血管平滑肌细胞自噬机制对动脉粥样硬化斑块形成影响及丹皮酚干预作用

Cell autophagy, which regulates inflammatory reaction, is a decisive factor of atherosclerosic plaque formation or rupture. Moderate autophagy inhibits inflammation and proliferation of vascular smooth muscle cells, while excessive autophagy leads to rupture of atherosclerotic plaque because of autophagic cell death. However, there is little scientific basis for vascular smooth muscle cells autophagy mechanism in developmental and pathological processes of atherosclerosis, and the corresponding drug intervention research is also extremely urgent. This project establishs a rabbit atherosclerosis model in vivo and a vascular endothelial cells - vascular smooth muscle cells coculture system in vitro based on the finished research of national natural science foundation. The aim is to investigate the processes of rabbit atherosclerosis and the effects of cell autophagy on vascular smooth muscle cells inflammation, proliferation, apoptosis under the stimulus of different concentration of oxidized low density lipoprotein. Cells culture, molecular biology, gene silencing and transfection are performed to explore the effects of autophagy deficiency or excess on PI3K/Akt/mTOR and PI3K/Beclin 1 signaling pathway. Simultaneously, the anti-atherosclerosis mechanisms of paeonol, a main active compound in radix of Cortex Moutan, are elucidated by interfering autophagy to increase plaque stability. This project provides scientific basis for new drug target of paeonol in clinical treatment on atherosclerosis.

细胞自噬对炎症有调控作用，是动脉粥样硬化（AS）斑块形成或不稳定的关键性因素。适度自噬可以抑制血管平滑肌细胞（VSMCs）炎症和增殖，而自噬的过度激活会导致细胞自噬样死亡引起斑块破裂，但VSMCs的自噬在AS发生发展中作用及具体调控机制至今鲜有科学依据，相应的药物干预机制研究更是亟待开展。本项目以已结题国家自然基金研究为基础，建立家兔AS模型及血管内皮细胞与平滑肌细胞体外共培养体系，探讨家兔AS模型的不同进程及ox-LDL不同浓度刺激下细胞自噬机制对VSMCs炎症、增殖和凋亡的影响；采用细胞培养、分子生物学、基因沉默和转染等技术研究自噬缺失或过度对PI3K/Akt/mTOR和PI3K/Beclin1通路的影响；同时研究清热解毒中药丹皮的有效成分丹皮酚通过干预自噬而减轻斑块或稳定斑块、发挥抗AS的分子机制，以期为有针对性使用药物治疗以及明确丹皮酚的作用靶点提供科学依据。

复制ApoE-/-小鼠动脉粥样硬化（AS）模型，观察丹皮酚（Pae）对AS不同阶段（斑块形成期和粥样斑块期）主动脉平滑肌细胞（VSMCs）增殖、凋亡和自噬影响。不同浓度ox-LDL诱导VSMCs增殖或凋亡，研究自噬在Pae对VSMCs异常增殖或凋亡作用和对AMPK/mTOR和 PI3K/Beclin 1通路调控，揭示自噬参与AS斑块形成过程病理机制及Pae调控VSMCs自噬分子机制。.1. 丹皮酚对AS小鼠主动脉斑块、VSMCs增殖/凋亡及自噬的影响.Pae减轻ApoE-/-小鼠主动脉（斑块形成期、粥样斑块期）斑块面积；减少VSMCs过度增殖/凋亡；增加VSMCsLC3II蛋白表达，促进自噬小体形成。.2. 丹皮酚抑制VSMCs异常增殖/凋亡的自噬机制.ox-LDL（50μg/mL）诱导VSMCs增殖，Pae减缓VSMCs细胞周期进程抑制增殖；显著诱导细胞内酸性囊泡生成，增加细胞内LC3II蛋白表达，加速p62蛋白降解，促进自噬小体数量增多，上调VSMCs自噬活性。自噬抑制剂氯喹并加入Pae后，可取消Pae抑制细胞增殖的作用。.ox-LDL（150μg/mL）诱导VSMCs凋亡，Pae降低VSMCs的凋亡率；增加LC3Ⅱ，减少p62、caspase-3表达。自噬抑制剂3-MA可抑制Pae诱导VSMCs自噬；3-MA并加入Pae后，能引起LC3Ⅱ蛋白水平表达下降、p62和caspase-3蛋白水平表达的上升和凋亡阳性细胞明显增多。.3. 丹皮酚调控VSMCs自噬的分子机制.Pae增加AS小鼠主动脉斑块形成期VSMCs内AMPK表达，抑制mTOR蛋白活性；Pae抑制体外VSMCs内mTOR蛋白表达，但AMPK抑制剂compound C并加入Pae，细胞内mTOR蛋白表达明显增加；siRNA沉默AMPKα2基因表达后，发现Pae诱导酸性囊泡形成、上调LC3II蛋白表达及加速p62降解作用减弱，并取消Pae抑制VSMCs异常增殖的作用。.Pae上调ox-LDL（150μg/mL）诱导VSMCs自噬通路ⅢPI3K和Becline-1蛋白的表达而激活该通路；对VSMCs进行PIK3c3和Becn-1的瞬时沉默，发现Pae降低LC3Ⅱ蛋白水平，增加p62和caspase-3蛋白水平，si-ⅢPI3K和si-Becline-1转染后可翻转丹皮酚抑制凋亡的作用。