基于组效关联的龙血通络胶囊抗缺血性脑中风的药效物质和作用机制研究

A new drug named “Longxuetongluo Capsule” (LTC) composed of total phenol extract of Chinese dragon’s blood has been successfully developed in our group, which has been proved to be safe and effective for the treatment of ischemic stroke on the basis of pharmacological experiments and clinical trials. However, the active material basis and action mechanisms of LTC against ischemic stroke remain unclear. In view of TCM characteristics of multiple components, multiple targets, and multiple pathways, we propose that a variety of active components of LTC may protect the brain from the cerebral ischemia injury through multiple targets and signaling pathways in terms of the pathophysiology of cerebral ischemia. The objective of the proposed research is to discover, characterize, and validate the active components and action mechanisms of LTC against ischemic stroke. This program uniquely combines chemistry with cutting edge chemometrics, computer aided drug design, and biomedical research. Specific Aim 1 will develop a research pattern of "Multiple active compounds found – Activity verified – Action mechanisms clarified" based on the combination of composition-effect correlation and serum pharmacochemistry. Specific Aim 2 will identify the potent active components of LTC on the basis of composition-effect correlation method, and characterize their chemical structures through LC-MS/MS-guided isolation and spectroscopic analysis. Specific Aim 3 will reveal the targets and signaling pathways of the active compounds of LTC at the cellular and molecular levels, and construct the molecular regulatory networks of LTC against ischemic stroke. Accomplishing these objectives will provide theoretical basis and scientific evidences for the clinical curative effects of LTC, afford new experimental evidences for the therapeutical principle of “promoting blood circulation to remove blood stasis” in treating ischemic stroke, and also provide the beneficial references for the study on the active material basis of other TCMs and compound prescriptions.

本课题在前期将龙血竭酚类提取物成功研发为中药二类新药“龙血通络胶囊”的基础上，进一步深入研究其药效物质和作用机制。采用组效关联和血清药物化学相结合的方法，揭示龙血通络胶囊针对脑缺血损伤多个病理环节的潜在药效成分；通过LC-MS导向分离制备，获得潜在药效成分，并对其进行活性确证，明确可靠、真实的药效成分；对药效成分进行作用靶点和信号通路研究，构建其抗缺血性脑中风的信号通路和分子调控网络。通过这种“药效组分发现－药效活性确证－药效机制阐明”层间递进的研究模式，系统阐明龙血通络胶囊治疗缺血性脑中风的药效物质基础和作用机制，为其临床疗效和合理用药提供理论依据和科学支撑，为创新药物的推广应用和市场培育提供科技支撑，也为缺血性脑中风“活血化瘀”的治则提供新的实验依据。同时，该研究思路和方法也将为其他中药及复方的药效物质基础研究提供有益的参考和借鉴，具有重要的学术价值和应用前景。

脑中风属于发病率、致残率、死亡率和复发率均很高的重大疾病，严重威胁着人类健康，寻找安全、有效的药物一直是全球医药领域研究的热点。龙血通络胶囊（LTC）是本课题组自主研发中药二类新药，用于治疗中风病中经络（轻中度脑梗死）恢复期血瘀证。本项目以LTC为研究对象，采用UHPLC-IT-TOF-MS技术对LTC中化学成分进行表征；通过硅胶色谱对LTC按化学成分极性分段，采用正交试验设计法将LTC制备成化学成分明显交叉、各成分含量不同的组分，并对各组分进行活性检测；采用灰色关联度与PLS两种方法将各组分的化学成分与活性测试结果进行组效关联分析，获得潜在活性成分；以LC-MS/MS为导向，对LTC的潜在活性成分进行分离、鉴定，并对活性成分进行活性验证；采用LC-MS/MS对LTC主要化学成分代谢动力学和组织分布进行研究；利用细胞生物学技术和代谢组学对LTC抗缺血性脑中风的作用机制进行研究，阐明了LTC抗缺血性脑中风药效物质和作用机制：通过组效关联技术、导向分离鉴定和活性验证，从LTC中分离鉴定了71个化合物，发现并确证了11个药效物质，这些成分均可快速吸收入血；作用机制研究表明，LTC分别从抗神经炎症、血管内皮细胞保护、神经细胞保护等多途径，通过抑制Caspase-3/9和MAPK等通路的激活、调控MAPKs/IKK/IκB/NF-κB信号通路、抑制神经元细胞内质网应激所介导的细胞凋亡，调节脂代谢、醚脂类代谢、花生四烯酸代谢、糖酵解等多途径发挥保护脑缺血再灌注损伤的作用。本项目系统阐明了LTC治疗缺血性脑中风的药效物质基础和作用机制，为其临床疗效和合理用药提供了理论依据和科学支撑。本项目基于组效关联技术，构建的“药效组分发现-药效活性确证-药效机制阐明”层间递进研究模式，为其他中药及复方的药效物质研究提供了有益的参考和借鉴，具有重要的学术价值和应用前景。本项目共发表论文14篇，其中SCI论文6篇，中文期刊6篇，会议论文2篇，获授权发明专利1项；培养博士研究生1名，硕士研究生3名。