Regulatory effects of Yixin Anshen Granules on neurotransmitters and receptor expression in insomnia model rodents

Yixin Anshen Granules (YX) has demonstrated notable clinical efficacy for insomnia, a prevalent sleep disorder with a complex and multifaceted pathogenesis. TCM offers unique advantages in its management, characterized by a favorable safety profile and potent therapeutic effects. Consequently, TCM plays an indispensable role in the therapeutic landscape of insomnia.

This study aims to investigate the sedative and hypnotic effects of YX, explore how they influence insomnia-prone mice induced by PCPA, and examine their impact on sleep architecture in a rat model of insomnia caused by chronic unpredictable mild stress (CUMS) and PCPA administration.

Locomotor activity and sleep patterns were assessed using the open field and pentobarbital sodium-induced sleep tests. The Morris water maze test was performed to evaluate the ameliorative effects of YX on learning and memory in insomniac mice. Neuronal damage in the cerebral cortex and hypothalamus was evaluated by hematoxylin and eosin (HE) staining. Brain levels of serotonin (5-HT), γ-aminobutyric acid (GABA), melatonin (MT), and orexin (OX) were measured by enzyme-linked immunosorbent assay (ELISA), while immunofluorescence was used to detect 5-HT and GABA levels specifically in the cerebral cortex and hypothalamus. Protein expression of 5-HT1A, GABA Aα1, GABA Aγ2, MT1, and OX1 receptors in brain tissue was determined by Western blot (WB). Based on these findings, the two doses exhibiting the most potent sedative-hypnotic effects were identified for sleep phase monitoring. Insomnia was induced in rats using CUMS combined with PCPA, and sleep architecture was assessed via electroencephalography (EEG). The innovation of this study lies in the fact that, in addition to conventional assessments including the open field test, pentobarbital-induced sleep test, and Morris water maze test, EEG/EMG-based sleep-stage analysis was further introduced to extend the pharmacodynamic evaluation to the level of objective sleep architecture, thereby enabling a systematic assessment of the effects of YX on insomnia-like behaviors, sleep, and cognitive function. Meanwhile, two insomnia-related models, namely PCPA-induced insomnia mice and CUMS-PCPA-induced insomnia rats, were employed for pharmacodynamic/mechanistic screening and sleep architecture validation, respectively. This design makes the insomnia models more closely aligned with clinical features and enhances the robustness, systematic nature, and persuasiveness of the findings.

Treatment with YX significantly ameliorated locomotor and exploratory deficits in insomniac mice, as demonstrated by increased total distance, duration, and average speed traveled in the open field test. Moreover, the granules promoted sleep by increasing the sleep onset rate, shortening sleep latency, and extending sleep duration in the PCPA-induced insomnia model. Furthermore, YX markedly ameliorated neuronal damage in the cerebral cortex and hypothalamus, as evidenced by reduced neuronal shrinkage and chromatin condensation, and decreased neuronal necrosis. ELISA results demonstrated that the granules increased brain levels of 5-HT, GABA, and MT, while reducing OX levels. Consistently, WB revealed upregulation of 5-HT1A, GABA Aα1, GABA Aγ2, and MT1 receptor proteins, alongside downregulation of OX1 receptor expression in brain tissue. Immunofluorescence further confirmed enhanced 5-HT and GABA fluorescence intensity in both the cerebral cortex and hypothalamus following treatment. Notably, EEG monitoring revealed that YXZ and YXG significantly reduced wakefulness (Wake) and prolonged non-rapid eye movement (NREM) sleep in insomniac rats. In addition, YXZ and YXG markedly reduce the bout occurrences of Wake and NREM and prolong the average duration of the NREM period. Additionally, at the 4th hour of recording, YXG significantly increased the delta band power in the NREM period of insomnia rats.

In conclusion, YX exerts significant sedative-hypnotic effects, markedly shortening Wake time and prolonging NREM sleep in rats, and reducing the frequency of Wake and NREM occurrences, and prolonging the average duration of the NREM period. Additionally, YXG increased the delta-band power during the NREM period in insomnia rats, promoting deep sleep. These effects may be mediated by enhancement of GABAergic and serotonergic neurotransmission, as well as by modulating central MT and OX levels and their receptor expression.