Neuroprotective Effects of AnMei Decoction on Hypothalamic Neuronal Injury and Circadian Rhythm Dysregulation in Murine Models of Insomnia.

ETHNOPHARMACOLOGICAL RELEVANCE: AnMei Decoction(AMD) is a traditional Chinese herbal formula historically employed in the management of insomnia. Notwithstanding its long-standing clinical application, the neurobiological mechanisms through which AMD modulates and restores the circadian sleep-wake rhythm remain to be fully elucidated. RESEARCH OBJECTIVE: This study aims to investigate the therapeutic efficacy of AMD in ameliorating circadian rhythm disturbances induced by p-chlorophenylalanine (PCPA) in Hcrt-Cre transgenic mice, while further elucidating the underlying regulatory mechanisms involving the orexin system and core circadian clock genes. MATERIALS AND METHODS: Insomnia was induced in Hcrt-Cre mice via intraperitoneal administration of PCPA. Sleep-wake architecture was meticulously monitored using EEG-EMG recordings, whereas anxiety-like behavior was assessed using the open-field test. To probe the mechanisms involved, we performed Nissl staining to examine the histomorphological integrity of orexin neurons. Quantitative analysis of key neurotransmitters (OXA, OXB, 5-HT, NE, CRH, ACh, and CORT) was conducted using ELISA. Furthermore, real-time PCR (RT-PCR) was utilized to profile the mRNA expression patterns of core circadian clock genes (Bmal1, Clock, Cry1, and Per1) and components of the hypocretin system (OXA, OXB, OX1R, and OX2R). RESULTS: AMD treatment significantly attenuated PCPA-induced sleep fragmentation, characterized by a marked prolongation of REM sleep duration and the restoration of normal NREM/REM cycle periodicity as confirmed by EEG-EMG monitoring. Concurrently with these behavioral improvements, AMD treatment, particularly at the medium and high doses, was associated with down-regulation of various arousal-promoting neurotransmitters within the hypothalamus-specifically OXA,OXB,NE,ACh,CRH,and CORT-while concurrently augmenting the levels of the inhibitory neurotransmitter 5-HT. Nissl staining revealed that AMD effectively restored the structural integrity of hypothalamic Nissl bodies, suggesting an ameliorative effect on the morphological alterations of hypothalamic neurons induced by sleep deprivation. Furthermore, AMD significantly attenuated the hyper-activation of the orexin signaling pathway and corrected the aberrant mRNA expression levels of core circadian clock genes (Bmal1, Clock, Cry1, and Per1) as measured at a single time point. Across multiple efficacy parameters, high-dose AMD demonstrated therapeutic outcomes that were either comparable or superior to the positive control, suvorexant. CONCLUSION: Our findings suggest that AMD exerts significant sleep-stabilizing effects, which may involve the modulation of the hypothalamic orexin system and core circadian clock genes. These results provide pharmacological evidence supporting the traditional use of AMD against insomnia and highlight its therapeutic potential as a candidate agent for addressing anxiety and cognitive impairments associated with sleep disorders.