白藜芦醇抑制血小板金属蛋白酶分泌和促进自噬蛋白表达的分子机制研究

Atherosclerosis is vascular inflammatory disease in aging. Platelets play a key role in development of atherosclerosis. In our previous studies we found that inhibition of platelet function might reduce plaque area in ApoE gene defective mice. Based on the studies, in the present study, we will focus on the effect of resveratrol on the expressions of metalloproleinase and TLR4 in ox-LDL-activated platelets. Sirt1 is an important molecule in cell including platelets. Resveratrol is an agonist of Sirt1. We also investigate that the effect of resveratrol onSirt1, MMP2, MMP3 and MMP9. Furthermore, we evaluate the effect of EX527 (Sirt inhibitor) on the expression of MMP2, MMP3 and MMP9 in ox-LDL-activated platelets. To determine the effect of resveratrol on autophage in ox-LDL-stimulated platelets we will examine the effect of resveratrol on the autophage-related protein mTOR, LC3II and BECN1 expression, and potent mechanism in ox-LDL-activated platelets. Final, to detect the protective effect of resveratrol on atherosclerosis, we will do animal experiments. Young and elder mice will be fed by high fat diet for eight weeks, Plasma MMP2, MMP3 and MMP9 will been measured by ELISA kit. Immunohistochemical assay, oil red O and HE staining will be used to measure inflammatory protein expression, collagen and plaque area in aorta of mice. The study will provide new evidence for prevention and treatment for atherosclerosis.

动脉粥样硬化是一个随年龄增长而发病的血管炎症性疾病，血小板在动脉粥样硬化炎症的启动过程中起十分重要的作用。我们在前期工作中证实，抑制血小板功能可以减少ApoE基因敲除小鼠主动脉斑块面积以及炎症蛋白表达。本研究以此为基础，探讨白藜芦醇抑制ox-LDL诱导的血小板活化，分析ox-LDL刺激血小板金属蛋白酶（MMP2、MMP3、MMP9）分泌，TLR4活化，Sirt1表达降低的现象，并使用白藜芦醇干预分析对血小板炎症蛋白及保护性分子的影响。本研究还将探讨ox-LDL对血小板自噬功能的影响，重点分析mTOR、 LC3II和BECN1等自噬相关蛋白的表达以及白藜芦醇的影响，并评价白藜芦醇对ox-LDL刺激血小板自噬功能的影响是否与STAT3活化和Sirt1相关。此外，我们还将评价白藜芦醇干预对高脂喂养年轻小鼠以及高龄小鼠动脉粥样硬化、金属蛋白酶表达的影响。本研究对于动脉粥样硬化的防治具有重要意义。

心血管疾病是目前全球性死亡率最高的疾病，动脉粥样硬化是心血管事件的重要病理基础，防治动脉粥样硬化的发生与进展仍是医学亟待解决的重要课题。近年研究表明，血小板在动脉粥样硬化血管炎症过程中起十分重要的作用，然而相关分子机制尚不完全清楚。ox-LDL是动脉粥样硬化的风险因子，MMPs水平增高促进血管胶原组分的降解，参与动脉粥样硬化过程。本研究重点探讨了ox-LDL诱导血小板炎症反应以及对MMPs的影响，并重点研究了白藜芦醇通过抑制血小板功能，减轻血管炎症，维护血管稳态的作用。研究结果显示，ox-LDL能够促进血小板TLR4高表达，增加MMP3, MMP9表达和分泌，而白藜芦醇抑制了这些炎症蛋白的表达。酪氨酸激酶Syk参与血小板的多种活化反应，炎症小体NLRP3是诱导IL-1β分泌的作用信号分子。我们的结果显示，白藜芦醇能够抑制TLR4/Syk/NLRP3通道活化，增强血小板自噬功能，减少血小板分泌MMP3, MMP9。动物实验表明，白藜芦醇能够降低小鼠血清MM3, MMP9水平、降低血管组织MM3, MMP9表达，能够抑制高脂饮食诱导小鼠血管细胞衰老变化，对血管胶原结构紊乱的病变具有保护作用。这些研究结果提示抑制血小板功能对于动脉粥样硬化的防治具有重要作用。