痰热清治疗急性肝损伤药效物质基础及作用机制研究

Acute liver injury (ALI) is a rare disorder marked by the sudden loss of hepatic function with multiple causes and high incidence. Traditional Chinese Medicine (TCM) has a long history as effective agents for treating liver disorders. Tangreqing injection belongs to the State category II new medicine, and also has satisfactory efficacy against ALI clinically. Its main effect is to clear away pathogenic heat, remove the toxin, and dissolve phlegm. Tangreqing injection is composed of five medicinal materials and characterized by complicated chemical compositions. The less knowledge on the bioactive constituents of Tangreqing injection had severely affected its quality control and its application in clinic. Our previous research completed the analysis methods of the medicinal material used in the Tanreqing injection and the finished product. Furthermore, pharmacological test indicated that Tangreqing injection exerted the hepatoprotective effect on CCl4, ANIT, D-galactosamine (D-GalN)-induced ALI in rats/mice, and suggested that this protective effect was likely due to decrease the level of total bile acid (TBA), ALT and AST activity of animals. However, the mechanisms of ALI and the hepatoprotective effect remains unknown. Therefore, the aim of research is to analysis the global chemome with HPLC/UPLC-DAD/MS, screen the effective constituents group or effective chemical compound, and then characterize the pharmacodynamic material basis of Tanreqing injection. The research will explore the mechanisms of hepatoprotective effect induced by CCl4, ANIT, D-GalN combined with the method of metabonomics, pharmacology and molecular biology. This work also can provide not only the theoretical base for the treatment of ALI and develop the potential medicines, but also the new ideas for traditional hepatoprotective herbs with modern theory.

急性肝损伤是多种因素引起的严重肝脏损害，病发率极高。中药治疗肝胆疾病历史悠久。痰热清属国家二类新药，临床研究表明痰热清治疗急性肝损伤疗效较好，可能与其清热、解毒、化痰之功效有关，但其组方药味较多，化学成分复杂，药效物质基础并不完全明晰。前期工作已经完成各单味药及痰热清复方中的化学物质研究，同时发现痰热清对四氯化碳、a-异硫氰酸萘酯、D-氨基半乳糖苷诱导的大鼠急性肝损伤模型具有预防保护作用，能明显降低谷丙转氨酶（ALT）和谷草转氨酶（AST）活性以及总胆汁酸（TBA）的水平，但其作用机制尚未清楚。鉴于此，本项目拟采用HPLC/UPLC-DAD/MS技术手段分析整体化学组，筛选有效化学成分群或有效化学成分，表征痰热清注射液的药效物质基础。结合代谢组学、药理学和分子生物学等研究方法，探究作用机理，为临床治疗急性肝损伤提供理论依据，挖掘潜在的有效治疗药物。

痰热清属国家二类新药，临床研究表明痰热清注射液治疗急性肝损伤疗效较好，可能与其清热、解毒、化痰的功效有关，但其组方药味较多，化学成分复杂，药效物质基础并不完全明晰。本项目首先采用HPLC-MS/MS分析技术对痰热清胶囊及5种中间体中的化学成分进行鉴定并归属，并对注射液和胶囊中的化学组分进行差异分析，建立了痰热清胶囊的化学指纹图谱和7个主要成分的含量测定方法，为后续药效研究奠定物质基础。通过胆汁酸代谢物靶标分析对痰热清胶囊不同剂量的保肝药效进行研究，结果表明牛磺猪去氧胆酸和牛磺胆酸可能为表征痰热清胶囊保肝作用的分子标记物。采用胆汁酸代谢物靶标分析和胆汁酸代谢通路相关基因的表达情况进行考察，结合血清生化指标和肝脏病理组织切片检查，初步确定痰热清中的有效成分群为熊胆粉中间体、山羊角中间体、连翘中间体和金银花中间体，肝脏中的牛磺酸结合型胆汁酸和血清中的游离型胆汁酸可能为表征其药效的胆汁酸分子标记物；进一步推测其有效成分可能为熊去氧胆酸、鹅去氧胆酸和总有机酸，血清中的牛磺酸结合型胆汁酸和游离型胆汁酸以及肝脏中的结合型胆汁酸可能为其表征活性的胆汁酸标记物，而保肝的作用机制可能与调控肝内NTCP，SULT2A1，UGT1A1，GSTA2A，CYP3A11，PXR和FXR等基因mRNA表达量而改善肝脏功能。说明胆汁酸的代谢可能为痰热清发挥保肝作用的重要靶点，本课题的实施有助于完善以活性成分为指标的痰热清质量标准，更加科学、客观地评价痰热清质量；痰热清保肝作用机制的研究有助于扩大痰热清的临床使用范围并提供依据。