Neferine improves sleep quality in chronically sleep-deprived mice by antagonizing OXRs in noradrenergic and serotonergic neurons.

Orexin receptors (OXRs) have emerged as promising therapeutic targets for insomnia. Our preliminary research suggested that neferine (NEF)-a bisbenzylisoquinoline alkaloid derived from Nelumbinis plumula-is a potential antagonist of OXRs in vitro. However, it remains poorly understood whether NEF affects sleep architecture and quality, as well as the underlying neurobiological mechanisms. To explore this, a chronic sleep-deprived (CSD) mouse model was established using a multi-platform method. The pentobarbital-induced sleep test and HomeCageScan system analysis showed that 3 weeks of CSD prolonged sleep latency, decreased sleep duration and reduced sleep behavior, and NEF treatment (80 mg/kg) could obviously reverse these effects. Telemetric EEG/EMG recordings revealed that NEF administration increased the delta power ratio (sleep depth) and reduced sleep fragmentation (sleep stability) in chronically sleep-deprived mice. Mechanistically, Ca2+ influx assays and immunofluorescence staining demonstrated that NEF effectively antagonized OXRs both in vitro and in vivo. Bioinformatics analysis revealed that OXRs are highly expressed in monoaminergic neurons within the locus coeruleus (LC) and the dorsal raphe nucleus (DRN). NEF treatment suppressed the activities of noradrenergic-LC and serotonergic-DRN neurons, and reduced the levels of their corresponding neurotransmitters, norepinephrine (NE) and serotonin (5-HT). Together, these findings indicate that NEF exerts desirable sleep-promoting effects by improving sleep depth and sleep stability in sleep-deprived mice, and these effects are potentially mediated by antagonizing OXRs in noradrenergic and serotonergic neurons. This study provides a theoretical basis for the application of NEF in insomnia and offers a new scaffold for insomnia drug development.