Guhan Yangsheng Jing alleviates sleep deprivation-induced neuronal injury via neurotransmitter rebalancing, mitochondrial protection, and inhibition of pyroptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Sleep deprivation is a growing public health burden linked to neuroinflammation, oxidative stress, and neurodegeneration. While conventional hypnotics provide transient relief, their long-term use is limited by tolerance and adverse effects. Guhan Yangsheng Jing (GHYSJ), a classical multi-herbal prescription from traditional Chinese medicine, has been clinically used to relieve insomnia and fatigue, yet its neuroprotective mechanisms remain unclear. OBJECTIVE: This study aimed to elucidate the protective effects and molecular mechanisms of GHYSJ against sleep-deprivation-induced neuronal injury, focusing on neurotransmitter balance, mitochondrial function, and pyroptosis regulation. METHODS: A PCPA-induced mouse model of sleep deprivation was established. Behavioral assays (righting-reflex and open-field tests), histological analyses, and ELISA quantifications of neurotransmitters and cytokines were performed. Integrative serum metabolomics and hippocampal transcriptomics were conducted to identify molecular pathways, and pyroptosis-related proteins were validated by immunohistochemistry, immunoblotting, and immunofluorescence. LPS/nigericin-induced pyroptosis in HT-22 neurons was used for in vitro validation. RESULTS: GHYSJ significantly shortened sleep latency, prolonged sleep duration, and restored locomotor activity. It rebalanced hippocampal 5-HTP, 5-HT, GABA, and Glu levels, preserved neuronal cytoarchitecture, and reduced degenerative signals. Multi-omics profiling revealed that GHYSJ modulated pathways related to the TCA cycle, lipid peroxidation, and innate immunity, reversing the aberrant expression of key genes such as Hspa1a, Plin4, Nod2, and Nkx2-4. GHYSJ suppressed hippocampal NLRP3, ASC, HMGB1, cleaved caspase-1, and GSDMD-N expression, decreased IL-1β/IL-18 release, and ameliorated mitochondrial swelling and cristae disruption. In HT-22 cells, GHYSJ-containing serum restored cell viability, alleviated G1/S arrest, stabilized mitochondrial membrane potential, increased ATP levels, and reduced pyroptosis-related membrane rupture. CONCLUSION: GHYSJ exerts multi-target neuroprotection through integrated neurotransmitter rebalancing, mitochondrial stabilization, and inhibition of NLRP3/caspase-1/GSDMD-mediated pyroptosis. These findings provide mechanistic insights supporting GHYSJ as a promising phytotherapeutic candidate for sleep disorders and related neurodegenerative diseases.