Frequency‐Dependent Effects of Transauricular Vagus Nerve Stimulation on Alleviating Visceral Hypersensitivity: Involvement of the Nesfatin‐1/ <scp>CRF</scp> / <scp>CRF1R</scp> Pathway

BACKGROUND: Visceral hypersensitivity (VH) is acknowledged as a critical pathogenic mechanism underlying functional gastrointestinal disorders (FGIDs), which is influenced by dysregulation of the gut-brain axis. Transauricular vagus nerve stimulation (taVNS) is a clinically used intervention for the relief of visceral pain; however, its underlying neural mechanisms remain poorly understood. Drawing on the pathogenesis of VH and our team's previous research, we hypothesized that taVNS mitigates VH through the nesfatin-1/corticotropin-releasing factor (CRF) pathway. This study aims to elucidate the potential central regulatory mechanisms that contribute to the analgesic effects of taVNS. METHODS: A validated VH model was established in Sprague-Dawley (SD) rats through maternal separation (MS) combined with acetic acid enema. The VH rats underwent taVNS at frequencies of 2 Hz, 10 Hz, and 15 Hz, whereas the sham group received 2 Hz electrical stimulation at the earlobe. The visceral pain threshold was evaluated using the abdominal withdrawal reflex (AWR) scoring and electromyographic activity of the external oblique muscle. Additionally, Western blot analysis was conducted to assess the expression levels of nesfatin-1, CRF, and its receptors 1 and 2 (CRF1/2R) in the hypothalamus. KEY RESULTS: The analyses of AWR and electromyogram (EMG) responses indicated that (a) the area under the curve (AUC) of the EMG response to colorectal distension (CRD) at four distension pressures (20, 40, 60, and 80 mmHg) was significantly elevated in the VH group. (b) The AUC was significantly reduced in the 2 Hz taVNS group across all four distension pressures. (c) The 10 Hz taVNS group exhibited a significant decrease in the AUC at 20 mmHg and 40 mmHg. Furthermore, the results of Western blot analysis demonstrated that (a) 2 Hz taVNS significantly lowered the expressions of nesfatin-1, CRF, and CRF1R, aligning with the findings in the sham group. (b) Both 10 Hz and 15 Hz taVNS significantly decreased CRF expression, whereas nesfatin-1 and CRF1R expression remained unchanged. (c) There was no significant downregulation of CRF2R expression among the intervention groups. CONCLUSION: This study demonstrated that taVNS alleviates VH, potentially via the nesfatin-1/CRF/CRF1R signaling pathway in the rat hypothalamus. Furthermore, the analgesic effect was frequency-dependent, with 2 Hz taVNS providing a more effective reduction in visceral pain.