Abstract: BACKGROUND: Research has demonstrated a close association between ferroptosis and vascular dementia. Tongmai Kaiqiao Pill has a certain effect on improving the cognitive function of vascular dementia patients, but its mechanism is unclear.
OBJECTIVE: To explore the interventional effects and molecular mechanisms of Tongmai Kaiqiao Pill for vascular dementia based on the regulation of ferroptosis by the nuclear factor erythroid-2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1)/glutathione peroxidase 4 (GPX4) signaling pathway. 
METHODS: Among eighty-four SD male rats, 12 rats were used as the sham-operated group, and the rest of them were prepared as a model of vascular dementia by the modified 2-VO method, and then randomly divided into the model group, the Tongmai Kaiqiao Pills high-, moderate-, and low-dosage (27.6, 13.8, and 6.9 g/kg) groups, the combined group (Tongmai Kaiqiao Pill high-dosage+ML385, 20 mg/kg), and the donepezil hydrochloride group (0.45 mg/kg). The drug was given once a day by intragastric administration. The combined group was also intraperitoneally injected Nrf2 inhibitor ML385, once a day, for 4 weeks. Morris water maze was used to detect the learning memory ability of rats. Hematoxylin-eosin staining was used to observe the histopathological changes in the hippocampus of rats in each group. Colorimetric assay was used to detect the content of reduced glutathione, ferrous ion (Fe2+), and malondialdehyde in the serum of rats. Prussian blue staining was used to detect the iron deposition in the hippocampal tissue of rats. Transmission electron microscopy was used to observe the ultrastructural changes of mitochondria in rat hippocampal tissues. Western blot assay was used to detect the protein expression levels of Nrf2, HO-1, GPX4, XCT, and ferritin heavy chain 1 (FTH1) in rat hippocampal tissues. 
RESULTS AND CONCLUSION: (1) In comparison to the sham operation, rats in the model group exhibited a significantly prolonged latency period (P < 0.05) and a reduced number of platform crossings (P < 0.05). Additionally, the hippocampal tissues of these rats displayed loosely organized structure, deeply stained cell nuclei, and solidified or lysed chromatin. Ferri ions aggregated in CA1 region. There were atrophied mitochondria with dissolved cristae and thickened mitochondrial membranes. Fe2+, malondialdehyde, and reduced glutathione levels in rat serum were found to be elevated (P < 0.05). A significant reduction in the expression of GPX4, HO-1, XCT, Nrf2, and FTH1 proteins was detected in the hippocampus (P < 0.05). (2) Compared to the model group, the average escape latency of the rats was significantly reduced following intervention with Tongmai Kaiqiao Pills and donepezil hydrochloride (P < 0.05), with an increased number of platform crossings (P < 0.05). Hippocampal neurons showed significant recovery. Notably, iron aggregation in the CA1 region was significantly reduced, and mitochondrial structure and function were improved. There were significant reductions in Fe2+ and malondialdehyde levels, while the levels of GPX4, HO-1, XCT, Nrf2, and FTH1 in rat hippocampal tissues, and reduced glutathione in serum were significantly increased (P < 0.05). (3) The high-dose Tongmai Kaiqiao Pills exhibited a treatment effect comparable to that of donepezil hydrochloride (P > 0.05), with a significant prolongation of water maze escape latency (P < 0.05), a reduced number of platform crossings (P < 0.05), and insignificant neuronal pathological changes in the CA1 area. However, the combined group showed increased iron deposition, elevated malondialdehyde and Fe2+ levels in blood serum (P < 0.05), reduced glutathione content (P < 0.05), hippocampal tissue mitochondrial atrophy, and reduced expression of Nrf2, XCT, HO-1, GPX4, and FTH1 proteins (P < 0.05). Within a certain range, higher doses of Tongmai Kaiqiao Pills demonstrated a more pronounced effect, comparable to the efficacy of high-dose donepezil hydrochloride. (4) It is concluded that Tongmai Kaiqiao Pills have been shown to mitigate histopathological changes in the rat hippocampus and enhance cognitive function in rats with vascular dementia. The mechanism of action is likely associated with the suppression of ferroptosis through the activation of the Nrf2/HO-1/GPX4 signaling pathway.

Key words: vascular dementia, neuron, Tongmai Kaiqiao Pills, nuclear factor erythroid-2-related factor 2, heme oxygenase-1, glutathione peroxidase 4, ferroptosis