BACKGROUND: Sleep disturbances in depressive disorders are closely associated with circadian rhythm disruption of the BMAL1-mediated clockophagy. Semen Ziziphi Spinosae (SZS) has demonstrated clinical efficacy in relieving depression-related sleep disorders. However, the specific mechanism by which SZS acts through the BMAL1-dependent regulation of clockophagy circadian rhythmicity remains undefined.
PURPOSE: To investigate the mechanistic role of SZS in ameliorating depression-associated sleep disturbances via modulation of the BMAL1-mediated circadian rhythm of the clockophagy axis.
METHODS: Depression mouse models were established using Chronic Unpredictable Mild Stress (CUMS), and sleep deprivation (SD) was employed to induce sleep disorder phenotypes. Different concentrations of SZS were administered to assess behavioral indices, including the Forced Swim Test (FST), Tail Suspension Test (TST), Open Field Test (OFT), Sucrose Preference Test (SPT), sleep-wake parameters, including sleep duration, latency, wake time, non-rapid eye movement sleep (NREMS) and rapid eye movement sleep (REMS) duration, sleep bout stability, and stage transitions, and circadian fluctuations in 5-hydroxytryptamine (5-HT). A cellular model of clockophagy circadian dysregulation was generated using the autophagy activator rapamycin. Western Blotting (WB) and immunofluorescence were employed to assess circadian rhythmicity of clockophagy proteins (ATG7, ATG5, LC3, P62, and BMAL1). Brain-penetrating components of SZS were identified, and molecular docking combined with Cellular thermal shift assay (CETSA) validated their direct binding to BMAL1. Bioinformatics analysis of GEO dataset highlighted the cAMP signaling pathway as a downstream effector of BMAL1, which is confirmed via functional validation in BMAL1 knockout and control cells. Animal studies further evaluated circadian dynamics of the cAMP pathway, including cAMP levels, p21-Activated Kinase 1 (PAK1), p-PAK1, Phosphorylated PAK1 (p-PAK1), cAMP-responsive element-binding protein (CREB), and Phosphorylated CREB (p-CREB).
RESULTS: SZS intervention significantly improved depressive-like behaviors and normalized sleep architecture in CUMS- and SD-exposed mice, manifesting as increased total sleep time, shortened latency, and restored NREMS, REMS duration, sleep bout organization, and stage transition kinetics. Mechanistically, SZS regulated circadian oscillations of 5-HT, core clockophagy proteins, and key nodes of the cAMP signaling cascade. SZS-containing serum recapitulated these effects on clockophagy and cAMP pathway rhythmicity in vitro. Structural docking and CETSA confirmed direct interaction between bioactive compounds from SZS and BMAL1.
CONCLUSIONS: SZS improves depression-related sleep disorders by regulating the circadian rhythm of the cAMP signaling pathway through BMAL1-mediated clockophagy..
