非酒精性脂肪发病新机制：PNPLA3参与炎症相关肝细胞损伤与凋亡的分子机制研究

PNPLA3 is a novel adipokine, and also belongs to the iPLA2 family. Its mainly function invovles in triglyceride synthesis. It has been reported that PNPLA3 was a major susceptibility gene for nonalcoholic fatty liver disease (NAFLD), its polymorphism I148M was significantly associated with liver fat content,ALT，liver fibrosis and necrosis,suggesting that PNPLA3 involved in the mechanisms of both liver fat deposition and liver damage. Therefore, PNPLA3 might play an important role in the onset and development of NAFLD. In this project we hypotheses that PNPLA3 mediates apoptosis of hepatocytes in NAFLD. Given that no reports were found about the PNPLA3 and hepatic damage and apoptosis so far, in we will firstly examine the effect of PNPLA3 on hepatic apoptosis and the regulatory mechanism of NF-kB on the PNPLA3 related apoptosis in NAFLD animal model and hepatocyte model via Molecular Biotechnology and gene knockout technology. This will reveal a new mechanism of NAFLD.

PNPLA3是一种主要参与甘油三酯的合成脂肪因子，也是iPLA2家族成员之一，是非酒精性脂肪肝（NAFLD）的主要易感基因，其基因多态性与肝脏脂肪含量、转氨酶、肝纤维化和坏死均关系密切，提示PNPLA3同时涉及肝脏脂肪沉积和肝脏损伤的分子机制，在NAFLD的发生发展中发挥重要作用。由于NAFLD相关肝细胞损伤与凋亡的病理基础为炎症，因此，我们设想在NAFLD中，PNPLA3介导了炎症导致的肝细胞损伤与凋亡。现关于PNPLA3参与NAFLD的肝细胞损伤和凋亡方面的研究，国内外均未见报道，本课题拟通过构建NAFLD的动物和细胞模型，利用分子生物学、基因敲除等技术，围绕参与炎症反应的关键因子NF-kB，首次探讨PNPLA3对细胞凋亡的影响及NF-kB对PNPLA3相关细胞凋亡的调控作用，从而揭示病理状态下肝脏脂肪沉积的新机制。

PNPLA3基因是非酒精性脂肪肝（NAFLD）的主要易感基因，与NAFLD全疾病谱均显著性相关，但致病机制不明。炎症反应参与NAFLD的 “二次打击”导致肝炎、纤维化、肝硬变等，提示PNPLA3可能参与炎症相关NAFLD机制。 本项目通过生物信息学分析，分子生物学、基因克隆、基因打靶等技术，在动物和细胞模型中，探讨了（1）PNPLA3是否是NF-kB的靶基因；(2)NF-kB调控PNPLA3的机制是否介导了PA诱导的内质网应激凋亡与炎症；（3）比较上述机制在PA干预的不同PNPLA3 I148M基因型HepG2细胞系中的差异性。.研究主要发现.1、发现NF-kB 参与PNPLA3基因的表达调控：高脂饮食诱导的脂肪肝大鼠模型和细胞过表达抑制实验探明PNPLA3与NF-kB表达的相关性.2、证实NF-kB 介导PA对PNPLA3的表达调控：明确了PA处理HepG2细胞中PNPLA3与NF-kB表达之间的剂量依赖关系，EMSA和CHIP证实PA干预增加NF-kB核蛋白与PNPLA3启动子结合.3、明确NF-kB对人PNPLA3基因调控的分子机制，即核NF-kB与人PNPLA3启动子-183~ -193bp结合激活转录：荧光素酶报告检测及EMSA证实人PNPAL3基因启动子-183至-193bp是NF-kB结合motif.4、明确PNPLA3148M/M 参与NAFLD的发病机制，即NF-kB调控PNPLA3 148M/M参与PA相关内质网应激诱导的凋亡与炎症：通过过表达和抑制、挽救实验，探明PNPLA3148M/M介导了NF-kB相关PA激活内质网应激凋亡（PERK-eIF2α-CHOP）和炎症（XBP1-MAPK/ERK1/2-TNFα）的机制.结论.1、人PNPLA3基因启动子-183~-193bp为NF-kB结合位点，该位点介导了NF-kB对人PNPLA3基因的转录调节.2、PNPLA3介导PA诱导的NF-kB相关内质网应激凋亡和炎症.研究意义. 国内外首次研究NF-kB对PNPLA3的调控作用及其参与内质网应激凋亡和炎症促进NAFLD发生发展的机制，为阐明NAFLD发病机制开辟了新思路；对不同PNPLA3 I148M基因型致病机制差异性的研讨，为解释携带PNPLA3 148M的人群更易患NALFD提供了理论依据，进而为挖掘PNPLA3相关治疗靶点提供奠定了基础