Shuangxia Decoction attenuates sleep disruption in 5×FAD mice through neuroinflammation inhibition: an integrative analysis of transcriptomic and molecular biology investigations.

Abstract

Ethnopharmacological relevance: Alzheimer's disease (AD) is a neurodegenerative disease characterized by memory and learning deficits. Circadian rhythm disruption-induced sleep disruption is frequently observed in AD patients. The Shuangxia Decoction (SXD) comprising Pinellia ternata (Thunb.) Breit. (Banxia) and Prunella vulgaris L. (Xiakucao), has been effectively used to treate sleep disruption for thousands of years. However, the mechanisms by which SXD treated AD through circadian rhythm-related pathways remain unexplored.

Aims of the study: This research sought to determine the efficacy, mechanisms, and active compounds of SXD in AD treatment via an integrative approach.

Materials and methods: We conducted a chronic jet lag (CJL) protocol in wild-type (WT) mice and monitored their rest/activity to compare their rest/activity period among WT, CJL, and CJD+SXD groups. In addition, we evaluated the impact of SXD on the cognitive and Aβ burden of 5×FAD mice by behavioral tests and Thioflavin staining. The underlying pathway analysis of SXD was revealed through transcriptomic and biology experimental validation. The active compounds of SXD were further analyzed using the UPLC-MS, molecular docking, and cellular thermal shift assay (CESTA).

Results: Our study demonstrated a rapid recovery of rest/activity period in CJL mice following SXD treatment. Additionally, SXD treatment alleviated Aβ plaque accumulation, subsequently preserving cognitive behavior and motor ability in 5×FAD mice. Moreover, SXD significantly enhanced neuronal synaptic plasticity dendritic plasticity in CA1 neurons of 5×FAD mice. Transcriptomic analysis showed upregulation of the neuroinflammation-related pathway in 5×FAD mice. Subsequent heatmap analysis indicated a suppression of inflammatory factor secretion (Cd68, Trem2, IL-6, IL-1β, Cxc3r1, Tnf, et al.) and an increase of anti-inflammatory factor secretion (IL4, Ccl19, Ccl21a et al.,) following SXD treatment in the 5×FAD mice. Meanwhile, SXD upregulated positive regulators involved in the circadian rhythm like Bmal1 and Clock, and downregulated negative regulators like Nr1d1. Moreover, microglia exhibited an amoeboid morphology characterized by few processes and rounded cell bodies in 5×FAD mice, whereas the age-matched SXD group maintained microglia with a ramified appearance. Additionally, our study identified 20 major components of SXD and identified 3-(3,4-Dihydroxyphenyl) lactic acid, Salviaflaside, and Ilexhainanoside D for further molecular docking with REV-ERBα (NR1D1), a commonly used circadian target. Salviaflaside further showed a strong bind with REV-ERBα via CESTA.

Conclusions: Our findings indicate that SXD may rescue circadian rhythm in 5×FAD mice through specifically binding to REV-ERBα in microglia to activate the BMAL1/ CLOCK pathway, thus inhibiting transcription of inflammatory factors, contributing to alleviating neuroinflammation and impeding AD progression. Our results offer a scientific foundation for developing SXD-based therapies in the early stage of AD, where sleep disruption precedes cognitive decline, offering potential leads for clinical trials to improve sleep quality thus delaying neurodegeneration in AD patients.