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.

　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.

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.

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