Transcutaneous auricular vagus nerve stimulation alleviates depressive- and anxiety-like behaviors in adolescent stressed rats via SIRT1/NRF2-mediated modulation of hippocampal redox and neuroimmune homeostasis.

AIMS: This study aimed to identify key molecular changes in the hippocampal subregions underlying the antidepressant effects of transcutaneous auricular vagus nerve stimulation (taVNS) in a rat model of chronic unpredictable mild stress (CUMS) during adolescence, and to explore the potential involvement of the SIRT1/NRF2/GPX4 signaling pathway in regulating oxidative stress and related ferroptosis- and pyroptosis-associated signaling.

METHODS: To investigate the antidepressant mechanism of taVNS, a CUMS rat model was established. One hundred postnatal day 21 male Sprague-Dawley rats were used. In Experiment 1, rats (n = 10 per group) were randomly assigned to Control (Con), CUMS model (Mod), CUMS+taVNS (taVNS), and CUMS+tnVNS (tnVNS) groups. After 21 days of CUMS exposure, rats received a 21-day taVNS or tnVNS intervention. In Experiment 2, rats (n = 10 per group) were randomly assigned to Con, Mod, taVNS, taVNS+DMSO, taVNS+ML385 (NRF2 inhibitor), and taVNS+EX527 (SIRT1 inhibitor) groups, with inhibitors administered intraperitoneally for 14 consecutive days. Behavioral tests, including sucrose preference test (SPT), open field test (OFT), elevated plus maze (EPM), and passive avoidance test, were performed. Hippocampal tissues and plasma were collected for ELISA, western blot, and immunofluorescence analyses. The expression of SIRT1, NRF2, GPX4, KEAP1, NLRP3, Caspase-1, GSDMD, COX-2, and β-actin was assessed, along with oxidative stress markers (MDA, SOD, CAT, GSH), inflammatory cytokines (IL-1β, TNF-α, IL-18, IFN-γ), and immunofluorescence staining for Iba-1/NeuN, PSD95/MAP2, BDNF/TREM2, and 4-HNE.

RESULTS: Compared with the Con group, CUMS-induced rats exhibited significant depressive- and anxiety-like behaviors, increased oxidative stress and neuroinflammation, and decreased expression of SIRT1, NRF2, and GPX4 in the hippocampus. taVNS treatment significantly reversed these behavioral abnormalities, reduced MDA, IL-1β, and TNF-α levels, and increased SOD, CAT, and GSH activities. taVNS also upregulated SIRT1, NRF2, and GPX4 expression, while downregulating KEAP1 and COX-2, and showed a downregulation trend in GSDMD-related pyroptotic effectors, without statistically significant changes in NLRP3 or Caspase-1 protein levels. Immunofluorescence showed that taVNS reduced Iba-1-positive microglia, increased NeuN/Iba-1 ratio, restored MAP2 and PSD95 expression, and decreased 4-HNE levels in the CA1, CA3, and DG regions. However, administration of ML385 or EX527 attenuated these protective effects, as evidenced by worsened behavioral performance, increased oxidative stress and inflammatory markers, and reduced expression of NRF2 and GPX4. No significant differences were observed between the taVNS and taVNS+DMSO groups.

CONCLUSION: This study demonstrates that the SIRT1/NRF2/GPX4 signaling pathway in the hippocampus is a critical regulatory node underlying CUMS-induced oxidative stress and the associated alterations in ferroptosis- and pyroptosis-related signaling in adolescent rats. taVNS exerts its antidepressant and anxiolytic effects by activating this pathway, thereby attenuating ferroptosis- and pyroptosis-related signaling, alleviating neuroinflammation, and enhancing neural plasticity.