Otophylloside T alleviates depression-like behaviors by directly targeting EP4: An integrated multi-strategy study.

BACKGROUND: Depression is closely linked to inflammation, driving interest in novel antidepressant targets. However, the inconsistent efficacy of current anti‑inflammatory agents underscores the urgent need for new mechanisms and targets. PURPOSE: This study aimed to evaluate the antidepressant effects of Qingyangshen (QYS) extract and its constituent Otophylloside T (Oto-T) on neuroinflammation-mediated depression and to elucidate their pharmacological mechanisms and molecular targets. METHODS: Depression-like models were established in mice via lipopolysaccharide (LPS) administration, in zebrafish larvae via reserpine treatment, and in Drosophila melanogaster via chronic unpredictable mild stress (CUMS). Antidepressant effects were evaluated using behavioral tests, enzyme-linked immunosorbent assay (ELISA) and histological examination. Furthermore, a multi‑method strategy was used to identify the direct target of Oto‑T, including RNA sequencing (bulk and single‑cell), machine learning‑based screening, molecular dynamics (MD) simulation, cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS), fluorescence correlation spectroscopy (FCS), solvent-induced protein precipitation (SIP), ERK1/2 phosphorylation assays, and EP4 knockdown mice. RESULTS: The nonsteroidal anti-inflammatory drug celecoxib exhibits antidepressant effects, suggesting that anti-inflammatory strategies represent a viable approach for developing antidepressant drugs. Consistently, the QYS extract significantly alleviated LPS-induced depression-like behaviors in mice, and its active constituent Oto-T demonstrated marked antidepressant efficacy in LPS-induced depression-like behavior mouse models, a reserpine-induced zebrafish larval model, and a chronic unpredictable mild stress (CUMS) Drosophila melanogaster model. Both QYS and Oto-T modulated inflammatory cytokines (IL-1β, IL-6, TNF-α, and IL-10), antioxidant enzymes (SOD and GSH-Px), hypothalamic neurotransmitter (DA, NA, and 5-HT), and as well as microglial activation. Through the integration of bioinformatics analyses (transcriptomics, single‑cell transcriptomics, and the EP4 Double‑Hit Model) with experimental validations (including CETSA, DARTS, SIP, FCS, Docking, and MD), the prostaglandin E2 receptor EP4 subtype (EP4) was identified as a direct target of Oto‑T. Further investigation revealed that Oto‑T acts as an EP4 antagonist, and its antidepressant effects were abolished in EP4‑knockdown mice. CONCLUSION: Oto-T, derived from QYS, represents a promising antidepressant candidate that acts by directly targeting EP4, highlighting this receptor as a novel therapeutic target for the treatment of depression.