Unraveling the neuroprotective mechanism of gastrodin against CRS-induced depression in adult male mice via Notch3: Integration of proteomics, transcriptomics, molecular docking, and cellular validation

Background: Major Depressive Disorder (MDD) affects hundreds of millions of people worldwide. Numerous studies have identified the medial prefrontal cortex (mPFC) as a key brain region involved in regulating depression. Clarifying how to modulate the mPFC can improve our understanding of its role in depression. Gastrodin (GAS), an active compound derived from Gastrodia elata, has attracted increasing attention for its ability to alleviate anxiety and depression. However, the impact of GAS on the mPFC and the mechanisms through which it may relieve depression still require further investigation. Understanding how GAS protects the mPFC may offer new insights into the mechanisms underlying its antidepressant effects and may contribute to the development of more targeted therapeutic strategies.

Purpose: To investigate the potential signaling pathways and targets through which the active compound GAS from Gastrodia elata reverses depression in chronic restraint stress (CRS)-induced mice by protecting mPFC neurons.

Study design and methods: Depressive-like behaviors (n = 11 mice/group) were induced in 8-week-old mice using CRS for 28 days. In the CRS model, the GAS-treated group received daily intraperitoneal injections of gastrodin for 28 days. After collecting mPFC brain region samples, hematoxylin and eosin (H&E) staining, Nissl staining (n = 5 mice/group), immunofluorescence staining (n = 5 mice/group), and qPCR (n = 3 mice/group) were used to observe the effects of GAS on the mPFC in CRS-induced mice. Transcriptomic and proteomic analyses of the mPFC (n = 3 mice/group) were then performed to elucidate the antidepressant signaling pathways of GAS. Molecular docking was conducted to assess the affinity of gastrodin for signaling pathway-related proteins. This was followed by in vitro validation using HT-22 cells, siRNA interference (si-Notch3), and Notch3 overexpression (Notch3 OE). Immunofluorescence and qPCR (n = 3) were used to detect Apoptosis markers, including Caspase-3, Bax, and Bcl-2, in HT-22 cells.

Results: GAS alleviated CRS-induced depressive behaviors. It reversed neuronal loss (Nissl⁺), microglial and astrocytic activation, and Apoptosis in the mPFC. Proteomic and transcriptomic analyses showed significant simultaneous downregulation of both protein and mRNA expression in the Notch signaling pathway in the GAS-treated group. Molecular docking identified Notch3 as the top binding candidates. CRS increased mPFC Notch3 expression (p < 0.05 vs. control), and gastrodin restored this change (p < 0.05 vs. CRS). In vitro, gastrodin reduced hydrocortisone/cortisol (Cort)-induced Apoptosis in HT-22 cells. Notch3 knockdown mimicked the anti-apoptotic effect of gastrodin, and Notch3 overexpression reversed the effect of gastrodin indicating that gastrodin acts as a functional antagonist of Notch3.

Conclusion: Gastrodin attenuates depression by suppressing neuronal Apoptosis through functional antagonism of Notch3 in the mPFC. This provides new evidence that Notch3 is a druggable target for gastrodin-based antidepressant therapy.

Chronic restraint stress; Depression; Gastrodin; Medial prefrontal cortex; Notch signaling pathway; Notch3.