基于OATP1B3/MRPs活性与FXR-OATP1B3/MRPs表达研究黄芩-黄连药对中黄连对黄芩黄酮转运过程影响机制

Radix Scutellaria (RS)-Rhizoma Coptidis (RC) herb couple (RS-RC) is the classical herb couple in traditional Chinese compound formulas, the scientific connotation of RS-RC has not yet been revealed. In the previous studies, RC had an influence on the pharmacokinetic characteristics of RS flavones, and drug transporter organic anion transporting polypeptides (OATP) 1B3 and multidrug resistance-associated protein (MRPs) mediated the transport of RS flavones in vivo. It is unclear whether RC has an influence on the activity and expression of OATP1B3 and MRPs, which induced the disposition difference of RS flavones in vivo. This project will study the influence of RC on the activity of transporters. High performance liquid chromatograph-Tandem mass spectrometry (LC-MS/MS) will be used to quantify the transport amount of RS flavones after administration of RS or RS-RC by rat study, tissue perfusion, OATP1B3-Human embryonic kidney 293 (HEK293) cell and MRPs membrane vesicles model. In order to research the influence of RC on the expression of transporters, PCR and Western Blot will be used to determine the expression levels of FXR, OATP1B3 and MRPs genes and proteins. This project will clarify the mechanism of influence of RC on the transport of RS flavones in vivo by “animals-tissue-cell-molecular” composite analysis, and reveal the scientific connotation of RS-RC from pharmacokinetics. The results will provide the new ideas and methods for the scientific research of Chinese medicine prescriptions.

黄芩-黄连是中药方剂中的经典药对，其配伍的科学内涵缺乏深入研究。前期研究发现，黄芩-黄连配伍后黄芩黄酮的药动学特征发生显著变化，而黄芩黄酮的体内过程由转运体OATP1B3和MRPs介导，黄连是否通过影响OATP1B3和MRPs的活性和表达而改变黄芩黄酮的体内过程有待探索。本课题拟建立大鼠整体、肠肝组织灌流、OATP1B3-HEK293细胞和MRPs膜囊模型，采用液质联用技术（LC-MS/MS）检测配伍黄连后黄芩黄酮转运量的变化，探索黄连对转运体活性的影响；运用PCR和Western blot技术，研究黄连对核受体FXR及其调控的OATP1B3和MRPs表达水平的影响，明确黄连对转运体表达的影响。从“整体-组织-细胞-分子”多层次综合分析黄芩-黄连配伍后黄连对黄芩黄酮体内转运过程的影响机制，从药动学角度揭示黄芩-黄连药对配伍的内涵，为探索和解析中药传统方剂配伍的科学内涵提供新思路和新方法。

黄芩-黄连为中药方剂中的常用配伍药对，其配伍的科学机制尚未明确，本研究基于黄芩黄酮的体内过程受药物转运体影响这一特征，从药动学及药物转运体角度对黄芩-黄连药对的配伍机制进行了探索，研究了黄连对黄芩主要成分黄芩黄酮体内过程的影响机制，主要研究内容与结果如下：（1）采用LC-MS/MS法检测大鼠口服给药黄连-黄芩药对提取物与单味黄芩提取物后体内黄芩黄酮含量的变化，配伍黄连后在大鼠小肠、门静脉血、肝脏、全身血及胆汁中，主要黄芩黄酮成分的暴露量均有不同程度的降低，且长期给药后，相较于单味黄芩组，黄连-黄芩配伍可上调大鼠肝脏OATP1B3和FXR的蛋白表达。（2）在HepG2细胞模型及HEK293-OATP1B3细胞模型中考察了黄连生物碱对黄芩黄酮细胞摄取的影响，结果表明黄连生物碱可通过抑制HEK293-OATP1B3活性减少黄芩苷和汉黄芩苷的细胞摄取量。（3）采用代谢组学技术研究发现，相较于单味黄芩，黄芩-黄连配伍给药后大鼠肝脏中约有38个代谢物含量发生显著变化，差异代谢物与ABC转运体代谢通路密切相关，除此之外还与泛酸和辅酶A的生物合成、嘧啶的代谢、β-丙氨酸代谢等代谢通路相关。本研究从动物整体、细胞及分子各水平初步阐明了黄连通过影响药物转运体OATP1B3的活性与蛋白表达及核受体FXR的蛋白表达，改变黄芩黄酮体内药动学特征机制，为今后深入研究黄芩-黄连配伍的科学内涵提供了参考和依据。