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中华脑血管病杂志(电子版)

中华脑血管病杂志(电子版) ›› 2025, Vol. 19 ›› Issue (05) : 418 -428. doi: 10.3877/cma.j.issn.1673-9248.2025.05.009

基础研究

基于数据非依赖性采集蛋白质组学探讨超早期经耳迷走神经刺激对缺血性脑卒中神经功能的影响
张霞1, 樊震峰1, 成柯岭1, 王志勇1, 陈乐文1, 蔡斌2, 倪隽3,†()   
  1. 1　350005　福建　福州，福建医科大学附属第一医院康复科
    2　350005　福建　福州，福建医科大学附属第一医院神经内科
    3　350212　福建　福州，福建医科大学附属第一医院滨海院区国家区域医疗中心康复科
  • 收稿日期:2025-05-06 出版日期:2025-10-01
  • 通信作者: 倪隽
  • 基金资助:
    国家自然科学基金面上项目(82172531); 福建省科技创新联合资金项目(2021Y9105)

Data-independent acquisition proteomics-based investigation of the impact of ultra-early transcutaneous auricular vagus nerve stimulation on neurological function in ischemic stroke

Xia Zhang1, Zhenfeng Fan1, Keling Cheng1, Zhiyong Wang1, Lewen Chen1, Bin Cai2, Jun Ni3,†()   

  1. 1 Department of Rehabilitation, the First Affiliated Hospital, Fujian Medical University, Fuzhou 350005, China
    2 Department of Neurology, the First Affiliated Hospital, Fujian Medical University, Fuzhou 350005, China
    3 Department of Rehabilitation, National Regional Medical Center, Binhai Campus of the First Affiliated Hospital, Fujian Medical University, Fuzhou 350212, China
  • Received:2025-05-06 Published:2025-10-01
  • Corresponding author: Jun Ni
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引用本文：

张霞, 樊震峰, 成柯岭, 王志勇, 陈乐文, 蔡斌, 倪隽. 基于数据非依赖性采集蛋白质组学探讨超早期经耳迷走神经刺激对缺血性脑卒中神经功能的影响[J/OL]. 中华脑血管病杂志(电子版), 2025, 19(05): 418-428.

Xia Zhang, Zhenfeng Fan, Keling Cheng, Zhiyong Wang, Lewen Chen, Bin Cai, Jun Ni. Data-independent acquisition proteomics-based investigation of the impact of ultra-early transcutaneous auricular vagus nerve stimulation on neurological function in ischemic stroke[J/OL]. Chinese Journal of Cerebrovascular Diseases(Electronic Edition), 2025, 19(05): 418-428.

目的

探讨超早期经耳迷走神经刺激（taVNS）对脑缺血再灌注引起的神经功能损伤的保护作用，并基于蛋白质组学探索其潜在的作用机制。

方法

将48只雄性SD大鼠随机分为模型组[短暂性大脑中动脉阻塞（tMCAO）组，16只]、假手术组（Sham组，16只）和taVNS组（16只）。taVNS组在脑缺血30 min后再灌注前给予1次taVNS干预，90 min后恢复血供。再灌注24 h后，应用改良神经功能缺损评分（mNSS）观察大鼠神经损伤程度，应用握力计测量前肢握力，采用2，3，5-三苯基氯化四氮唑（TTC）染色法观察脑梗死面积。此外，进行数据非依赖性采集（DIA）蛋白质组学分析，采用蛋白质印迹法检测裂解的半胱氨酸蛋白酶3（cleaved Caspase-3）、B淋巴细胞瘤-2（Bcl-2）和Bcl-2相关X蛋白（Bax）等凋亡蛋白的表达变化，应用酶联免疫吸附试验（ELISA）法检测白细胞介素（IL）-1β、IL-6和肿瘤坏死因子（TNF）-α的表达水平。采用独立样本t检验比较2组间差异，采用配对样本t检验比较组内不同时间点的差异，3组间差异比较采用单因素方差分析，组间两两比较采用LSD-t检验。

