α-亚麻酸植物甾醇酯基于AMPK相关信号通路调控脂代谢改善非酒精性脂肪肝

Non-alcoholic fatty liver disease (NAFLD) is a clinical-pathologic syndrome which is characterized by steatosis of more than 5% of the liver parenchyma in the absence of alcohol consumption. Nowadays, the prevalence of NAFLD is increasing worldwide. However, there are no licensed therapies for NAFLD. Diet and physical exercise are considered the first line of treatment for patients with NAFLD. Our previous study show that α-linolenic acid ester of plant sterol (ALA-PS)）is the potential intervention for the prevention and treatment of NAFLD, however, the molecular mechanism is not clear. Lipid metabolism dysfunction leaded to cholesterol (CHO) and triglycerides (TG) accumulation in human hepatocytes plays an important role in the pathogenesis of NAFLD. AMP-activated protein kinase (AMPK) has become an attractive target to prevent NAFLD due to regulating lipid metabolism via mulitiple signal pathways. We hypothesized that ALA-PS improved lipid metabolism through AMPK pathway and then prevented NAFLD. In present study, NAFLD mice model induced by high-fat and high-cholesterol diet are supplemented with ALA-PS. In vitro, the cultured hepatic cells are incubated with ALA-PS, AMPK activator and inhibitor. RNA interference and over expression technology is also used to further explore the mechanism. After different treatments, the expressions of AMPK and the AMPK-related regulatory factors which play vital role in CHO metabolism, mitochondrial biogenesis/fatty acid decomposition and endoplasmic reticulum stress/TG synthesis such as SREBP-2, FXR, LXRα, PGC-1α, PPARα, PERK and SREBP-1c are analyzed. Further, these regulatory factors’ down-stream key moleculars and enzymes involving CHO de novo synthesis (HMGCR), synthesis of bile acids (CYP7A1), CHO excretion (ABCG5, ABCG8), fatty acid β-oxidation (CPT1A) and de novo TG synthesis (ACC, FAS) are examined in the level of mRNA, protein or enzyme activities. Finally, the ameliorative effect of ALA-PS on NAFLD and the underlying mechanisms by which ALA-PS promotes lipid metabolism via multiple AMPK-associated signaling pathways will be revealed. The results of this study will provide new dietary strategy and an effective target for the prevention and treatment of NAFLD.

非酒精性脂肪肝（NAFLD）呈全球化流行趋势，治疗尚无特效药，预防是关键。本课题组初步研究显示，α-亚麻酸植物甾醇酯（ALA-PS）具有改善NAFLD的潜力，但机制不明。磷酸腺苷活化蛋白激酶（AMPK）可多路径调控脂代谢，已成为防治NAFLD的靶点。本项目提出“ALA-PS调控AMPK信号通路改善脂代谢防治NAFLD”的假说，拟通过高脂膳食诱导小鼠NAFLD并进行ALA-PS干预，同时结合体外培养肝细胞，给予AMPK激活剂、抑制剂、过表达及siRNA，分析AMPK变化对胆固醇代谢、线粒体生物合成与脂肪酸β-氧化、内质网应激与甘油三酯合成关键调节因子SREBP-2、FXR、LXRα、PGC-1α、PPARα、PERK和SREBP-1c的影响。以阐明ALA-PS基于AMPK信号通路调控脂代谢改善NAFLD的相关机制。此项目的实施和成果取得可为NAFLD的防治提供新的膳食策略和有效的分子靶点。

近年来，非酒精性脂肪性肝病（NAFLD）呈全球流行趋势，已成为第一大慢性肝病和肝功能异常的首要原因。但临床治疗效果有限，预防是关键。本项目基于植物甾醇防治NAFLD的优点和不足，采用植物来源的n-3多不饱和脂肪酸α-亚麻酸将植物甾醇酯化合成α-亚麻酸植物甾醇酯（ALA-PS），研究其改善NAFLD的作用。并以脂代谢失衡为切入点，以AMPK为作用靶点，揭示ALA-PS调控脂代谢，防治NAFLD的分子机制。主要研究结果如下：.1、动物实验发现：①ALA-PS干预显著改善高脂膳食诱导的肝脏脂肪变性以及血脂紊乱、氧化应激、肝脏损伤和炎症反应，效果明显好于植物甾醇和α-亚麻酸单独干预。②在分子机制方面，ALA-PS显著减少内质网应激标志物GRP78和CHOP蛋白和mRNA表达水平，调节内质网稳态；抑制内质网应激引起的胆固醇合成关键调节因子（SREBP-2、HMGCR）和脂肪酸从头合成关键调节因子（SREBP-1c、FAS）蛋白和mRNA表达水平增加；抑制内质网应激诱导的IRE1α/XBP1s信号通路的活化及其引起的NLRP3炎症小体激活，减轻炎症反应。同时，ALA-PS显著增加PGC-1α、Nrf1和Tfam蛋白和mRNA表达，促进线粒体生物合成。并通过增加PPARα和CPT1A的蛋白表达，促进脂肪酸酸β-氧化分解。③ALA-PS显著增加肝脏磷酸化AMPK的蛋白表达水平。.2、细胞实验发现：①ALA-PS显著改善油酸诱导的HepG2细胞脂肪变性和氧化应激，效果明显好于植物甾醇和α-亚麻酸单独干预。②在分子水平，ALA-PS显著增加磷酸化AMPK、Nrf2和PGC-1α的蛋白表达水平，并相应地改善HepG2细胞脂肪变性和氧化应激。而给予AMPK的特异性抑制剂Compound C以后，ALA-PS增加磷酸化AMPK、Nrf2和PGC-1α蛋白表达的作用显著降低，改善HepG2细胞脂肪变性和氧化应激的作用明显减弱。说明，ALA-PS具有改善NAFLD的作用，其分子机制与激活AMPK，改善脂代谢和抑制氧化应激相关。.本项目的研究阐明了ALA-PS改善NAFLD的作用，丰富了ALA-PS的研究资料，为ALA-PS的营养作用提供理论支撑。同时，对深化我国油脂加工副产物植物甾醇和特色油料资源α-亚麻酸的利用，开发食源性健康活性天然成分，提高我国国民健康水平具有重要意义。