Tongren Niuhuang Qingxin Wan attenuates chronic stress-induced anxiety-like behavior by modulating adrenergic signaling and synaptic plasticity.

Chronic stress is a major precipitating factor for anxiety disorders, yet current pharmacotherapies are limited by delayed onset of action and adverse effects, highlighting the need for safer and more effective alternatives. Tongren Niuhuang Qingxin Wan (NHQXW), a traditional Chinese medicine formulation, has been used clinically to alleviate symptoms including palpitations, insomnia, and anxiety-related discomfort; however, its potential to mitigate stress-induced anxiety-like behaviors in preclinical models, along with the underlying molecular mechanisms, remains largely unexplored. In this study, using the chronic unpredictable mild stress (CUMS) mouse model, we assessed anxiety-like behaviors via the open field, elevated plus maze, and light-dark box tests. Neurotransmitter levels were measured by LC-MS/MS, and associated molecular alterations were investigated using non-targeted proteomics, followed by RT-qPCR and western blotting for validation. NHQXW attenuated the development of CUMS-induced anxiety-like behavioral alterations and reduced the stress-associated elevation of norepinephrine in the PFC. Proteomic analysis identified 107 differentially expressed proteins in the PFC of high-dose NHQXW-treated mice compared to CUMS controls, primarily enriched in adrenergic signaling, synaptic plasticity, and stress-related pathways. Subsequent validation showed that NHQXW upregulated the expression of α2A-adrenergic receptor (ADRA2A) and its coupled G protein subunit GNAI2, normalized glucocorticoid receptor expression, and protected synapse-related proteins. These findings suggest that the protective effects of NHQXW may be associated with modulation of prefrontal noradrenergic signaling and synaptic plasticity. This study provides a molecular rationale for the therapeutic potential of NHQXW as a novel, multi-targeted intervention for stress-related anxiety disorders, paving the way for future translational research and clinical applications.