基于血管平滑肌细胞自噬探究“瓜蒌-薤白”药对抗动脉粥样硬化斑块形成的作用机制

In the book Synopsis of Golden Chamber (Jin Kui Yao Lue), the pair of Trichosanthes kirilowii (TK) and Allium macrostemon (AM) possesses the effects such as “ Activating Yang and Dispersing blood stasis , Regulating Qi and Resolving Phlegm”, is the classical traditional Chinese medicine prescriptions in treating chest distress. Atherosclerosis (AS) is closely connected with diseases in TCM such as chest distress, heartache, and so on. AS is also the pathological base of many cardio-cerebrovascular disease like coronary heart disease, myocardial infarction, etc. The pair of TK and AM has favorable clinical efficacy for AS, but its functionary mechanism is still unknown. Abnormal proliferation of vascular smooth muscle cells (VSMCs) is the main pathological process to form AS plaque. Autophagy is particularly important for the regulation of cell proliferation and function. Therefore, the present study intends to explore the mechanism that the pair of TK and AM against the formation of AS plaque via the autophagy pathway of VSMCs. ApoE-/- mouse AS model and the in vitro abnormal proliferation of VSMCs were studied respectively. Molecular biology, gene silencing and transfection were used to investigate the formation of AS plaque and the proliferation of VSMCs that caused by autophagy deletion or excessive autophagy. The role autophagy plays on proliferation of cells and the intervention effect of the pair of TK and AM are investigated. At the same time, the study on the mechanism of the pair of TK and AM alleviating the plaque by interfering with autophagy and exerting the anti-AS molecular mechanism are carried out. We hope to find out the new targets for the treatment of AS, provide scientific and objective experimental evidence for the treatment of chest distress by using the pair of TK and AM and explain the scientific connotation of traditional Chinese medicine compound.

瓜蒌-薤白药对载于《金匮要略》治疗胸痹的具有通阳散结、理气化痰功效的瓜蒌-薤白类经典方剂。动脉粥样硬化(AS)多与中医之胸痹等病证相联系，是冠心病、心肌梗死等众多心脑血管病的主要病理基础。瓜蒌-薤白药对AS有较好临床疗效，但具体机制不明。血管平滑肌细胞（VSMCs）增殖是AS斑块形成的主要病理过程，自噬对细胞增殖及功能的调控尤为重要。本研究拟从VSMCs自噬途径探讨瓜蒌-薤白药对抗AS斑块形成作用机制；以ApoE-/-小鼠AS模型，采用分子生物学、基因沉默和转染等技术研究自噬缺失或过度对AS斑块的影响，探讨自噬对细胞增殖的作用及瓜蒌-薤白药对的干预作用；同时研究其通过干预自噬减轻斑块而发挥抗AS的分子机制，以期发现瓜蒌-薤白药对治疗AS的新靶点，为其临床安全、有效使用瓜蒌-薤白类方治疗胸痹提供科学客观的实验依据，诠释中药复方的科学内涵。

瓜蒌-薤白药对载于《金匮要略》治疗胸痹的具有通阳散结、理气化痰功效的瓜蒌-薤白类经典方剂。动脉粥样硬化(AS)多与中医之胸痹等病证相联系，是冠心病、心肌梗死等众多心脑血管病的主要病理基础。瓜蒌-薤白药对AS有较好临床疗效，但具体机制不明。. 本研究从VSMCs自噬途径探讨瓜蒌-薤白药对抗AS斑块形成作用机制，分别以ApoE-/-小鼠AS模型及体外VSMCs异常增殖为研究对象，探讨自噬对细胞增殖的作用及瓜蒌-薤白药对的干预作用；同时研究其通过干预自噬减轻斑块而发挥抗AS的分子机制，以期发现瓜蒌-薤白药对治疗AS的新靶点。. 1、“瓜蒌-薤白”对AS小鼠主动脉斑块形成、VSMCs增殖及自噬的影响. “瓜蒌-薤白”药对减少AS小鼠主动脉斑块及脂质累积面积，减少小鼠主动脉内VSMCs标志性蛋白α-SMA及增殖相关蛋白PCNA表达，增加AS小鼠主动脉VSMCs内自噬相关蛋白LC3表达，促进小鼠主动脉VSMCs内自噬小体形成。. 2、“瓜蒌-薤白”通过上调细胞自噬抑制VSMCs异常增殖. 体外培养VSMCs，利用ox-LDL诱导VSMCs增殖；“瓜蒌-薤白”药对可抑制ox-LDL诱导的VSMCs增殖；“瓜蒌-薤白”促进体外VSMCs内自噬小体形成，增加自噬相关蛋白LC3表达且减少p62表达；自噬抑制剂氯喹可反转“瓜蒌-薤白”抑制VSMCs增殖的作用，表明“瓜蒌-薤白”抑制VSMCs异常增殖，与自噬机制密切相关。. 3、“瓜蒌-薤白”诱导VSMCs自噬的分子机制. 体外实验证明，“瓜蒌-薤白”抑制VSMCs的mPI3K、mAKT及mTOR蛋白磷酸化，PI3K激活剂740 Y-P的加入可反转“瓜蒌-薤白”对mAKT及mTOR蛋白的抑制作用，表明“瓜蒌-薤白”通过抑制PI3K/AKT/mTOR信号通路诱导VSMCs自噬。. 此研究揭示“瓜蒌-薤白”治疗AS的新机制，为其临床安全、有效使用瓜蒌-薤白类方治疗胸痹提供科学客观的实验依据，诠释中药复方的科学内涵。