疏利气机法抑制热毒血瘀证中血管内皮损伤的分子机制研究

Noxious Heat and Blood Stasis Syndrome (NH-BS) is a type of syndromes in which noxious heat invade into the yingfen and xuefen syndrome, and frequently occures in the development of most infectious diseases and internal diseases in the veterinary clinic. "Disorder of Qi" and "Development of Noxious Heat" are always accompanied in the process of NH-BS, and one leads to the other, which cause characteristic pathological changes that is the lesion of vascular endothelial injury. Activation of PC-EPCR signaling pathway can protect endothelial cells from anti-inflammatory, anti apoptosis and enhacing vascular barrier function. In this project, firstly, we will study whether blocking the binding of PC and EPCR can caused the vascular endothelial injury in the rat and cell model of NH-BS, using EPCR neutralizing antibodies, gene overexpression and other means. And then, we will study the effect of traditional Chinese medicine "Xiang Qi Tang (XQT)” on straightening of Qi through the orthogonal test and uniform design experimentation. The effect of XQT on the rate of PC-EPCR bingding and activating will followingly be determined by flow cytometry, qPCR, WB and ELISA. The above researches are helpful to enrich the connotation of straightening of Qi, and to interpret its therapeutic mechanism from the perspective of modern molecular biology, which provide theoretical and experimental basis for the development and rules of application of Qi straightening.

热毒血瘀证是热毒邪气侵入到营血分时出现的证候类型，在兽医临床上，绝大多数的传染病和许多内科杂病在发展过程中常出现这种证侯类型。“气机不畅”和“热毒滋长”在该病证中，相伴而生，又互为因果，最终引起以血管内皮损伤为核心的病变。PC-EPCR信号途径激活后可以从抗炎、抗细胞凋亡和增强血管屏障功能方面保护血管内皮。本项目首先以热毒血瘀证大鼠和细胞模型为对象，利用EPCR中和抗体、基因过表达等手段研究PC-EPCR结合受阻是否引发热毒血瘀证血管损伤，进而在阐明中药“香芪汤”疏利气机作用的基础上（正交试验、均匀设计），通过流式细胞术、荧光定量PCR、WB、ELISA等技术考察 “香芪汤”对PC-EPCR结合率和活化率的影响。研究结果有助于丰富疏利气机法的内涵，并从现代分子生物学角度阐释其治疗作用机理，为拓展疏利气机法的运用范围、探索疏利气机法的运用规律提供理论依据和实验基础。

热毒血瘀证是兽医临床上常出现的证候类型。本项目首先利用临床病案的分析统计结合数学建模的方式建立了热毒血瘀证大鼠模型评价体系，包括用以判断动物模型建立是否成功的评判标准和用以评估动物模型证候严重程度的评分方法两个方面。接着比较了三种热毒血瘀证大鼠模型建立方法的差异，我们发现单次腹腔注射LPS是热毒血瘀证大鼠模型建立的最佳方法。在此基础上，进一步考察了中药香芪汤治疗大鼠热毒血瘀证的疗效观察，结果发现，香芪汤可以缓解热毒血瘀证大鼠目征、耳部症状、舌象的血瘀症状，显著性增加体重、降低肛温、减少呼吸频率；能维护热毒血瘀证大鼠血液系统中物质水平和血液凝固系统的稳定；能预防治疗热毒血瘀证大鼠主动脉的病理损伤，显著性减少主动脉中炎性蛋白TNF-α的表达，显著性降低主动脉BAX/BCL-2的凋亡比率，显著性降低屏障功能蛋白S1PR1的比率，表明香芪汤能有效阻断PC-EPCR的结合。通过香芪汤治疗大鼠热毒血瘀证的拆方分析发现，香附组的血瘀症状分值最低；用ELISA法检测各组大鼠血清中炎性因子水平，香芪汤预处理治疗后IL-1β、IL-6和NO水平显著性降低，比较三味药后得出香附在香芪汤预防治疗大鼠热毒血瘀证过程中起主要作用，说明疏利气机法对治疗热毒血瘀证十分关键。在细胞实验方面，香芪汤对10%热毒血瘀证大鼠血清介导的热毒血瘀证细胞模型有一定治疗作用，透射电镜镜下观察到能够改善细胞形态与结构的受损程度。进一步研究显示，香芪汤能抑制热毒血瘀证细胞上EPCR、TM脱落，并减少炎症因子IL-1β、TNF-α的分泌，维护Bcl-2并下调BAX的蛋白及基因表达，促进细胞SPHK1和S1PR1的基因和蛋白表达。香芪汤抑制血管内皮细胞上EPCR脱落的同时还一定程度促进其在细胞内的表达，并能提高APC的活化效率。在PC-EPCR信号途径阻断后，香芪汤对热毒血瘀证细胞可以起到一定的抗凋亡与保护细胞屏障的效果，包括提高Bcl-2并下调BAX的基因与蛋白表达，增加SPHK1、S1PR1的基因与蛋白表达。