the size of lipid cord of AS plague and alleviate the inflammatory process which is consistent with the concept of inhibiting foam cells pyroptosis. Protein-ligand docking reveals some components in this Decoction match the structures of some proteins in NLRP3 mediate signal pathway. Therefore, the hypothesis "Interrupting the NLRP3 inflammasome mediate signal pathway to inhibit foam cells pyroptosis is the potential mechanism of Huang Lian Jie Du Decoction to decrease lipid cord in AS plague and to alleviate inflammatory process of AS plaque development." was raised. This decoction was administrated to apoE-/- mice of AS model and foam cell model to elucidate its role in suppressing AS development and anti-pyroptosis in vivo. This mechanism will also be examined in vitro and potential targets would be screened by high throughput liquid phase chip, multiple process protein function antagonistic and agonistic agents. The outcome of our research will enrich the theory of "Heat-scavenging and Detoxicating Remedy" and the involved signal proteins can be used as targets for AS prevention and treatment.

动脉粥样硬化(AS)是以泡沫细胞在动脉内膜聚集为特征的慢性炎症，严重威胁人类健康。近期发现：激活NLRP3炎性小体信号通路导致泡沫细胞“焦亡”，是扩大AS斑块脂核、加剧炎症的主因。中医认为“热毒内蕴”是当今AS的重要病机并以清热解毒法论治。前期研究发现：该法代表方“黄连解毒汤”无降脂功效，却可减小斑块脂核、减轻斑块炎症，这与抑制泡沫细胞焦亡的效果不谋而合；分子对接提示该方有效成分与上述信号蛋白结构域匹配。据此提出假说：干预NLRP3炎性小体信号通路抑制泡沫细胞焦亡，是黄连解毒汤减小斑块脂核、减轻斑块炎症潜在机制，并以该方干预apoE-/-小鼠AS模型、泡沫细胞模型，在体明确其抗AS作用与抗焦亡的相关性；离体确证其抗泡沫细胞焦亡药效，并以高通量液相芯片、多环节蛋白功能拮抗与激动相结合，阐明药物作用靶点，验证假说。本研究结果可丰富清热解毒法的理论内涵，所涉及的信号蛋白有望成为防治AS重要靶点。

动脉粥样硬化(AS)是以泡沫细胞在动脉内膜聚集为特征的慢性炎症。近期发现“泡沫细胞焦亡”是扩大AS斑块脂核、加剧斑块炎症的主因。中医认为“热毒内蕴”是AS的重要病机，并与炎症密切关联，常以清热解毒法论治。前期研究发现：清热解毒代表方“黄连解毒汤”，虽无降脂功效，却可减小斑块脂核、减轻斑块炎症，这与抑制泡沫细胞焦亡的效果不谋而合。综上，提示抑制泡沫细胞焦亡可能是黄连解毒汤抑制斑块炎症进而减轻AS的机制。为了验证上述假说，本研究循序渐进完成了如下两部分研究：①在体实验明确黄连解毒汤是否具有抗AS并抑制泡沫细胞焦亡的药效：以高脂饲养的雄性apoE-/-小鼠为研究对象复制AS模型，以黄连解毒汤为干预药物，发现该方药可减小AS斑块面积和斑块脂核、抑制炎细胞浸润，首先确证了黄连解毒汤抗AS的药效。在此基础上，以蛋白印迹法检测焦亡经典通路关键酶Caspase-1蛋白的表达，以液相芯片法检测焦亡效应因子--血清 IL-1β、IL-18和HMGB1的水平，结果显示黄连解毒汤可以抑制Caspase-1蛋白的表达，减少焦亡效应分子IL-1β、IL-18和HMGB1的释放，确证了黄连解毒汤具有抑制泡沫焦亡的药效。②oxLDL 既是诱导巨噬细胞焦亡的毒性物质，也是诱导其形成泡沫细胞的物质基础。因此，培养apoE-/-小鼠腹腔巨噬细胞，以ox-LDL孵育巨噬细胞复制泡沫细胞的焦亡模型。研究发现黄连解毒汤载药血清可以抑制泡沫细胞LDH的流出、下调Caspase-1的表达。加入NLRP3炎症小体抑制剂MCC950，反向验证药物靶点。结果显示黄连解毒汤抑制泡沫细胞焦亡的机制与NLRP3炎症小体预激活无关，而是通过影响NLRP3炎症小体组装或活性发挥，从而降低Caspase-1水平，阻止IL-1β、IL-18、GSDMD N端释放，最终抑制细胞焦亡的发生并降低相关炎症因子的水平。本研究的结果建立了热毒内蕴和炎性细胞死亡在动脉粥样硬化中的关联，而干预NLRP3炎症小体的活化而非预激活而发挥了抑制焦亡及其引发的炎症是清热解毒的代表方“黄连解毒汤”通过抑制炎症抗AS的重要机制。