基于 P-糖蛋白基因表达评价尼罗罗非鱼体内恩诺沙星代谢“首过效应”

摘要

为从药物酶的角度建立一种客观评价鱼类“首过效应”的方法,利用荧光定量 PCR 法测定尼罗罗非鱼(Oreochomis niloticus Linn)肝、肾组织中 P-糖蛋白(P-gp)基因表达量,分析了单剂量(40 mg·kg−1)口服给药恩诺沙星(enrofloxacin, ENR)后,尼罗罗非鱼肠道、肝组织中 mRNA 水平的相对表达量与 ENR 血药浓度的时实相关性.实验结果显示：在尼罗罗非鱼肠道、肝组织中, P-gp 基因在分子量127 bp 处出现了与预期大小相符的特异性扩增片段.对尼罗罗非鱼口灌给药 ENR 后, ENR 能迅速通过肠道进入血浆,其在肠道、肝和血浆中的消除速度较快,其药物时量曲线关系符合一级吸收的二室开放动力学模型.当血浆中 ENR 浓度达到最高达峰时(1 h),实验组肠道和肝中 P-gp 基因的相对表达量相对于对照组均表现出显著性差异(P<0.05)；当肠道中 ENR 浓度达到最高峰时(2 h),实验组肠道 P-gp 基因的相对表达量则表现出极显著差异(P<0.01)；当肝中 ENR 浓度达到最高峰时(2 h),实验组肝P-gp 基因的相对表达量与对照组相比则表现出显著性差异(P<0.05).该结果证实了鱼类 P-gp 基因参与药物代谢过程,提供了一种从分子水平揭示水产动物体内药物代谢规律的思路.%A fluorescence quantitative PCR method for detecting the P-glycoprotein gene in Oreochomis niloticus Linn was established to evaluate the first-pass effects. After the oral gavage at a dosage of 40 mg·kg-1 for O. niloticus, the relative expression quantity of P-glycoprotein in the intestinal and liver tissues was associated with the concentration of enrofloxacin in the plasma. The results showed that P-glycoprotein fragments (127 bp) were successfully amplified in both the intestinal and liver tissues of O. niloticus by using specific primers. The concentrations of enrofloxacin were determined by HPLC. Enrofloxacin can be rapidly absorbed in the intestine and infiltrated into the plasma. The concentration versus time in the intestine, liver and plasma of O. niloticus were well described by a two-department open model with first-order absorption. The relative expression quantity of P-glycoprotein of both intestinal and liver tissues in the first hour were higher than the control group (P<0.05) while that of the intestine in the second hour was significantly higher (P<0.01). The relative expression quantity of P-glycoprotein of liver tissues in the second hour were higher than the control group (P<0.05). These results sug-gest that P-glycoprotein may be involved in the enrofloxacin metabolism process in O. niloticus, providing a novel model for the potential utility of gene expression and drug metabolism studies in aquatic animals.