Aurantii Fructus alleviates depression via modulating hippocampal caspase-4-linked neurogenesis and synaptic plasticity in mice.

Ethnopharmacological Relevancy: Aurantii Fructus (AF) is known for its ability to regulate qi and alleviate stagnation. In Traditional Chinese Medicine (TCM), qi stagnation is considered a key factor in the development of major depressive disorder (MDD). Scientific research needs to further reveal the exact neural mechanisms of AF's antidepressant effects.

Aim Of The Study: The aim of this research was to explore the effect of AF on neurogenesis and synaptic plasticity by inhibiting caspase-4 in the hippocampus.

Methods And Materials: Ultra-performance liquid chromatography (UPLC) was employed to determine the component content of AF and assess the stability of its extracts. We started by evaluating AF's antidepressant effects and effective dose using mouse models of depression induced by chronic unpredictable mild stress (CUMS) and verificated by chronic restraint stress (CRS), along with behavioral tests such as the sucrose preference test (SPT) and tail suspension test (TST). Transcriptomics was utilized to identify the specific targets of AF's antidepressant effects, and verified the protein and gene expression of this molecule in the hippocampus using Western blot analysis and RT-PCR. Via hippocampus-specific viral overexpression combined with behavioral and immunofluorescence analyses, we verified the role of this target in AF's antidepressant effects concerning neuronal excitability, neurogenesis, and synaptic proteins.

Results: In this study, we systematically characterized the antidepressant potential of AFand delineated its underlying neurobiological mechanism. UPLCquantification of its aqueous extract identified five major bioactive constituents-hesperidin, naringin, neohesperidin, nobiletin, and tangeretin-whose batch-to-batch concentrations were consistent (CV < 5 %), confirming extract stability. Using two well-validated mouse models of depression-CUMS and CRS-we demonstrated that a single acute oral dose of AF (2 g/kg) rapidly ameliorated depressive-like behaviours within 24 h. In the forced-swim test, AF reduced immobility time to the same extent as the fast-acting antidepressant ketamine (5 mg/kg, i.p.). Similarly, anhedonia-like behaviour, assessed by the sucrose-preference test, was completely reversed by AF in both CUMS and CRS cohorts. At the molecular level, CRS induced increase in hippocampal caspase-4 mRNA and in active-caspase-4 protein relative to unstressed controls (P < 0.01). AF treatment normalized these elevations to baseline, indicating potent suppression of caspase-4 expression and activity. Site-specific over-expression of caspase-4 in the dorsal hippocampus (AAV-casp4-eGFP) abolished AF's behavioural efficacy, confirming that its antidepressant action is contingent upon hippocampal caspase-4 inhibition. Functional consequences of caspase-4 down-regulation were examined 24 hR post-treatment. Immunohistochemistry revealed a increase in c-FOS neurons in the dentate gyrus (DG) and CA3 subfield (P < 0.001), indicative of elevated neuronal excitability. Concurrently, the number of Ki67/BrdU/DCX newborn neurons in the subgranular zone of the DG (P < 0.01), demonstrating robust enhancement of adult hippocampal neurogenesis. Synaptic integrity was assessed by Western blot and immunofluorescence. AF elevated postsynaptic density protein-95 (PSD95) and presynaptic synaptophysinin hippocampal homogenates (P < 0.01 for both). Confocal imaging further showed increase in co-localized PSD95/synaptophysin, reflecting strengthened synaptic connectivity. Importantly, these synaptic gains were fully reversed when caspase-4 was ectopically activated, underscoring a causal link between AF-mediated caspase-4 inhibition and synaptic plasticity.

Conclusions: AF alleviates depression by inhibiting hippocampal caspase-4, thereby enhancing neurogenesis, neuronal excitability and synaptic transmission, establishing caspase-4 as a key target for its antidepressant action.