Qian Zhanyang, Xia Mingjie, Zhao Tianyu, Li You, Li Guangshen, Zhang Yanan, Li Haijun, Yang Lei
Department of Orthopedics, Taizhou School of Clinical Medicine, Taizhou People's Hospital of Nanjing Medical University, Nanjing Medical University, Taizhou, China.
Department of Spine Surgery, Nantong First People's Hospital, The Second Affiliated Hospital of Nantong University, Nantong, China.
Clin Transl Med. 2024 Apr;14(4):e1661. doi: 10.1002/ctm2.1661.
Spinal cord injury (SCI)-induced neuroinflammation and oxidative stress (OS) are crucial events causing neurological dysfunction. Aconitate decarboxylase 1 (ACOD1) and its metabolite itaconate (Ita) inhibit inflammation and OS by promoting alkylation of Keap1 to induce Nrf2 expression; however, it is unclear whether there is another pathway regulating their effects in inflammation-activated microglia after SCI.
Adult male C57BL/6 ACOD1 mice and their wild-type (WT) littermates were subjected to a moderate thoracic spinal cord contusion. The degree of neuroinflammation and OS in the injured spinal cord were assessed using qPCR, western blot, flow cytometry, immunofluorescence, and trans-well assay. We then employed immunoprecipitation-western blot, chromatin immunoprecipitation (ChIP)-PCR, dual-luciferase assay, and immunofluorescence-confocal imaging to examine the molecular mechanisms of ACOD1. Finally, the locomotor function was evaluated with the Basso Mouse Scale and footprint assay.
Both in vitro and in vivo, microglia with transcriptional blockage of ACOD1 exhibited more severe levels of neuroinflammation and OS, in which the expression of p62/Keap1/Nrf2 was down-regulated. Furthermore, silencing ACOD1 exacerbated neurological dysfunction in SCI mice. Administration of exogenous Ita or 4-octyl itaconate reduced p62 phosphorylation. Besides, ACOD1 was capable of interacting with phosphorylated p62 to enhance Nrf2 activation, which in turn further promoted transcription of ACOD1.
Here, we identified an unreported ACOD1-p62-Nrf2-ACOD1 feedback loop exerting anti-inflammatory and anti-OS in inflammatory microglia, and demonstrated the neuroprotective role of ACOD1 after SCI, which was different from that of endogenous and exogenous Ita. The present study extends the functions of ACOD1 and uncovers marked property differences between endogenous and exogenous Ita.
ACOD1 attenuated neuroinflammation and oxidative stress after spinal cord injury. ACOD1, not itaconate, interacted with p-p62 to facilitate Nrf2 expression and nuclear translocation. Nrf2 was capable of promoting ACOD1 transcription in microglia.
脊髓损伤（SCI）诱导的神经炎症和氧化应激（OS）是导致神经功能障碍的关键事件。乌头酸脱羧酶1（ACOD1）及其代谢产物衣康酸（Ita）通过促进Keap1的烷基化以诱导Nrf2表达来抑制炎症和OS；然而，尚不清楚在SCI后炎症激活的小胶质细胞中是否存在另一条调节其作用的途径。
成年雄性C57BL/6 ACOD1小鼠及其野生型（WT）同窝小鼠接受中度胸段脊髓挫伤。使用qPCR、蛋白质免疫印迹、流式细胞术、免疫荧光和Transwell实验评估损伤脊髓中的神经炎症和OS程度。然后，我们采用免疫沉淀-蛋白质免疫印迹、染色质免疫沉淀（ChIP）-PCR、双荧光素酶实验和免疫荧光共聚焦成像来研究ACOD1的分子机制。最后，用Basso小鼠评分量表和足迹实验评估运动功能。
在体外和体内，ACOD1转录受阻的小胶质细胞均表现出更严重的神经炎症和OS水平，其中p62/Keap1/Nrf2的表达下调。此外，沉默ACOD1会加重SCI小鼠的神经功能障碍。给予外源性Ita或4-辛基衣康酸可降低p62磷酸化。此外，ACOD1能够与磷酸化的p62相互作用以增强Nrf2激活，这进而进一步促进ACOD1的转录。
在此，我们鉴定出一条未报道的ACOD1-p62-Nrf2-ACOD1反馈回路，其在炎性小胶质细胞中发挥抗炎和抗OS作用，并证明了ACOD1在SCI后的神经保护作用，这与内源性和外源性Ita的作用不同。本研究扩展了ACOD1的功能，并揭示了内源性和外源性Ita之间明显的性质差异。
ACOD1减轻脊髓损伤后的神经炎症和氧化应激。ACOD1而非衣康酸与p-p62相互作用以促进Nrf2表达和核转位。Nrf2能够促进小胶质细胞中ACOD1的转录。
