20-Hydroxyeicosatetraenoic Acid Regulates the Src/EGFR/NF-κB Signaling Pathway Via GPR75 to Activate Microglia and Promote TBI in the Immature Brain

IF 3.7 3区医学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Neurochemical Research Pub Date : 2024-11-14 DOI:10.1007/s11064-024-04260-3

Zhihui Ma, Yalei Ning, Xiaoli Chen, Shan Zhao, Jie Yan, Bo Wang, Changhong Li, Ruobing Gao, Xing Chen, Nan Yang, Yan Peng, Ping Li, Shiyu Shu

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引用次数: 0

Abstract

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.

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20-羟基二十碳四烯酸通过 GPR75 调节 Src/EGFR/NF-κB 信号通路，激活小胶质细胞并促进未成熟脑的创伤性脑损伤。

20-羟基二十碳四烯酸（20-HETE）与未成熟大脑创伤性脑损伤（TBI）的二次损伤有关。小胶质细胞的活化在这一过程中起着关键作用。然而，其潜在的作用机制仍然未知。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 是否能通过与 GPR75 结合来激活未成熟脑 TBI 中的小胶质细胞。本研究采用药物亲和力反应分子靶标稳定性（DARTS）测定法、环己亚胺（CHX）追逐测定法和自动对接测定法来分析20-HETE与GPR75之间的相互作用。通过 Western 印迹和多重免疫荧光染色观察了未成熟脑 TBI 模型中激活的小胶质细胞中细胞色素 P450 4A（CYP4A）和 GPR75 的表达水平。通过酶联免疫测定（ELISA）观察了不同水平的 20-HETE 表达和慢病毒介导的 GPR75 基因沉默对 20-HETE 诱导的 BV-2 细胞炎性因子释放的影响。采用 Western 印迹法评估了下游 Src 激酶、表皮生长因子受体（EGFR）和核因子（NF）-κB 的磷酸化水平。细胞活力和细胞凋亡通过 CCK-8 和末端脱氧核苷酸转移酶 dUTP缺口末端标记（TUNEL）检测法进行检测。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，过表达 GPR75 则会产生相反的效果。此外，未成熟脑创伤后，20-HETE 和 GPR75 在小胶质细胞中的表达水平上调，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 的合成则会产生相反的效果。20-HETE通过GPR75调节Src/EGFR/NF-κB信号通路，激活小胶质细胞，促进未成熟脑创伤性脑损伤。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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来源期刊

Neurochemical Research 医学-神经科学

CiteScore

7.70

自引率

2.30%

发文量

320

审稿时长

6 months

期刊介绍： Neurochemical Research is devoted to the rapid publication of studies that use neurochemical methodology in research on nervous system structure and function. The journal publishes original reports of experimental and clinical research results, perceptive reviews of significant problem areas in the neurosciences, brief comments of a methodological or interpretive nature, and research summaries conducted by leading scientists whose works are not readily available in English.