糖皮质激素编程子代CYP3A表达改变及其药代动力学意义

Our previous studies reported that prenatal exposure to xenobiotics (nicotine, caffeine, and ethanol) can causes consistent changes of cytochrome P450 (CYP) 3A mRNA expression in offspring rats, and high intrauterine glucocorticoids (GCs) were observed in all those animal models. As it has been known, GCs play a key role in fetal programming and leads to continuous change of genes by epigenetic regulation. Based on preliminary experiment, we hypothesized that GCs and glucocorticoid receptor (GR) binding complex programs sustained transcription activation of pregnane X receptor (PXR) through cross talk of nuclear receptors, and then upregulates expression of CYP3A. To prove the hypothesis, we will use dexamethasone as positive control, observe the fetal programming effect of GCs on expression of PXR and CYP3A in offspring rats, then confirm the epigenetic and molecular mechanism under GCs programming (linkage among GCs and GR binding complex, epigenetic regulation of PXR, transcription activation of PXR and alteration of CYP3A expression). Knockout mice were used to verify the cross-talk effect of GR and PXR on programming effect of GCs on CYP3A expression. In addition, we will study the effect of intrauterine GCs exposure on pharmacokinetics characteristic of anti-hypertension and anti-hyperlipidemia agents in adult rats. This study is in order to clarify the fetal programming effect of GCs on CYP3A variation in adult and its pharmacokinetic significance, and provide experimental evidence and a novel idea for precision individualized medicine.

本室前期研究显示孕期外源物暴露（尼古丁、咖啡因、乙醇）均可致大鼠宫内和成年后CYP3A表达增加，并存在胎仔过暴露于母源性糖皮质激素(GCs)的共性改变。GCs有胎儿编程作用，即在宫内生长发育关键时期，通过表观遗传修饰影响胎儿代谢或结构功能改变以至持续到出生后甚至终生。结合预实验结果，我们提出宫内GCs与其受体（GR）结合后通过交互作用（cross-talk）编程胎肝孕烷X受体（PXR）的持续转录活化，最终调控成年后CYP3A高表达。本项目拟在整体动物观察GCs对PXR和CYP3A的编程现象，细胞实验和基因敲除鼠证实GCs与GR结合—PXR表遗传修饰/转录活化—CYP3A表达改变的表遗传编程机制，在此基础上研究宫内GCs暴露对子代代谢综合征治疗药物药代动力学的影响，以期阐明GCs介导CYP3A差异的胎儿编程机制及其药代动力学意义，为实现CYP3A介导的精准个体化给药提供新思路和新数据。

细胞色素P450（cytochrome P450, CYP）是肝脏中主要的I相药物代谢酶，其中CYP3A是肝脏所有CYP中含量最高的亚型。越来越多的研究表明，成年个体CYP表达的差异可能受到宫内时期发育编程的影响，但机制尚不清楚。胎儿暴露于母源性高糖皮质激素（glucocorticoid, GC）是宫内不良环境下的共性表现，本项目以乙醇和地塞米松（dexamethasone, DEX）分别作为引起母源性高GCs 的外源物代表和阳性药建立宫内高GC动物模型，探索GC对子代肝CYP3A表达的编程改变和药物代谢的影响，并在大鼠原代细胞、肝脏特异性GR敲出鼠、人源细胞系上验证其分子和表观遗传机制。Wistar大鼠孕中晚期（孕9-20天）乙醇暴露可明显增加子代宫内和成年早期CYP3A和其他CYP亚型的基因表达，但CYP蛋白表达、活性和代谢能力降低。在大鼠原代细胞和人源细胞系上均未观察到蛋白和基因水平的不一致。提示除GC编程CYP转录表达外，乙醇暴露还存在转录后调控机制。而孕中晚期DEX暴露雄性子代肝脏CYP3A1持续高表达，成年后CYP3A体内外代谢辛伐他汀和硝苯地平的能力增强，再次DEX处理后显示出诱导记忆效应。DEX暴露子代组成型雄甾烷受体（constitutive androstane receptor，CAR）持续激活，CAR启动子区组蛋白多个位点高乙酰化，组蛋白乙酰化酶P300表达增加。新生大鼠肝原代细胞经DEX处理后呈现同样的改变，而宫内敲除肝脏的糖皮质激素受体（glucocorticoid receptor, GR）后，其成年子代出现CAR、CYP3A表达缺陷。通过在人源细胞系上分别拮抗或沉默GR、P300和CAR，证实DEX可通过GC激活GR招募P300，使CAR持续高表达并介导CYP3A的表达增高，与内源性GC效应一致。以上结果提示，P300引起的CAR持续激活是宫内高GC环境下子代肝CYP3A表达编程改变的机制，并可能影响成年子代个体的药物代谢差异。本研究为响应国家对发展精准医疗和个性化用药的需求和号召，从发育源性疾病的热点问题——孕期GCs对胎儿的编程效应入手，为临床制定个性化用药方案提供了新的思路和依据。