基于VSMCs自噬信号通路PI3K/AKT/mTOR探讨复方中药单体生物可降解支架对小型猪冠状动脉内膜增殖的影响

Aiming at the risks of 10％～20％restenosis and thrombosis in the drug eluting stent after which were implanted in coronary artery, we prepared the biodegradable stents have been coated with Paclitaxel and Hirudin which come from Taxus Chinensis and leech respectively, the herbs that effects is for detoxicating, expelling wind-evil and removing extravasated blood, according to the theory of “Stirring of endogenous lateral wind”on atherosclerosis. Using the miniature swine coronary artery stent model, we prepare to study the inhibition of the biodegradable stent to the injury , autophagy and endarterium hyperplssia of artery. At the same time, test the activation and expression of PI3K/Akt/mTOR signaling relating genes. By using human coronary artery smooth muscle cells (HCASMC) proliferation induced by thrombin inhibitors of VSMCs, combined with autophagy downstream of the PI3K/Akt/mTOR pathway related molecules in different conditions, then the correlations were studied between the activation of PI3K/Akt/mTOR signaling pathway relating downstream molecules and the proliferation and migration of HCASMC. The effect of Paclitaxel and Hirudin to the activated HCASMC and related indices were observed. It would be investigated that the effectiveness and molecular mechanism of the biodegradable stents coated with Paclitaxel and Hirudin to prevent the restenosis and thrombosis in the drug eluting biodegradable stent after PCI.

针对药物支架仍有10％～20％的再狭窄率及血栓形成风险，我们依据络风内动学说，选择解毒祛风、活血破瘀的红豆杉和水蛭组成复合物，用其单体成份紫杉醇和水蛭素，制备复方中药单体生物可降解支架。本团队既往发现，此支架能通过抗VSMCs增殖起到抗再狭窄作用，该过程可能与细胞自噬有关，然而具体机制未明。我们基于预实验结果，提出假说：复方中药单体生物可降解支架抗再狭窄作用是通过调控自噬PI3K/Akt/mTOR通路，抑制VSMCs增殖迁移实现。本研究采用球囊拉伤+高脂饲养法制备小型猪冠心病模型，于病变部位置入对应支架，探究该支架对诱导血管损伤及内膜增生的抑制作用并观测PI3K/Akt/mTOR通路表达变化。另用凝血酶诱导HCASMC增殖，联合自噬因子抑制剂探讨自噬通路下游分子在不同条件下的活化状态与SMC增殖、迁移的相关性，揭示复方中药单体生物可降解支架防治PCI术后再狭窄的有效基础及分子机理。

本研究是针对当前生物可吸收支架（BRS）植入后支架内再狭窄（ISR）及支架血栓发生率高的现状开展。ISR的形成机制复杂，其中平滑肌细胞的表型转变、迁移、增殖起到了重要的作用。多种因素（如凝血酶等）可通过PI3K/Akt/mTOR信号通路影响自噬，诱导平滑肌细胞的过度迁移、增殖，形成ISR。王显教授基于“络风内动”病机学说，提出了“络风宁0号（LFN-0）”冠脉支架洗脱药物涂层，用于抑制支架植入后ISR取得了良好的效果，但其应用于抑制BRS相关ISR的疗效及安全性需进一步确认，且其抑制ISR的机制需进一步探讨。.本研究中将络风宁0号药物涂层涂覆于BRS基质表面，制备了复方中药单体涂层生物可吸收支架作为试验用支架，同时制备用于阳性对照的依维莫司涂层支架及裸支架。通过向小型猪冠状动脉模型中植入三种支架诱导ISR形成，并在随访终点造影，病理取材等评估抑制ISR的疗效；基于动物在体实验与细胞离体实验两部分，通过检测自噬相关分子标记物及PI3K/Akt/mTOR信号通路关键分子表达，探讨其抑制ISR的机制。.研究结果显示： LFN-0涂层经改良后与BRS基质相容性良好，成功制备LFN-0 BRS。LFN-0 涂层可有效抑制BRS植入后ISR，抑制BRS植入术后平滑肌细胞增生及局部炎症反应；除外程序性因素，LFN-0 BRS表现出良好的安全性；机制上，BRS-ISR中自噬水平降低，可能伴有自噬流阻滞；LFN-0可上调BRS植入后ISR病变组织中的自噬水平，可能是通过抑制PI3K/Akt/mTOR信号通路中的PI3KR1与上游位点的结合或其他上游机制发挥通路抑制作用，从而激活自噬。.本研究的实际意义在于为临床中BRS植入后预后不良的问题提出了一种可能的解决方案，为后续开展LFN-0 BRS的临床研究提供了数据支持。