COTL1通过调节ALOX5影响非酒精性脂肪性肝病发生发展的研究

Nonalcoholic fatty liver disease (NAFLD) is a recently prevalent liver disease that impairs public health. Recent study shows that 5-lipoxygenase/leukotriene pathway plays an important role during NAFLD progression, especially the transition from simple steatosis to nonalcoholic hepatitis.Key enzyme ALOX5 catalyzes leukotrienes (LTs) formation from Arachidonic acid. We previously found that COTL1 protein was consistently up-regulated at nonalcoholic simple fatty liver (NAFL), nonalcoholic steatohepatitis (NASH), NASH with fibrosis stages in mouse liver by proteomic technology. As a ALOX5 interacting protein, COTL1 can stablize ALOX5 and up-regulate ALOX5 activity. However, the contribution of COTL1 in NAFLD haven't been reported. In this study, we will firstly enhance or inhibit COTL1 expression through recombinant Lentivirus vector and RNAi, and then examine the influence of COTL1 over- or under-expression on mouse phenotype in various NAFLD stages by in vivo experiments and hepatocyte phenotype by in vitro experiments. At the same time, hepatic expression and activity of ALOX5, ALOX5 product LTs, effector molecules, and insulin resistance level will be assessed.Results may reveal that COTL1 can influence NAFLD progression through regulating ALOX5. The successful implementation of the project will provide in-depth understanding of the molecular mechanisms of NAFLD, and find a new therapeutic target for disease treatment.

非酒精性脂肪性肝病（NAFLD）是近年来影响人民健康的常见肝病之一。最新研究发现，参与花生四烯酸（AA）代谢的ALOX5通路在NAFLD发病过程，尤其在单纯性脂变向非酒精性脂肪性肝炎转变中发挥重要作用。我们前期运用蛋白质组学方法发现NAFLD各阶段小鼠肝脏COTL1表达上调。作为ALOX5相互作用蛋白，COTL1能稳定ALOX5、上调ALOX5活性，但COTL1在NAFLD中的作用尚未见文献报道。本研究拟采用重组慢病毒和RNAi等手段，分别探讨COTL1表达增强或抑制对体内实验NAFLD各阶段小鼠表型和体外实验肝实质细胞表型的影响，同时通过检测小鼠肝脏ALOX5表达和活性及其产物LTs和效应分子、胰岛素耐受水平，揭示COTL1可能通过ALOX5影响NAFLD发生发展。本课题的顺利实施将为深入认识NAFLD的分子机制，发现新的治疗靶标做出有益尝试。

目前非酒精性脂肪性肝病（Nonalcoholic fatty liver disease, NAFLD）发病率不断增高，然而其中的发病机制仍然不明。前期研究中，我们对MCD造模的NAFLD模型小鼠用双向差异凝胶电泳（difference gel electrophoresis, DIGE）发现一些差异表达蛋白，在NAFLD发生发展中找到96个差异表达蛋白（≥2.0或 ≤0.5, P<0.05），这些分子参与细胞骨架、抗氧化反应以及一些代谢过程等。其中，骨架蛋白COTL1在NAFLD单纯性脂变、脂肪性肝炎和轻度纤维化中表达上调。我们亦在人肝脏组织中验证COTL1表达上调。因此，我们研究敲低小鼠肝脏COTL1对于小鼠的NAFLD发生发展有无影响。首先，我们通过尾静脉高压注射siRNA成功建造了COTL1敲低小鼠模型，其次，我们发现敲低COTL1下调肝脏脂肪酸摄取分子CD36，脂肪酸合成酶FAS，和上调了参与β氧化的分子包括CPT, ACADL和 ACOX1等，这可以部分解释COTL1敲低小鼠肝脏脂肪含量和血清甘油三酯水平下降的表型；敲低COTL1下调一些炎症因子(MAC2, MCP1, TNFα and IL-1α)，能部分解释COTL1敲低小鼠血清ALT、AST水平降低即肝脏炎症减轻的表型。综上，首先， COTL1敲低减轻了肝脏脂肪变和肝脏炎症；其次，COTL1敲低减少了其相互作用蛋白ALOX5的水平，及其产物LTB4和一些脂肪代谢分子、促炎因子水平。此外，体外敲低AML12细胞COTL1并未改变细胞脂肪变程度。由此可见：NAFLD与COTL1过表达相关，敲低异常表达的COTL1能够减轻肝脏脂肪变和肝损伤，而单一的肝脏实质细胞难以发挥这个作用。