活性多糖TY772通过PPARα信号通路调控脂质稳态延缓动脉粥样硬化斑块形成的分子机制

降脂是治疗动脉粥样硬化（AS）的有效手段。前期工作表明：多糖粗品（CMPS）显著促进胆固醇逆向转运（RCT），下调小鼠血脂水平，减少apoE(-/-)小鼠主动脉和肝脏脂质蓄积；CMPS促RCT抗AS主要活性成分为TY772，已获得其模拟结构；生物芯片数据提示上述功效与PPARα信号通路介导的脂稳态有关，具体机制不明。我们提出TY772通过PPARα信号通路抑制胆固醇合成与吸收、促进脂肪酸氧化代谢和RCT，从而调控脂质稳态并延缓AS斑块形成的科学假说。本项目拟借助糖苷酶和现代分析技术阐明TY772的精细结构，采用多种细胞模型、CETP-tg小鼠、拟人化基因工程仓鼠、CETP-tg×apoE(-/-)小鼠，结合siRNA技术与PPAR拮抗剂和激动剂，在分子、细胞与生理学水平上验证上述假说。期待锁定TY772调脂抗AS的靶蛋白，为开发我国自主知识产权、构-效关系明确的多糖类降脂药物提供理论支撑。

The patients with cardiovascular and cerebrovascular disease (CCVD) increase year by year, and atherosclerosis (AS) induced by hyperlipidemia is the basic pathological change of CCVD, while lipid-lowering is an effective way for prevention and treatment of AS. Our preliminary experiments indicated that the polysaccharides from Cordyceps militaris (CMPS, 50 mg/Kg) could decrease the lipid level and increase the 3H-labelled cholesterol transport in C57BL/6 mice, reduce the lipid accumulation in the aorta and liver of the apolipoprotein E knockout (apoE-/-) mice, and the in vitro 3H-labelled cholesterol efflux activity screening experiment showed that TY772 with molecular weight of 772 kDa is the most effective one among the polysaccharides separated from CMPS, which elevates reverse cholesterol transport (RCT) in cholesterol ester transfer protein (CETP) transgenic (CETP-tg) mice (CETP is closely related to lipid metabolism in human, while mice lack the CETP gene; provided by Dr. Jiang Xian-cheng at SUNY University, New York, America). We have presumed the structure of TY772 with methylation and 1D,2D-NMR analysis. Furthermore, the microRNA and mRNA biological chips indicated that the effect of CMPS may be associated with its lipids regulating and influence on PPARα signaling pathways. Based on our preliminary results, we hypothesis that TY772 could inhibit the synthesis and adsorption of cholesterol, improve β-oxidation and metabolism of fatty acids, and elevate RCT. However, the fine molecular characteristics of TY772 and the involved mechanisms are not clear. This item is designed to elucidate the fine structure and its characteristic oligosaccharides of TY772 by 1 and 2 dimensional nuclear magnetic resonance (1D, 2D-NMR), methylation analysis, and selective degradation with mild acid and glycosidase; clarify the lipid-lowering mechanisms of TY772 mediated by PPARα signaling pathways with the help of siRNA and PPARs agonist and antagonist, based on multiple cell models and CETP-tg mice (CETP is closely related to lipid metabolism in human, while mice lack the CETP gene; provided by Dr. Jiang Xian-cheng at SUNY University, New York, America) and genetic engineered hamster (with similar lipid profile with human being, provided by Dr. Liu Guo-qing at Peking Universtiy) models, combining with molecular biological techniques (RT-PCR, Western blot), lipidomics by LC-MS/MS with LipidViewTM software (Version 1.2, ABI SCIEX), proteomics by LC-MS/MS after dimethyl labelling, and biochips provided by related companies; verify the protective effect and mechanisms of TY772 on the basic CCVD pathological change “AS” by apoE(-/- )×CETP-tg mice and genetic engineered hamster. The expected results could clarify the regulating targets of TY772 for lipid-lowering and AS protection, provide lipid-lowering drug with specific structure-activity relationship and attain independent intellectual property rights.