Yue Jianhe, Mo Lijuan, Zeng Guotao, Ma Ping, Zhang Xiaolin, Peng Yuhang, Zhang Xiang, Zhou You, Jiang Yongxiang, Huang Ning, Cheng Yuan
Joint Project of Pinnacle Disciplinary Group, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China.
Department of Neurosurgery, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China.
J Neuroinflammation. 2025 Mar 18;22(1):87. doi: 10.1186/s12974-025-03395-6.
Neutrophils and neutrophil extracellular traps (NETs) have been identified as crucial contributors in several neuroinflammatory models, such as stroke and traumatic brain injury, but their role in sepsis-associated encephalopathy (SAE) has not been thoroughly investigated.
In this study, we established an SAE model using cecal ligation puncture (CLP) surgery to examine neutrophil infiltration and NETs formation. A protein arginine deiminase 4 (PAD4) inhibitor, GSK484, was employed to suppress NETs release. To assess changes in hippocampal gene expression induced by GSK484 treatment in CLP mice, we utilized RNA sequencing (RNA-Seq) combined with bioinformatics analysis. Additionally, the Elisa, cognitive function test, western bolt and immunofluorescence staining were used to measured hippocampal inflammatory cytokine, cognitive function, and the protein levels of tight junctions (TJs) and adherens junctions (AJs) in SAE mice. We also established a Transwell™ co-culture system using bEnd.3 cells and bone marrow-derived neutrophils to examine the effects of GSK484 on endothelial cell function. This comprehensive approach allowed us to evaluate the impact of NETs inhibition on neuroinflammation, cognitive function, and the underlying molecular mechanisms in the CLP-induced SAE model.
Our findings revealed that neutrophils were significantly overactivated, releasing abundant NETs in the hippocampus of CLP-induced SAE mice. Inhibition of NET formation using GSK484 led to reduced neuroinflammatory responses, improved blood-brain barrier (BBB) integrity, and enhanced survival rates and cognitive function in SAE mice. RNA-Seq and bioinformatics analyses identified the Wnt signaling pathway as the most significant pathway affected. Subsequent experiments demonstrated that NETs inhibition alleviated BBB damage primarily by increasing the expression of Occludin, a TJs protein, and promoting the formation of the VCL/β-catenin/VE-cadherin complex at AJs, mediated by the Wnt3/β-catenin/TCF4 signaling pathway.
Our results suggest that inhibition of NETs may protect BBB permeability and cognitive function through the Wnt3/β-catenin/TCF4 signaling pathway in the context of CLP-induced SAE, which provides a promising strategy for SAE therapy.
中性粒细胞和中性粒细胞胞外诱捕网（NETs）已被确定为多种神经炎症模型（如中风和创伤性脑损伤）中的关键促成因素，但其在脓毒症相关性脑病（SAE）中的作用尚未得到充分研究。
在本研究中，我们采用盲肠结扎穿刺（CLP）手术建立了SAE模型，以检测中性粒细胞浸润和NETs形成。使用蛋白精氨酸脱亚氨酶4（PAD4）抑制剂GSK484抑制NETs释放。为了评估GSK484治疗CLP小鼠诱导的海马基因表达变化，我们利用RNA测序（RNA-Seq）结合生物信息学分析。此外，采用酶联免疫吸附测定（Elisa）、认知功能测试、蛋白质免疫印迹法（western bolt）和免疫荧光染色来检测SAE小鼠海马中的炎性细胞因子、认知功能以及紧密连接（TJs）和黏附连接（AJs）的蛋白质水平。我们还使用bEnd.3细胞和骨髓来源的中性粒细胞建立了Transwell™共培养系统，以研究GSK484对内皮细胞功能的影响。这种综合方法使我们能够评估NETs抑制对CLP诱导的SAE模型中神经炎症、认知功能和潜在分子机制的影响。
我们的研究结果显示，在CLP诱导的SAE小鼠海马中，中性粒细胞显著过度活化，释放大量NETs。使用GSK484抑制NET形成可导致SAE小鼠的神经炎症反应减轻、血脑屏障（BBB）完整性改善、存活率提高和认知功能增强。RNA-Seq和生物信息学分析确定Wnt信号通路是受影响最显著的通路。随后的实验表明，NETs抑制主要通过增加紧密连接蛋白Occludin的表达，并促进由Wnt3/β-连环蛋白/TCF4信号通路介导的AJs处VCL/β-连环蛋白/VE-钙黏蛋白复合物的形成，从而减轻BBB损伤。
我们的结果表明，在CLP诱导的SAE背景下，抑制NETs可能通过Wnt3/β-连环蛋白/TCF4信号通路保护BBB通透性和认知功能，这为SAE治疗提供了一种有前景的策略。
