摘要
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。
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-αwere 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.
作者
张霞
樊震峰
成柯岭
王志勇
陈乐文
蔡斌
倪隽
Zhang Xia;Fan Zhenfeng;Cheng Keling;Wang Zhiyong;Chen Lewen;Cai Bin;Ni Jun(Department of Rehabilitation,the First Affiliated Hospital,Fujian Medical University,Fuzhou 350005,China;Department of Neurology,the First Affiliated Hospital,Fujian Medical University,Fuzhou 350005,China;Department of Rehabilitation,National Regional Medical Center,Binhai Campus of the First Affiliated Hospital,Fujian Medical University,Fuzhou 350212,China)
出处
《中华脑血管病杂志(电子版)》
2025年第5期418-428,共11页
Chinese Journal of Cerebrovascular Diseases(Electronic Edition)
基金
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)。
关键词
缺血性脑卒中
经耳迷走神经刺激
超早期
功能结局
Ischemic stroke
Transcutaneous auricular vagus nerve stimulation
Ultra-early
Functional outcome
