杜仲提纯物通过泛素蛋白酶通路途径在动脉粥样硬化机制中发挥的作用

Atherosclerosis (AS) is a common pathogenesis of coronary heart disease, hypertension, stroke and other common diseases, which could seriously affects health. We have found the Chinese medicine Qingre Huayu traditional prescription could prevent the expression of inflammatory protein in atherosclerotic mouse model by ubiquitin protease system, and the pre-experimental study showed Eucommia ulmoides extract could reduce the progress of atherosclerosis. This study was to further investigate the effect of Eucommia ulmoides extract on the inflammatory response of coronary atherosclerosis in atherosclerotic Apo E knockout mice and we detected the expression of the related factors such as nuclear transcription factor-1 (MMP-9), nuclear factor-1, matrix metalloproteinase-9 (MMP-9), matrix metalloproteinase-9 (MMP-9) (NF-KB), tumor suppressor gene-53 (P53), intercellular adhesion molecule (ICAM), tumor suppressor gene-27 (P27) and tumor necrosis factor-related activator protein (CD40L)by tissue staining, Real-Time PCR, Western Blot, fluorescence staining, micro-ultrasound, immunohistochemistry and Pathological morphology. To explore the molecular mechanism how Eucommia ulmoides extract affects atherosclerosis through ubiquitin protease system. The aim of this project was to identify the effect of Eucommia ulmoides extract on coronary atherosclerotic ubiquitin protease pathway in atherosclerotic Apo E knockout mice and elucidate the mechanism of inflammatory molecular mechanism to provide intervention and treatment for atherosclerosis.

动脉粥样硬化是冠心病、高血压、中风等常见多发病的病理学基础，严重影响人类健康。我们在前期研究发现清热化瘀中药方可通过泛素蛋白酶体系阻止动脉粥样硬化小鼠模型炎症蛋白表达，预实验提示贵州道地药材杜仲提取物可抑制动脉粥样硬化。故本研究旨在进一步观察杜仲提取物对动脉粥样硬化Apo E基因敲除小鼠冠状动脉粥样硬化炎症反应的影响。评估小鼠大动脉病理形态学改变及冠状动脉周围的炎症浸润程度，通过组织染色，Real-Time PCR, Western Blot，荧光染色，显微超声技术，免疫组织化学等方法测定基质金属蛋白酶-9(MMP-9)、核转录因子(NF-KB)、抑癌基因-53(P53)、细胞间黏附分子(ICAM)、抑癌基因-27(P27)、肿瘤坏死因子相关激活蛋白(CD40L)的表达。本项目旨在研究杜仲提取物对动脉粥样硬化Apo E基因敲除小鼠冠状动脉粥样硬化泛素蛋白酶通路的影响，阐明其影响炎症分子机制。

泛素蛋白酶体系统(ubiqutin proteasome system, UPS)是泛素蛋白酶途径是ATP依赖性的，细胞内非溶酶体途径蛋白降解通路，在生理及病理生理中均发挥重要作用。贵州道地药材杜仲是杜仲科植物杜仲的干燥树皮，自古以来被列为滋补药物上品，谓之能“补中益气、强志、坚筋骨，久服轻身耐老”，故能防病治病，对于心血管疾病的预防及治疗亦有很好的作用。.. 本研究应用杜仲提取物及蛋白酶抑制剂，分别阻止泛素蛋白酶信号通路，通过ELISA、免疫印迹法、western blot、免疫荧光及定量PCR方法干预相关炎症因子基质金属蛋白酶-9(MMP-9)、核转录因子(NF-KB)、抑癌基因-53(P53)、细胞间黏附分子(ICAM)、抑癌基因-27(P27)、肿瘤坏死因子相关激活蛋白(CD40L)的表达情况，探讨杜仲提取物在动脉粥样硬化血管中对于泛素蛋白酶体系炎症因子的影响，原位杂交以及蛋白质印迹技术从基因及蛋白水平探讨泛素蛋白酶在动脉粥样硬化斑块内的表达情况，通过腺病毒转染表达，以及 RNA干扰技术siRNA载体作用以进一步明确杜仲提取物通过泛素蛋白酶体系在抑制动脉粥样硬化中的作用。结果提示：经过杜仲提取物干预后，治疗组2、4月组小鼠CH较高脂组显著减低(P<0.05)，MMPS、CD54/ICAM-1、p53、CD40L/CD154、p27蛋白表达程度在干预后1、2、4月呈现降低趋势）。结论：杜仲提取物能一定程度上降低APOE-/-小鼠血清胆固醇浓度，其途径可能通过抑制UPP相关蛋白表达实现。