IL-17A介导Th17细胞促非酒精性脂肪性肝炎肝损伤的效应及机制研究

Nonalcoholic steatohepatitis or NASH is a critical pathological stage in the progession of hepatic steatosis to liver fibrosis and cirrhosis. Because hepatic inflammation is one of the most distinguished features of NASH, hepatic immune responses may also play critical roles in the pathogenesis of NASH.The recently discovered Th17 cells have been characterized as improtant players in modulating immune responses, and expanded Th17 cells have been shown in NASH patients and animals to promote inflammatory liver injury. However, the underlying mechanisms are largely unknown. As IL-17A is the major mediator secreting by Th17 cells, this study thus aims to: (1) determine the role of IL-17A in the pathogenesis of NASH using IL-17A gene knockout mice as a NASH model; (2) explore the molecular mechanisms of IL-17A-mediated liver injury by subjecting the primary hepatocytes and kuppfer cells from IL-17A-/- mice and the wild type mice to proteomic and transcriptomic analysis and characterizing the differentially expressed genes for critical cell functions and signaling pathways; (3) As the differentiation of Th17 cells is induced upon IL-6 and its recruitment enhanced upon CCL-20, two cytokines abundantly present in injured liver and are induced by FOXO1, we will determine the role of FOXO1 in promoting Th17 cell-mediated liver injury using liver-specific transgenic FOXO1 mice, and test the underlining molecular mechanisms with a focus on CCL-20 and IL-6 in the expansion of Th17 cells. Results from this study are expected to help understanding the pathogenesis from simple steatosis to steatohepatitis, and developing new strategies in counteracting NASH and/or advanced liver diseases in patients with NAFLD.

非酒精性脂肪性肝炎(NASH)是单纯脂肪肝向肝纤维化及肝硬化进展的重要阶段，以炎细胞浸润为主要特征，炎症免疫在NASH发病中有重要作用。近年发现，辅助性T细胞Th17是免疫调节的重要成分，在NASH肝脏中数量增多，对NASH肝损伤起促进作用，但机制尚不清楚。IL-17A是Th17分泌的特征性细胞因子,本课题将在（1）IL-17A-/-基因敲除小鼠模型上观察对NASH肝损伤的影响；（2）采用蛋白质组及转录组学技术, 鉴定IL-17A-/-小鼠与野生小鼠肝细胞和枯否细胞中炎症损伤效应基因,从而揭示IL-17A介导Th17细胞免疫效应促进肝损伤的分子机制；（3）以Th17细胞分化和趋化为线索,探讨NASH肝组织FOXO1高表达与Th17细胞数量增多之间通过FOXO1-CCL20/IL6-Th17的内在联系机制，并在肝脏FOXO1转基因小鼠上加以验证。研究结果对NASH发病机制及防治有重要意义。

随着肥胖人口的增多，非酒精性脂肪性肝病（NAFLD）的发病率和患病人口数量激增。 非酒精性脂肪性肝炎（NASH）是单纯脂肪肝向肝纤维化、肝硬化发展的关键病变过程。近年发现，炎症免疫及其重要调节因子IL-17A在NASH发生发展中起重要作用，但机制尚未阐明。 本研究以IL-17A基因敲除小鼠为工具，通过长程高脂膳食（HF）诱导脂肪肝，并以一次性腹腔注射低剂量细菌脂多糖（LPS）给以急性炎症打击，观察在肝脂肪变性的基础状态下，IL-17A基因缺陷（KO）对肝脏的炎性反应效应及肝功能损伤的影响。研究从动物肝损伤表型到细胞分子机制开展了三个层次的研究工作：首先，通过检测血清谷丙转氨酶（ALT）及谷草转氨酶（AST）水平评估肝脏损伤情况，通过肝脏及血清中三种主要炎症因子TNF-α、IL-1β、IL-6评估机体炎症水平，通过肝脏病理形态观测评估肝组织炎细胞浸润和肝细胞损伤情况；进一步通过对肝组织基因表达谱及蛋白质组的检测分析，寻找IL-17A基因缺失在HF+LPS诱导的肝损伤中发挥作用的相关分子信号机制；并在离体培养的L02细胞和原代培养的肝细胞及Kupffer细胞中对关键分子信号功能加以验证。我们的结果表明：（1）IL-17A KO能显著降低脂肪肝小鼠在急性LPS诱导下的炎症损伤效应。与野生型（WT）小鼠相比， KO小鼠血清中ALT、AST及TNF-α、IL-1β、IL-6炎症因子水平显著降低，肝组织中浸润的炎性细胞及凋亡细胞数量均显著降低；（2）KO小鼠肝脏中减少的炎性细胞主要是噬中性细胞和单核细胞，同时肝脏中CCL2、CCL7、CCL8，CXCL1、CXCL10及CXCL14等多种噬中性细胞和单核细胞趋化因子的mRNA表达水平显著降低；（3）分子机制的分析结果显示IL-17A基因缺陷至少从三个方面对肝损伤起保护作用：肝脏脂质分解代谢显著降低（p<10-8）、氧化应激水平降低而Nrf2及多种抗氧化酶水平升高（GPX1、IDH1及ME1）、巨噬细胞的活化显著降低。. 由于肥胖患者血清中LPS及IL-17A水平均显著升高，并与NAFLD的进展密切相关，本研究结果提示IL-17A升高可直接促进肝脏脂质代谢、加强肝脏氧化应激损伤，同时通过募集和激活炎性细胞（特别是巨噬细胞），形成脂代谢-氧化应激-炎性损伤的恶性循环。本研究对肥胖相关NAFLD的发病机制及防治具有重要意义。