基于核受体PPARβ与网络药理学研究淫羊藿苷调节老年脂代谢的机制

Lipid metabolism disorder seriously affects the health of the elderly. Lipid-lowering drugs of western medicine have more side effects and more likely to rebound after withdrawal. Our previous studies found that icariin can improve the abnormal lipid metabolism in aging and has long-lasting effect, and indicated the effect is related to the PPARβ. Combined with the previous base, we put forward the hypothesis that icariin regulates lipid metabolism directly by "drug -target" to activate PPARβ, and regulate lipid metabolism indirectly by multi-target network to have a lasting effect..This program will be based on chemical biology and network pharmacology approach. First, surface plasmon resonance (SPR) technology will be used to determine the affinity between icariin and PPARβ receptor. Second, reporter gene technology will be applied to clarify its function. Finally, the isobaric tags for relative and absolute quantitation (iTRAQ) technology will be employed to study its biological effects. This study will illustrate the mechanism of icariin lowering mechanism from "drug - target". Moreover, network pharmacology technology will be applied to research the network mechanisms of icariin regulating lipid metabolism from the "multitarget - network". The program will interpret the scientific connotation of icariin regulating lipid metabolism in aging by the molecules recognize each other between drug and biological target, the transmission of information, biological function and networks. It is expected to work out new mechanisms of lipid-lowering drugs. The program give full play the advantages of Chinese medicine regulating lipid metabolism and has a major significance for preventing and treating cardiovascular and cerebrovascular diseases of the elderly.

脂代谢紊乱性疾病严重影响老年人的身心健康，现代医学降脂药副作用多，停药后易反弹。而上一基金发现补肾药淫羊藿苷能改善衰老的脂代谢，且有疗效持久的优势。分子对接提示与核受体PPARβ有关，结合前期基础提出：淫羊藿苷可能通过PPARβ而直接调节衰老脂代谢，并通过多靶点及网络的间接作用重塑脂代谢而产生持久的疗效。.本计划拟应用表面等离子体共振生物传感技术检测淫羊藿苷与PPARβ的亲和力，确定能否结合；以报告基因技术明确其功能性质-激活或抑制；以同位素标记相对和绝对定量研究其生物效应；从药物-靶点论证淫羊藿苷基于PPARβ的降脂机制。同时应用网络药理学技术，从多靶点-网络角度揭示淫羊藿苷疗效持久的机理。本计划充分发挥中医药调节脂代谢的优势，从药物与生物分子的相互识别、信息传递、生物效应及网络药理诠释淫羊藿苷调节衰老脂代谢的科学内涵，有望研究出新作用机制的调脂新药，对于老年心脑血管疾病的防治意义重大。

本项目提出淫羊藿苷（Icariin, ICA）可能通过PPARβ而直接调节脂代谢，并通过多靶点及网络的间接作用重塑脂代谢而产生持久疗效的假说。由于ICA主要以代谢物淫羊藿次苷Ⅱ（Icarisid Ⅱ，ICSⅡ）吸收，并且体内ICA的含量显著低于ICSⅡ。根据ICA的药代动力学特征，从ICA及其代谢物ICSⅡ开展研究。.从直接和间接两方面研究ICA的调节脂代谢机制。首先研究发现低剂量ICA可抑制3T3-L1细胞脂质沉积，提高PPARβ的表达量，而高剂量具有相反作用。通过SPR检测发现ICA与PPARβ无结合作用，利用双荧光素酶报告基因发现ICA对PPARβ无激活作用，提示ICA对PPARβ无直接作用机制。其次发现低剂量ICA可逆转PPARβ抑制剂诱导的脂质沉积，并提高PPARβ的表达，证实ICA通过PPARβ间接调控脂代谢的机制。采用转录组、蛋白组、蛋白互作等技术发现低剂量ICA可通过Wnt信号通路间接调控PPARβ mRNA。选取Wnt信号通路中关键基因β-Catenin，通过抑制剂验证了ICA通过Wnt信号通路调控PPARβ，诠释了ICA间接调控脂代谢的作用机制。为了阐明不同剂量ICA具有相反调脂作用的机制，以转录组、蛋白组学检测高、低剂量的差异基因/蛋白，发现低剂量ICA对PPAR mRNA的剪切（α、β、γ三种亚型）产生了影响，提高PPARβ的表达而有利于调节脂代谢。此外研究还发现ICSⅡ也具有调脂作用，其机制与ICA类似，与ICA不同的是ICSⅡ的调脂作用有剂量依赖性。.模拟ICA体内药代动力学特征，选取低剂量ICA+高剂量ICSⅡ对3T3-L1细胞进行干预，与模型组相比，结果发现ICA+ICSⅡ组脂滴较单药组减少更明显，证实二者之间具有协同作用。另外网络药理学研究发现ICA可能通过参与花生四烯酸代谢、胰岛素抵抗、脂肪消化吸收、氨基酸代谢等多条信号通路改善脂代谢。提示ICA降脂疗效持久的机制除与ICA的多靶点网络作用有关外，还与ICA药代动力学特征有关。.本项目阐释了ICA基于PPARβ间接调节脂代谢的机制，还发现与代谢物ICSⅡ有协同降脂作用，将中药疗效机理研究推进到代谢物层次，实现中药药理与药代动力学的紧密结合；有望挖掘出基于PPARβ的降脂新药，对于老年心脑血管疾病的防治意义重大。