经皮耳迷走神经刺激通过内皮VWF/PI3K-Akt轴调节小胶质细胞极化减轻卒中后吞咽困难

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中文摘要

卒中后吞咽困难是一种常见并发症，治疗选择有限。经皮耳迷走神经刺激（ta-VNS）已显示出神经保护作用。本研究探讨ta-VNS是否通过调节小胶质细胞M1/M2极化改善吞咽功能并减轻神经炎症，并检验血管性血友病因子（VWF）在内皮-小胶质细胞交互对话中的作用。建立大鼠短暂性双侧大脑中动脉闭塞（MCAO）模型。大鼠接受ta-VNS治疗3周，评估吞咽功能、神经功能缺损和炎症标志物。通过免疫荧光检测白质中小胶质细胞M1（CD16）和M2（CD206）极化。采用RNA-seq鉴定候选基因。体外采用氧糖剥夺/复氧（OGD/R）模型，将hCMEC/D3内皮细胞与HMC3小胶质细胞共培养，并使用shRNA敲低VWF以评估其对小胶质细胞极化和炎症的影响。ta-VNS显著改善短暂性大脑中动脉闭塞大鼠的体重、进食/饮水量和吞咽功能（缩短潜伏期并增加吞咽频率）。ta-VNS降低TNF-α、IL-6、IL-1β和IL-10水平，减轻小胶质细胞活化，抑制M1（Iba-1+CD16+）小胶质细胞，并增强大鼠白质中M2（Iba-1+CD206+）极化。RNA-seq显示ta-VNS后VWF下调，并富集于PI3K-Akt通路。体外实验中，OGD/R上调VWF、ICAM-1、VCAM-1、内皮细胞凋亡和通透性。敲低内皮细胞中的VWF使共培养的小胶质细胞从M1向M2表型转变，并降低TNF-α、IL-6和IL-1β，同时增加IL-10。ta-VNS可通过促进M2小胶质细胞极化改善卒中后吞咽困难并减轻神经炎症。内皮VWF可能是该过程的关键下游介质。

英文摘要

Post-stroke dysphagia is a common complication with limited treatment options. Transcutaneous auricular vagus nerve stimulation (ta-VNS) has shown neuroprotective effects. This study investigated whether ta-VNS improves swallowing function and alleviates neuroinflammation by modulating microglial M1/M2 polarization, examining the role of von Willebrand factor (VWF) in endothelial-microglial crosstalk. A rat model of transient bilateral middle cerebral artery occlusion (MCAO) was established. Rats received ta-VNS for 3 weeks, and swallowing function, neurological deficits, and inflammatory markers were assessed. Microglial M1 (CD16) and M2 (CD206) polarization in the white matter was examined by immunofluorescence. RNA-seq was used to identify candidate genes. In vitro, an oxygen-glucose deprivation/reoxygenation (OGD/R) model was applied to hCMEC/D3 endothelial cells co-cultured with HMC3 microglia, and VWF was knocked down using shRNA to evaluate its effect on microglial polarization and inflammation. ta-VNS significantly improved body weight, food/water intake, and swallowing function (reduced latency and increased swallowing frequency) in transient middle cerebral artery occlusion rats. ta-VNS reduced levels of TNF-α, IL-6, IL-1β, and IL-10, attenuated microglial activation, suppressed M1 (Iba-1+CD16+) microglia, and enhanced M2 (Iba-1+CD206+) polarization in rat whiter matter. RNA-seq revealed downregulation of VWF after ta-VNS, with enrichment in the PI3K-Akt pathway. In vitro, OGD/R upregulated VWF, ICAM-1, VCAM-1, endothelial apoptosis, and permeability. Knockdown of VWF in endothelial cells shifted co-cultured microglia from M1 toward M2 phenotype and reduced TNF-α, IL-6, and IL-1β while increasing IL-10. ta-VNS can improve post-stroke dysphagia and reduce neuroinflammation by promoting M2 microglial polarization. Endothelial VWF may be a key downstream mediator of this process.