基于络脉理论从AMPK-SIRT1-NK-kB信号途径研究灸法抗血管内皮细胞损伤防治动脉粥样硬化形成机制

researches have revealed that collaterals have close relationship with the structures and functions of blood vessel, also phlegm, stagnation and toxin are the significant mechanism and critical link of cardiovascular diseases. Therefore, treating cardiovascular diseases from collaterals is very crucial. Atherosclerosis (AS) is the important pathological basis and critical link of cardiovascular diseases. Vascular endothelial cell damage is the initial factors of AS, while the silent information regulator 1 (SIRT1) signal pathway plays vital regulating effects in vascular endothelial damage. Previous studies indicate that moxibustion at suitable thermal stimulation can enhance activity of AMP-activated protein kinase (AMPK) in the vascular endothelial cells which relate to collaterals, facilitate secretion of SIRT1 to achieve the biological effects of dredging collaterals, promoting circulation of blood and anti-aging. This project catches hold of the anti-atherosclerosis formation mechanism in AS researches about inhibiting vascular endothelial injury, in the project AS model rabbit as research object and moxibustion on Shenque (CV8) as stimulating method. Then we are going to ascertain the entire action effect and target spot of moxibustion that treat AS at the integral level through small animal living imaging, which based on "stimulation outside to generate regulation inside". Finally, we will apply biological technological means to study the mechanism how moxibustion inhibit vascular endothelial damage to realize anti-atherosclerosis from signal pathway of AMPK-SIRT1- NF-κB, including immunohistochemical, FQ-PCR and Western blotting. We hope this project can provide some novel thinking to enrich the collateral theory and intensify preventing and treating AS with moxibustion.

研究揭示络脉与血管在结构和功能上密切相关，而痰瘀毒阻络是心血管病重要机制，由此从"络"论治至关重要。动脉粥样硬化(AS)是心血管疾病的关键环节，其中血管内皮细胞损伤是AS病变的始动因素，沉默因子（SIRT1）信号途径在血管内皮细胞损伤中起重要调控作用。前期研究表明灸法适宜温热刺激能增强络脉相关血管内皮细胞的AMP激活的蛋白激酶（AMPK）的活性，促进SIRT1分泌达到通络行血、抗衰老等生物学效应。本项目抓住抑制血管内皮损伤抗AS形成机制，以AS模型家兔为研究对象，神阙灸为刺激方法，用小动物活体成像技术从整体层面探明灸法治疗AS的"刺激于外，调节于内"的整体作用效应和靶点；使用免疫组化、FQ-PCR）和Western blotting等生物学技术手段从AMPK-SIRT1- NF-κB信号途径研究灸法抑血管内皮损伤抗AS形成机制，为丰富络脉理论和深化灸法防治AS提供新的思路。

本项目抓住抑制血管内皮损伤抗AS形成机制，以AS模型家兔为研究对象，神阙灸为刺激方法，用免疫组化等生物学技术手段从AMPK-SIRT1- NF-κB信号途径研究灸法抑血管内皮损伤抗AS形成机制。第一部分，通过观察温和灸对动脉粥样硬化家兔一般情况、血脂系列，主动脉弓部、主动脉内膜形态学及超微结构，血清相关炎性因子TNF、MCP-1、VCAM-1含量，家兔主动脉组织AMPK-SIRT1-NFκB信号通路中相关因子的基因表达及蛋白表达，结果表明，温和灸可改善动脉粥样硬化家兔活动情况、食欲情况、皮毛情况及大便形态等一般情况，对胆固醇代谢具有调节作用，对主动脉组织形态及超微结构有一定修复作用，降低动脉粥样硬化家兔血清TNF-α、VCAM-1及MCP-1含量，减轻炎性损伤，促进内膜修复；上调SIRT1含量，促进SIRT1 mRNA和蛋白表达上调，抑制炎症，促进内膜修复，降低NF-κB含量，下调NF-κB mRNA和蛋白表达，减轻内皮损伤，揭示温和灸抗动脉粥样硬化作用途径可能通过AMPK- SIRT1-NF-κB实现，为温和灸促进主动脉内膜损伤修复、减轻炎性损伤的作用机制提供了一定的实验基础,为温和灸发挥生物学效应提供理论依据。第二部分观察不同温度刺激对络脉相关血管内皮细胞生长活力变化，ET-1、TM、PGI2含量，脐静脉血管内皮细胞Caspase-3 表达的影响，结果灸法的温热刺激可下调脐静脉血管内皮细胞CasPase － 3 表达、降低血管内皮细胞ET-1的释放，促进血管内皮细胞TM、PGI2的释放，增强AMPK活性，促进AMPK基因及蛋白表达上调，激活能量开关，为温和灸产生生物学效应奠定一定基础。第三部分为循经取穴电针“内关”、“通里”穴对稳定型心绞痛患者临床症状及血清MMP-9、TIMP-1与DSCT中对斑块值的影响，结果表明循经取穴电针“内关”、“通里”穴能显著减少患者心绞痛发作次数，发作疼痛程度，降低患者血清MMP-9含量、升高血清TIMP-1含量与降低CACP体积、CACP当量块、CACS的趋势，可能通过影响血清MMP-9与TIMP-1含量变化参与发挥稳定AS斑块及抗AS进程，为基础研究选用内关、通里等穴位作为研究助学提供临床依据。