Developmental Regulation of Drug Metabolism by Targeting the Gut Microbiome

描述(由申请人提供)：对肝脏中药物代谢酶和转运体(统称为药物处理基因[DPG])的发育调节知之甚少，使新生儿和儿童面临更高的药物不良反应(ADR)风险。利用RNA-Seq，我们已经证明药物代谢是无菌小鼠整个肝脏转录组中差异最大的调节途径，这表明肠道微生物组和肝脏DPG之间存在着一种新的相互作用。肠道微生物组的关键功能之一是产生次级胆汁酸(BAs)，它可以激活肝脏中两个最关键的异物敏感核受体，即孕烷X受体(PXR)和构成雄烷受体(CAR)。在发育过程中，肠道细菌和次级BA谱发生了深刻的变化，表明肠道微生物群至少在一定程度上参与了肝脏DPG的发育调节。目前还没有系统的研究来表征肠道微生物组在发育过程中对所有DPG的调节，也很少知道抗生素或益生菌如何靶向肠道微生物组重新编程肝脏中DPG的个体发育。因此，这样做的目标是 研究将利用多学科方法，包括GF和基因工程小鼠、BA代谢组学、下一代测序和人类粪便样本，以揭示肠道微生物区系在调节PXR和CAR信号以及随后肝脏DPG的个体基因重新编程中的作用。我们的中心假设是：肠道微生物组的发育变化至少部分参与了肝脏DPG个体发育的调节，通过改变肠道中的次级BA来改变肝脏中的PXR和/或CAR信号。我们将在两个目标中验证我们的假设：目标1A将使用RNA-Seq来量化6个发育年龄的GF和常规(Conv)小鼠肝脏中281个关键DPG的mRNAs，并验证差异调控的DPG的蛋白质和活性。我们还将使用CHIP-SEQ来定量肠道细菌如何调节PXR/CAR DNA与某些DPG的结合，并将DPG个体发育与肠道微生物组(元基因组学)和BA图谱(UPLC-MS/MS)的个体发育相关。目的1B在不同年龄段的PXR、Car基因敲除和人源化转基因的GF小鼠中引入次级BAs，以验证我们的假设，即次级BAs可以恢复某些DPG的正常个体发育。目的利用不同发育年龄段的人粪便细菌定植的GF小鼠，研究抗生素和嗜酸乳杆菌在重组人微生物个体发育过程中的作用，以及宿主DPG在肝脏发育过程中的变化。这项拟议的工作将揭示肠道微生物组的个体发生与发育过程中药物处理能力的发育变化之间的新联系，并将通过建立一个新的概念来考虑儿童的不良反应，即除了已知的“药物-药物”和“食物-药物”相互作用之外的“细菌-药物”相互作用，从而导致儿科药理学的范式转变。

DESCRIPTION (provided by applicant): Very little is known about the developmental regulation of drug-metabolizing enzymes and transporters (together called "drug-processing genes" [DPGs]) in liver, placing newborns and children at a much higher risk of adverse drug reactions (ADRs). Using RNA-Seq, we have shown that drug metabolism is the top most differentially regulated pathway in the entire liver transcriptome of germ-free (GF) mice, suggesting that there is a novel interaction between gut microbiome and hepatic DPGs. One of the key functions of gut microbiome is to produce secondary bile acids (BAs), which can activate two most critical xenobiotic-sensing nuclear receptors in liver, namely the pregnane X receptor (PXR) and constitutive androstane receptor (CAR). During development, profound changes occur in the intestinal bacteria and the secondary BA profiles, suggesting that gut microbiome may at least in part contribute to the developmental regulation of DPGs in liver. No systematic studies have been performed to characterize the regulation of all DPGs by gut microbiome during development, and little is known regarding how targeting the gut microbiome by antibiotics or probiotics re- programs the ontogeny of DPGs in liver. Therefore the goal of this research is to utilize multidisciplinary approaches, including GF and genetically-engineered mice, BA metabolomics, Next-Generation Sequencing, and human fecal samples, to unveil the role of gut microbiota in modulating PXR and CAR signaling and the subsequent ontogenic re-programming of DPGs in liver. Our central hypothesis is: the developmental changes in the gut microbiome at least in part contribute to the regulation of the ontogeny of DPGs in liver, through altering secondary BAs in the gut to modify the PXR and/or CAR signaling in liver. We will test our hypothesis in 2 Aims: Aim 1A will use RNA-Seq to quantify mRNAs of 281 critical DPGs in livers of GF and conventional (Conv) mice at 6 developmental ages, and validate the proteins and activities of differentially regulated DPGs. We will also use ChIP-Seq to quantify how gut bacteria modulate PXR/CAR DNA binding to certain DPGs, and correlate DPG ontogeny with the ontogeny of gut microbiome (metagenomics) and BA profiles (UPLC-MS/MS). Aim 1B will introduce secondary BAs to GF mice in various PXR- and CAR-knockout and humanized transgenic) at various ages to test our hypothesis that secondary BAs restore the normal ontogeny of certain DPGs. Aim 2 will use GF mice colonized with human fecal bacteria from various developmental ages, to determine the roles of antibiotics and the probiotic L. acidophilus in re-programming the ontogeny of human microbiome and the subsequent changes in the host DPGs during liver development. The proposed work will unveil a novel link between the ontogeny of gut microbiome and the developmental changes of drug- processing capacities during development, and will lead to a paradigm shift in pediatric pharmacology, by establishing a new concept in considering ADRs in children, which are the "bug-drug" interactions, in addition to the known "drug-drug" and "food-drug" interactions.