Dual targeting of FSP1 and xCT: Potential mechanism of anthocyanins in alleviating neuronal ferroptosis in vascular dementia.

Background: VaD, the second most prevalent type of dementia in the elderly following Alzheimer's disease, is marked by significant cognitive and motor deficits, with few effective treatment options currently available. Ferroptosis, a type of regulated cell death driven by iron-mediated lipid peroxidation, has recently emerged as a key pathological mechanism in the development of VaD. Ferroptosis drives neuronal damage in VaD, making it a promising therapeutic target to reduce neuronal death and preserve cognitive function. ACN, a group of polyphenolic compounds recognized for their strong antioxidant properties, have demonstrated potential in reducing ferroptosis and alleviating neuronal damage.

Objective: The aim of this study was to explore the neuroprotective effects of ACN in reducing ferroptosis and mitigating cognitive impairments associated with VaD, focusing on the dual modulation of the FSP1 and xCT/GPX4 pathways. This novel dual-target approach provides an innovative strategy to reduce neuronal damage and oxidative stress in VaD.

Methods: A combination of in vitro and in vivo experiments was conducted to assess the protective effects and underlying mechanisms of ACN in mitigating ferroptosis associated with VaD. In vitro, a neurotoxicity model was established by inducing PC12 cells with Glu. Cell viability was determined using the CCK-8 assay, and various markers, including ROS levels, MDA, LPO, and GSH levels, were measured to evaluate the protective effects of ACN. Additionally, the expression of ferroptosis-related proteins, such as FSP1, xCT, and GPX4, was analyzed through Western blotting, RT-qPCR, and immunofluorescence. In vivo, a VaD rat model was established by performing bilateral common carotid artery occlusion (2-VO). The rats were divided into four groups: control, model, ACN-treated (with varying doses), and ALA-treated (positive control). The intervention lasted for 28 days. Cognitive functions were assessed using the Morris water maze and novel object recognition tests. Histological analyses, including HE staining and Nissl staining, were carried out to examine neuronal pathology. Moreover, electron microscopy was employed to evaluate mitochondrial ultrastructure integrity. Brain levels of iron, lipid peroxidation markers, and the expression of FSP1, xCT, and GPX4 were measured to elucidate the molecular mechanisms underlying the observed effects.

Results: Systematic in vitro and in vivo experiments demonstrated the significant neuroprotective effects of ACN against ferroptosis associated with VaD. In the Glu-induced PC12 cell model, ACN significantly improved cell viability, reduced ROS levels, restored GSH levels, and decreased the accumulation of MDA and LPO. Notably, ACN upregulated the expression of key ferroptosis-suppressing proteins, FSP1, xCT, and GPX4, through dual activation of these pathways, highlighting its powerful protective role against oxidative stress and ferroptosis. In the 2-VO VaD rat model, high-dose ACN significantly improved cognitive function, as shown by reduced escape latency in the Morris water maze and increased platform crossings. Moreover, ACN treatment enhanced the discrimination index in the novel object recognition test, suggesting improved learning and memory. Histopathological analyses revealed that ACN significantly alleviated neuronal disorganization, increased Nissl body counts, and restored mitochondrial integrity, with reduced swelling, rupture, and vacuolation observed under electron microscopy.

Conclusion: ACN exerts significant neuroprotective effects in VaD by dual regulation of the FSP1 and xCT/GPX4 pathways, effectively inhibiting ferroptosis and alleviating oxidative stress. This "dual-target" mechanism not only expands the current understanding of ACN's neuroprotective effects but also emphasizes its unique role in inhibiting ferroptosis. Overall, this study provides experimental evidence supporting the potential use of ACN in treating ferroptosis-related neurodegenerative diseases and highlights its promising prospects for clinical application.