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Cholesterol lights the fire of NASH

胆固醇点燃 NASH 之火

基本信息

批准号：
9138870
负责人：
George Ioannou
金额：
--
依托单位：
VA PUGET SOUND HEALTHCARE SYSTEM
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-04-01 至 2021-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9138870
关键词：
Age Animal Model Atherosclerosis Cell Aggregation Cell Culture Techniques Cells Characteristics Cholesterol Chronic Cirrhosis Coculture Techniques Crystallization Data Dental crowns Development Diabetes Mellitus Diet Dietary Cholesterol Dietary Fats Epidemic Ethnic Origin Fatty Liver Fatty acid glycerol esters Fibrosis Fire - disasters Freezing Gender Genes Genetic HepG2 Hepatic Hepatocyte High Fat Diet Histologic Human In Vitro Inflammasome Inflammation Inflammatory Intake Interleukin-1 beta Knockout Mice Kupffer Cells Lead Lipids Liver Mediating Molecular Mus Obesity Oral Pathogenesis Patients Pharmaceutical Preparations Pharmacology Population Prevalence Process Production Race Recruitment Activity Reporting Research Resolution Risk Factors Role Specimen Sterility Structure Testing Therapeutic Tissues Wild Type Mouse biobank chemokine cytokine design experimental study hepatoma cell hypercholesterolemia improved in vivo inhibitor/antagonist macrophage mouse model non-alcoholic fatty liver nonalcoholic steatohepatitis novel novel therapeutic intervention patient subsets prevent public health relevance translational pipeline uptake

项目摘要

DESCRIPTION (provided by applicant): We reported that dietary cholesterol induces progression from simple steatosis to NASH. We subsequently discovered that cholesterol crystals were present within the lipid droplets of steatotic hepatocytes in both patients with NASH and in mouse models of NASH induced by high-fat, high-cholesterol (HFHC) diet, but not in patients or mice with simple steatosis. We described that Kupffer cells (KCs) selectively surrounded and processed steatotic hepatocytes containing cholesterol crystals forming characteristic "crown-like structures" (CLS). Thus, both hepatocytes and KCs become exposed to cholesterol crystals, which have recently been shown to activate the NLRP3 inflammasome in animal models of atherosclerosis. We demonstrated NLRP3 activation in the KCs that form CLSs thus providing a mechanism by which exposure of hepatocytes and KCs to cholesterol crystals can lead to the chronic "sterile inflammation" of NASH. Collectively these results support our novel hypothesis that hepatic cholesterol crystallization causes NASH via activation of the NLRP3 inflammasome. We will pursue this hypothesis in both mouse models and humans with the following specific aims: SA1. Determine whether development of hepatocyte cholesterol crystals and KC-CLSs is the trigger that induces NLRP3 activation and progression from simple steatosis to NASH. a. In vitro experiments: HepG2 (human hepatoma) cells will be induced to develop large LDs either with or without cholesterol crystals and then co-cultured with THP1 macrophages or primary mouse KCs in contact or non- contact cocultures. We hypothesize that THP1 cells or KCs will demonstrate uptake of cholesterol crystals and NLRP3 activation only when co-cultured with HepG2 cells that have cholesterol crystals within their LDs. We will delineate the molecular mechanisms by which cholesterol crystals activate NLRP3 and induce IL-1β. b. In vivo experiments: Wild-type (Wt) C57BL/6J mice and hypercholesterolemic mice (Ldlr (-/-) and APOE2ki) will be exposed to high-fat diets ranging in cholesterol concentration from 0% to 1%. We hypothesize that all three mouse models will develop NLRP3 activation and histological NASH at that particular threshold dietary cholesterol concentration that leads to hepatic cholesterol crystals and CLSs in each mouse model, irrespective of other cholesterol-related factors. This will demonstrate that cholesterol crystallization is the critical switch that initiats NASH via NLRP3 activation. SA2. Determine whether genetic inactivation (global, KC-specific, or hepatocyte-specific) or pharmacological inhibition of the NLRP3 inflammasome prevent the development of cholesterol crystal-induced NASH. We will use global Casp1(-/-) and Nlrp3(-/-) KO mice to demonstrate that inactivation of the NLRP3 inflammasome inhibits cholesterol crystal-induced NASH. We will compare conditional KOs in which the Nlrp3 gene is selectively deleted either in KCs (Nlrp3KC(-/-)) or hepatocytes (Nlrp3Hep(-/-)) to distinguish whether NLRP3 activation in hepatocytes (causing pyroptosis) or KCs (causing release of cytokines, chemokines and inflammatory recruitment) or both is contributing to cholesterol crystal-induced NASH. We will determine whether a novel, oral, pharmacological NLRP3 inhibitor can prevent or reverse cholesterol crystal-induced NASH in Wt mice on a HFHC diet, with potential therapeutic implications for human NASH. SA3. Determine the prevalence and risk factors of hepatic cholesterol crystallization in human NAFLD/NASH and evaluate whether hepatic cholesterol crystallization independently predicts NLRP3 activation and development of NASH vs simple steatosis. We will use data and stored frozen liver tissue specimens from an existing VA biorepository of well- characterized patients with NAFLD/NASH (n=166).