基于LC/MS-IT-TOF技术和代谢组学研究方法探讨长梗喉毛花抗肝损伤作用的PK/PD相关性

Traditional medicines used for treatment of hepatitis and liver injury plays features and advantages in Tibetan medicine. There are plenty of teraputics and natural plant medicins, such as Houmaohua. Houmaohua is a medicinal herb from original plant Comastoma Pedunlulatum found in highland areas, with elevation levels between 3000 and 4800 meters above sea level. It is used as traditional Tibetan medicines Zangyinchen mainly in Tibet region, Qinghai, and Sichuan province, and it has a wide range of application and exact effect on treatment for hepatitis, liver fibrosis, cholecystitism, and some other hepatobiliary diseases. It possesses many medicinal benefits, some of which include antiflammatory, hepatoprotective, antioxidative, and gallbladder normalizing properties, recorded in the 12th century book on Traditional Tibetan Medicine.The results of previous research on anti-hepatitis effective material of Houmaohua show that Houmaohua exibite the exact protective effect on experimental liver injury, and its main active ingredients are xanthones and their glycosides. Based on these research results, the topic project is proposed to systematically study the anti-liver injury mechanism of Houmaohua from the viewpoint of metabolomic research ideas and through LC/MS-IT-TOF and other modern analysis technologies. This topic is propose to make use of classical experimental liver damage animal model with Tibetan medicine Houmaohua as the object of study, investigate the metabolic products and pharmacodynamic material of Houmaohua active ingredients and their metabolic pathways and laws, what's more, to analysis the dynamic change of endougenous metabolites and its rules in animal models under the intervention of Houmaohua, discover pharmacodynamic biomarkers and analysis their dynamic variation. The project is also going to investigate the relationship between the active ingredients of Houmaohua and the dynamic variation of endougenous metabolites and biomarkers. This project will provide experimental clues and scientific explanation for the relationship of active constituents and anti-liver injury activies of Houmaohua, and will provide experimental basis for the clinical reasonable application.

传统藏医药学对"青乃丹吾"等肝胆疾病的治疗积累了独特的诊疗方法和丰富的临床经验与方药，本课题前期完成国家自然科学基金青年基金项目研究表明：长梗喉毛花对实验性肝损伤有确切的保护作用，其主要有效成分为口山酮及其苷类成分。本课题在前期工作基础上，以藏医药学整体观为指导，拟采用现代高效液相色谱质谱联用（LC/MS-IT-TOF）分析技术和代谢组学研究思路，考察长梗喉毛花有效成分在实验动物体内的代谢产物及其代谢途径和规律等药物代谢动力学过程，识别体内主要有效成分，分析长梗喉毛花干预后实验动物内源性代谢物的动态变化和规律，发现其抗肝损伤作用的药效生物标志物，并进一步探讨长梗喉毛花体内有效成分与内源性代谢物及药效生物标志物的动态变化规律之间的关联性，以期揭示药物有效成分与生物标志物间的对应关系，为藏医药药效物质基础研究及其多成分协同作用关系研究，也为藏医药临床合理应用奠定实验依据和基础。

本课题对传统藏医药学治疗肝胆疾病的诊疗经验与方药进行文献梳理，结合前期长梗喉毛花化学成分研究的工作基础，应用基于现代高效液相色谱质谱联用分析技术和代谢组学研究思路，结合整体实验动物药效评价模式，从整体角度和代谢层面系统考察了长梗喉毛花提取物对急性化学性肝损伤的保护作用，检测分析了实验动物肝组织病理学、血浆和肝组织生化指标，分析了长梗喉毛花有效成分在实验动物体内的代谢规律，筛选出其调控的代谢标志物，揭示了长梗喉毛花提取物对急性化学性肝损伤保护作用的代谢调控机制，并对主要有效成分的药动学（PK）与抗肝损伤作用药效学（PD）之间的相关性进行了初步评价。研究结果表明：长梗喉毛花提取物对四氯化碳诱导急性化学性肝损伤大鼠有明确的肝保护作用，可明显缓解实验大鼠肝组织的大面积水肿、炎性细胞浸润及局灶样坏死等急性损伤，显著降低实验大鼠血浆和肝组织中异常升高的ALT和AST水平，明显升高GSH和SOD、降低MDA水平，增强抗氧化能力，减少GSH耗竭以抵抗肝细胞氧化应激损伤，TUNE染色与Caspase-3检测可明显抑制实验动物肝细胞凋亡，通过降低细胞因子TNF-1α和IL-1β过表达发挥抗炎作用；检测分析了长梗喉毛花提取物中1, 8-二羟基-2, 6-二甲氧基口山酮、1,3,6,7-四羟基-口山酮-3-O-β-D-葡萄糖苷等3个主要化学成分的体内代谢规律，分别得到单成分与多组分药动学参数和药时曲线；代谢组研究结果显示长梗喉毛花提取物明显逆转四氯化碳致急性肝损伤大鼠血浆代谢组改变，系统调控模型动物的整体代谢紊乱，发现与药效相关的6个潜在生物标记物，代谢通路分析提示长梗喉毛花提取物主要通过调控甘油磷脂代谢（影响因子0.16）、色氨酸（影响因子0.16）和甘油脂代谢（影响因子0.11）代谢通路来改善实验动物的代谢紊乱，初步揭示了长梗喉毛花抗急性化学性肝损伤保护的代谢机制；本研究为长梗喉毛花临床合理应用提供实验依据，为从藏药资源中研究和开发具有应用前景的保肝药物奠定基础。