p53/PXR调控肝细胞脂质生成诱导NAFLD发病的作用及分子机制

Nonalcoholic fatty liver disease (NAFLD) is one of the chronic liver diseases with high morbidity, but its pathogenesis is poorly understood. Our previous studies revealed that the p53 expression level in the hepatocytes from NAFLD mouse model was increased and inhibition of p53 expression could decrease the hepatic steatosis, meanwhile the activity of hepatic pregnane X receptor (PXR) was inhibited. However, the pathogenic mechanism of p53/PXR-induced pathogenesis of NAFLD remains unknown. Recent studies found that the activity of PXR is regulated by p53, whereas the knockdown of PXR results in elevation of the expression for aldo-keto reductase 1B10 (AKR1B10) and its target gene acetyl-CoA carboxylase (ACC), which is a key lipid synthesis enzyme. In this research project, by using target gene overexpression and siRNA-dependent silence or knockout, dual-luciferase reporter gene as well as NAFLD cell models and mouse models, we try to research and determine the roles and mechanisms that p53 antagonizes PXR activity to remove its inhibition effect on AKR1B10 and ACC,which involved in the pathogenesis of NAFLD. The findings of this project will possess high originality and clinical significance.

非酒精性脂肪性肝病（NAFLD）是高发慢性肝病，其发病机制尚未完全清楚。我们前期研究发现NAFLD小鼠肝细胞p53表达水平增高、抑制p53显著减轻肝细胞脂变，并且NAFLD小鼠肝脏中孕烷X受体（PXR）活性下降，但机制不明。近年报道p53可拮抗肝脏中PXR，且PXR沉默可引起醛-酮还原酶1B10（AKR1B10）及其下游脂质合成酶乙酰辅酶A羧化酶（ACC）的表达上升。本项目拟采用靶基因过表达与siRNA沉默或敲除、双荧光素酶报告基因等技术及NAFLD细胞模型和小鼠模型，探讨并阐明p53通过拮抗PXR，解除对其下游基因AKR1B10的抑制，来上调脂质合成酶ACC表达及活性，从而参与NAFLD发病的作用及分子机制，为p53/PXR相关的NAFLD发病分子机制提供新观点。

研究背景与研究目的：.P53作为肿瘤抑制基因，近年来被报道可能参与非酒精性脂肪性肝病(NAFLD)的发病过程中。孕甾烷X受体(PXR)是来自核受体超家族，被发现能够诱导脂质合成基因SCD1的表达从而也参与NAFLD当中。P53与PXR在肠道细胞中也被报道存在相互作用。本研究目的是阐明肝细胞中p53-PXR相互作用调控SCD1参与NAFLD的发病过程。.方法：.雄性p53野生型与敲除小鼠（8-10周）分别随机分为普通饲料（SCD）和高脂饲料（HFD）喂养处理组以建立体内NAFLD模型。HepG2细胞与小鼠原代肝细胞以棕榈酸（PA）处理以建立NAFLD体外模型。肝内/胞内甘油三酯水平及油红O染色用来评价NAFLD严重程度。P53激动剂Nutlin-3a在体内和体外被用来激活p53通路。小分子干扰siRNA在细胞水平用来敲低p53和PXR的表达。29T细胞上进行免疫共沉淀试验来证明P53与PXR之间的蛋白相互作用。荧光素酶报告基因试验用来检测SCD1启动子区域PXR结合元件的转录活性。.主要结果：.与野生型小鼠相比，P53敲除小鼠呈现减轻的NAFLD症状。在细胞模型中，p53敲低也可以显著逆转PA诱导的脂变。NAFLD造模体内及体外均诱导p53与PXR的核转位，然而在p53敲除小鼠或者p53敲低的细胞中无此变化。在HepG2细胞上证实p53与PXR有蛋白相互作用。P53激动剂在体内体外均可诱导PXR的核转位，而在p53敲除小鼠或p53敲低的细胞中无此变化 。p53激动剂能诱导SCD1启动子PXRE区域的转录活性，该作用可被PXR敲低所阻断。在细胞模型中，PXR敲低可阻断p53敲低对PA诱导的脂变的改善作用。.结论：.P53可通过与肝脏PXR相互作用，诱导SCD1的表达从而促进NAFLD的发生发展。