Yueju pill exerts rapid antidepressant effects via sequential activation of hippocampal GLP-1 receptor and PACAP...

Yueju pill (YJ), a classical Chinese medicine formula first documented in 'Danxixinfa by Zhu Danxi for treating "six stagnations" (Liu Yu: qi, blood, phlegm, fire, dampness, and food), has been clinically prescribed for mood disorders characterized by qi stagnation and depression for centuries. Despite its historical application for relieving emotional constraint (Yujie) and treating depressive disorders, the rapid antidepressant mechanism and molecular targets of YJ remain incompletely elucidated.

This study aimed to investigate the rapid antidepressant-like effects of YJ and uncover the underlying mechanism involving hippocampal GLP-1 receptor (GLP-1r) and pituitary adenylate cyclase-activating polypeptide (PACAP) signaling.

The chemical stability of YJ was evaluated by quantifying active constituents. A chronic unpredictable mild stress (CUMS) mouse model was employed to assess rapid antidepressant effects of YJ through acute administration using behavioral paradigms including novelty-suppressed feeding (NSF), tail suspension (TST), forced swim (FST), and sucrose preference tests (SPT). Hippocampal transcriptome sequencing was performed 30 min post-treatment to identify key targets. Protein and gene expressions were validated by Western blot, immunofluorescence, and quantitative PCR. Pharmacological interventions using exendin (9-39) (GLP-1r antagonist) and PACAP6-38 (PACAP antagonist) were conducted to establish signaling hierarchies. Additionally, HT22 hippocampal neuronal cells were utilized to examine direct effects on synaptic proteins.

Four active constituents (shanzhiside methylester, geniposide, ferulic acid, and gentiobioside) were identified in YJ with stable and reproducible concentrations. Acute administration of YJ (2 g/kg, as the final extract) rapidly ameliorated depressive-like behaviors in CUMS mice, with effects comparable to ketamine. Hippocampal transcriptome sequencing identified 461 differentially expressed genes following YJ treatment, with GLP-1r and Adcyap1 (PACAP) notably upregulated compared to the CUMS group. Western blot, immunofluorescence and PCR confirmed that YJ elevated protein and gene expression of GLP-1r and PACAP in the dentate gyrus within 30 min. Pharmacological blockade of GLP-1r with exendin9-39 abolished YJ's rapid antidepressant effects and prevented YJ-induced PACAP upregulation. Conversely, intra-dentate gyrus injection of PACAP6-38 blocked YJ's behavioral effects without affecting GLP-1 expression, indicating that PACAP acts downstream of GLP-1. In HT22 cells, YJ dose-dependently upregulated GLP-1r and PACAP, while also improving synaptic protein expression (pCaMKII, synapsin-1, PSD95, and BDNF).

These findings demonstrate that YJ produces rapid antidepressant effects through activation of hippocampal GLP-1r-dependent PACAP signaling, providing scientific evidence for the traditional "resolving depression" (Jieyu) function of this classical formula and identifying a novel polyherbal strategy for rapid-acting antidepressant therapy.