髓样分化蛋白-2（MD2）在糖尿病肾病（DN）中的作用及MD2抑制剂防治DN的机制研究

Chronic inflammation plays an important role in the progress and development of diabetic nephropathy (DN). TLR4/MD2 complex is a direct mediator in endotoxin (LPS)-induced inflammation. Recently, studies on DN patients and animal showed the overexpression of TLR4 in renal issues and the in vitro evidences demonstrated that TLR4 was involved in high glucose (HG)-induced inflammation. However, whether MD2 plays a role in HG-inflammation and DN is yet unkown. . In the previous studies on anti-inflammatory drug design and evluation, our lab found three novel molecules (A12, C12, and D33), which directly targeted MD2 protein and possessed excellent anti-inflammatory activity in LPS-stimulated models. Importantly, oral administration of compound D33 was found to significantly attenuate DN in type 1 diabetic rats. . In view of these results, we hypothesize here, that MD2 may play an important role in the development of diabetic renal inflammation and DN, and inhibition of MD2 may contribute to the treatment of DN. . A series of studies are designed to test and perform our hypothesis. These include, 1) to investigate the TLR4/MD2-dependent mechanism by which D33 inhibits HG-induced inflammation in renal epithelial cells and macrophages, 2) to probe the MD2-mediated pharmacology and pharmacodynamics of three molecules (A12, C12, and D33) in type 1 diabetic nephropathy mice, 3) and to demonstrate the important role of MD2 in DN development using MD2-/- mice. . There is an urgent demand to develop more reliable drug target and treatment in diabetic comlications containing DN. Completion of this challenge project not only has high scientific impact on understanding the MD2-involved mechanism in diabetic renal inflammation and DN, but also provides new target and strategy for the treatment of DN.

慢性炎症是糖尿病肾病（DN）的主要病理因素之一。TLR4/MD2复合物是内毒素LPS急性炎症的直接介导物，也有研究表明TLR4在DN肾中高表达，并介导高血糖诱导的肾组织炎症反应。但是MD2是否参与其中？关于MD2在DN炎症中的作用尚无报道。本实验室前期工作寻找到三个可以直接结合和抑制MD2，并具有优秀抗内毒素炎症活性的探针小分子；其中小分子D33可以显著缓解1型糖尿病小鼠肾损伤。基于此，我们假设：MD2在DN的炎症病理进程中有着重要的介导作用，抑制MD2可以缓解DN的发生发展。本项目中，我们拟利用细胞实验探讨MD2在D33抑制高糖炎症反应中的介导作用其对TLR4的依赖；利用动物实验探讨三个探针小分子通过抑制MD2-炎症通路缓解DN的药理药效；利用MD2-/-小鼠，探讨MD2靶点在DN发生发展中的重要性。项目实施将明确MD2介导的炎症反应在DN发生发展中的重要作用，为DN防治提供新的靶点。

慢性炎症是糖尿病肾病的主要病理因素之一。TLR4/MD2复合物是内毒素LPS急性炎症的直接介导物，也有研究表明TLR4在糖尿病肾病中高表达，并介导高血糖诱导的肾组织炎症反应。但是MD2是否参与其中？关于MD2在糖尿病肾病炎症中的作用尚无报道。在本课题中，我们首先利用多种分子生物学手段确证了L6H21是一个MD2特异性小分子抑制剂，并且阐明了L6H21与MD2蛋白的结合机制；在体外L6H21可以抑制LPS诱导的MAPKs磷酸化和NF-κB的激活，在体内L6H21可以缓解小鼠脓毒性死亡和脓毒症诱导的肺损伤。接着，我们在体外培养大鼠肾小管上皮细胞NRK-52E，确证了L6H21可以抑制高糖诱导的肾细胞炎症、黏附分子和趋化因子表达以及巨噬细胞浸润；并且在动物体内灌胃给予L6H21可以缓解糖尿病引起的肾损伤，其保护作用是通过抑制糖尿病引起的肾脏炎症和巨噬细胞浸润。进一步我们利用小干扰RNA技术和MD2抑制剂发现沉默MD2可以抑制高糖诱导的肾素-血管紧张素（RAS）系统的激活，且依赖于MD2/TLR4-MyD88信号通路；在体内我们发现灌胃给予MD2小分子抑制剂L6H9后缓解糖尿病引起的RAS系统激活和肾损伤；为了完全确证MD2对糖尿病肾病发病机制的介导作用，我们用MD2基因敲除小鼠发现MD2基因敲除后可缓解糖尿病引起的RAS系统激活以及肾损伤。本项目的实施不仅明确了MD2介导的炎症反应在糖尿病肾病发生发展中的重要作用，为糖尿病肾病防治提供新的靶点，同时为糖尿病肾病的治疗提供新的候选药物。