Spinosin's Hypnotic Effect Mediated by Adenosine A2A Receptors in Male Mice

Insomnia is a widespread sleep disorder, and current hypnotic medications often carry safety concerns, driving a critical need for safer, effective natural product-derived sleep aids. Spinosin, a key active component of Semen Ziziphi Spinosae, is known for its sedative and hypnotic properties. The adenosine receptor (AR) pathway is a recognized therapeutic target for insomnia, particularly the A2AR subtype, which plays a crucial role in sleep regulation. However, the precise mechanism by which Spinosin exerts its hypnotic effects via A2ARs has remained largely unexplored, representing a significant gap in understanding its therapeutic potential.

In male mice, researchers investigated Spinosin's hypnotic effects and the involvement of A2ARs. The study utilized caffeine, a known A2AR antagonist, and compared A2AR-wild-type (WT) mice with A2AR-knockout (KO) mice. Diazepam was included as a positive control to validate the experimental model. Primary endpoints included assessment of non-rapid eye movement (NREM) sleep and locomotor activity. Neuronal activity in the nucleus accumbens (NAc) and lateral hypothalamus (LH) was evaluated using c-Fos immunostaining. Additionally, molecular docking and molecular dynamics (MD) simulations were performed to confirm the interaction between Spinosin and the A2AR at a molecular level.

The hypnotic effects of Spinosin were effectively antagonized by caffeine, a known A2AR blocker. Crucially, compared to A2AR-wild-type (WT) mice, Spinosin-induced non-rapid eye movement (NREM) sleep and diminution of locomotor activity were significantly reduced in A2AR-knockout (KO) mice, strongly implicating A2ARs in its sleep-promoting actions. This indicates that the absence of functional A2ARs substantially diminishes Spinosin's ability to induce sleep and reduce activity.

Spinosin significantly increased the activity of γ-aminobutyric acid (GABA)ergic medium spiny neurons (MSNs) in the nucleus accumbens (NAc) and significantly decreased the activity of orexin neurons in the lateral hypothalamus (LH), as revealed by c-Fos immunostaining. These observed changes in neuronal activity, critical for sleep-wake regulation, were significantly reversed by caffeine pretreatment or in A2AR-KO mice, further solidifying the A2AR-mediated mechanism. Finally, molecular docking and MD simulations provided compelling evidence that Spinosin exhibits good binding potential and strong binding stability with the A2AR.