MEF2a在脓毒症时单核巨噬细胞内毒素耐受中的作用与机制研究

Endotoxin tolerance of monocyte/macrophage is one of major causes of sepsis-induced immunosuppression. However, its underlying mechanism remains unknown. Myocyte enhancer factor 2A(MEF2a), a newly identified transcriptional regulatory factor, is involved in inflammatory response regulation. Our preliminary experiment found that the endotoxin reactivity of monocyte/macrophage correlates with MEF2a expression. MEF2a knockdown can increase endotoxin reactivity in LPS-tolerized macrophage. Thus, we speculate that MEF2a might participate in the endotoxin tolerance regulation of monocyte/macrophage during sepsis. Our research aimed to clarify the role and mechanism of MEF2a in endotoxin tolerance of monocyte/macrophage during sepsis. We will firstly examine the expression of MEF2a and endotoxin reactivity in monocyte from septic patients. We will then investigate the expression of MEF2a and its signal pathway and epigenetic mechanism in LPS-tolerized macrophage in vitro. The production of cytokines and negative regulator variation will also be measured through gene transfection of MEF2a in macrophage. We will also explore the potential molecule mechanism via ChIP-seq and luciferase reporter gene. Eventually, the role of MEF2a will be validated by in vivo study. Our program will provide theoretical and experimental evidence for sepsis therapy, expecially for immunosuppression prevention in sepsis.

单核巨噬细胞内毒素耐受是脓毒症患者发生免疫抑制的重要原因，机制仍不清楚。新近发现转录调节因子MEF2a参与炎症调节。我们前期在脓毒症免疫抑制患者和内毒素耐受巨噬细胞模型中发现：单核巨噬细胞内毒素反应性与MEF2a表达相关，干扰MEF2a可提高巨噬细胞的内毒素反应性。我们推测MEF2a参与调控脓毒症时单核巨噬细胞内毒素耐受。本项目拟就MEF2a在脓毒症单核巨噬细胞内毒素耐受中的作用及机制进行研究。通过测定脓毒症患者单核细胞MEF2a表达与内毒素反应性；体外研究内毒素耐受巨噬细胞MEF2a的表达变化及其信号通路、表观遗传学机制；检测过表达或干扰MEF2a后，炎性因子产生、负性调控分子表达的变化，采用ChIP-seq及荧光素酶报告基因等手段，探索 MEF2a调控内毒素耐受的分子机制；并在体验证干预MEF2a对脓毒症发生发展的影响；以期为脓毒症免疫抑制的防治提供理论和实验依据。

内毒素耐受是目前重症感染/脓毒症患者死亡危险因素之一。转录因子Mef2a参与调节脓毒症导致的单核巨噬细胞内毒素耐受的发生发展中起着非常关键的作用。本研究发现脓毒症患者外周血PBMC中Mef2a的表达水平显著高于对照组，且其表达水平与反应脓毒症患者疾病的状态的指标(HLA-DR, APACHE II评分，MODS评分 )呈线性相关。此外，体外研究也表明内毒素耐受的巨噬细胞Mef2a的表达水平显著高于Sham组；NF-κB的抑制剂PDTC抑制内毒素耐受中mef2a的表达增高水平；Mef2a基因启动子区域羟甲基化水平参与调控内毒素耐受巨噬细胞Mef2a的表达水平的表达。体外干扰/过表达巨噬细胞Mef2a通过调节A20的基因转录调节巨噬细胞分泌细胞因子的功能，从而参与内毒素耐受发生发展。在体输入mef2ahi的巨噬细胞降低TNF-α细胞因子的分泌，增加小鼠外周血的细菌负荷；静脉给予mef2alo巨噬细胞能够改善脓毒症小鼠的生存率，能够降低小鼠机体的细菌负荷，增加促炎细胞因子，降低抑炎细胞因子水平。能够减轻内毒素耐受导致的多脏器的损伤。