基于Keap1-Nrf2/ARE信号通路研究五味子-甘草配伍调节脂代谢作用机制

Hyperlipidemia and fatty liver induced by lipid metabolism disorder is the current research focus. Chinese medicinal preparations containing Schisandrae Fructus are commonly applied for lowering lipid and protecting liver. However, high doses of Schisandrae Fructus could cause hyperlipidemia and fatty liver induced by lipid metabolism disorder, which was proved by our previous studies, the phenomenon is contrary to the clinical effect. Based on clinical practice and previous experiments, the combination of Schisandrae Fructus and Licorice could significantly improve the increased serum/hepatic lipid induced by Schisandrae Fructus. Schisandrin B (Sch B) could significantly increase serum/hepatic lipid. It may be closed to that Licorice could change the process of Sch B in vivo and regulate the genes and proteins expression in Keap1-Nrf2/ARE signaling pathway. Therefore, the pharmacokinetics, metabolic pathway and tissue distribution changes of Sch B in Schisandrae Fructus are determined after the compatibility of Licorice; and the correlation of above indexes with serum/hepatic lipid on corresponding time points will be analyzed; clarifying the process in vivo of Schisandrae Fructus-Licorice compatibility in lipid lowering and liver protecting. Then, the proteins and genes related to lipid metabolism in Keap1-Nrf2/ARE signaling pathway are measured by modern molecular biological technology on basis of serum pharmacology. In order to elucidate the synergistic molecular mechanism of Schisandrae Fructus-Licorice compatibility; reveal the scientific connotation of Schisandrae Fructus-Licorice compatibility; provide scientific basis for the clinical rational use of Schisandrae Fructus.

脂代谢紊乱诱发高脂血症和脂肪肝是当前研究的热点，含五味子的中药制剂是临床常用的降脂保肝药，但课题组前期研究发现大剂量五味子可致脂代谢紊乱、诱发高脂血症和脂肪肝，与临床疗效相悖。基于临床实践和前期实验发现，五味子-甘草配伍可显著改善大剂量五味子升高血/肝脂的现象，或与甘草改变五味子中具显著升高血/肝脂的五味子素B（Sch B）体内过程和调控Keap1-Nrf2/ARE信号通路中基因和蛋白表达发挥降脂保肝作用有密切关系。据此，本项目拟通过检测五味子配伍甘草后Sch B的药代动力学、代谢途径和组织分布的变化，并与相应时间点的血/肝脂指标进行关联分析，阐明五味子-甘草配伍协同降脂保肝的体内过程；然后基于血清药理学，采用现代分子生物学技术检测Keap1-Nrf2/ARE信号通路及脂代谢关键蛋白和基因，阐释两药配伍的协同作用机制，揭示五味子-甘草配伍的科学内涵，为临床合理使用五味子提供科学依据。

现代药理学研究表明五味子具有降血脂保肝的作用，然而课题组前期研究发现其主要活性成分五味子素B（Sch B）大剂量可显著升高正常小鼠血清/肝脏中TG和TC含量，诱导高脂血症和脂肪肝，进而引发思考：临床上五味子超剂量应用存在诱发高脂血症和脂肪肝的潜在风险。本课题基本按照计划完成预定任务。第一部分，基于体内外实验证实了大剂量五味子（醇提物和水提物）可诱发高脂血症，且这一现象可被甘草缓解，配比1:1效果较好。第二部分，基于多通路多靶点作用特点探索五味子-甘草配伍调节脂代谢的作用机制，结果发现高剂量的五味子一方面增加TG的合成速度，其机制可能是增加肝脏中SREBP-1c的蛋白表达，上调脂肪酸合成过程的限速酶ACC活性和TG合成过程中关键酶GPAT含量；另一方面，降低脂肪酸的β氧化，增加TG在肝脏中的沉积，其机制可能为下调肝脏PPARα蛋白的表达，降低脂肪酸β-氧化途径的关键酶ACO的活性。通过以上两种途径联合增加肝脏中TG的合成，诱发高脂血症，而甘草配伍五味子后可通过调节TG合成途径相关蛋白，降低五味子诱发的高脂血症风险，同时调控PPAR-α、PPAR-γ、Fabp1/2、SREBP1c、ACCα、FAS基因表达来缓解大剂量五味子造成的脂质堆积；但五味子对假说中的Keap1-Nrf2/ARE信号通路上的蛋白Keap1、Nrf2、HO-1、NQO-1、GCLC无明显影响，说明此通路并非主要机制。第三部分，探索五味子-甘草配伍对诱发高脂血症的主要成分Sch B体内代谢的影响。①基于分子对接技术发现Sch B与CYP2A6、CYP2C8、CYP2C19、CYP2E1、CYP3A4、CYP2C9、CYP2B6均有较高的结合力；②通过研究Sch B在肝微粒中的代谢进一步验证了CYP2A6、CYP2C19、CYP2E1参与了Sch B的代谢；③正在进行五味子-甘草配伍对参与Sch B代谢的CYP2A6、CYP2C19、CYP2E1的影响研究。第四部分，探索五味子-甘草配伍对Sch B药代动力学、组织分布和生物转化过程分析，样本已经收集，课题组正在进行前期条件摸索。通过以上研究内容，本课题揭示了五味子-甘草配伍的科学内涵，为临床合理使用五味子提供了数据支撑。