治疗NAFLD的icaritin小分子衍生物设计及其药理机制研究

Developing the efficient and low toxic drugs for the non-alcoholic fatty liver disease (NAFLD) is an urgent problem. Our previous study approved that icaritin regulated the lipid metabolism and the glucose metabolism in rats, and mediated the lipidosis by regulating the GLUT4 intracellular distribution and autophagy in NAFLD model cells. Icaritin is a main metabolite of the traditional Chinese medicine icariin. The intracellular distribution of GLUT4 and autophagy level in liver cells are closely related to the pathological process of NAFLD. This project intends to study the details of icaritin regulating the GLUT4 intracellular distribution and autophagy, and to design the highly efficient and low toxic drugs for NAFLD and to reveal their pharmacological mechanism. We would identify the target proteins of icaritin, analysis the crystal structures of the target proteins with their bound small molecule compounds, and design a series of icaritin derivatives as the small molecule drugs in this project. After screening of the small molecule compounds with the high binding ability to the target proteins and low cytotoxicity, the NAFLD model cells and animals would be used to evaluate the primary effect of the small molecule drugs for NAFLD. According to the existing cell signaling pathways of target proteins, we would investigate the effects of the drugs on GLUT4 distribution and autophagy in NAFLD model cells, and reveal their pharmacological mechanism for NAFLD.

研发高效低毒的非酒精性脂肪肝(NAFLD)治疗药物是一个急需解决的问题。课题组前期研究发现中药淫羊藿苷代谢产物淫羊藿素(icaritin)具有调节大鼠脂代谢和糖代谢的功效；并且icaritin调节NAFLD模型细胞内GLUT4分布和细胞自噬。肝脏细胞内GLUT4分布和细胞自噬与NAFLD病理进程紧密相关。本项目拟详细研究icaritin对细胞内GLUT4分布和细胞自噬的调控作用，设计高效低毒的NAFLD治疗药物并揭示其药理作用机制。项目将鉴定icaritin作用的靶点蛋白，根据小分子与靶点蛋白结合的晶体结构信息，设计合成icaritin衍生小分子药物。筛选与靶蛋白结合能力强和细胞毒性小的药物作用NAFLD细胞模型和动物模型，评估其治疗NAFLD的初步效果。结合靶点蛋白现有信号通路研究药物对NAFLD模型细胞内GLUT4分布和细胞自噬调节作用，揭示它们治疗NAFLD的药理作用机制。

项目按照研究计划完成如下既定目标：.1.鉴定出淫羊藿素（icaritin）治疗NAFLD的靶蛋白AMP依赖的蛋白激酶（AMPK）和雌激素受体α（ERα）；.2.以icaritin为基础，筛选出五个具有良好降脂效果的小分子药物（淫羊藿素、柚皮素、槲皮素、补骨脂素、罗通定）.3.揭示以上小分子药物对AMPK的直接激活作用和通过ERα间接上调AMPK活性，调控氧化应激和线粒体自噬，进而缓解NAFLD的分子机制。.本项目主要围绕icaritin对NAFLD进展的两个关键点——肝内脂代谢和氧化应激诱发的肝细胞损伤，开展机制探索和药物作用验证。.项目通过高脂饮食建立NAFLD大鼠模型和小鼠模型，油酸钠和棕榈酸钠构建高脂压力的L02和Huh7肝细胞脂质沉积模型，用于药物对细胞内脂质堆积的药效研究；应用3T3-L1脂肪细胞和C2C12多核肌管细胞模型，探索药物对NAFLD相关胰岛素抵抗的调控作用；通过测试细胞内ROS水平、线粒体形态和数量、细胞自噬水平等进行亚细胞水平的药物作用探索；结合以上功能关键分子NQO1、SOD1、SOD2、SIRT1、PGC-1α、TFAM等的检测，阐释药物作用的可能细胞信号通路；通过AMPK 抑制剂、ERα调节剂、siRNA敲降AMPK和ERα等方法，验证药物通过这两个关键分子发挥效应。为了进一步阐明药物对ERα的作用，应用摘除双侧卵巢的去势手术后雌性小鼠的NAFLD模型进行药物与雌激素的类比研究；同时制备肝脏特异性Ers1（ERα）基因敲除小鼠，用于药物作用的验证。.研究结果表明，icaritin等药物改善NAFLD大鼠和小鼠模型肝脏气球样病变和肝细胞内脂质沉积，且此作用使依赖于AMPK和ERα。icaritin等药物经由靶蛋白促进细胞的葡萄糖摄取，缓解胰岛素抵抗；增强线粒体自噬，促进细胞内脂质和损伤线粒体的分解；降低细胞内氧化应激，减缓NAFLD病程。本项目也说明了AMPK和ERα等关键分子在肝内脂质堆积，增强线粒体自噬和新生，缓解氧化应激，阻滞NAFLD发展等方面具有良好的调控作用。.项目的执行完成既定研究目标，发表期刊论著6篇及综述2篇，申报专利3项，参与出版学术专著1部（负责编撰其中1个章节）；培养毕业硕士研究生3人，博士研究生4人；1位主研人晋升为副教授。