Rescue of CUMS-induced HPA axis hyperfunction and hypothalamic synaptic deficits by Citrus aurantium L. cv. Daidai...

Traditional Chinese medicine has historically used Citrus aurantium L. cv. Daidai to alleviate anxiety and improve mood. Essential oils are the primary active component of Citrus aurantium L. cv. Daidai, but little research has been conducted on the active substances and mechanisms of the antidepressant effect of Citrus aurantium L. cv. Daidai essential oil (CEO).

This research used network pharmacology, molecular docking, transcriptomics, and in vitro and in vivo experimental validation to assess the effectiveness and therapeutic mechanism of CEO.

We used gas chromatography‒mass spectrometry (GC-MS) to identify and quantify CEO components and brain-penetrating chemicals. CEO was given to chronic and unpredictable mild stimulation (CUMS) mice for 4 weeks. Depression was assessed using sucrose preference, forced swimming, tail suspension, elevated plus-maze, and open field tests. Analyzing brain homogenates and serum biochemistry data simultaneously revealed neurotransmitter and hormone alterations. Tissue samples were collected for histological and protein analysis. Furthermore, transcriptome and network pharmacology were used to investigate the mechanism by which CEO alleviates depression, and in vitro glutamate (Glu)-induced PC12 cell injury assays were conducted to validate this new mechanism.

CEO inhalation altered neurotransmitter and hormone expression and improved CUMS-induced weight and depression-like behavior in mice. Compared with CUMS mice, CEO mice presented less pathological brain damage, as demonstrated by HE staining, immunohistochemistry, Golgi staining, transmission electron microscopy, and immunofluorescence staining. We discovered that 13 of the active chemicals in CEO act on 522 targets, 96 of which are linked to depression. PRKACA was identified as the core target by a modular analysis of the PPI network. Network pharmacology and transcriptomics revealed that CEO influences depression via the cAMP/PKA/Grin2b pathway and Glu synaptic modulation. In vivo studies indicated that CEO administration reduced PKA and Grin2b phosphorylation in CUMS mice, inhibited the cAMP/PKA/Grin2b pathway, protected against synaptic deficits, and restored HPA axis function. In vitro investigations revealed that the survival rate of PC12 cells treated with CEO increased, the apoptotic rate decreased, and the expression of LDH, Ca²⁺, and MDA decreased. Western blot and immunofluorescence staining indicated that CEO inhibits the cAMP/PKA/Grin2b pathway to regulate Glu-induced PC12 cells and that 8-Bromo-cAMP pretreatment reduces the protective effect of CEO.

The active chemicals in CEO can inhibit the cAMP/PKA/Grin2b pathway, reduce anxiety and depression, alleviate excitotoxicity caused by Glu synaptic overactivation, protect against hypothalamic synaptic deficits, and restore HPA axis function. This research could serve as a reference for the field of illness prevention and complementary therapies for depression.