Vascular dementia (VaD) is a common cause of cognitive decline resulting from cerebrovascular disease and chronic cerebral hypoperfusion. Current clinical management focuses on risk-factor control and symptom relief, with no established disease-modifying drugs. Recent work has identified cuproptosis, a copper-driven regulated cell death pathway characterized by mitochondrial dysfunction and aggregation of lipoylated proteins, as relevant to neurological disorders. Key regulators include the copper importer SLC31A1 and FDX1, which reduces Cu2+ to the more toxic Cu+ and influences lipoylation of mitochondrial enzymes such as DLAT.
Tanshinone IIA (TSA), the major liposoluble active constituent of Salvia miltiorrhiza, has documented anti-inflammatory, antioxidant and mitochondrial-protective properties and has improved cognitive outcomes in prior VaD models by multiple mechanisms. The present study asked whether TSA’s neuroprotective effects involve modulation of copper homeostasis and inhibition of cuproptosis through the SLC31A1/FDX1 axis.
The investigators used the permanent bilateral common carotid artery occlusion (2-VO) rat model to induce chronic cerebral hypoperfusion and mimic aspects of subcortical ischemic VaD. This model is well established for producing white matter damage, neuronal loss in hippocampus and cortex, and spatial learning and memory deficits. Complementary in vitro experiments employed HT-22 hippocampal neuronal cells subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) to simulate ischemia-reperfusion injury in vitro.
Cognitive performance in rats was evaluated by the Morris water maze, a standard test of spatial learning and memory. Neuronal injury and loss in the hippocampus were assessed histologically using hematoxylin–eosin (HE) staining and Nissl staining. Transmission electron microscopy (TEM) was used to examine mitochondrial ultrastructure in hippocampal neurons.
Western blot analyses quantified expression levels of cuproptosis-related proteins, including SLC31A1, FDX1, lipoic acid synthetase (LIAS) and dihydrolipoamide transacetylase (DLAT). These markers were chosen to evaluate both copper transport and the downstream lipoylation-related events central to cuproptosis. TEM images provided ultrastructural evidence of mitochondrial damage and its modulation by treatment.
In HT-22 cells subjected to OGD/R, cell viability was measured by CCK-8 assay. Intracellular reactive oxygen species (ROS) levels were assessed, and intracellular copper accumulation was detected using the fluorescent probe Coppersensor-1. These assays tested whether TSA could reduce oxidative stress and copper overload in an ischemia-reoxygenation context.
TSA treatment markedly alleviated cognitive deficits in the 2-VO VaD rats, as shown by improved performance in the Morris water maze. Histologically, TSA reduced neuronal damage and loss in the hippocampus on HE and Nissl staining and preserved mitochondrial ultrastructure on TEM compared with untreated VaD animals.
At the molecular level, TSA reversed abnormal changes in the expression of cuproptosis-related proteins observed in VaD rats, modulating the SLC31A1/FDX1 pathway and related markers (LIAS, DLAT). These changes were consistent with reduction of copper-dependent mitochondrial injury.
In vitro, TSA improved HT-22 cell viability after OGD/R, decreased intracellular ROS, and lowered cellular copper accumulation detected by Coppersensor-1 staining, supporting a role for TSA in attenuating copper-dependent oxidative stress and cell injury.
The combined in vivo and in vitro findings indicate that TSA’s neuroprotective effects in this VaD model are at least partly mediated by restoration of copper homeostasis, mitigation of mitochondrial damage, and suppression of cuproptosis-related signaling via the SLC31A1/FDX1 axis. By downregulating pathways that favor intracellular copper accumulation and downstream lipoylation-driven proteotoxic stress, TSA reduced markers of neuronal injury and improved cognitive outcomes in the 2-VO model.
These results provide preclinical evidence linking modulation of copper transport and cuproptosis regulation to improved neuronal survival and function in a chronic hypoperfusion model of VaD.
The 2-VO rat model reproduces many features of subcortical ischemic VaD but does not capture all human vascular risk factors or small-vessel pathologies; cerebral blood flow declines acutely after ligation, which differs from some clinical courses. The present summary reports the main experimental approaches and outcomes; detailed dosing regimens, exact statistical values and full methodological parameters are provided in the manuscript and supporting information. The study suggests that targeting cuproptosis and copper transport proteins such as SLC31A1 and FDX1 could inform development of novel therapeutic strategies for VaD, but further validation and translational work are required.
Tanshinone IIA attenuated cognitive impairment and neuronal injury in a rat model of vascular dementia and protected HT-22 cells from OGD/R injury. The protective effects were associated with reduced oxidative stress, decreased copper accumulation, preservation of mitochondrial integrity, and modulation of the SLC31A1/FDX1 cuproptosis axis. The authors propose that TSA may provide a mechanistic basis for cuproptosis-targeted drug development in VaD. For full experimental detail and raw data, refer to the manuscript and supporting information.