雷公藤红素调控PPARγ/Wnt5a信号通路介导的细胞自噬在抗动脉粥样硬化中的作用及机制

Atherosclerosis (AS) is the main pathological basis of ischemic cerebrovascular diseases such as coronary heart diseases. In the concept of traditional Chinese medicine, syndrome of intermingled phlegm and blood stasis is the main pathological mechanism to AS with lipid deposition as the initiative factor. While the imbalance of intracellular cholesterol transport is an essential factor in the development of AS and the capacity of reverse cholesterol transport plays a key role in determining the process and outcome of AS, autophagy is a primary way to regulate intracellular RCT. Recent reports have revealed that celastrol as traditional Chinese medicine monomer exhibits good druggability and it can play the anti-atherosclerosis role by promoting RCT. However, so far the underlying mechanism remains unclear. Our previous study has confirmed that celastrol can enhance autophagy in lipid-loaded cells. Hence our present proposal is based on the hypothesis that celastrol can promote RCT to exert the role of anti-atherosclerosis by activating autophagy. Specifically speaking, we believe that celastrol can target PPARγ/Wnt5a to induce autophagy via PKC/mTOR/TFEB signaling pathway and as a result accelerate RCT. This project will establish lipid-loaded cell and AS mice models, and then focus on observing the effect of celastrol on autophagy and RCT at the cell and the whole level. Thereafter, it will endeavor to identify the underlying targets of celastrol by using molecular docking analysis and pull-down assays. Furthermore, it will clarify the potential mechanism by small molecule interference or agonist in established stable cell lines using retroviral vector infected systems. By accomplishing this proposal, we expect to identify potential targets and new mechanisms for celastrol as a traditional Chinese medicine, to advance the anti-atherosclerosis drug development, and to provide novel approaches in developing traditional Chinese medicines.

动脉粥样硬化(AS)是冠心病等缺血性心脑血管疾病的重要病理基础。中医学“痰瘀互结”是AS主要病理机制，其始动因素表现为脂质沉积。胆固醇逆向转运(RCT)的能力决定AS的进程，而细胞自噬是RCT调节的重要方式。近年报道具有良好成药性的中药单体雷公藤红素（CeT）通过促进RCT抗AS，但机制未明。前期发现，CeT可诱导荷脂细胞发生自噬，故提出“CeT介导的细胞自噬促进RCT”的思路：即CeT靶向PPARγ，转录调控Wnt5a，经PKC/mTOR/TFEB信号通路，诱导细胞自噬，促进RCT。本研究拟建立荷脂细胞及apoE敲除小鼠AS模型，分别在细胞和整体水平观察CeT对自噬和RCT的影响；采用分子对接分析和pull-down技术，鉴定其作用靶点；采用病毒载体构建稳定细胞株，予以小分子干扰或激动剂阐明分子机制。该研究将揭示CeT作为传统中药单体抗AS的潜在靶点和新机制，为中药研发提供新的靶点和思路

动脉粥样硬化（atherosclerosis，AS）可引起缺血性心脏病、缺血性脑卒中和外周动脉疾病等，其主要病理特征是动脉内膜斑块的形成。血管平滑肌细胞和巨噬细胞来源的泡沫细胞积极参与脂质摄取和加工，极大地促进了斑块的产生。其中，胆固醇逆向转运（reverse cholesterol transport，RCT）是调控AS发展和进程的关键。同时，血管平滑肌细胞异常增殖引起的内膜增生在血管重塑的发病机制中扮演着重要作用。近年来，雷公藤红素展现出显著的抗动脉粥样硬化作用，但其机制尚不明确。本项目在国家自然基金的资助下，从脂质代谢和细胞增殖角度分别探讨雷公藤红素抑制动脉粥样硬化和血管再狭窄的作用及相关机制的研究。首先，通过建立泡沫细胞模型，采用雷公藤红素以及工具药等予以干预，发现雷公藤红素可通过促进LXR/ABCA1信号通路和激活自噬，减少血管平滑肌细胞和巨噬细胞衍生的泡沫细胞形成，从而抑制动脉粥样硬化。此外，通过建立血管平滑肌细胞增殖细胞模型及血管内膜增生小鼠模型，发现雷公藤红素可通过抑制Wnt5a/PKC/mTOR信号通路，促进平滑肌细胞自噬进而降解c-MYC和p62形成的结合物，最终减少血管平滑肌细胞的异常增殖及血管内膜增生，抑制血管再狭窄的发生发展发挥抗动脉粥样硬化作用。上述提示，雷公藤红素可作为防治心血管疾病的潜在药物，其相关靶点和通路可用于进一步的新药研究。此外，我们拓展领域发现雷公藤红素通过诱导LXR/ABCA1通路促进肾透明细胞癌中脂自噬，并诱导CAV-1/LOX-1通路减弱肾癌的脂质蓄积和细胞干性，抑制肾癌发生发展等。该项目的成果以科研论文、项目资助和青年优秀论文报告等形式提供等形式提供，已经在《Biochemical and Biophysical Research Communications》、《International Journal of Biological Sciences》、《Acta Pharmacologica Sinica》、《Frontiers in Pharmacology》等国内外核心期刊发表论文十余篇，培养研究生多名