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目的建立hPXR介导的CYP2B6、CYP2C9药物诱导剂的体外筛选体系,分析硝苯地平诱导CYP2B6、CYP2C9的分子机制。方法利用双荧光素酶报告基因系统,将包含hPXR蛋白识别和结合调控元件的CYP2B6和2C9启动子序列插入报告基因上游,将表达载体和报告载体共转染HepG2细胞,以10μM利福平为阳性对照,用1、5或10μM硝苯地平处理48h后裂解细胞进行双荧光素酶活性检测;或用10μM硝苯地平分别处理细胞12、24和48h后进行双荧光素酶活性检测。结果硝苯地平激活hPXR的起始浓度为1～5μm,在5μm浓度下,硝苯地平诱导CYP2B6和2C9表达增加3.93和3.59倍,与DMSO溶媒组差异存在显著性(P<0.001),在10μm硝苯地平作用下,CYP2B6和2C9表达分别增加6.23和5.24倍,与溶媒对照组和5μm浓度组差异都存在显著性(P<0.001)。10μm硝苯地平处理12h后即能显著地诱导CYP2B6和CYP2C9表达增强,但其诱导倍数与24和48h组差异存在显著性(P<0.001)。结论该研究成功构建了hPXR介导的CYP2B6和CYP2C9药物诱导剂的体外筛选体系;硝苯地平通过激活hPXR介导了CYP2B6和2C9的表达上调,并表现出明确的剂量-效应关系。
Objective To establish an in vitro screening system of hPXR-mediated CYP2B6 and CYP2C9 drug inducer and analyze the molecular mechanism of the induction of CYP2B6 and CYP2C9 by nifedipine. Methods The dual luciferase reporter gene system was used to insert the promoter region of CYP2B6 and 2C9 containing the recognition and binding regulatory elements of hPXR into the upstream of the reporter gene. The expression vector and the reporter vector were co-transfected into HepG2 cells with 10 μM rifampicin as positive The control cells were lysed with 1, 5 or 10 μM nifedipine for 48 h, then the cells were lysed to detect the dual luciferase activity, or the cells were treated with 10 μM nifedipine for 12, 24 and 48 h, respectively. Results The initial concentration of hPXR activated by nifedipine was 1 ~ 5μm. At 5μm, the expression of CYP2B6 and 2C9 increased by 3.93 and 3.59 times, respectively. The difference between nifedipine and DMSO was significant (P <0.001) The expression of CYP2B6 and 2C9 increased by 6.23 and 5.24 fold, respectively, with the effect of nifedipine, which was significantly different from that of vehicle control and 5 μm (P <0.001). The results showed that the expression of CYP2B6 and CYP2C9 was significantly induced after treated with 10μm nifedipine for 12h, but the difference between the induction times and 24th and 48th hour groups was significant (P <0.001). Conclusion The in vitro screening system of hPXR mediated CYP2B6 and CYP2C9 drug inducer was successfully constructed in this study. Nifedipine up-regulated the expression of CYP2B6 and 2C9 by activating hPXR, and showed a definite dose-response relationship.