髓样分化蛋白-2（MD2）在胰岛素抵抗（IR）中的作用及以MD2为靶点缓解IR的机制研究

Elevation of serum free fatty acid (FFA) -induced chronic inflammation plays an important role in the progress and development of diabetic insulin resistance (IR). Compared with endotoxin LPS, FFA also has effective long-chain fatty acid.TLR4/MD2 complex is a direct mediator in endotoxin (LPS)-induced inflammation. Recently, studies on diabetic patients and animals showed the overexpression of TLR4 in hepatic and adipose issues and evidences also demonstrated that TLR4 was involved in high FFA-induced insulin resistance. However, whether MD2 plays a role in high FFA-induced diabetic IR is yet unkown. In the previous studies on anti-diabetes drug design and evluation, our lab found a novel compound (NH-66), which directly targeted MD2 protein and possessed excellent anti-inflammatory activity in LPS-stimulated models. Importantly, oral administration of compound NH-66 was found to significantly attenuate IR in high fat diet-induced type 2 diabetic rat. In view of these results, we hypothesize that, MD2 may play an important role in the development of diabetic insulin resistance, and inhibition of MD2 may contribute to the treatment of IR. A series of studies are designed to test and confirm our hypothesis. These include, 1) to confirm the selective inhibition of three novel molecules on MD2 protein in macrophages, 2) to investigate the TLR4/MD2-dependent mechanism by which NH-66 inhibits high FFA-induced insulin resistance in hepatic cells and adipocytes, 3) to probe the MD2-mediated pharmacology and pharmacodynamics of NH-66 in fatty diet-induced diabetic IR, 4) and to demonstrate the important role of MD2 in IR development using MD2-/- mice. There is an urgent demand to develop more reliable drugs target and treatment in diabetic syndromes containing hepatic and adipose IR. Completion of this challenge project not only has high scientific impact on understanding the MD2-involved mechanism in diabetic IR, but also provides a new target and strategy for the treatment of IR.

高血脂（FFA）引起的慢性炎症是糖尿病胰岛素抵抗（IR）的主要病理因素之一。有研究表明Toll样受体（TLR）4在炎症介导IR过程中发挥关键作用，TLR4/MD2复合物是LPS急性炎症的直接介导物。但是MD2在IR中的作用尚无报道。课题组前期工作找到可以直接结合和抑制MD2，并具有优秀抗炎活性的探针小分子NH-66；并且可以显著缓解2型糖尿病小鼠肝脏IR。基于此，我们假设：MD2在IR的病理进程中有着重要的介导作用，抑制MD2可以缓解IR的发生发展。本项目中，我们拟利用细胞实验确证NH-66小分子探针对MD2的靶点性，探讨MD2在NH-66抑制FFA诱导炎症-IR反应中的介导作用；利用2型糖尿病小鼠实验探讨NH-66通过抑制MD2缓解IR的药理药效；利用MD2-/-小鼠，探讨MD2靶点在IR发生发展中的重要性。项目实施将明确MD2在糖尿病IR发生发展中的重要作用，为IR防治提供新的靶点。

胰岛素抵抗（Insulin resistance, IR）通常是指机体在胰岛素刺激下葡萄糖吸收和利用能力降低或丧失的一种病理表现。2型糖尿病患者胰岛素抵抗发生率超过80％，因此抑制胰岛素抵抗可以缓解T2DM及其并发症的发生发展。髓样分化蛋白2（MD2）是TLR4的共受体，靶向MD2的抑制剂可以缓解脂多糖LPS诱导的急性炎症，但是MD2在慢性疾病尤其是胰岛素抵抗中的作用尚无人报道。在本课题中，我们首先利用多种分子生物学手段确证了L6H21和L6H9是MD2特异性小分子抑制剂，并且阐明了L6H21和L6H9与MD2蛋白的结合机制；在体外L6H21与L6H9可以抑制LPS诱导的MAPKs磷酸化和NF-κB的激活，在体内L6H21和L6H9可以缓解小鼠脓毒性死亡和脓毒症诱导的肺损伤。随后我们用高脂喂养或者尾静脉注射棕榈酸给小鼠导致高血糖、高胰岛素血症、脂质紊乱和改变调节肝脏中糖脂代谢的基因。高脂饮食诱导的这些改变与肝组织中的促炎症状态有关。在MD2基因敲除小鼠中高脂饮食不能诱导肝炎和胰岛素抵抗。用MD2抑制剂抑制MD2能够阻止高脂饮食或者棕榈酸诱导的胰岛素抵抗和肝组织炎症。而且，在肝细胞和巨噬细胞中棕榈酸诱导的促炎症信号是MD2信号通路依赖的。我们的实验结果表明MD2调节肝胰岛素抵抗的关键蛋白，并且MD2是一个重要的治疗靶点。