基于热毒血瘀证模型动物体内过程和谱效关系探索赤芍清热凉血散瘀的物质基础及作用部位

Paeoniae Radix Rubra (PRR) is a commonly used traditional Chinese medicine (TCM) for removing pathogenic heat from blood. Researches have demonstrated that it not only can treat noxious heat and blood stasis syndrome clinically, but also can prevent the pathologic changes of animals with noxious heat and blood stasis syndrome, and it contains anti-endotoxin constituents. Although many chemical constituents of PRR have numerous pharmacological activities, the effective substances and action sites of its traditional efficacy of removing pathogenic heat from blood and dissipating blood stasis are still not clear. In this program, we will do research in a multi-disciplinary manner by using the methods of TCM analysis, pharmacokinetics, pharmacology, and chemistry. Firstly, we will apply PRR to treat the animals with noxious heat and blood stasis syndrome, and study its in vivo process in the animals simultaneously by LC-MS. Secondly, the potential effective substances of PRR will be proposed by analyzing the relationship between the fingerprints of biosamples and the efficacies of PRR, and the action sites can be inferred from the distribution characteristics of PRR. Thirdly, the potential effective substances will be isolated and prepared from PRR or biosamples guided by LC-MS. Finally, the pharmacological effects of potential effective substances will be verified by in vitro and in vivo syndrome models. This program will not only clarify the in vivo process of PRR in animals with noxious heat and blood stasis syndrome, but also clarify the effective substances and action sites of its traditional efficacy of removing pathogenic heat from blood and dissipating blood stasis. Furthermore, this program will provide ideas for the research on effective substances of TCM’s traditional efficacy, and will provide a solid basis for screening quality evaluation indicator of PRR based on its traditional efficacy.

赤芍为常用清热凉血药。中医药临床和药理研究表明，赤芍能治疗热毒血瘀证，能阻止热毒血瘀证动物病理变化，并含有抗内毒素成分。虽然已发现赤芍许多化学成分具有众多的药理活性，但是其传统功效“清热凉血散瘀”的物质基础却不清楚。因此，本课题将采用中药分析、中药药代动力学、中药药理、中药化学等多学科的方法进行研究。采用赤芍对符合中医药理论的多种热毒血瘀证模型动物进行实验治疗，并同时采用液质技术研究治疗期间赤芍在动物体内的过程。通过对生物样品指纹谱和效应之间的关系进行分析，推测功效的潜在物质基础。通过分布特征，探索赤芍的作用部位。随后在液质的引导下，对潜在的物质基础进行分离制备，最终在离体和整体模型上进行验证。本课题除将阐明赤芍在热毒血瘀证动物体内的过程外，还将阐明其传统功效“清热凉血散瘀”的物质基础和作用部位。本课题将为中药功效的物质基础研究提供思路，还将为基于传统功效确定赤芍的质量评价指标奠定基础。

赤芍为常用清热凉血药，能治疗热毒血瘀证。虽然已发现赤芍许多化学成分具有众多的药理活性，但是其传统功效“清热凉血散瘀”的物质基础却不清楚。本项目：（1）首先采用中医诊断学标准，对脂多糖/角叉菜胶所致大鼠热毒血瘀证模型进行了证候演变分析，发现其可分为三个阶段，即热盛瘀轻、瘀热共存和瘀血证阶段；（2）随后采用代谢组学技术对其进行了研究，发现可以分为同样的三个阶段，和中医诊断学结果一致。从代谢组学的角度揭示了证候演变的生物学基础：烟酸烟酰胺代谢受到抑制，导致SIRT1激活，诱导产生炎症因子，并导致能量代谢紊乱和氨基酸代谢紊乱，最终导致瘀血证；（3）发现涉及到9条代谢通路，其中烟酸烟酰胺代谢和炎症反应密切相关，且烟酸烟酰胺代谢可能是热毒血瘀证区别于其他血瘀证的特征代谢通路；而组氨酸代谢异常可能是导致后期演变成严重瘀血证的主要原因。发现了7个热毒血瘀证演变的证候标志物isocitric acid、glucose、3-dehydroxycarnitine、N-acetylglutamine、N-acetylglutamine、iduronic acid、homocysteinesultinic acid；（4）采用药理学及代谢组学技术，阐明了赤芍在热盛瘀轻阶段和瘀血证阶段的药效作用及机制。在热盛瘀轻阶段，赤芍的功效为清热凉血，它能降低炎性因子IL-6、TNF-α的水平，降低大鼠额温及全血黏度；发现了热盛瘀轻阶段的10种证候标志物及赤芍的4种药效生物标志物。在瘀血证阶段，赤芍的功效为化瘀，它能显著降低大鼠全血黏度，使PT值增大，改善血液凝聚状态，还可减轻肝、肺组织损伤；发现了瘀血证阶段的10种证候标志物及赤芍的2种药效生物标志物；（5）发现了热盛瘀轻阶段与赤芍功效相关的药效物质17种，瘀血证阶段与赤芍功效相关的药效物质9种。共确定了赤芍“清热凉血散瘀”功效的物质基础20种，其中16种为代谢产物；（6）此外本项目还阐明了枯芩和子芩功效不同的原因，并阐明了若干黄酮类和萘醌类成分的代谢特点。本项目证明了药-证动态相关的客观存在，为中药功效物质基础的精准动态研究及热毒血瘀证新药的研发提供了科学基础。