磷脂酶PLA2G12B调控极低密度脂蛋白合成与分泌的分子机制及在非酒精性脂肪肝病的作用研究

Nonalcoholic fatty liver diseases (NAFLD/NASH) is becoming more prominent in China. Our previous study indicated that PLA2G12B, a target gene of HNF-4a, is the only secreted phospholipase A2 family member essential for hepatic VLDL secretion. PLA2G12B knockout mice develop severe hepatosteatosis despite low serum TG level, phenotypically copying a human disease familial hypobetalipoproteinemia. As a phospholipase without demonstrated enzymatic activity, we propose a novel hypothesis that PLA2G12B functions as a phospholipid chaperone affecting VLDL maturation and secretion. This study aims to investigate: 1) how PLA2G12B interacts with and delivers phospholipids and lipid droplets to nascent ApoB100 particles; 2) how dysregulation of PLA2G1B contributes to progression of NAFL to NASH. We would clarify the subcellular localization of PLA2G12B within the secretory pathway and structural activity relation as a phospholipid chaperone. Additionally, we would utilize high fat and high glucose diet to enhance the rates of fatty acid absorption and de novo lipid synthesis to exacerbate lipid flux into the hepatic VLDL secretory pathway, investigating how defective PLA2G12B activity contributes to lipid stress, inflammation, insulin resistance and fibrosis.

我国非酒精性脂肪肝病（NAFLD/NASH）的发病趋势愈见严重，一些降脂药物副作用及基因遗传病揭示极低密度脂蛋白（VLDL）代谢失调是其发生的重要因素，高血脂并不是NAFLD发生的必要条件。我们前期研究发现PLA2G12B是唯一参与调控VLDL的分泌型磷脂酶A2，其功能缺失导致血脂降低和肝细胞脂肪变性。本项目继续重点研究PLA2G12B作为磷脂伴侣参与调控VLDL组装合成与分泌的分子新机制，及其功能缺失导致NAFLD/NASH发生发展的作用机理。本项目将明确PLA2G12B的亚细胞定位、蛋白/磷脂/脂滴相互作用等分子机制，同时利用敲除老鼠及高糖高脂诱导模型，研究脂质过氧化、NASH、胰岛素耐受的发生机理和相关信号通路。本研究提出VLDL合成分泌必须需要PLA2G12B作为磷脂伴侣参与调控的分子新机制，且与NAFLD/NASH的疾病发展密切相关，具有重要的科学创新和临床参考价值。

我国非酒精性脂肪肝病（NAFLD/NASH）的发病趋势愈见严重，肝脏极低密度脂蛋白（VLDL）合成分泌异常是导致肝脂代谢紊乱的重要因素。代谢性核受体ERRα、FXR、HNF4α是肝脂代谢的重要调控因子，本项目主要研究发现核受体通过调控PLA2G12B影响VLDL合成分泌和肝脂代谢的作用机制。首次发现性别差异性的VLDL合成分泌可能与PLA2G12B等VLDL相关基因在肝脏表达的性别差异相关，通过染色质免疫共沉淀、荧光素酶报告系统等方法明确PLA2G12B受到ERRα直接转录调控，并阐明Estrogen-ERa-ERRα-PLA2G12B/ApoB/Mttp的新分子调控通路。ERRα肝脏特异敲除(ERRαLKO)小鼠中发现PLA2G12B/ApoB/Mttp等VLDL相关基因表达下降、VLDL分泌减少和明显NAFLD，而高糖/高胆固醇饮食处理ERRαLKO雌鼠显著增加NASH发生。通过ERa选择性抑制剂Tamoxifen、ERRα拮抗剂C29等药物处理以及卵巢切除术处理雌鼠等多个体内实验，进一步证实Estrogen-ERa-ERRα的信号扰动导致PLA2G12B等VLDL相关基因表达异常、VLDL分泌减少及肝脂异常积累。腺病毒过表达ERRα/PLA2G12B/Mttp 在以上不同动物模型的拯救实验可显著提高VLDL分泌、改善NAFLD。此外，研究发现FXR激动剂通过干扰HNF4a转录活性调控PLA2G12B的表达从而影响肝脏VLDL合成分泌；细胞荧光实验初步验证PLA2G12B定位在内质网上并在一定状态下可能定位于脂滴表面。因此，我们研究明确了PLA2G12B影响VLDL合成分泌的新分子机制，揭示性别差异影响NAFLD/NASH发生发展的潜在新机理，对脂代谢紊乱相关疾病的研究和防治具有重要意义。