RIPK3调控糖酵解在脓毒症时单核巨噬细胞功能障碍的作用研究

Monocyte/macrophage dysfunction is associated with abnormal energy metabolism during sepsis-induced immunosuppression. RIP3 is a critical regulator of programmed cell death and its role in metabolic pathway has been revealed recently. Our preliminary experiment data shows that expression of RIP3 in monocyte/macrophages from sepsis mice models was up-regulated. RIP3-depletion mice showed improved survival in a sepsis mice model. Moreover, RIP3-depletion led to up-regulation of metabolism enzymes and their upstream regulator HIF-1a. Thus, our hypothesis is RIP3 may influence the process of sepsis by regulating glycolysis-related enzymes. Herein, in this project we are about to explore three major aspects through patient sample analyzation, in vitro cell-based studies and transgenic animal studies: relationship of monocyte/macrophage function and metabolism malfunction during sepsis; the role RIP3 in regulating monocyte/macrophage metabolism during sepsis; the detailed mechanism of above two questions. From the new point of view of energy metabolism, this study will deepen the understanding of the pathophysiological mechanism of sepsis and provide experimental evidence for RIP3-targeted treatment of sepsis.

单核巨噬细胞能量代谢异常伴随细胞功能障碍是脓毒症患者后期产生免疫抑制的重要机制。RIPK3蛋白是调控细胞程序性坏死的关键分子，近期研究发现其对细胞能量代谢也有重要的调控作用。在前期研究工作中我们发现，在脓毒症模型中：小鼠腹腔单核巨噬细胞RIPK3表达显著升高；RIPK3基因敲除小鼠生存时间较野生型小鼠明显延长；RIPK3基因敲除小鼠腹腔巨噬细胞糖酵解相关酶表达明显降低伴随细胞因子表达下调；RIPK3基因敲除小鼠腹腔巨噬细胞HIF-1α表达下调。由此我们推测，RIPK3可能通过调节单核巨噬细胞糖酵解途径参与调控脓毒症时机体免疫反应。本项目拟分析比较脓毒症患者外周血单核细胞RIPK3表达与糖酵解水平的关系；利用小鼠脓毒症模型，进一步分析RIPK3缺失后对脓毒症时机体能量代谢、免疫反应的影响；利用体外巨噬细胞模型探究RIPK3对糖酵解相关信号通路的调控机制；探究RIPK3作为治疗干预靶点可行性。

脓毒症是机体对入侵的病原微生物呈现异常炎症、免疫等反应，导致的严重的器官功能障碍，往往继发于严重创伤、烧伤或大手术，是临床危重患者死亡的重要原因之一。单核巨噬细胞能量代谢异常伴随细胞功能障碍是脓毒症患者后期产生免疫抑制的重要机制。RIPK3蛋白是调控细胞程序性坏死的关键分子，近期研究发现其对细胞能量代谢也有重要的调控作用。在本研究项目中我们发现，在部分脓毒症或早期大面积烧伤患者RIPK3表达上调，同时存在糖酵解异常，HK2表达上调。在小鼠模型中：盲肠结扎穿孔模型小鼠腹腔单核巨噬细胞RIPK3表达显著升高；RIPK3基因敲除小鼠生存时间较野生型小鼠明显延长；RIPK3基因敲除小鼠腹腔巨噬细胞糖酵解相关酶表达明显降低伴随细胞因子表达下调；RIPK3基因敲除小鼠腹腔巨噬细胞HIF-1α表达下调。在体外BMDM实验：RIPK3敲除后，BMDM细胞的糖酵解水平下降，而线粒体代谢无明显变化；利用RNA-seq和PCR检测发现，LPS刺激BMDM后，RIPK缺失导致糖酵解相关酶表达明显下调，尤其是HK2。本研究表明RIPK3通过调控HIF-1α促进HK2，从而调控糖酵解水平。在体给予RIPK3抑制剂GSK872后，CLP小鼠生存率改善，提示RIPK3可作为脓毒症的治疗干预靶点。.在本项目执行期间，共发表SCI文章1篇，综述1篇，在投SCI论文1篇及中文文章3篇，拟投稿SCI论文2篇。申请专利3项，参编专著1部（2021年出版），培养硕士研究生2名。