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

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。

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.