回医七藤脉宁方对颈动脉斑块形成大鼠脑缺血微血管损伤和HMGB1 /TLR4 /NF-κB 炎症通路的影响

Based on the study of the "Mucous Mass" theory of Hui Medicine and the research of Mai Ning pill being recorded in the medical literature of Hui Hui Prescription, Qi Teng Mai Ning recipe （QTMN recipe）of Hui Medicine was composed and used. It was showed that patients’ carotid artery plaques were suppressed. Results of clinical observation confirmed that the plaques greater than 19.0 mm x 3.5 mm was reduced and less than 9.6 mm x 1.7 mm were removed after the treatment of QTMN recipe. By the other animal experiments, it was showed that QTMN recipe performed the significant effect on the resistance of rats’thrombosis. On the basis of preliminary clinical and animal experiments, scientific hypothesis will be proposed in this study. Vine herbs were the main drug in QTMN recipe, which could perform the function of dredging the collateral vessel, eliminating rheumatism and stabling artery. The first scientific hypothesis is that QTMN recipe could perform the effects on resisting the carotid plaque and thrombosis by inhibiting inflammatory cytokines. The second scientific hypothesis is that QTMN recipe performed the role of Traditional Chinese Medicine in removing the wind, viscosity and reducing the viscosity, which may be related to the intervention of inflammatory pathway of High-Mobility Group Box1/ Toll-Like Receptors 4/Nuclear Factor-kappa B（HMGB1/TLR4/NF-κB）, and then to reduce the inflammatory mediators. In the next step, vascular endothelial injury would be inhibited which were mediated by Protein Kinase C（PKC）signaling molecules. As the results, the cerebral ischemia capillaries injury being caused by carotid plaque could be reduced by QTMN recipe. The subject would takes rats as the research object and will be performed by three steps. The first is to confirm the inhibition to rats’carotid plaque and thrombosis of QTMN recipe being based on the animal model replication of carotid plaque formation （single model）. The second is to study the role of QTMN recipe in protecting the brain capillaries against injury after cerebral ischemia. Rats’ischemia model（double model）was prepared on the carotid plaque model, and the effects of QTMN recipe on the cerebral ischemia capillaries basement membrane and Matrix Metalloproteinase （MMPs ）were observed. The third is to explore the effects of QTMN recipe on HMGB1/TLR4/NF- kappa B inflammatory pathway and PKC signal molecules. The study can provide scientific evidence for the atherosclerosis treatment of classic prescription of Hui Medicine.

基于回医“黏液质”理论和《回回药方》“脉宁丸”研究而组成的七藤脉宁方，可抑制患者颈动脉斑块形成，减小19mm×3.5mm以上、清除9.6mm×1.7mm以下斑块；实验发现其抗血栓形成作用明显。本课题在临床和动物实验基础上，提出科研假说：以“通络消痹宁脉”藤类药为主的七藤脉宁方可通过抑制炎症因子而发挥抗颈动脉斑块和血栓形成作用；该方可减轻颈动脉斑块形成后脑缺血微血管损伤，其机制可能与“祛风除湿降黏”干预HMGB1/TLR4/NF-κB 炎症通路，减少炎性介质，进而抑制PKC介导的血管内皮损伤有关。课题以大鼠为研究对象，分为三步：复制颈动脉斑块形成动物模型（单模），证实该方抑制颈动脉斑块和血栓形成作用；颈动脉斑块模型上制备脑缺血模型（双模），观察对脑微血管基底膜和MMPs的作用；观察对HMGB1/TLR4/NF-κB炎症通路和PKC信号分子的影响。研究可为回医经典方剂治疗动脉粥样硬化提供依据。

目的 研究七藤脉宁方防治颈动脉斑块对脑缺血损伤是通过影响炎症通路相关因子活化而发挥脑保护作用。.方法.①本次实验所用的SPF级雄性SD(Sprague Dawley)大鼠全部由宁夏医科大学提供。将大鼠随机分为七藤脉宁方组、阿司匹林组、阿托伐他汀钙组、模型组、假手术组和正常组，每组大鼠10只，除正常组外，其余各组大鼠分别完成颈动脉斑块模型制备，除假手术组和正常组，其余大鼠在颈动脉斑块模型基础上再完成中动脉缺血模型制备；颈动脉斑块模型完成后的第6周，将正常组和模型组大鼠，分别用兽用B超和HE染色检测模型制备状态。.②模型制备：模型一用高脂饲料完成颈动脉斑块模型大鼠制备，及时注射维生素D3辅助斑块形成。模型二采用线栓法完成大脑中动脉闭塞(Middle cerebral artery occlusion, MCAO)模型大鼠于缺血2h后缓慢拔出线栓再灌注。.③各组大鼠于造模前禁食12h， 造模完成后，模型组和假手术组用生理盐水灌胃，其余三组大鼠分别用对应药物灌胃，并分别于药物干预后的1d、3d、7d取材；.④实验中各大鼠参照文献报道的8分评分标准进行神经功能损伤测评；用苏木精伊红（HE）染色观察神经细胞的形态。用尼氏染色观察神经元凋亡的情况。应用脑含水量计算公式，对各组大鼠脑组织含水量进行统计分析。应用免疫组化法分析MMP-2/MMP-9的炎性表达。用Western Blot检测颈动脉斑块形成大鼠脑缺血再灌注中HMGB1/TLR4/NF-κB信号通路、PKC/PKC、MMP-2/MMP-9蛋白表达；用ELISA检测颈动脉斑块形成大鼠脑缺血再灌注损伤后脑组织TNF-α、IL-1β、IL-6的含量。.结果.1.根据神经功能测评法测定，与正常组和假手术组比较，各手术组神经功能测评损伤显著（P＜0.01），提示手术组模型制备成功；.2.HE染色：与正常组相比，模型组血管内膜增厚，内皮细胞脱落，炎性细胞浸润，伴钙化形成、脂质沉积及大量脂肪泡沫细胞及条状纹路，提示模型制备成功；.3.B超状态：与正常组相比，模型组颈动脉见血管粗糙，斑块回声较大，分布较模糊，走形较差，斑块长度及面积变化较明显，证明模型制备过程进展顺利；.4.脑组织HE染色：与空白组和假手术组相比，模型组的大鼠脑组织则大面积梗死，脑组织染色变浅，大量的神经元细胞骨架破坏，形态皱缩，空泡结构出现、细胞质中的嗜酸性较前增强、细