NAFLD逐渐进展的新机制：高表达Fibronectin诱导ER stress加剧肝细胞脂肪变性及损伤

NAFLD is a chronic liver disease which closely related to metabolism, stress and shows trends in the incidence of globalization. In China, NAFLD has become the primary cause of abnormal liver function. In recent years, people have found that the risk of NAFLD decreased in rats with Fibronectin (FN) deficiency. On the contrary, the uptake of lipids by liver cells was obviously increased in high expression FN, but its mechanism was not clear enough. Studies have confirmed that sustained ERS played a critical role in inducing development of NAFLD. This project will investigate the relationships between ERS and NAFLD, FN and ERS, hypothesize that FN is the endogenous factor inducing ERS and could aggravated NAFLD disease progression via ERS signals pathways regulating lipid intake and apoptosis. We plan to isolate and culture liver cells from NAFLD model rats, induce the pathological process of NAFLD in vitro and explored the mechanism by which FN induces ERS from molecular level. Furthermore, the project will clarify the mechanisms that FN aggravates hepatocyte fatty degeneration, activates the specific signaling pathways including JNK and CHOP, and promotes cell damage. We aim to lay scientific foundation for improving the pathogenesis of NAFLD and provide new target spot for intervention treatment of NAFLD.

NAFLD是与代谢、应激密切相关的慢性肝病，呈全球化扩展趋势，已成为我国肝功能异常的首要病因。近来研究发现，Fibronectin(FN)缺失的小鼠罹患NAFLD的危险性降低，相反，高表达FN的肝细胞脂质摄取能力却明显增强，但具体作用机制尚不清楚。研究证实：持续的ERS是导致NAFLD进展的重要机制。本课题从ERS与NAFLD，FN与ERS的联系入手，提出FN是导致肝细胞发生ERS的内源性因素，并通过ERS相关信号通路影响肝细胞脂质摄取和凋亡,从而加剧NAFLD病程进展的假说。项目拟从NAFLD模型大鼠中分离培养肝细胞，更好的在体外实验中还原NAFLD的病理过程,并从分子水平探讨FN诱导ERS的可能途径与机制。阐明FN加剧肝细胞脂肪变性，激活JNK、CHOP等信号通路，促进细胞损伤的作用机制，为进一步完善NAFLD的病理机制奠定理论基础，并为干预和治疗NAFLD提供新的靶点。

研究背景：非酒精性脂肪肝病（NAFLD）是以肝实质细胞弥漫性脂肪病变为基本病理特征，并导致过量的脂质沉积的慢性肝病，呈全球化扩展趋势，已成为我国肝功能异常的首要病因。以往研究证实，纤维粘连蛋白（Fibronectin, FN）是肝脏分泌的细胞因子，参与调节胞外基质形成，损伤修复，炎症反应等重要生物学事件。值得注意的是，含EDA剪切区的FN（EDA+FN）在高脂负荷的肝细胞中呈特异性高表达，且加剧了肝组织的纤维化进展，但具体作用机制尚不清楚。本课题聚焦于EDA+FN与NAFLD发生的关联研究，及其与NAFLD相关的代谢性内质网应激（ER stress）中调控的分子机制研究。研究内容及结果：首先，通过横断面研究分析血清中EDA+FN含量与NAFLD的相关性，初步评价其对NAFLD的预测价值。67例基线无NAFLD的受试者血清中EDA+FN浓度为3.25 μg/ml，而106例NAFLD患者血清中EDA+FN浓度为5.01 μg/ml，显著高于正常对照组。ROC曲线分析显示血清EDA+FN水平对NAFLD有较好的预测价值（曲线下面积为0.82, 95%CI 0.76-0.88）。此外，我们还在动物及细胞水平检测了EDA+FN和ER stress标志蛋白GRP78, ATF4以及CHOP的表达及关联，明确ER stress对EDA+FN表达及被动分泌的调控作用。在细胞实验中，我们进一步阐明ER stress 引起UPR过程中，CHOP扮演了“双刃剑”的角色，通过促进肝细胞坏死，加剧了EDA+FN释放。结论：1、人体血清EDA+FN有望成为预测NAFLD的新型血清标志物；2、肝脏脂肪变性引起内质网应激及EDA+FN的表达上调，活化CHOP信号通路，诱发肝细胞坏死，导致EDA+FN释放入外周血。在此基础上，我们推测EDA+FN作为了一种重要的介质，参与了NASH炎症微环境中肝细胞，kupffer细胞以及肝星状之间的crosstalk，促进了纤维化进展。