高酮体代谢减少急性脊髓损伤巨噬细胞M1亚型及抑制炎症反应的分子机制研究

Previously, we reported that both ketogenic diet (KD) and calorie restriction (CR), which result ketonemia, can improve neurological recovery after acute SCI in rats, but the underlying mechanisms are not fully understood; moreover, clinical application of KD and CR is difficult due to their strict diet restriction, especially in the context of SCI. BD-AcAc2 can significantly increase serum ketone level without diet restriction, but its role in SCI is still unknown. Inflammatory reaction is one of the major factors that cause secondary injury after spinal cord injury (SCI), macrophage is the key element of post-SCI inflammation. Activated macrophages differentiate into two primary subsets, M1 and M2. M1 promotes inflammation, causing secondary neural damage; while M2 exhibits anti-inflammatory properties and improves neural regeneration.NF-κB signaling pathway is one of the main pathways that control macrophage polarization. Recently, ketonemia has been showed to decrease the expression of NF-κB in different tissues. Our preliminary data showed that high βHB, regulated the expression of inflammatory genes in a rodent model of SCI; meanwhile, immunofluorescence staining of the injury spinal cord from the same cohort animals at 7 days after injury showed decreased M1/M2 ratio when compared to the control group. Base on the above findings, we hypothesize that through regulating the activity of NF-κB signaling pathway, ketonemia reduces the expression of pro-inflammatory cytokine genes and inhibits macrophages from differentiating to M1 subtype after SCI, hence protects neurological function from inflammatory damage. This study is designed to investigate: 1) the effect of ketonemia on the expression of NF-κB signaling pathway related genes, as well as inflammatory related genes after spinal cord injury in rodent; 2) the effect of ketonemia on macrophage polarization in spinal cord tissue at different time points after SCI; 3) whether increasing βHB levels by BD-AcAc2 exhibit neuroprotective effect after SCI. We believe such study will shed light on the mechanism behind the neuroprotective effect of CR/IF, and benefit new treatment strategies development for SCI patients.

我们前期研究证实生酮饮食（KD）及热量限制（CR）引起的高酮体代谢对脊髓损伤（SCI）有神经保护作用，但机制不清；且临床中KD和CR患者均不易耐受。口服1,3-丁二醇乙酰乙酸酯（BD-AcAc2）可提高体内酮体代谢，但对SCI的作用不明。由巨噬细胞参与的炎症反应是SCI后继发神经损害的重要因素。已证实SCI后巨噬细胞激活并分化为M1和M2亚型，其中M1促进炎症反应，增加神经损害。NF-κB是诱导M1分化的主要信号通路。近期研究发现高酮体代谢可抑制外周细胞NF-κB信号传导。预实验发现，KD可调节SCI后炎症基因转录水平，减少M1比例。由此，我们假设：酮体代谢通过下调NF-κB通路，减少M1比例，降低炎症损伤，保护神经功能。本项目将研究：1）酮体代谢对SCI后NF-κB信号通路及相关炎症基因转录表达的影响；2）酮体代谢对巨噬细胞激活与分化的调节；3）BD-AcAc2对SCI的神经保护作用。

本项目主要探讨酮体代谢对脊髓损伤（SCI）神经保护作用的机制。我们按照建立脊髓损伤动物模型-建立高酮体代谢模型-评估高酮体代谢对SCI神经保护机制的线路设计并完成实验。主要成果包括：1、对位移控制的大鼠半侧颈脊髓损伤模型进行了规范化，并将成果发表于Int J Clin Exp Med上；率先建立了位移控制的小鼠的颈脊髓半侧损伤模型，并将成果发表于《中国临床解剖学杂志》；此外课题组还将啮齿类动物的SCI模型进一步系统化，并对比不同打击模式对产生的颈脊髓损伤在行为学、组织学上、电生理、活体磁共振现象上的差异，该部分文章正在撰写中；2、课题组增加了脊髓血管造影作为评估伤后KD神经保护作用的新方法，并展示了SCI后中远期的病灶周边血管分布情况及对比了不同造影剂进行脊髓3D血管造影的效果，成果分别发表于World Neurosurgery和Contrast Media & Molecular Imaging杂志上。3、探讨了高酮体代谢对于SCI后神经保护机制的多样性，揭示酮体代谢可调节SCI后氧化应激反应，该成果发表于Neuroscience杂志上。4、观察到生酮饮食（KD）及正常饮食配合1, 3-丁二醇（BD）灌胃均可提高大鼠血清酮体水平，且发现两者对于SCI后促炎因子的基因表达及蛋白合成具有抑制作用，提示两者均可抑制SCI后炎症反应；此外KD及BD组伤后巨噬细胞M1亚型标志物iNOS表达抑制，M2亚型标志物Arg1表达升高，免疫荧光染色可见M2/M1比例增加，提示其抑制炎症反应与调控巨噬细胞分化相关。但BD组在伤后12周的行为学测试及组织染色中没有观察到与KD相似的神经保护作用。目前该结果部分数据已在投稿中，课题组将利用本课题结余经费优化或深入该部分研究，拟再发表SCI文章2-3篇。综上，项目组通过本课题的开展所取得的成果，为脊髓损伤的基础研究提供了有效、重复性高及经济的动物模型，对酮体代谢的神经保护作用，尤其是对于伤后炎症反应的抑制及氧化应激的调节提出证据，对开展生酮饮食治疗脊髓损伤提供理论基础。对于正常饮食添加BD对SCI的神经保护展开讨论，为后续研究打下一定基础。