髓样分化蛋白2（MD2）特异性抑制剂L6H21通过激活AMPK缓解糖尿病心肌病的药理作用与机制研究

Hyperglycemia induced chronic inflammation and energy metabolic disorder, mutual interaction and regulation of each other, take a pivotal role in the pathological process of diabetic cardiomyopathy (DCM). Although it was found that metabolic disorder caused by AMPK dysfunction is closely associated with MD2/TLR4, the underlying mechanism is still unclear. Myeloid differentiation protein 2 (MD2), an accessory protein of Toll-like receptor 4 (TLR4), is key in recognizing the pathogenic ligand to regulate innate immunity system. Our previous study indicated that oral administration of MD2 specific inhibitor L6H21 significantly alleviates myocardial injury by suppressing inflammation in type 1 diabetic mice, accompanied by activation of AMPK. So, we hypothesize that MD2 mediates the development of DCM by regulating AMPK activity through inflammatory dependent or independent pathway. In this project, we will employ gene editing technique, transcriptome analysis and molecular biological methods to explore both upstream and downstream molecular mechanisms of AMPK that regulated by MD2, and pharmacological effects of L6H21 in vitro. MD2-/- knockout mice and adeno-associated virus based tissue specific knockdown methods were used to make confirm in vivo. Through the present study, we will demonstrate a novel mechanism link innate immune system to energy metabolism in metabolic diseases. It will also provide a crucial experimental evidence and a candidate drug for treatment of DCM.

慢性炎症与能量代谢紊乱是糖尿病心肌病（DCM）的两种重要病理过程，两者相互影响和调控。研究表明代谢性疾病中AMPK功能失调引起的能量代谢紊乱与Toll样受体4（TLR4）密切相关，但其中的分子机制尚不清晰。MD2是TLR4识别病原配体调节天然免疫的关键辅助蛋白。我们前期研究发现口服MD2特异性抑制剂L6H21阻抑炎症反应，显著缓解1型糖尿病小鼠心肌病且伴随心肌AMPK的激活，提示MD2可能通过炎症或非炎症通路调控AMPK介导DCM的发生发展。本项目拟利用基因编辑、转录组分析和细胞生物学技术，在细胞层面探索MD2调控AMPK活性的上下游分子机制，阐明L6H21的药理作用；在动物水平利用MD2-/-基因敲除小鼠和基于腺相关病毒的心肌特异性启动子驱动沉默技术进行验证。通过本项目的研究有望发现DCM中天然免疫系统对能量代谢的调控新机制，为DCM的临床治疗提供理论依据与候选新药。

慢性炎症反应是糖尿病心肌病（DCM）的重要病因。髓样分化蛋白（MD2）和Toll样受体4介导的固有炎症免疫通路的异常活化是关键病理机制。本项目的前期工作发现，MD2特异性抑制剂L6H21除了能有效阻抑高糖（HG）引起的心肌炎症反应以外还伴随着AMPK激活，提示MD2可能通过炎症或非炎症通路调控AMPK而介导DCM的发生发展。本研究旨在明确MD2调控AMPK介导DCM的非炎症性机制，同时确证MD2抑制剂L6H21对DCM的疗效。我们利用链脲佐菌素分别构建了野生型和MD2-/-基因敲除小鼠糖尿病模型，通过灌胃的方式给予L6H21，之后应用转录组测序分析（RNA-seq）、小动物心脏超声、流式细胞术和组织学方法等进行综合研究。RNA-seq数据的基因集富集分析（GSEA）表明，糖和脂质代谢相关基因在MD2-/-糖尿病鼠中富集。进一步的体外和体内数据表明，在HG条件下AMPK 通路受损是由p38MAPK激活介导的。MD2敲除或应用p38MAPK抑制剂能有效挽救AMPK的信号传导。另外，MD2抑制剂L6H21可显著缓解糖尿病小鼠的心肌损伤和功能障碍，主要表现为心脏射血分数改善、血清心肌酶降低、心肌细胞凋亡和组织纤维化减少等。本研究明确了L6H21对DCM 的治疗作用与p38MAPK抑制和AMPK通路激活相关。除了减少炎症反应之外，MD2抑制还可通过p38MAPK-AMPK轴对DCM产生治疗作用。因此，本项目确证了MD2是治疗DCM非常有希望的靶点，同时提供了L6H21作为候选先导化合物。