结果

taVNS组大鼠缺血再灌注后24 h的mNSS评分比缺血再灌注后3 h显著降低[（8.67±1.21）分 vs（10.00±0.63）分]，差异有统计学意义（t=4.029，P=0.010）。与tMCAO组相比，taVNS组大鼠前肢握力评分显著增高[（15.43±2.57）N vs （11.50±1.80）N]，差异有统计学意义（t=3.060，P=0.012）；脑梗死面积显著减小[（14.46±3.71）% vs （33.08±4.19）%]，差异有统计学意义（t=8.146，P<0.001）；cleaved Caspase-3和Bax的蛋白表达水平均显著减少，Bcl-2的蛋白表达水平显著增加，IL-1β、IL-6和TNF-α的表达水平均显著下降，差异均有统计学意义（P均<0.05）。DIA蛋白质组学结果提示，超早期taVNS对缺血性卒中大鼠的改善作用可能是通过调控凋亡信号网络和炎症相关通路实现的。

结论

超早期taVNS通过抑制大鼠缺血性脑卒中诱导的细胞凋亡和炎症反应，减轻大鼠缺血性脑损伤，发挥神经保护作用。

关键词: 缺血性脑卒中, 经耳迷走神经刺激, 超早期, 功能结局
Objective

To explore the protective effects of transcutaneous auricular vagus nerve stimulation (taVNS) against neurological functional impairment caused by cerebral ischemia-reperfusion injury, and to investigate the underlying mechanisms using proteomics approaches.

Mesthods

　Forty-eight male SD rats were randomly divided into three groups: model group [transient middle cerebral artery occlusion (tMCAO) group, n=16], Sham group (n=16), and taVNS group (n=16). The taVNS group received a single taVNS intervention 30 minutes after cerebral ischemia induction, prior to reperfusion. After 90 minutes of ischemia, blood flow was restored. At 24 hours after reperfusion, neurological impairment was assessed using the modified Neurological Severity Score (mNSS), forelimb muscle strength was measured with a grip strength meter, and cerebral infarction area was visualized with 2,3,5-triphenyltetrazolium chloride (TTC) staining. Additionally, data-independent acquisition (DIA) proteomics analysis was conducted, and Western blotting was performed to detect changes in the expression of apoptosis-related proteins including cleaved Caspase-3, B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax). Enzyme-linked immunosorbent assay (ELISA) was used to measure the expression levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α. Statistical analyses included independent samples t-test for intergroup comparisons, paired t-test for intragroup temporal comparisons, one-way ANOVA for multi-group comparisons, and LSD-t test for post-hoc pairwise analyses.

Results

In the taVNS group, the mNSS score after 24 h of ischemia-reperfusion was significantly lower than that after 3 h of ischemia-reperfusion [(8.67±1.21) points vs (10.00±0.63) points, t=4.029, P=0.010]. Compared to the tMCAO group, the taVNS group exhibited significantly greater forelimb grip strength [(15.43±2.57)N vs (11.50±1.80)N, t=3.060, P=0.012], significantly reduced cerebral infarct area [(14.46±3.71)% vs (33.08±4.19)%, t=8.146, P<0.001], and significantly decreased protein expression levels of cleaved Caspase-3 and Bax (all P<0.05). Conversely, the expression level of Bcl-2 was significantly increased (P<0.05). Additionally, the levels of IL-1β, IL-6 , and TNF-α were significantly reduced in the taVNS group (all P<0.05). DIA proteomics results suggested that the neuroprotective effects of ultra-early taVNS in ischemic stroke rats may be mediated through the regulation of apoptotic signaling networks and inflammation-related pathways.

Conclusion

Ultra-early taVNS exerts neuroprotective effects by against ischemic brain injury in rats, potentially through suppression of ischemia-induced apoptosis and inflammatory responses.

