Deng Yifan, Xuan Ruoheng, Qiu Zhuolin, Xiang Ping, Guo Yue, Xu Lejia, Zhang Xiaohan, Mai Haiyan, Li Xiang
Department of Anesthesiology, the Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou, Guangdong Province 510630, China.
Department of Neurosurgery, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong Province 510000, China.
Brain Behav Immun. 2025 Mar;125:92-109. doi: 10.1016/j.bbi.2024.12.152. Epub 2024 Dec 23.
Glial cell-induced neuroinflammation in the spinal cord is the critical pathology underlying complete Freund's adjuvant (CFA)-induced inflammatory pain. Previously, we showed that spinal glial cells undergo ferroptosis after CFA injection, which may contribute to the development of neuroinflammation and inflammatory pain. However, the mechanism underlying the occurrence of ferroptosis during inflammatory pain remains unclear. The aim of this study was to investigate the molecular factors involved in the occurrence of ferroptosis during the development of inflammatory pain. Bulk and single-cell RNA sequencing were performed to identify the key genes involved in the ferroptosis of spinal astrocytes, microglia, and oligodendrocytes in rats. We identified nuclear receptor 4A1 (NR4A1) as a common ferroptosis-related gene present in all three types of glial cells. Western blotting and immunostaining revealed increased NR4A1 levels in the spinal glial cells of the CFA-treated rats. Moreover, intrathecal injection of DIM-C-pPhOH (an NR4A1 inhibitor) effectively alleviated mechanical and thermal hypersensitivity in the CFA-treated rats by attenuating ferroptosis and neuroinflammation in spinal glial cells. Proteomic analysis revealed that mitogen-activated protein kinase 3 (MAPK3) may be the target protein of NR4A1. In addition, the combined results of chromatin immunoprecipitation and dual-luciferase assays indicated that NR4A1 can bind to the promoter region and promote transcription of MAPK3, ultimately leading to lipid peroxidation. In conclusion, this study demonstrated that increased expression of NR4A1 promotes the progression of CFA-induced inflammatory pain by enhancing ferroptosis through the transcriptional activation of MAPK3 and subsequent lipid peroxidation. Furthermore, inhibition of NR4A1 was found to suppress ferroptosis and reduce the release of pro-inflammatory cytokines in the spinal cord of rats with inflammatory pain. Collectively, these findings outline a novel pathological mechanism and identify potential therapeutic targets for the treatment of inflammatory pain.
脊髓中胶质细胞诱导的神经炎症是完全弗氏佐剂（CFA）诱导的炎性疼痛的关键病理学基础。此前，我们发现CFA注射后脊髓胶质细胞会发生铁死亡，这可能促进神经炎症和炎性疼痛的发展。然而，炎性疼痛期间铁死亡发生的机制仍不清楚。本研究的目的是调查炎性疼痛发展过程中参与铁死亡发生的分子因素。进行了批量和单细胞RNA测序，以鉴定大鼠脊髓星形胶质细胞、小胶质细胞和少突胶质细胞铁死亡相关的关键基因。我们确定核受体4A1（NR4A1）是存在于所有三种胶质细胞类型中的常见铁死亡相关基因。蛋白质免疫印迹和免疫染色显示，CFA处理大鼠的脊髓胶质细胞中NR4A1水平升高。此外，鞘内注射DIM-C-pPhOH（一种NR4A1抑制剂）可通过减轻脊髓胶质细胞中的铁死亡和神经炎症，有效缓解CFA处理大鼠的机械性和热超敏反应。蛋白质组学分析表明，丝裂原活化蛋白激酶3（MAPK3）可能是NR4A1的靶蛋白。此外，染色质免疫沉淀和双荧光素酶测定的联合结果表明，NR4A1可与启动子区域结合并促进MAPK3的转录，最终导致脂质过氧化。总之，本研究表明，NR4A1表达增加通过MAPK3的转录激活和随后的脂质过氧化增强铁死亡，从而促进CFA诱导的炎性疼痛进展。此外，发现抑制NR4A1可抑制铁死亡并减少炎性疼痛大鼠脊髓中促炎细胞因子的释放。这些发现共同概述了一种新的病理机制，并确定了治疗炎性疼痛的潜在治疗靶点。
