Chronic co-treatment with geniposide and shanzhiside methyl ester elicits antidepressant effects via PACAP signaling in stressed male mice

Yueju, an herbal medicine, has demonstrated rapid antidepressant effects, primarily attributed to its iridoid constituents geniposide (GP) and shanzhiside methyl ester (SM). This study investigates the sustained antidepressant efficacy of chronic co-treatment with GP and SM (GS) and elucidates the underlying molecular mechanisms, with a focus on hippocampal neurogenesis and PACAP signaling. Using a 4-week chronic unpredictable stress (CUS) mouse model, the effects of chronic co-treatment with GS on depression-like behaviors were examined. Adult male C57/BL6J mice received either vehicle or GS at a high dose, known to induce rapid antidepressant effects or a lower dose during the final two weeks of CUS. Chronic GS administration significantly alleviated depression-like behaviors, as evidenced by reduced immobility time in the tail suspension test, increased sucrose preference in the sucrose preference test, and reduced latency in the novelty-suppressed feeding test. Notably, only high-dose GS enhanced hippocampal neurogenesis, indicated by increased number of Ki67-and DCX-positive cells, and upregulated protein expression of PACAP and mTOR signaling, and synaptic proteins such as PSD95 in the hippocampus. Importantly, the antidepressant effects of high-dose GS were abolished when PACAP knockdown in the dentate gyrus. These findings demonstrate that the sustained antidepressant effects of high-dose GS depend on persistent activation of PACAP signaling within the dentate gyrus and are closely linked to enhanced hippocampal synaptic plasticity. These findings suggest that GS offers a promising therapeutic strategy for long-term treatment of depression, acting through a mechanistic pathway distinct from conventional neurogenesis-dependent interventions.