Key words: Ischemic stroke, Transcutaneous auricular vagus nerve stimulation, Ultra-early, Functional outcome
表1 线栓法诱导的大鼠脑缺血模型建立前后局部脑血流量变化（%，
±s）
组别 只数 建模前 建模后 差值 t值 P值
tMCAO组 12 99.84±0.44 38.17±3.08 61.66±2.86 74.576 <0.001
taVNS组 12 99.80±0.45 37.57±3.03 62.23±2.87 70.320 <0.001
t值 -0.483
P值 0.843
图1 建立短暂性大脑中动脉阻塞大鼠模型在插栓前（图a）、后（图b）通过激光斑点监测仪监测脑血流
表2 3组大鼠行为学指标比较（
±s）
组别 只数 mNSS评分（分） 前肢握力评分（N）
3 h 24 h t值 P值
Sham组 6 0 0 - - 23.42±2.58
tMCAO组 6 10.67±1.03 10.83±0.98 -0.542 0.611 11.50±1.80a
taVNS组 6 10.00±0.63 8.67±1.21 4.029 0.010 15.43±2.57ab
F值 40.137
P值 <0.001
图2 各组大鼠（n=6）脑缺血再灌注后24 h的TTC染色结果 注：Sham为假手术；tMCAO为短暂性大脑中动脉阻塞；taVNS为经耳迷走神经刺激。a，bSham组和taVNS组分别与tMCAO组比较，差异均有统计学意义（t=18.780、8.146，P均<0.001）
图3 各组大鼠（n=3）脑缺血再灌注后24 h苏木精-伊红（HE；图a）染色和尼氏染色结果（Brain：100 μm；Cortex：60 μm；图b） 注：Sham为假手术；tMCAO为短暂性大脑中动脉阻塞；taVNS为经耳迷走神经刺激；Brain为大脑；Cortex为皮质
图4 各组大鼠蛋白差异表达的火山图。图a为tMCAO组与Sham组的差异表达蛋白火山图；图b为taVNS组与tMCAO组的差异表达蛋白火山图 注：Sham为假手术；tMCAO为短暂性大脑中动脉阻塞；taVNS为经耳迷走神经刺激。显著上调的蛋白质以橙色标注[表达倍数（fold change，FC）>1.5且P.adj<0.05]，显著下调的蛋白质以蓝色标注（FC<0.67且P.adj<0.05），无差异的蛋白质标注为灰色
图5 经耳迷走神经刺激组与短暂性大脑中动脉阻塞组大鼠的组间差异蛋白GO功能富集分析（图a）和KEGG通路富集分析（图b） 注：MF为分子功能；CC为细胞组分；BP为生物过程
图6 各组大鼠（n=3）凋亡蛋白表达水平。图a为各组蛋白质印迹法测量cleaved Caspase-3、Bcl-2、Bax、tublin蛋白的化学发光条带图像；图b为各组cleaved Caspase-3/tublin的蛋白质印迹法结果分析；图c为各组Bcl-2/tublin的蛋白质印迹法结果分析；图d为各组Bax/tublin的蛋白质印迹法结果分析 注：Sham为假手术；tMCAO为短暂性大脑中动脉阻塞；taVNS为经耳迷走神经刺激；Bcl-2为B淋巴细胞瘤-2；Bax为B淋巴细胞瘤-2相关X蛋白。a，bSham组和taVNS组分别与tMCAO组比较，差异均有统计学意义（t=8.945、6.337，P均<0.001；c，dSham组和taVNS组分别与tMCAO组比较，差异均有统计学意义（t=4.637、4.619，P=0.008、<0.001）；e，fSham组和taVNS组分别与tMCAO组比较，差异均有统计学意义（t=10.230、7.990，P均<0.001）
图7 各组大鼠（n=3）炎症因子的表达水平。图a为各组IL-1β的酶联免疫吸附测定（ELISA）结果分析；图b为各组IL-6的ELISA结果分析；图c为各组TNF-α的ELISA结果分析 注：Sham为假手术；tMCAO为短暂性大脑中动脉阻塞；taVNS为经耳迷走神经刺激；IL为白细胞介素；TNF为肿瘤坏死因子。a，bSham组和taVNS组分别与tMCAO组比较，差异均有统计学意义（t=2.867、4.533，P=0.04、=0.039）；c，dSham组和taVNS组分别与tMCAO组比较，差异均有统计学意义（t=11.110、4.247，P<0.001、=0.006）；e，fSham组和taVNS组分别与tMCAO组比较，差异均有统计学意义（t=5.072、4.394，P=0.002、=0.009）
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