IRG1调节铁离子应答元件结合蛋白1参与心肌梗死后巨噬细胞炎症应答的机制研究

Excessive macrophage inflammatory response aggravates the cardiac injury at early stage of myocardial infarction (MI). Aconitate decarboxylase(IRG1), mainly expressed in macrophages, was shown to be increased in the early phase after MI. Our previous work uncovered that IRG1 and its product, itaconate, inhibit HIF2α protein levels and promote M1 phenotype macrophages through using proteomics. We found a common feature of these enzymes inhibited by IRG1 (mitochondrial aconitase, cytoplasmic aconitase (IRP1) and succinate dehydrogenase), which are iron-sulfur cluster-dependent enzymes. Especially, after losing iron-sulfur cluster, IRP1 will bind to RNA stem-loops containing an iron response element (IRE) in the 5’UTR to inhibit translation of HIF2α mRNA. Based on those discoveries, we hypothesize that IRG1/itaconate inhibits the protein translation of HIF2α and promote the M1 macrophages polarization by destroying the iron-sulfur cluster of IRP1, while over-expression of IRG1 after MI may aggravate myocardial damage. In order to prove our hypothesis, we will use molecular biological techniques to verify effects of IRG1 on IRP1 activity and HIF2α level. Through an IRG1 gene knockout mouse model with MI, we will try to investigate new therapeutic strategy to decrease cardiac injury.

心肌梗死后初期巨噬细胞（Mφ）过度的炎症应答反应会造成心肌损伤加重。我们前期工作发现心肌梗死患者的单核巨噬细胞高表达顺乌头酸脱羧酶（IRG1），蛋白质组和分子生物学证实IRG1及其代谢产物衣康酸抑制HIF2α蛋白水平并促进Mφ向促炎表型M1极化，但机制不明。IRG1可以抑制线粒体乌头酸酶，胞浆乌头酸酶（IRP1，也叫铁应答元件结合蛋白1）和琥珀酸脱氢酶的活性，此三者的共性为铁硫簇依赖的酶。而IRP1是具有双重功能的蛋白，即失去铁硫簇后会结合HIF2α的mRNA上的铁应答元件序列，并抑制HIF2α翻译。由此提出课题假说，IRG1通过破坏IRP1的铁硫簇，从而抑制HIF2α的蛋白翻译，并促进Mφ向M1极化，心肌梗死后过度表达IRG1可能造成心肌损伤加重。拟通过分子生物学手段，明确IRG1/衣康酸，IRP1和HIF2α三者调控机制。通过对小鼠IRG1基因的干预，探索心肌梗死后抗炎治疗的新途径。

脂多糖刺激的巨噬细胞高表达一种乌头酸脱羧酶（IRG1，也称为ACOD1），导致内源性代谢物衣康酸的积累。然而，衣康酸水平升高改变巨噬细胞功能的确切机制尚不清楚。我们假设衣康酸通过某种不确定的机制影响巨噬细胞功能。依据衣康酸刺激巨噬细胞的转录组学和蛋白质组学联合分析，确定了衣康酸可能调节巨噬细胞的IL-1β分泌和铁代谢。同样，IRG1敲除THP-1细胞系降低了LPS促进的IL-1β分泌和铁代谢相关蛋白表达。无论是增加铁离子浓度还是铁剥夺都加剧衣康酸促进的caspase-1激活和IL-1β分泌。只有半胱氨酸和抗氧化剂(儿茶素)能抑制衣康酸促进的caspase-1激活和IL-1β分泌。进一步发现衣康酸处理巨噬细胞后乌头酸酶活性降低。结果证明线粒体乌头酸酶(ACO2，一种TCA循环酶)或细胞质乌头酸酶(ACO1或IPR1，一种铁调节蛋白)的过表达分别对衣康酸促进的IL-1β分泌和改变铁代谢有抵消作用。衣康酸抑制乌头酸酶活性通过硫醇攻击破坏它们的铁硫簇，使其失活。以上结果表明，IRG1和衣康酸通过乌头酸酶应激性铁硫簇破坏和铁代谢的再平衡对巨噬细胞进行免疫调节。