豁痰解毒通络方干预实验性AS家兔颈动脉PTCA术后内膜增生的金属硫蛋白-内质网应激-自噬机制研究

Coronary artery endovascular stent angioplasty after restenosis has become a hot topic and difficult in the field of cardiovascular research. Its mechanism is complex, is still not clear. The preliminary work of this group found that phlegm detoxification Tongluo can significantly inhibit the carotid artery balloon injury after neointimal proliferation. Based on this study, we established real-time fluorescence quantitative qPCR, Western Blot, immunofluorescence staining, scanning electron microscopy, and so on. Based on the establishment of rabbit hyperhomocysteinemia and carotid artery atherosclerosis induced by balloon injury in the early stage To study the key mechanism of endothermic metallothionein-endoplasmic reticulum stress-autophagy after PTCA in experimental AS rabbits, and to demonstrate the mechanism of phlegm and blood stasis , Which is related to the progress of the "endoplasmic reticulum stress-autophagy-target organ damage" event chain proposed by modern medicine. Which provides a new idea for better prevention of restenosis after PTCA. And to lay the theoretical foundation and experimental basis for the prevention and treatment of restenosis after percutaneous transluminal coronary angioplasty.

经皮冠状动脉腔内血管成形术后再狭窄成为心血管领域的研究热点和难点。其机制复杂，目前仍不明确。本课题组前期工作发现，豁痰解毒通络饮能够显著抑制大鼠颈动脉球囊损伤后新生内膜增殖。基于此，本项目在前期建立兔高同型半胱氨酸血症及结合球囊损伤颈动脉致动脉粥样硬化模型基础上，采用实时荧光定量qPCR、Western Blot、免疫荧光染色、扫描电镜等细胞分子生物学技术，研究豁痰解毒通络饮对实验性AS家兔颈动脉PTCA术后内膜增生的金属硫蛋白-内质网应激-自噬的关键机制，并论证“痰瘀伏络、蕴结成毒、毒损脉络”为科学假说，拟与现代医学提出的“内质网应激-自噬-靶器官损害”事件链的进展相关。为更好的防范PTCA术后再狭窄提供了新的思路。同时为经皮冠状动脉腔内血管成形术后再狭窄中药防治奠定理论基础和实验依据。

经皮冠状动脉腔内血管成形术后再狭窄成为心血管领域的研究热点和难点。其机制复杂，目前仍不明确。本课题组前期工作发现，豁痰解毒通络饮能够显著抑制大鼠颈动脉球囊损伤后新生内膜增殖。基于项目研究基础，建立蛋氨酸喂养加颈总动脉球囊损伤术后的AS模型，模拟RS的形成，研究内质网应激-自噬对于此模型的作用机制；研究中药豁痰解毒通络饮对于此模型及内质网应激-自噬的干预作用。实验采用了2%L-蛋氨酸喂养加球囊损伤兔颈总动脉成功建立AS模型，应用血管超声检测、HE染色、Masson染色观察颈总动脉内膜增生情况；透射电镜观察兔颈总动脉内膜超微结构；酶联免疫吸附测定法（ELISA）检测兔血清中同型半胱氨酸（Hcy）的含量；实时荧光定量聚合酶链反应（Real-time PCR）检测：①各组兔颈总动脉组织中的内质网应激标志分子GRP78、PERK、eIF2a、IRE1α、XBP1的mRNA水平；②各组动脉组织中自噬标志分子beclin1、Atg12、ULK1的mRNA水平。5.蛋白免疫印记法（Western blot）检测：ERS标志分子GRP78、PERK、eIF2a、p-eIF2a 、IRE1α、XBP1以及自噬标志分子beclin1、Atg5、Atg12、ULK1在颈总动脉组织中的蛋白分子的表达水平。实验结果表明：2%L-蛋氨酸喂养加球囊损伤兔颈总动脉成功建立AS模型，成模率高，病变恒定，可用于模仿人类PTCA术后再狭窄的机制研究。豁痰解毒通络饮可以明显改善兔颈动脉粥样硬化，对球囊损伤后的内膜增生有明显抑制作用。豁痰解毒通络饮能够降低兔血清中Hcy的含量。内质网应激(ERS)-自噬信号传导通路分子在球囊损伤术后AS模型中的过度表达，豁痰解毒通络饮能够抑制ERS-自噬通路的过度激活。豁痰解毒通络饮是以痰瘀伏络学说为理论基础组方而成，而此方对于AS模型有明显的干预作用，说明PTCA术后AS与痰瘀伏络学说密切相关，为中药防治PTCA术后再狭窄提供新的治疗靶点和理论基础、实验依据。