What does this research mean for the field?

The combination of tetrahydroberberine and protopanaxadiol significantly improves sleep latency and duration in PCPA-induced insomnia in rats, while also restoring neurochemical balance and gut microbiota homeostasis. Novelty: ClaimNovelty.NOVEL_FINDING. Consensus alignment: ConsensusAlignment.NEUTRAL.

What question did this study set out to answer?

The aim is to explore how THB combined with PPD affects insomnia induced by PCPA in rats and elucidate underlying mechanisms.

March 3, 2026Open Access

Synergistic Therapeutic Effects of Tetrahydroberberine Combined with Protopanaxadiol on PCPA-Induced Insomnia in Rats: Involvement of the Microbiota–Gut–Brain Axis and Regulation of PI3K/AKT/AGE-RAGE Pathways

Key Points

The aim is to explore how THB combined with PPD affects insomnia induced by PCPA in rats and elucidate underlying mechanisms.
Rats divided into treatment groups: normal, model, diazepam, THB monotherapy, PPD monotherapy, THB + PPD combination.
Evaluations included sleep tests, histological analysis, cytokine measurements, gut microbiota sequencing, and signaling pathway analysis.
Analysis of neurotrophic factors and neurotransmitter levels was conducted.
THB + PPD combination shortened sleep latency by 56.2% and prolonged sleep duration by 112.8% compared to the model group.
Histological improvements included reduced neuronal damage and more intact neurons in the dentate gyrus.
Molecular improvements included increased BDNF and GDNF, restored neurotransmitter levels, and reduced inflammatory cytokines.

Abstract

Aim: This study investigated the synergistic therapeutic effects and underlying mechanisms of tetrahydroberberine (THB) combined with protopanaxadiol (PPD) on p-chlorophenylalanine (PCPA)-induced insomnia in rats. Methods: Rats were randomly divided into normal, model, diazepam, THB monotherapy, PPD monotherapy, and THB + PPD combination groups. Evaluations included the pentobarbital sleep test, HE staining, ELISA, 16S rRNA sequencing, metabolomics, and Western blot. Results: Results demonstrated that the THB + PPD combination exhibited significant synergistic effects compared with monotherapies: the combination shortened sleep latency by 56.2% (vs. 44.2% for THB alone and 20.7% for PPD alone) and prolonged sleep duration by 112.8% (vs. 70.2% for THB and 59.6% for PPD) relative to the model group, while effectively restoring body weight gain. Histologically, combined treatment significantly alleviated hippocampal neuronal damage and increased the number of intact neurons in the dentate gyrus. Molecularly, it upregulated brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) levels, restored neurotransmitter balance (serotonin, dopamine, and glutamate), suppressed overactivation of the hypothalamic–pituitary–adrenal (HPA) axis (reducing corticotropin-releasing hormone and corticosterone), and decreased pro-inflammatory cytokine expression. Gut microbiota analysis revealed that the combination restored microbial homeostasis (increasing beneficial bacteria such as *Lactobacillus*) and modulated the glycine–serine–threonine metabolic pathway. Mechanistically, THB + PPD synergistically activated the PI3K/AKT neurotrophic pathway (p-PI3K and p-AKT expression increased by 1.9-fold and 2.5-fold, respectively, vs. model), inhibited the AGE/RAGE pro-inflammatory axis (RAGE expression decreased by 31.8%), and enhanced blood–brain barrier integrity by upregulating tight junction proteins (ZO-1, Occludin). Conclusions: THB combined with PPD exerts synergistic anti-insomnia effects through multi-level regulation of the microbiota–gut–brain axis, neurochemical balance, and key signaling pathways, providing a promising foundation for developing safe natural product-based combination therapies.

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