KLF2调控糖代谢在脓毒症单核巨噬细胞功能低下中的作用和机制研究

Dysfunction of monocyte/macrophage is one of the major causes of immunosuppression during sepsis. Cell metabolism refers to the intracellular chemical reactions that convert nutrients and endogenous molecules into energy biomass. Emerging evidence highlights an intimate interaction between glucose metabolism and immunity. Kruppel-like transcription factor 2(KLF2),a newly identified transcription regulator, is involved in negatively modulating monocyte/macrophage function. However,the role of KLF2 in glucose metabolism regulation in monocyte/macrophage during sepsis remains unknown. Our preliminary experiments showed that the expression KLF2 in human peripheral blood monocytes in patients with sepsis was decreased. Our in vitro study indicated that glucose metabolism in KLF2-deficent macrophage was elevated, with an improvement in its function. In order to explore the role of KLF2 in glucose metabolism regulation in monocyte/macrophage during sepsis, we will examine the expression of KLF2 and determine glucose metabolism in monocyte/macrophage from septic patients. The correlations between the expression of KLF2 and glucose metabolism of monocyte/macrophage will be determined. We will further investigate the role of KLF2 in macrophage dysfunction and glucose metabolism as well as the underlying mechanism by modulating the KLF2 expression in macrophage. Our current proposal may provide new experimental evidence for therapy of sepsis-induced immunosuppression, raising KLF2 as a potential target for glucose metabolism modulation during sepsis.

单核巨噬细胞功能低下是脓毒症免疫抑制的重要原因，糖代谢是维持细胞功能的重要能量方式。KLF2是新近发现负性调控单核巨噬细胞活化的转录因子。我们在脓毒症患者和离体巨噬细胞中发现单核巨噬细胞糖酵解水平和KLF2表达相关，干预KLF2表达改善了巨噬细胞糖酵解水平。为此，我们提出科学假设：KLF2参与调控脓毒症单核巨噬细胞糖代谢异常，进而影响其功能。我们拟通过脓毒症患者外周血分析KLF2表达与单核细胞糖代谢水平及功能的相关性；利用RNA干扰、腺病毒转染等手段干预巨噬细胞KLF2表达，分析KLF2对巨噬细胞糖代谢的调控作用及功能的影响；并探讨KLF2调控糖代谢异常的信号机制；最后，通过输注KLF2低表达或高表达巨噬细胞在体验证干扰KLF2表达对脓毒症小鼠病情发展及单核巨噬细胞糖代谢的影响。本课题从糖代谢这个新角度探讨单核巨噬细胞功能障碍，为脓毒症免疫抑制的防治提供新的思路。

脓毒症时单核巨噬细胞功能低下障碍，而细胞功能的维持依赖于糖代谢提供能量。已知转录因子KLF2负性调控单核巨噬细胞功能。本研究通过临床患者试验发现脓毒症时单核细胞中KLF2表达显著上调，且单核细胞糖酵解水平降低，说明KLF2表达与单核细胞糖代谢存在一定的负性相关；随后体外利用RNA干扰技术构建KLF2低表达小鼠腹腔巨噬细胞，并发现KLF2被干扰的巨噬细胞呈现升高的糖酵解水平，表现为乳酸，ATP生成增多，糖酵解关键酶mRNA水平表达增加及分泌促炎因子（TNF-ɑ 、IL-6）的能力增强，提示KLF2能够负性调控巨噬细胞的功能（与文献报道一致）及糖代谢；接着体外分别使用不同糖代谢水平的培养基（乏糖、正常糖和正常糖+2-DG）培养小鼠腹腔巨噬细胞，LPS刺激6h后发现乏糖和正常糖+2-DG组巨噬细胞分泌的TNF-ɑ 、IL-6均降低及吞噬荧光微球的能力均减弱，表明糖代谢水平对巨噬细胞的功能有影响；深入机制研究发现KLF2可能是通过影响PI3K/Akt激酶信号和HIF-1ɑ转录因子活性调控巨噬细胞糖酵解水平；最后在体验证，将正常腹腔巨噬细胞和KLF2低表达巨噬细胞CLP术后即刻尾静脉输入小鼠，观察发现输入KLF2低表达巨噬细胞的脓毒症小鼠7d生存率显著提高，肝肺损伤明显改善及促炎因子生成下降，对脓毒症小鼠有一定的治疗价值。本研究从糖代谢这个新角度探讨了转录因子KLF2对单核巨噬细胞功能障碍的调控，为脓毒症免疫抑制的防治提供新的思路。