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蛋白（统称为“药物加工基因”[DPG]）的发育调控知之甚少，这使新生儿和儿童发生药物不良反应（ADR）的风险更高。使用RNA-Seq，我们已经表明药物代谢是无菌（GF）小鼠整个肝脏转录组中最具差异调节的途径，这表明肠道微生物组和肝脏DPG之间存在新的相互作用。肠道微生物组的关键功能之一是产生次级胆汁酸（BAs），其可以激活肝脏中两种最关键的异生物质敏感核受体，即胆烷X受体（PXR）和组成型雄烷受体（CAR）。在发育过程中，肠道细菌和次级BA谱发生了深刻的变化，这表明肠道微生物组可能至少部分有助于肝脏中DPG的发育调节。尚未进行系统的研究来表征发育期间肠道微生物组对所有DPG的调节，并且关于抗生素或益生菌靶向肠道微生物组如何重新编程肝脏中DPG的个体发生知之甚少。因此， 研究是利用多学科的方法，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，以揭示肠道微生物群在调节PXR和CAR信号传导中的作用，以及随后肝脏中DPG的个体发生重编程。我们的中心假设是：肠道微生物组的发育变化至少部分地有助于通过改变肠道中的次级BA来调节肝脏中的PXR和/或CAR信号传导，从而调节肝脏中DPG的个体发生。我们将在2个目标中测试我们的假设：目标1A将使用RNA-Seq定量6个发育年龄的GF和常规（Conv）小鼠肝脏中281个关键DPG的mRNA，并验证差异调节的DPG的蛋白质和活性。我们还将使用ChIP-Seq来量化肠道细菌如何调节PXR/CAR DNA与某些DPG的结合，并将DPG个体发育与肠道微生物组的个体发育（宏基因组学）和BA谱（UPLC-MS/MS）相关联。目的1B将在不同年龄的不同PXR和CAR敲除和人源化转基因的GF小鼠中引入二级BA，以测试我们的假设，即二级BA恢复某些DPG的正常个体发育。目的2：利用不同发育年龄的人粪便细菌定植的GF小鼠，研究抗生素和益生菌L。嗜酸乳杆菌在重编程人类微生物组的个体发生和肝脏发育期间宿主DPG的后续变化中的作用。拟议的工作将揭示肠道微生物组的个体发育与开发期间药物加工能力的发育变化之间的新联系，并将通过建立考虑儿童ADR的新概念，导致儿科药理学的范式转变，除了已知的“药物-药物”和“食品-药物”相互作用之外，还将考虑“昆虫-药物”相互作用。

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.