昼夜节律紊乱与环境污染物引起肝脏毒性的独立/协同效应及基于核受体CAR的机制研究

Circadian misalignment or disorder and environmental pollutants, as potential risk factors for Non-alcoholic fatty liver disease/ Non-alcoholic steatohepatitis (NAFLD/ NASH) and related cardio-metabolic disorders show a high prevalence in general population, and the co-exposure is very common in some workplaces, e.g. doctors and nurses in hospitals, policemen, taxi drivers and industrial workers. The causal relationships between circadian disorder, environmental pollutants and NAFLD/NASH are not clear. We design animal tests, epidemiological studies and in vitro cell assays to assess the independent/synergistic effects of circadian misalignment and environmental pollutants on hepatotoxicity (NAFLD/NASH) and explore constitutive androstane receptor (CAR)-mediated mechanism..In animal (C57BL/6J mice) tests light/dark cycle is to be inverted once a week to change the central clock time , and shifting eating to the circadian rest phase changes the liver clocks. Both methods could induce circadian misalignment between central clock and liver clock. CdCl2 and PCBS (PCB126 and Aroclor 1260) as representative environmental pollutants will be used to induce toxicant-associated fatty liver disease (TAFLD) or more severe form toxicant-associated steatohepatitis (TASH). Liver gene transcripts, metabolites, and associated metabolic pathways and functions are assessed. The joint effects will be observed in orthogonal design tests for circadian misalignment and environmental pollutants exposure. To elucidate the role of nuclear receptor CAR in the above mentioned toxic effect, the mRNA accumulation of CAR target genes will be measured and CAR-/- mice will also be used. .We will design two epidemiological studies: a hospital-based case-control studies and a cross-sectional studies in nurses to explore the relationships between night shift work, chronotypes and environmental pollutants with NAFLD/NASH and its related metabolic disorders. In the case-control study, newly diagnosed NAFLD/NASH patients are selected as cases and hospital-based controls are selected. The total sample size is 1400 with equal cases and controls. In the cross-sectional study 1400 doctors and nurses are enrolled in hospitals. Questionnaires are used to collect information on shift work, chronotype and potential confounders, such as diet, sports and sleep. Urine samples are used to analyze Cd and PCBs. Annual physical examination results of the doctors and nurses including B-mode images of liver, liver function indexes, plasma lipids, glucose, insulin and inflammatory factors are collected and used as outcomes..In vitro cell studies will be performed to explore the activation mode of nuclear receptor CAR and its effects on CAR target genes induced by caffeine, nobiletin, Cd and PCBs (PCB126 and Aroclor 1260). Both caffeine and nobiletin are small molecules targeting the molecular oscillator in cells to enhance circadian rhythms.

昼夜节律紊乱与环境污染物暴露在普通人群十分普遍，且在职业人群存在联合暴露，而非酒精因素引起的脂肪性肝病（NAFLD/NASH）发病率不断升高，病因未明。本项目从动物、人群和体外细胞三个层面研究昼夜节律紊乱和Cd、PCBs与NAFLD/NASH的因果关联。动物实验观察光照改变中枢节律、睡眠期饮食改变肝脏节律及Cd、PCBs经口暴露对小鼠肝脏一般毒性、糖脂代谢、炎症反应的影响以及核受体CAR及其靶基因的表达；人群研究包括以医院为基础的病例-对照研究和面向医护人员的横断面调查，用问卷收集轮班作业、昼夜作息制度以及饮食、运动、睡眠等混杂因素信息，采集尿样检测污染物水平，收集体检和疾病信息，分析轮班作业、作息类型及污染物暴露与NAFLD/NASH及相关代谢、炎症指标的关联性；体外细胞实验观察能够调节细胞节律的天然小分子物质咖啡因、蜜桔黄素和Cd、PCBs活化核受体CAR的方式及对CAR靶基因的调控。

本项目旨在探索昼夜节律紊乱与环境污染物暴露对肝脏糖脂代谢的独立/联合效应及可能的分子机制。首先模拟轮班作业的夜间光照，调控实验室12h：12h的光照/黑暗周期构建C57BL/6J小鼠昼夜节律紊乱模型，发现光照周期改变可升高小鼠空腹血糖、改变胰岛素敏感性和糖耐量的24小时节律，扰乱肝脏昼夜节律核心基因及糖脂代谢基因的表达节律。在此基础上，结合职业与环境卫生实践，观察了苯及双酚A与昼夜节律紊乱对小鼠肝脏糖脂代谢的联合效应，并对交互作用的基因表达机制进行了初步探索。随着学术界对昼夜节律影响健康分子机制研究的深入，“时间毒理学”的概念重新受到重视。小鼠光照期（静止期）或黑暗期（活动期）暴露于环境因素的毒性效应可能存在差异。我们观察到光照期、黑暗期C57BL/6J小鼠暴露于双酚A或高脂饮食对肝脏胰岛素敏感性、脂质沉积的影响存在差异，提出BMAL、Rev-erbα直接调控肝细胞转录因子Pparα、Srebp1c、Chrebp，进而影响肝脏糖脂代谢基因Lpl、Acc1、Hmgcr表达的分子机制，并在体外培养的原代细胞、HepG2细胞中得到验证。为了回应公众对双酚A安全限值的质疑，我们观察了生命早期双酚A暴露联合成年后高脂饮食对子代肝脏糖脂代谢的影响，发现上述不良效应存在性别差异，为后续研究提供了线索。最后，利用前期建立的出生队列，观察了孕期昼夜相关行为、尿液重金属、尿液双酚A及其同系物（进行中）、尿液褪黑素水平（进行中）与妊娠期糖尿病、出生体重、出生端粒长度的关联性及婴儿性别的修饰效应。综上，本项目以肝脏糖脂代谢紊乱为核心，利用动物模型、人群队列及体外细胞实验，观察了昼夜节律紊乱与常见环境污染物暴露对肝脏糖脂代谢的独立及联合效应，提出了BMAL调控肝细胞转录因子Pparα、Srebp1c及Chrebp，进而调控肝脏糖脂代谢基因表达的分子机制。