调肝导浊方调控内皮细胞AMPK通路防治动脉硬化的机制

Endothelial cell function is regulated by AMPK pathway，under the action of the oxidative modification of lipoprotein ( OX-DL )，Inhibition of the AMPK pathway, leading to endothelial dysfunction, then initiating of atherosclerosis ( AS ) and cardiovascular events. Preliminary studies and clinical data showed" Tiaogandaozhuo Decoction" with the protection of endothelial cell function, preventing and treating of AS, but the mechanism has not been elucidated. So putting forward a hypothesis that preventing the happening of AS. "Tiaogandaozhuo Decoction" regulating of" endothelial dysfunction" on the AMPK pathway.In vivo and in vitro experiments, establishment of ApoE4 AS knockout mouse model and OX-DL induced endothelial cell injury model. Study of" Tiaogandaozhuo Decoction" is through activation of the AMPK pathway upstream material threonine kinase ( LKBl ), increased endothelial AMP / ATP ratio, so as to adjust the channel, improving endothelial cell function. Application of AMPK pathway inhibitor Compound C and agonist AICAR further role in the pathway, from positive and negative two respects verified" Tiaogandaozhuo Decoction" is by adjusting the AMPK pathway, improving endothelial cell function. The subject's aim is to elucidate Tiaogandaozhuo Decoction prevention and treatment of AS's the mechanism, has the important value to provide evidence that preventing of cardiovascular and cerebrovascular events.

内皮细胞功能受AMPK通路调节，在氧化性修饰脂蛋白（OX-DL）作用下，抑制AMPK通路，导致内皮细胞功能失调，启动动脉粥样硬化（AS）及心脑血管事件发生。前期研究和临床资料显示"调肝导浊方"具有保护内皮细胞功能、防治AS发生，但机制尚未阐明。提出"调肝导浊方"基于AMPK通路，调节 "内皮功能失调"，防治AS的假说。在体和离体实验相结合，建立ApoE4基因敲除小鼠AS模型和OX-DL致内皮细胞损伤模型，研究"调肝导浊方"是否通过激活AMPK通路上游物质苏氨酸激酶（LKBl），增加内皮细胞AMP/ATP比值从而调节该通路、改善内皮细胞功能；并运用AMPK通路抑制剂Compound C和激动剂AICAR进一步作用于该通路，从正反两个方面验证"调肝导浊方"是否通过调节AMPK通路，改善内皮细胞功能。课题对于阐明调肝导浊中药防治AS的作用机制，为其预防心脑血管事件发生提供证据，具有重要价值。

动脉粥样硬化（AS）是多种心脑血管疾病的病理生理基础，而内皮细胞损伤是动脉粥样硬化发生的始动因素。内皮细胞具有强大的分泌功能，其分泌的保护因子对心血管疾病具有保护效应。研究证实AMPK通路与内皮细胞内分泌功能密切相关，一旦内皮细胞的分泌功能失调，AMPK通路将被抑制，促进AS以及进一步的心血管事件发生。本课题运用调肝导浊方干预动脉粥样硬化内皮功能失调这一关键阶段，通过激活AMPK信号通路，旨在调节失调的内皮功能。.本课题以自发动脉粥样硬化的ApoE-/-小鼠为研究对象，经调肝导浊方干预后，①运用小动物超声心动检测血管相关功能和结构改变；②运用HE，油红O检测血管和肝脏的形态学变化；③运用生化法检测血清和肝脏匀浆血脂相关指标；④运用基因芯片方法，检测调肝导浊方作用靶点。结果证实调肝导浊方可以防治AS，其作用机制与调节能量代谢、脂代谢、炎症，抗氧化等有关.在调肝导浊方疗效确定基础上，Elisa法检测内皮功能失调相关指标，运用RT-PCR和蛋白印迹，以血管和肝脏为靶器官，分别检测AMPK通路相关指标，结果证实调肝导浊方可以激活AMPK通路，调节紊乱的内皮功能，防治AS。为进一步验证调肝导浊方调节内皮功能是否依赖于AMPK通路，体外实验以人主动脉内皮细胞株为研究对象，通过OX-LDL，TNF-α诱导内皮功能失调，并以AMPK激动剂和抑制剂进行反面验证，观察调肝导浊方调节AMPK通路对内皮细胞功能失调的影响。结果证实：调肝导浊方具有调节内皮功能紊乱作用，这一作用与激活AMPK信号通路有关。结论：调肝导浊方可以调节内皮功能紊乱防治AS，其机制与激活AMPK通路有关。