Citrus-caspase-4-serotonin axis: Aurantii Fructus rapidly reverses chronic-stress-induced despair via suppression of hippocampal caspase-4.

Abstract

ETHNOPHARMACOLOGICAL RELEVANCE: The citrus-derived flavonoid-rich fraction Aurantii Fructus (AF) has been traditionally used in East Asian folk medicine to relieve mood-related complaints, yet its antidepressant profile and mechanism remain ill-defined. AIM OF THE STUDY: To evaluate the antidepressant-like activity of AF in a chronic unpredictable mild stress (CUMS) mouse model and to identify the molecular targets involved. MATERIALS AND METHODS: Chemical fingerprinting was performed by LC/MS. Male C57BL/6 mice were subjected to 4-week CUMS and treated orally with AF (0.5, 0.75, 1 g/kg) or fluoxetine (10 mg kg) for 7 days. Behavioral tests (TST, FST, SPT, OFT) were conducted. Network pharmacology, RNA-seq, and hippocampal over-expression of caspase-4 by adeno-associated virus were employed to dissect mechanisms. RESULTS: LC/MS identified naringin, hesperidin, neohesperidin, naringenin and nobiletin as major constituents. AF dose-dependently reversed CUMS-induced despair-like behavior and anhedonia without altering locomotion. Integrated network pharmacology and transcriptomics highlighted the serotonergic synapse and caspase-4 as top targets. AF restored hippocampal SERT, 5-HT and 5-HT1A levels while down-regulating caspase-4 mRNA and protein. Caspase-4 over-expression abolished both the behavioral benefits and the serotonergic effects of AF. CONCLUSIONS: AF produces antidepressant-like actions in CUMS mice, mechanistically linked to inhibition of hippocampal caspase-4 and subsequent potentiation of serotonergic neurotransmission. These data validate AF as a promising botanical lead for mood disorders and identify caspase-4 as a previously unrecognized therapeutic node.