牛黄解毒片中甘草对雄黄致肝损伤具有保护作用的物质基础和作用机制研究

Niuhuang Jiedu Tablet (NJT) is one common prescription of traditional Chinese medicine (TCM) prepared from Realgar, Bovis Calculus Artificialis, Borneolum Synthcticum, Gypsum Fibrosum, Rhei Radix et Rhizoma, Scutellariae Radix, Platycodonis Radix and Glycyrrhizae Radix et Rhizoma. The dosage of realgar recommended by the Chinese Pharmacopoeia (2010) ranges from 0.05 g to 0.1 g per day when it is used alone. However, as a component of NJT, the dosage of realgar recommended by the Chinese Pharmacopoeia (2010) ranges from 0.3 g to 0.35 g per day. Pharmaceutical studies indicated that realgar had toxicological effects on liver and Glycyrrhizae Radix et Rhizoma had protective effects on liver injury. Thus, it is suggested that Glycyrrhizae Radix et Rhizoma had protective effect to realgar-induced liver injury in NJT. In this study, serum pharmacochemistry combined with serum pharmacology and modern chromatographic separation technologies are applied to screen, separate and identify the active ingredients haven protective effects on realgar-induced liver injury in Glycyrrhizae Radix et Rhizoma and perform pharmacokinetics study. Metabonomics and “Nrf2/Keap1-ARE” pathway are studied to clarify the mechanism of protective effects of Glycyrrhizae Radix et Rhizoma on realgar-induced liver injury in metabolic network and genetics. The study will be helpful to clarify the scientific connotation of the prescription of NJT, provide theoretical basis for guidance of its rational clinical use and promote the modernization of TCM.

牛黄解毒片是家庭常备药，每日服用量中雄黄含量高达0.3-0.35 g。雄黄有很强的肝毒性，而甘草有保肝作用，因此提出牛黄解毒片中甘草对雄黄致肝损伤具有保护作用。本研究采用血清药物化学结合血清药理学和现代色谱分离技术，筛选、分离、鉴定在牛黄解毒片中甘草对雄黄致肝损伤具有保护作用的活性成分，并对活性成分进行药动学研究，揭示牛黄解毒片中甘草对雄黄致肝损伤具有保护作用的物质基础。采用代谢组学策略结合“Nrf2/Keap1-ARE”通路，分别从代谢网络和基因通路角度探讨牛黄解毒片中甘草对雄黄致肝损伤具有保护作用的机制。本研究以血清药物化学-代谢组学-“Nrf2/Keap1-ARE”通路为研究主线，全方位、多角度地揭示牛黄解毒片中甘草对雄黄致肝损伤具有保护作用的物质基础及作用机制，为阐明牛黄解毒片处方配伍的科学内涵、指导临床合理用药和推进中药现代化建设提供理论依据。

本课题以家庭常备药牛黄解毒片为研究对象，通过血清药物化学、药代动力学、代谢组学和Nrf2通路对牛黄解毒片中甘草对雄黄所致肝损伤具有解毒作用的物质基础和作用机制进行了研究。建立了牛黄解毒片全方、去甘草方和甘草药材的化学指纹图谱，对牛黄解毒片全方中来源于甘草药材的色谱峰进行了归属。采用UPLC-MS技术研究了甘草药材水提取物的化学成分。建立了牛黄解毒片的血清指纹图谱，得到了牛黄解毒片中来源于甘草的4个入血成分，并对主要入血成分甘草酸和甘草次酸进行了药代动力学研究。对灌服牛黄解毒片全方，雄黄甘草药对和雄黄大鼠血中砷的药动学进行了研究，揭示了牛黄解毒片和甘草中其他成分抑制雄黄中砷的吸收。建立了雄黄致肝损伤动物模型，并采用此动物模型评价了甘草次酸对雄黄致肝损伤的保护作用，明确了牛黄解毒片中甘草对雄黄致肝损伤具有保护作用的物质基础。建立了雄黄致肝损伤小鼠血浆、尿液和肝组织的代谢指纹谱及肝组织内源性微量元素的靶标代谢谱寻找到甘草次酸干预肝损伤小鼠代谢网络变化的潜在生物标记物，阐明了牛黄解毒片中甘草通过调节雄黄致肝损伤小鼠体内能量代谢、胆碱代谢、肠道菌群代谢和氧化平衡代谢紊乱从而起到保护作用。以甘草次酸改善雄黄致肝损伤小鼠体内氧化还原代谢为出发点，进一步揭示了牛黄解毒片中甘草药效成分甘草次酸通过激活Nrf2通路相关物质、酶和蛋白对雄黄致肝损伤发挥保护作用。本研究完成了课题的预期目标，为阐明牛黄解毒片处方配伍的科学内涵、指导临床合理用药和推进中药现代化建设提供了理论依据。