人参炔三醇短时别构于CYP3A4酶结合位点、长时作用于核受体PXR/CAR调控通路的药物相互作用机制

The difference effect of Panaxytriol (PXT) short-time pretreatment on MDZ 4-hydroxylation and 1’- hydroxylation was found in rat and human hepatocytes in our previous study. However, prolonged intervention resulted in a time-dependent inhibition on both metabolic pathways of midazolam, which eventually led to the downregulation of CYP3A4 activity. It may be an underlying interaction mechanism of drug-drug about the short-time effect on enzyme binding sites and the long-time effect on the nuclear receptor of PXR and CAR that controls the regulation of CYP3A4 enzyme. This project will study the enzyme kinetics mechanism of the differential effect of PXT on the both hydroxylation of midazolam metabolic pathway by the building of atypical model of enzyme kinetics and conformation analysis of CYP3A4 binding sites；and explore the internal mechanism of effect of PXT on CYP3A4 expression and activity regulated by PXR/CAR using cell culture, plasmid construction and gene report methods at genetic level; and investigate the change of CYP3A1/2 enzyme expression and midazolam hydroxylation in rats after the pretreatment of a single and muitiple dose of PXT for the explanation of the effect mechanism of PXT on the activity of CYP3A1/2 and metabolism of midazolam in rats. The research of our project is helpful to establish the effective theoretical basis for clinical drug safety.

PXT短期干预对大鼠及人肝细胞中MDZ的4-羟化及1’-羟化存在差异影响，但长时干预对两种代谢通路均呈现时间依赖性抑制作用，导致CYP3A4活性下调。PXT与药物间可能存在短时別构于CYP3A4酶结合位点、长时作用于PXR/CAR调控通路进而影响酶蛋白表达的相互作用机制。课题将通过非经典酶动力学模型构建及空间构象分析，从酶分子水平探讨PXT对CYP3A4酶结合位点的短时別构效应及其介导的咪达唑仑不同羟化代谢通路的差异影响机制；采用细胞培养、质粒构建及瞬时转染基因报告技术，研究PXT不同时间干预后PXR、CAR、CYP3A4酶蛋白表达及其活性改变，从分子生物学水平探讨PXT对核受体调控通路的干预机制；同时研究单、多次给予PXT后大鼠肝、肠CYP3A酶表达及其活性的变化，从整体水平阐明PXT对CYP3A酶表达及其介导的咪达唑仑羟化代谢的影响机制，为临床安全用药提供实验依据。

人参炔三醇（PXT）是红参提取物及参麦注射液的重要活性组分。前期研究表明，PXT短期干预对大鼠及人肝细胞中MDZ的4-羟化及1’-羟化存在差异影响，但长时干预对两种代谢通路均呈时间依赖性抑制作用，导致CYP3A4活性下调。PXT与药物间可能存在短时别构于CYP3A4酶结合位点、长时作用于PXR/CAR调控通路进而影响酶蛋白表达的相互作用机制。本课题重点研究了PXT长-短时干预对正常HepG2和高表达hPXR的HepG2细胞中PXR、CYP3A4 mRNA和蛋白表达的影响及其对PXR-CYP3A4调控通路的影响机制。并通过高表达hCAR的HepG2和沉默hCAR的HepG2细胞模型，研究了PXT对CAR-CYP3A4调控通路的影响机制。结果发现：PXT短时间干预1h，对HepG2细胞中PXR和CYP3A4的mRNA的表达有上调作用，但对其蛋白表达无明显影响；长时间干预24h，对PXR和CYP3A4的mRNA和蛋白的表达均有上调作用，且PXT对PXR与CYP3A4二者影响趋势一致。提示PXT长-短时干预对HepG2细胞中CYP3A4表达的影响可能与PXR通路有关。报告基因法研究进一步证实，PXT对HepG2细胞中CYP3A4上调作用主要是通过激活PXR来实现的。此外，PXT在高浓度（80μM）时对CYP3A4的诱导作用还与CAR有关，但在低中浓度（10-40μM）不会通过CAR来调控CYP3A4的表达，此时PXT对CYP3A4的诱导作用主要由PXR介导。且随着CAR的高表达，PXT基于PXR通路对CYP3A4的诱导作用被拮抗。反之，将HepG2细胞中CAR沉默后，基于PXR介导的PXT对CYP3A4的诱导作用又显著呈现出来。结果提示，PXT干预对CYP3A4的调控作用中CAR和PXR存在交互对话，CAR对PXT干预后PXR-CYP3A4的调控通路有抑制作用。总之，本课题从分子生物学水平探索了PXT长-短时间干预对核受体调控CYP3A4的影响机制，为临床药物相互作用研究提供了理论依据。