20-羟二十碳四烯酸通过 GPR75 调节Src/EGFR/NF-κB 信号通路激活小胶质细胞并促进未成熟大脑中的 TBI。
20-Hydroxyeicosatetraenoic Acid Regulates the Src/EGFR/NF-κB Signaling Pathway Via GPR75 to Activate Microglia and Promote TBI in the Immature Brain.
机构信息
Department of Anesthesiology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, 400010, China.
Department of Army Occupational Disease, State Key Laboratory of Trauma, Burns and Combined Injury, Research Institute of Surgery, Daping Hospital, Army Medical University, Chongqing, 400042, China.
出版信息
Neurochem Res. 2024 Nov 14;50(1):7. doi: 10.1007/s11064-024-04260-3.
20-Hydroxyeicosatetraenoic acid (20-HETE) is associated with secondary damage in traumatic brain injury (TBI) of the immature brain. Microglial activation is pivotal in this process. However, the underlying mechanism of action remains unknown. While 20-HETE interacts with G protein-coupled receptor 75 (GPR75) in some pathological processes, their interaction in brain tissue remains uncertain. This study aimed to investigate whether 20-HETE can activate microglia by binding to GPR75 in TBI of the immature brain. Drug affinity responsive molecular target stability (DARTS) assays, cycloheximide (CHX) chase assays, and auto-dock assays were employed to analyze the interaction between 20-HETE and GPR75. The expression levels of cytochrome P450 4A (CYP4A) and GPR75 in activated microglia in an immature brain TBI model were observed by western blot and multiple immunofluorescence staining. The effects of different levels of 20-HETE expression and lentivirus-mediated GPR75 gene silencing on 20-HETE-induced inflammatory factor release from BV-2 cells were observed by enzyme-linked immunoassay (ELISA). The phosphorylation levels of the downstream Src kinase, epidermal growth factor receptor (EGFR), and nuclear factor (NF)-κB were assessed using western blot. Cell viability and apoptosis were detected by CCK-8 and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays. 20-HETE bound to the GPR75 protein and inhibited its degradation. GPR75 gene silencing reversed the 20-HETE-induced inflammatory activation of BV-2 cells, effectively inhibiting the activation of the Src/EGFR/NF-κB pathway and the effects of 20-HETE on cell viability and the apoptosis rate. In contrast, overexpression of GPR75 had the opposite effect. In addition, after immature brain TBI, the 20-HETE and GPR75 expression levels were upregulated in microglia, with significant activation of the Src/EGFR/NF-κB pathway. Inhibition of 20-HETE synthesis with N-hydroxy-N'-(4-n-butyl-2-methylphenyl) formamidine (HET0016) produced the opposite effect. 20-HETE regulates the Src/EGFR/NF-κB signaling pathway via GPR75 to activate microglia, promoting immature brain TBI. These findings offer a novel target for promoting the brain injury effect of 20-HETE.
20-羟二十碳四烯酸（20-HETE）与未成熟脑创伤性脑损伤（TBI）中的继发性损伤有关。小胶质细胞的激活在这个过程中起着关键作用。然而，其作用机制尚不清楚。虽然 20-HETE 在某些病理过程中与 G 蛋白偶联受体 75（GPR75）相互作用，但它们在脑组织中的相互作用仍不确定。本研究旨在探讨 20-HETE 是否可以通过与未成熟脑 TBI 中的 GPR75 结合来激活小胶质细胞。采用药物亲和反应分子靶标稳定性（DARTS）测定、环己酰亚胺（CHX）追踪测定和自动对接测定来分析 20-HETE 与 GPR75 之间的相互作用。通过 Western blot 和多重免疫荧光染色观察到未成熟脑 TBI 模型中小胶质细胞中细胞色素 P450 4A（CYP4A）和 GPR75 的表达水平。通过酶联免疫吸附试验（ELISA）观察不同水平的 20-HETE 表达和慢病毒介导的 GPR75 基因沉默对 20-HETE 诱导 BV-2 细胞炎症因子释放的影响。采用 Western blot 检测下游 Src 激酶、表皮生长因子受体（EGFR）和核因子（NF）-κB 的磷酸化水平。通过 CCK-8 和末端脱氧核苷酸转移酶 dUTP 缺口末端标记（TUNEL）测定检测细胞活力和细胞凋亡。20-HETE 与 GPR75 蛋白结合并抑制其降解。GPR75 基因沉默逆转了 20-HETE 诱导的 BV-2 细胞炎症激活，有效抑制了 Src/EGFR/NF-κB 通路的激活以及 20-HETE 对细胞活力和凋亡率的影响。相反，GPR75 的过表达则产生相反的效果。此外，在未成熟脑 TBI 后，小胶质细胞中 20-HETE 和 GPR75 的表达水平上调，Src/EGFR/NF-κB 通路被显著激活。用 N-羟基-N'-(4-正丁基-2-甲基苯基)甲脒（HET0016）抑制 20-HETE 合成产生了相反的效果。20-HETE 通过 GPR75 调节 Src/EGFR/NF-κB 信号通路来激活小胶质细胞，从而促进未成熟脑 TBI。这些发现为促进 20-HETE 的脑损伤效应提供了一个新的靶点。
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