高原低氧对核受体PXR和CAR介导的CYP450和UGT1A1的转录调节作用

The activity and expression of drug metabolism enzymes were changed significantly in special environment of high altitude hypoxia. PXR and CAR are main factors to regulate the transcription of drug metabolism enzymes, so it is necessary to further study the regulation of high altitude hypoxia on transcription of CYP450 and UGT1A1 mediated by nuclear PXR and CAR. The project will make use of special geographic environment of Qinghai-Tibet plateau, and regard CYP1A2, CYP2B1, CYP2C11, CYP3A1 and UGT1A1 as research objects. The study include: ① To analyse the dependability between the expression of five metabolic enzymes and nuclear receptors, the mRNA and protein expression of five metabolic enzymes, PXR and CAR will be determined by Real-time PCR and Western Blot in rat's liver of plain control, acute hypoxia and chronic hypoxia group, respectively. ②HepG2 cells was adopted to further study the effect of hypoxia on the mRNA and protein expression of five metabolic enzymes, PXR and CAR. ③HepG2 cells and transient co-transfected dual luciferase reportor assay were adopted to study the regulation of hypoxia on transcription of CYP450 and UGT1A1 mediated by nuclear PXR and CAR. The project will provide the guidance to explain drug's metabolism in special environment of high altitude hypoxia, and give valuable references to clinical reasonable medication in plateau areas.

在高原低氧特殊环境中，药物代谢酶的活性和表达发生显著变化，而核受体PXR和CAR是药物代谢酶基因表达调控的重要转录因子，高原低氧对PXR和CAR介导的CYP450和UGT1A1转录是否具有调节作用值得进一步去探讨。 利用青藏高原独特的地理条件，将低氧与药物代谢相结合，以CYP1A2、2B1、2C11、3A1和UGT1A1为研究目标，①采用定量PCR和Western blot分别测定平原、急性和慢性缺氧大鼠肝脏中五种酶及PXR和CAR在mRNA和蛋白水平的表达，分析高原低氧对酶和核受体表达的影响及二者之间的相关性；②在HepG2细胞中，进一步验证低氧对五种酶及PXR和CAR在mRNA和蛋白水平表达的影响；③在HepG2细胞中，采用瞬时共转染报告基因实验探讨低氧对PXR和CAR介导的五种酶的转录调节作用。 期望能为合理解释高原低氧环境中的药物代谢起指导作用，并对高原地区临床用药提供参考依据。

项目研究了高原低氧对CYP450、UGT1A1、PXR、CAR蛋白和mRNA表达的影响，并探讨了低氧条件下PXR和CAR对CYP450和UGT1A1的转录调节作用。动物实验分为平原组、中度海拔急性和慢性缺氧组、高海拔急性和慢性缺氧组。细胞实验分为对照组、PXR抑制剂组（酮康唑，KCZ）、CAR抑制剂组（视黄酸，RA）、PXR激动剂组（利福平，RIF）组、CAR激动剂组（CITCO）、2%、5%、10%低氧24h组、5%低氧2h、6h、12h、24h、48h组。大鼠和细胞CYP450、UGT1A1、PXR、CAR的蛋白及mRNA表达分别采用ELISA和qPCR测定。构建pGL3-CYP450和pGL3--UGT1A1质粒载体，双荧光素酶活性检测低氧对CYP450和UGT1A1启动子活性的影响；利用RNAi和抑制剂沉默PXR和CAR的基因表达，研究PXR和CAR对CYP450和UGT1A1的转录调节作用；采用qPCR测定PXR和CAR的激动剂对CYP450和UGT1A1的mRNA表达的影响，并采用双荧光素酶活性观察不同氧气浓度下激动剂对CYP450和UGT1A1的启动子活性的影响。.高原低氧条件下，大鼠和HepG细胞CYP450、UGT1A1、PXR、CAR的蛋白和mRNA表达显著降低。双荧光素酶活性研究表明，低氧显著降低CYP450和UGT1A1的启动子活性。另外，采用RNAi或抑制剂沉默PXR和CAR基因后，CYP450和UGT1A1 mRNA表达明显降低。给予PXR和CAR激动剂后发现，RIF诱导24h后PXR、CYP450和UGT1A1的mRNA表达升高，CITCO诱导2h后CAR、CYP450和UGT1A1的mRNA表达升高，RIF和CITCO共处理24h后，PXR、CAR、CYP450和UGT1A1的mRNA表达升高。在2%和5%氧气浓度下，RIF和CITCO对CYP450和UGT1A1启动子活性无明显影响，而在10%氧气浓度下，RIF和CITCO使CYP450和UGT1A1启动子活性升高。.项目研究证实高原低氧对核受体PXR和CAR介导的药物代谢酶CYP2C9、CYP2E1、CYP3A4和UGT1A1的转录调节作用，发现了高原低氧特殊条件下药物代谢酶活性和表达发生变化的机制，为合理解释高原低氧环境中的药物代谢和高原地区合理用药起一定的指导作用。