Regulation of Hepatobiliary Bile Acid Homeostasis in Neonatal Obstructive Cholestasis

项目摘要 胆道闭锁（BA）导致胆汁酸稳态的严重破坏，是引起胆道梗阻的主要疾病。 新生儿阻塞性胆汁淤积症核激素受体法尼醇调节胆汁酸的稳态 X受体（FXR）通过激活肠和肝激素，成纤维细胞生长因子19（FGF 19）。 FGF 19抑制肝细胞色素P450 7a 1（Cyp 7a 1）的胆汁酸合成，Cyp 7a 1是经典肝纤维化的关键酶。 胆汁酸合成在健康婴儿中，FGF 19的表达主要在肠中，在肝脏中的表达较低。在 相反，BA婴儿的肝脏FGF 19升高，以补偿肠胆汁酸诱导的FGF 19损失 分泌物BA婴儿肝脏FGF 19和胆汁酸蓄积的增加表明， 通常抑制肝Cyp 7a 1和胆汁酸合成的FGF 19信号传导。胆汁的第二来源 肝细胞和胆管细胞中的酸合成是通过Cyp 27 a1的替代途径。在我们的世界里， 了解Cyp 7a 1和Cyp 27 a1如何通过FXR-FGF 19信号传导调节胆汁酸 新生儿阻塞性胆汁淤积的体内平衡。本书的目的是了解适应性，时间- 肝脏补偿改变的肠道胆汁酸信号传导的依赖机制，以及这种机制是如何产生的。 代偿通过经典途径和替代途径影响胆汁酸合成。我们的核心假设是 肝脏对阻塞性胆汁淤积的适应导致肝细胞FXR-FGF 19信号传导增加，但 Cyp 7a 1抑制或Cyp 27 a1补偿受损，以维持胆汁酸蓄积。在目标1a中，我们 将表征胆汁淤积性肝损伤、胆汁酸稳态和细胞特异性肝细胞的个体发生， 胆管细胞基因表达特征在一个新开发的新生猪模型的胆管结扎（BDL）。 在目标1b中，我们将量化胆汁中FXR和FGF 19信号通路的分子机制。 人培养肝细胞和胆管细胞在长期胆汁酸暴露期间的酸合成 类器官在目标2a中，我们将测试FGF 19输注是否剂量依赖性地抑制Cyp 7a 1表达， BDL后早期和晚期时间点。在目标2b中，我们将测试FXR-FGF 19信号的早期激活是否能保护 抗BDL诱导的胆汁淤积。本研究将有助于了解胆汁酸的调节机制 在新生儿阻塞性胆汁淤积症的背景下的体内平衡和测试潜在的治疗干预。 职业发展和环境：提高我作为独立工作者未来成功的潜力 我将接受道格拉斯伯林博士的指导和稳定同位素动力学使用方面的培训 建模、质谱分析、RNA-seq管道分析和可视化以及肝类器官 培养。本次培训将在儿童营养学优秀的研究环境中进行 研究中心（CNRC）与NIDDK消化疾病中心技术核心和专家教师的支持 贝勒医学院的监督委员会这个项目将给我的科学专业知识和研究 在儿科肝病领域开展独立研究的技能。

PROJECT SUMMARY Biliary atresia (BA) results in severe disruption of bile acid homeostasis and is the predominant disease causing neonatal obstructive cholestasis. Bile acid homeostasis in regulated by the nuclear hormone receptor Farnesoid X Receptor (FXR) by activation of the intestinal and hepatic hormone, fibroblast growth factor 19 (FGF19). FGF19 suppresses bile acid synthesis of hepatic cytochrome P450 7a1 (Cyp7a1), the key enzyme for classical bile acid synthesis. In healthy infants, the FGF19 expression is mainly in the intestine and low in the liver. In contrast, hepatic FGF19 is elevated in BA infants to compensate for loss of intestinal bile acid-induced FGF19 secretion. The increase in hepatic FGF19 and bile acid accumulation in BA infants suggests an uncoupling of FGF19 signaling that normally suppresses hepatic Cyp7a1 and bile acid synthesis. A secondary source of bile acid synthesis in hepatocytes and cholangiocytes is the alternative pathway via Cyp27a1. There is a gap in our knowledge of how the regulation of Cyp7a1 and Cyp27a1 via FXR-FGF19 signaling contributes to bile acid homeostasis during neonatal obstructive cholestasis. This proposal aims to understand the adaptive, time- dependent mechanism by which the liver compensates for altered intestinal bile acid signaling and how this compensation effects bile acid synthesis via the classical and alternative pathways. Our central hypothesis is that hepatic adaptation to obstructive cholestasis leads to increased hepatocyte FXR-FGF19 signaling, but with impaired suppression of Cyp7a1 or compensation by Cyp27a1 to sustain bile acid accumulation. In Aim 1a we will characterize the ontogeny of cholestatic liver injury, bile acid homeostasis, and cell-specific hepatocyte and cholangiocyte gene expression signatures in a newly developed neonatal pig model of bile duct ligation (BDL). In Aim 1b, we will quantify the molecular mechanisms of FXR and FGF19 signaling pathways involved in bile acid synthesis during long-term bile acid exposure using human cultured hepatocyte and cholangiocyte organoids. In Aim 2a, we will test whether FGF19 infusion dose-dependently suppresses Cyp7a1 expression at early and late time points after BDL. In Aim 2b, we will test if early activation of FXR-FGF19 signaling protects against BDL induced cholestasis. This study will provide understanding of the mechanism that regulate bile acid homeostasis in the context of neonatal obstructive cholestasis and test potential therapeutic interventions. Career development and environment: To enhance my potential for future success as an independent researcher I will receive mentorship from Dr. Douglas Burrin, PhD and training in use of stable isotope kinetics modeling, mass spectrometry analysis, RNA-seq pipeline analysis and visualization, and hepatic organoid culturing. This training will be conducted in an outstanding research environment of the Children’s Nutrition Research Center (CNRC) with support of NIDDK Digestive Disease Center technical cores and an expert faculty oversight committee at Baylor College of Medicine. This project will give me the scientific expertise and research skills to launch an independent research career in the field of pediatric liver diseases.