从中性粒细胞胞外诱捕网（NETs）的形成与降解探讨冠心病血瘀证证型特点的分子机制

Atherosclerosis (AS) is the main pathological basis for coronary heart disease, and its pathogenesis is complex. Neutrophil extracellular traps (NETs) exerts cytotoxic and prothrombotic effects, possibly providing a causative link between inflammation and coagulation. NETs are defined as large web-like structures composed of decondensed chromatin and neutrophil-derived nuclear and granular proteins, which are capable of ensnaring and killing pathogens. According to the different sources of stimulation, the formation mechanism and effect of NETs are different. Blood stasis syndrome is one of the most common syndromes of coronary heart disease. Studies have shown that blood stasis syndrome of coronary heart disease is closely related to blood circulation, coagulation and fibrinolytic system and inflammatory immune function. Our previous study found that plasma level of MPO, one of the major proteins released by NETs, is higher in blood stasis syndrome patients with unstable angina pectoris than in non-blood stasis syndrome, and it was gradually decreased with the qi deficiency and blood stasis syndrome, qi stagnation and blood stasis syndrome, phlegm and blood stasis syndrome. In view of the similarity association with inflammatory and coagulation system between blood stasis syndrome and NETs, we explore the biological basis of coronary heart disease blood stasis syndrome based on NETs. Meanwhile, we take the different mechanisms formed by NETs as a breakthrough point to explain the molecular mechanisms of syndrome differentiation and classification of coronary heart disease blood stasis syndrome, which provides a scientific basis for the diagnosis of treatment of coronary heart disease with blood stasis syndrome.

动脉粥样硬化(AS)是冠心病的主要病理基础，中性粒细胞胞外诱捕网(NETs)的细胞炎性和促血栓形成的效应为动脉粥样硬化的发生发展提供新机制。NETs是中性粒细胞激活时释放的一种网状结构，根据刺激来源的不同，其诱导机制及效应结果也不同。血瘀证是冠心病最常见的证候之一，研究表明其与血液循环状态、凝血纤溶系统和炎症免疫功能密切相关。我们前期研究发现，冠心病不稳定型心绞痛血瘀证患者血浆MPO（NETs释放的主要蛋白之一）水平较非血瘀证患者升高，且随气虚血瘀、气滞血瘀和痰瘀互阻的证候变化逐渐降低，又鉴于血瘀证和NETs均与AS两大病理机制--炎症免疫和凝血系统关联的相似性，本课题研究冠心病血瘀证和NETs形成与降解的相关性，初步探寻血瘀证的生物学基础，为中医证候要素的客观化提供新线索；并以NETs诱导形成的不同机制为切入点，阐明冠心病血瘀证不同亚型证型特点的分子机制，为中医辨证论治理论提供科学依据。

中性粒细胞胞外诱捕网（NETs）的细胞炎性和促血栓形成效应为动脉粥样硬化（AS）的发生发展提供新视角，冠心病血瘀证也与血液循环状态、凝血纤溶系统和炎症免疫功能密切关联，因此本研究以不稳定型心绞痛血瘀证为代表，探讨其与NETs形成的相关性，并依据NETs诱导生成的不同机制，进一步阐释冠心病血瘀证不同证候亚型的分子基础。.研究表明，不稳定型心绞痛组NETs标志物dsDNA的血浆水平明显高于健康对照组，其中血瘀证组dsDNA和citH3的血浆水平均高于非血瘀证组，且气滞血瘀证组citH3的血浆水平明显高于其它证候亚型组，阳虚/寒凝血瘀证组dsDNA和痰瘀互阻证组MPO-DNA复合物的血浆水平均明显高于健康对照组，初步揭示了NETs的生成可能与不稳定型心绞痛血瘀证的证候分型相关。NETs诱导机制及效应结果根据刺激来源的不同而有差异，因此本研究针对AS病理机制的三种重要学说：炎症、脂质浸润和血栓源性学说分别选择LPS、oxLDL 和激活的血小板作为诱导因子，PMA为阳性对照，观察血瘀证不同证候亚型间NETs的生成情况。免疫荧光共染结果显示，PMA诱导多为自杀式NETosis，而oxLDL诱导还存在活性细胞NETosis，此现象在不稳定型心绞痛阳虚/寒凝血瘀证组和痰瘀互阻证组更为明显，其具体机制仍需更大样本量和进一步研究进行验证。同时发现，PMA诱导NETs分泌dsDNA的水平在气滞血瘀证组和痰瘀互阻证组明显高于其它证候亚型组；oxLDL诱导NETs分泌dsDNA的水平在阳虚/寒凝血瘀证组和痰瘀互阻证组明显高于其它证候亚型组；凝血酶激活的血小板诱导NETs的水平在气滞血瘀证组明显高于其它证候亚型组；LPS诱导NETs分泌dsDNA的水平在不稳定型心绞痛血瘀证四个证候亚型组间未发现统计学差异。.本研究采用病证结合的诊疗模式，辨证论治的基本原则，基于NETs 参与动脉粥样硬化进展这一新机制，初步探寻不稳定型心绞痛血瘀证的证候本质；并以NETs诱导形成的不同机制为切入点，探索冠心病血瘀证辨证分型的生物学基础，为冠心病血瘀证的客观化及个体化诊疗提供科学依据。