衣康酸调节巨噬细胞活化改善脓毒症诱导的心肌功能障碍及其机制研究

The mortality rate of patients with sepsis-induced cardiac dysfunction (SIMD) is as high as 70%, which is the main cause of sepsis death. At present, there is no clinically effective treatment. Over-infiltration of macrophages (Mφ) into the heart, over-polarization to M1 and release of inflammatory factors are the main causes of SIMD. Therefore, effective regulation of macrophage activation is a potential target for SIMD therapy. The applicant observed a significant increase in Mφ in the heart of SIMD mice. GC-MS analysis of the situation cells in the heart showed that itaconic acid was the most significant metabolite. No expression of itaconic acid was detected in cardiomyocytes, indicating that itaconic acid was mainly produced in Mφ. The applicant injecting dimethyl itaconate (DI) via tail vein could significantly improve the survival rate of SIMD mice, reduce the infiltration of Mφ in the heart and improve cardiac function. The improvement of cardiac function seemed to be dose-dependent on DI. On the basis of confirming that itaconic acid can reduce the mortality rate of SIMD mice, the applicant intends to use IRG1 knockout mice to down-regulate the expression of itaconic acid, Nrf2 knockout mice and Mφ-eliminated mice to explore the possible molecular mechanisms, from phenotype to molecular mechanism, to progressively prove the mechanism of itaconic acid in the treatment of SIMD, and to explore its clinical value as a therapeutic target for SIMD.

脓毒症诱导的心肌功能障碍（SIMD）患者死亡率高达70%，是脓毒症死亡的主要原因，目前临床上缺乏有效治疗措施。巨噬细胞（Mφ）向心脏过度浸润、向M1过度极化和释放炎性因子是SIMD形成的主要原因，因此有效的调节Mφ活化是SIMD治疗的潜在靶点。申请者观察到SIMD小鼠心脏中Mφ显著增多，对心脏中Mφ进行GC-MS（气相色谱-质谱联用仪）分析，发现衣康酸是升高最显著代谢物，心肌细胞未测出衣康酸的表达，可见衣康酸主要在Mφ中产生。申请者通过尾静脉注射衣康酸二甲酯（DI），能够显著提高SIMD小鼠的存活率，减少心脏中Mφ浸润，改善心功能，心功能改善与DI似乎呈剂量依赖性关系。申请者拟在证实衣康酸能够减少SIMD小鼠死亡率基础上，利用IRG1-/-小鼠下调衣康酸表达，Nrf2-/-小鼠、Mφ去除小鼠探索分子机制，层层递进地证明衣康酸在SIMD治疗中的机制，探讨其作为SIMD治疗靶点的临床价值。

研究背景：由免疫应答基因1（IRG1）编码的酶产生的衣康酸盐通过多种机制发挥抗炎和抗氧化作用。但是，IRG1/衣康酸盐在脓毒症引起的心脏功能障碍（SIMD）中的潜在作用仍不清楚。.主要研究内容：向小鼠注射脂多糖（LPS）（10mg/kg）以在体内产生实验性脓毒症模型。在患有SIMD的小鼠中评估了衣康酸/IRG1的心脏水平。构建IRG1敲除（IRG1−/−）小鼠和野生型小鼠的败血症模型。我们通过外源性补充进一步研究了衣康酸4-辛酯（4-OI）在SIMD中的作用。此外，为了确定4-OI在体外的作用和机制，我们在LPS给药前用4-OI和红细胞衍生核因子2相关因子2（NRF2）-小干扰RNA处理骨髓来源的巨噬细胞和RAW264.7细胞。通过流式细胞术、蛋白质印迹分析和定量实时聚合酶链反应（qPCR）确定4-OI的作用。.研究结果、数据和科学意义：SIMD期间心脏中的衣康酸显著增加。此外，IRG1基因敲除加重了脓毒性心肌损伤，恶化了心脏功能，增加了心肌组织的炎症反应，增加了心肌巨噬细胞的浸润。流式细胞分析显示，心肌组织中M1型巨噬细胞的极化增加，而M2型巨噬细胞极化减少。此外，外周血单个核细胞从脾脏向心肌组织的浸润增加。相反，4-OI给药显著保留了心脏功能并减轻了全身炎症。从机制上讲，4-OI通过激活体外NRF2/HO-1信号通路抑制巨噬细胞的炎症并促进其极化。IRG1表达是SIMD期间抵抗过度炎症反应的天然保护机制，外源性补充4-OI通过调节巨噬细胞极化在SIMD中起保护作用。IRG1/衣康酸通过激活巨噬细胞中的NRF2信号通路，提高了SIMD患者的存活率并减轻了心脏功能障碍。