Parishin A alleviates insomnia by regulating hypothalamic-pituitary-adrenal axis homeostasis and directly targeting orexin receptor OX2.

Parishin A (PA), a bioactive compound derived from Gastrodia elata Blume, has been used as a herbal remedy for insomnia. Nevertheless, the mechanism underlying the effect of PA on promotion of sleep and its potential targets remain to be elucidated. This study aimed to investigate the potential of PA in ameliorating insomnia, probing into its interactions with the orexin receptor 2 (OX2), antagonists of which are used clinically for the treatment of sleep disorders. We employed an array of methodologies, including in vivo experiments involving the assessment of the impacts of PA on sleep behavior in a p-chlorophenylalanine (PCPA)-induced insomnia mouse model, and the detection of neurotransmitters, inflammatory factors, and hypothalamic-pituitary-adrenal (HPA) axis-related hormones. In vitro experiments, such as extracellular signal-regulated kinase (ERK) 1/2 phosphorylation assay, drug-receptor binding stability assay (DARTS), cellular thermal shift assay (CETSA), solvent-induced protein precipitation (SIP), and molecular docking, were performed to validate the interaction between PA and OX2. The results showed that PA relieved insomnia in mice by effectively increasing the content of 5-hydroxytryptamine (5-HT) while reducing those of dopamine (DA), norepinephrine (NE) and glutamine/γ-aminobutyric acid (Glu/GABA), as well as the inflammatory factor tumor necrosis factor-alpha (TNF-α) in the hypothalamus. PA also improved the morphological changes in the hippocampus of insomnia mice and decreased the levels of HPA axis-related hormones. Furthermore, OX2 was found to be a potential direct target of PA. In conclusion, PA might be an antagonist of OX2 because of its ability to inhibit OX2-induced ERK 1/2 activation. These findings provide valuable insights into the therapeutic potential of PA in insomnia.