背景:神经炎症是创伤性脑损伤(TBI)后继发性脑损伤中最重要的病因之一。在整个TBI患者的脑组织中发现了形成中性粒细胞的中性粒细胞胞外诱捕器(NETs),并且升高的血浆NET生物标志物与较差的结果相关。然而,NETs在TBI诱导的神经损伤中的生物学功能和潜在机制尚未完全了解。这里,我们用了Cl-脒,一种NETs的选择性抑制剂,用于研究NETs在TBI后神经损伤中的作用。
方法:采用控制性皮质撞击模型建立TBI。Cl-脒,2\'3\'-cGAMP(刺激干扰素基因(STING)的激活剂),C-176(选择性STING抑制剂),和Kira6[一种选择性磷酸化的肌醇需求酶-1α[IRE1α]抑制剂]被施用以探索NETs促进TBI后神经炎症和神经元凋亡的机制。肽基精氨酸脱亚胺酶4(PAD4),中性粒细胞胞外诱捕网形成所必需的酶,在TBI前1天在小鼠的皮质中过表达腺病毒。短期神经行为测试,磁共振成像(MRI),激光散斑对比成像(LSCI),伊文思蓝外渗试验,Fluoro-JadeC(FJC),TUNEL,免疫荧光,酶联免疫吸附测定(ELISA),西方印迹,在这项研究中进行了定量PCR。
结果:嗜中性粒细胞在TBI后3天在循环和脑中形成呈现的NETs。NETs抑制剂Cl-脒治疗改善短期神经功能,脑损伤体积减少,减少脑水肿,TBI后恢复脑血流量(CBF)。此外,Cl-脒通过减弱BBB破坏发挥神经保护作用,抑制免疫细胞浸润,减轻TBI后的神经元死亡。此外,Cl-脒处理在TBI后3天抑制小胶质细胞/巨噬细胞促炎极化并促进抗炎极化。机械上,STING配体2\'3\'-cGAMP在TBI后通过IRE1α/ASK1/JNK信号通路消除了Cl-脒的神经保护作用。重要的是,PAD4的过表达通过IRE1α/ASK1/JNK信号通路促进TBI后的神经炎症和神经元死亡。然而,STING抑制剂C-176或IRE1α抑制剂Kira6有效消除了TBI后PAD4过表达的神经破坏作用。
结论:总而言之,我们是第一个证明用Cl-脒抑制NETs改善神经炎症的人,神经元凋亡,TBI后通过STING依赖性IRE1α/ASK1/JNK信号通路出现神经功能缺损。因此,Cl-脒治疗可以为TBI的早期管理提供有希望的治疗方法。
BACKGROUND: Neuroinflammation is one of the most important pathogeneses in secondary brain injury after traumatic brain injury (TBI). Neutrophil extracellular traps (NETs) forming neutrophils were found throughout the brain tissue of TBI patients and elevated plasma NET biomarkers correlated with worse outcomes. However, the biological function and underlying mechanisms of NETs in TBI-induced neural damage are not yet fully understood. Here, we used Cl-amidine, a selective inhibitor of NETs to investigate the role of NETs in neural damage after TBI.
METHODS: Controlled cortical impact model was performed to establish TBI. Cl-amidine, 2\'3\'-cGAMP (an activator of stimulating Interferon genes (STING)), C-176 (a selective STING inhibitor), and Kira6 [a selectively phosphorylated inositol-requiring enzyme-1 alpha [
IRE1α] inhibitor] were administrated to explore the mechanism by which NETs promote neuroinflammation and neuronal apoptosis after TBI. Peptidyl arginine deiminase 4 (PAD4), an essential enzyme for neutrophil extracellular trap formation, is overexpressed with adenoviruses in the cortex of mice 1 day before TBI. The short-term neurobehavior tests, magnetic resonance imaging (MRI), laser speckle contrast imaging (LSCI), Evans blue extravasation assay, Fluoro-Jade C (FJC), TUNEL, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), western blotting, and quantitative-PCR were performed in this study.
RESULTS: Neutrophils form NETs presenting in the circulation and brain at 3 days after TBI. NETs inhibitor Cl-amidine treatment improved short-term neurological functions, reduced cerebral lesion volume, reduced brain edema, and restored cerebral blood flow (CBF) after TBI. In addition, Cl-amidine exerted neuroprotective effects by attenuating BBB disruption, inhibiting immune cell infiltration, and alleviating neuronal death after TBI. Moreover, Cl-amidine treatment inhibited microglia/macrophage pro-inflammatory polarization and promoted anti-inflammatory polarization at 3 days after TBI. Mechanistically, STING ligand 2\'3\'-cGAMP abolished the neuroprotection of Cl-amidine via
IRE1α/ASK1/JNK signaling pathway after TBI. Importantly, overexpression of PAD4 promotes neuroinflammation and neuronal death via the
IRE1α/ASK1/JNK signaling pathway after TBI. However, STING inhibitor C-176 or
IRE1α inhibitor Kira6 effectively abolished the neurodestructive effects of PAD4 overexpression after TBI.
CONCLUSIONS: Altogether, we are the first to demonstrate that NETs inhibition with Cl-amidine ameliorated neuroinflammation, neuronal apoptosis, and neurological deficits via STING-dependent
IRE1α/ASK1/JNK signaling pathway after TBI. Thus, Cl-amidine treatment may provide a promising therapeutic approach for the early management of TBI.