降血脂活性天然产物荜茇宁特异性靶标的确定及其作用机理研究

Piperlonguminine has significant lipid-lowering activity. However, its mechanism of action is still not clear. The aim of this project is to identify the targets of piperlonguminine in different cell types in different species and study the mechanism of antihyperlipidemic effect using activity-based protein profiling, biological mass spectrometry, and RNA interference technology. We will use chemical probes of piperlonguminine to enrich its protein targets in liver cells and macrophages of human, mouse and rat, and analyze and identify specific protein targets in different cells by mass spectrometry. Further, we will validate the identified targets of piperlonguminine by RNA interference and illustrate their function during cellular lipid synthesis, absorption and efflux, and explore the possible mechanism and cellular networks of the targets. This research will shed light on the mechanism of antilipidemic activity of piperlonguminine and provide substantial knowledge for natural product-based drug development in the future.

荜茇宁（piperlonguminine）具有显著的降血脂作用，但其作用机理还不清楚。本项目拟利用活性蛋白表达谱分析（Activity-Based Protein Profiling，ABPP）技术、生物质谱分析技术、RNA干扰技术等手段确定不同物种、不同细胞内荜茇宁的靶标及其降血脂作用机理。通过利用荜茇宁化学探针，亲和富集荜茇宁靶标蛋白群并运用生物质谱分析，来研究人、小鼠、大鼠的肝脏细胞和巨噬细胞内荜茇宁的靶标谱，确定荜茇宁在不同组织中的靶标蛋白组。进一步地，利用RNA干扰技术，验证荜茇宁靶标在细胞内脂肪合成、摄取及流出过程中的功能，实现系统阐述荜茇宁降血脂作用的机理及其细胞通路的目标，为开发高效、安全的天然产物降血脂药物提供重要的线索和理论依据。

荜茇宁（piperlonguminine）是中药荜茇中的有效成分，具有显著的降血脂作用。为了阐明荜茇宁的作用机制，本项目设计、合成并表征了四种基于生物活性的荜茇宁小分子探针，可用于活性蛋白表达谱分析（Activity-Based Protein Profiling，ABPP）法确定荜茇宁的靶标蛋白组合。我们确定了荜茇宁ABP探针的光学反应性，并采用不同探针在不同物种、不同细胞内荜茇宁的可能靶标进行了分析。结果显示，荜茇宁ABP探针在细胞内外均能够与蛋白质进行交联反应，但是由于ABP探针中荜茇宁活性基团部分过于小，难于检测到特异性靶标。SDS-PAGE分析显示，荜茇宁ABP探针与对照探针（不含荜茇宁基团的ABP探针）在总蛋白中交联产生的富集物差异不大。本项目执行期内发表了6篇第一标注的论文。