基于PXR-CYP酶/ABC转运体通路探索熊果酸体内代谢与肠道吸收转运机制的研究

Ursolic acid, as a triterpenoid compound, has widely pharmacological activities of antitumor, protect liver, et al. Scholars of the world have paid attention to it in recent years. But the study on its metabolic characteristics and intestinal transport mechanism in vivo is not clear.Preliminary studies shown that CYP induction agents could promote the metabolism of ursolic acid in liver microsomes, and ABC transporter inhibitor could significantly affect the intestinal absorption. Accordingly, we speculate that CYP relevant subtypes enzymes and ABC transporter members may be involved in ursolic acid metabolism and intestinal transport respectively. Considering PXR as a key transcription factor, We take this as a breakthrough point and put forward through constructing PXR-CYP enzyme/ABC transporter pathways model so as to prove which cytochrome P450 enzymes and ABC transporter members-mediated ursolic acid metabolism and intestinal transport on the molecular level, and explore the metabolism and transport characteristics. At the same time, through silencing or knocking out PXR gene, interference the channels and hindering the expression of CYP enzymes and ABC transporter members, so as to intervente drug metabolism and intestinal efflux process and ultimately improve the intestinal absorption and bioavailability of the drug. This subject aims to explore the metabolism and intestinal transport properties and regulation of intestinal absorption of ursolic acid in vivo, cellular and molecular level. So as to promote the development and clinical application of traditional Chinese medicine monomer of ursolic acid.

熊果酸为五环三萜类化合物，具有抗肿瘤、护肝等广泛药理学效应而备受国内外学者关注，但有关其体内代谢特性及肠道转运机理尚不明确。我们前期研究发现CYP酶诱导剂可促进熊果酸在肝微粒体中的代谢，且ABC转运体抑制剂可明显影响其肠道吸收，据此，我们推测CYP酶与ABC转运体相关成员可能参与熊果酸的代谢及肠道转运。鉴于PXR作为关键的转录调控因子，我们以此为切入点，提出通过构建PXR-CYP酶／ABC转运体通路模型，从分子水平证实是何种CYP亚型酶及ABC转运体成员介导熊果酸的代谢与肠道转运，以及它们分别介导其代谢及转运的特性；同时，通过PXR基因的沉默或敲除，干扰通路的形成，阻碍CYP酶和ABC转运体的表达，以期干预熊果酸的体内代谢和肠道外排过程，最终改善肠道吸收而提高药物的生物利用度。本研究旨在体内、细胞及分子水平探索熊果酸的代谢特性与肠道吸收转运机制，促进熊果酸中药单体的开发与临床应用研究。

该课题研究了熊果酸在转基因细胞模型的转运动力学和代谢动力学特性，其次在大鼠中研究了5-孕烯-3β-醇-20-酮-16α-碳腈（PCN）激活孕烷X受体（PXR）受体后熊果酸的药代动力学特征和脏组织中ATP结合盒转运体（ABC转运体）和细胞色素P450（CYP）酶的表达变化。在Caco2-siRNA-PXR细胞中，PXR沉默后，p-糖蛋白，多糖蛋白多药耐药相关蛋白2（MRP2）和CYP2C9的蛋白含量降低。Caco2细胞中，熊果酸的浓度为10，20和50µM时表观渗透率（PDR）的值分别为2.19±0.44、1.40±0.17和1.40±0.17 ，而在Caco2-siRNA-PXR细胞中分别为1.85±0.36,1.24±0.11和 1.19±0.04。PXR–RXRα会显著激活ABC转运蛋白在CaCO 2细胞中的表达。与CaCO 2细胞相比，熊果酸的浓度分别为10，20和50µM时，PXR激活后的Caco2–PXR–RXRα细胞中PDR值随浓度的增加而增加：1.60±0.31vs1.97±0.21，1.46±0.08vs2.01±0.19，1.32±0.26vs2.09±0.22。同时，PXR-RXRα可激活CYP2C9代谢的表达。 对熊果酸在Caco2细胞和Caco2-PXR-RXR细胞中的CYP代谢酶动力学进行研究，结果发现K m值分别为81.99±44.32和60.05±29.62µg/ml，V max值为3.77±0.86和3.41±0.96µg•ml−1•min−1。然而，在人体CYP代谢酶中，我们发现CYP2C9和CYP34A都参与了熊果酸的代谢，CYP3A4和CYP2C9的V m和K m值分别为为3.57±1.12µg•ml−1•min−1和81.71±18.38µg/ml，3.85±1.46µg•ml−1•min−1和62.18±14.56µg/ml。PCN作为小鼠pxr的强激动剂，可以显著影响熊果酸在大鼠体内的药代动力学，对组织中cyp和转运体RNA的表达影响存在差异影响。