TLR4参与糖尿病动脉粥样硬化的分子机制研究

Recent, some studies showed that TLR4 expression increase,platelets function change and endothelial cell function injury in diabetes. However, It is needed to clear that the effects of TLR4 expression on platelet function change and endothelial cell injury in diabetes with atherosclerosis.We have found that the high dose of glucose increased TLR4 expression on platelets and endothelial cells. Simultaneously, our result showed that the high concentration of glucese induced PF4 and CD40L release, and PECAM-1 expression in platelets. Similar results were obtained in high glucose treated endothelial cells. The high dose of glucose also challenged PECAM-1 expression in HUVECs. Furthermore, we found that gikgolide B, an inhibitor of PAF receptor,inhibited TLR4 expression induced by high dose of glucose in platelets and HUVECs, respectively. PAF （platelet activating factor）is an endogenous proinflammatory cytokine, which involved in the multifarious inflammatory responses. Therefore, we speculate that PAF receptor might be involved TLR4 activation in platelets and endothelial cells. In the present study, we attempt to explore that the effect of PAF on TLR4 expression in glucose-treated platelets and endothelial cells. To explore the relation of signaling molecule Akt, p38MAPK, and Erk, we will determine the phosphorylation of Akt, p38MAPK, and Erk in glucose-treated platelets or endothelial cells. Meanwhile, we will observe the effect of ginkgolide B on TLR4 expression and above molecules as well as. In addtion , we will perform animal expreienment to investigated the atherogenesis in the ginkgolide B treated db/db mice (a leptin receptor defective mice) and positive mice.We suggested that inhibiting TLR4 expression in platelets and endothelial cells might be a potential therepeutic stategy for preventing atherosgenesis in diabetes.

糖尿病时出现TLR4增高、血小板功能亢进、内皮细胞功能损伤等现象，然而，TLR4表达增加对血小板功能、内皮细胞功能以及对糖尿病动脉粥样硬化的影响尚不清楚。我们在前期工作中发现高糖增加TLR4在血小板和内皮细胞表达，还观察到高糖刺激血小板炎症蛋白释放、内皮细胞粘附蛋白表达，而抑制PAF受体则减少高糖刺激的TLR4表达。因此，本课题提出PAF受体参与高糖刺激TLR4活化的假说，分析PAF受体在高糖刺激TLR4活化中的作用；探讨Akt、p38MAPK、Erk等信号分子对TLR4表达的调控作用；探讨银杏内酯B抑制PAF受体对上述信号分子的影响。本课题还将利用db/db基因缺陷小鼠，建立2型糖尿病动脉粥样硬化小鼠模型。通过分析各组小鼠的斑块面积、TLR4、PECAM-1、VCAM-1等炎症蛋白表达，评价银杏内酯B干预对糖尿病小鼠动脉粥样硬化的影响。本课题对于糖尿病动脉粥样硬化的防治研究具有重要意义。

本课题探讨了TLR4参与糖尿病动脉粥样硬化的分子机制，TLR4为TLR家族的成员，参与天然免疫和获得性免疫，在机体多种细胞均有表达。在免疫性细胞的研究表明TLR4是一个革兰氏阴性杆菌代谢产物脂多糖的特异性受体，外来抗原感染时诱导TLR4活化，通过MyD88、TRIF接头蛋白的信号转道，激活转录因子NF-KB炎症通道活化，产生炎症蛋白。近年研究显示TLR4不仅对外来刺激产生炎症反应，对内源性有害产物的刺激同样能够产生炎症反应。动脉粥样硬化是一个系统性血管炎症性疾病，主要表现在大动脉出现粥样斑块，以及局部炎症。可见到大量巨噬细胞浸润、泡沫细胞形成，平滑肌细胞增生，血小板黏附、血管结构紊乱，纤维帽形成等多种改变。动脉粥样硬化的早期主要为内皮细胞功能损伤以及内皮细胞的炎症蛋白表达增加。然而，分子机制尚不完全清楚。糖尿病患者是动脉粥样硬化的高发人群，本研究重点高糖是如何诱导内皮细胞炎症反应以及免疫应答的分子机制。本研究按照研究计划在细胞水平探讨了高糖损伤内皮细胞的炎症表型变化以及介导的分子机制，并探讨了PAF受体抑制剂银杏内酯B对高糖损伤内皮细胞的保护作用。研究结果提示高糖处理内皮细胞有明显的炎症表征，使用银杏内酯B干预能够逆转高糖损伤的内皮细胞炎症，其分子机制与抑制TLR4/NF-κB/STAT3通道活化相关。本研究还发现高糖诱导信号分子Akt, p38 MAPK磷酸化，银杏内酯B能够抑制这些信号分子的磷酸化。此外，我们还发现银杏内酯B能够抑制高糖诱导的内皮细胞凋亡。动物实验也显示，银杏内酯B对db/db糖尿病小鼠的血管炎症蛋白表达具有抑制作用。提示银杏内酯B是一个多靶点药物，对高糖刺激内皮细胞功能损伤具有保护作用。