YangXue QingNao Wan ameliorates vascular dementia in 2-VO rats via inhibition of ferroptosis.

ETHNOPHARMACOLOGICAL RELEVANCE

YangXue QingNao Wan (YXQN) is a compound Chinese medicine comprising of 11 traditional Chinese medicinal herbs, including Angelica sinensis, Ligusticumstriatum, and Paeonia lactiflora, etc. Previous studies in our laboratory have demonstrated that YXQN improved cerebral microcirculation in hypertensive rats. However, its efficacy and underlying mechanisms in treating vascular dementia (VaD) remain unclear.

AIM OF THE STUDY

This study aims to investigate the therapeutic effects and underlying mechanisms of YXQN on VaD using a rat model of bilateral common carotid artery occlusion (2-VO).

MATERIALS AND METHODS

Male Wistar rats (10-12 weeks old) were randomly divided into seven groups: Sham, YXQN, 2-VO model, 2-VO with low-, medium-, or high-dose YXQN, and 2-VO with donepezil as a positive control. The 2-VO model was established by bilateral common carotid artery ligation, followed by 36 consecutive days of oral YXQN administration. Cognitive function was assessed using the Morris water maze and Y-maze tests, while hippocampal neuronal damage was evaluated by Nissl staining. Western blot analysis was performed to examine the types of neuronal cell death in the hippocampus. Transcriptome sequencing was conducted to explore potential molecular targets and mechanisms. In addition, in vitro experiments using HT22 cells were carried out to further validate the effects and mechanisms of YXQN.

RESULTS

Our results showed that YXQN alleviated learning and memory impairments, as well as hippocampal neuronal structural damage in 2-VO rats. Western blot analysis indicated that ferroptosis occurred in the hippocampal neurons of 2-VO rats, characterized by a significant downregulation of GPX4 and NRF2, upregulation of TF, TFR and ACSL4, increased levels of Fe, MDA, 4-HNE and GSSG, decreased levels of GSH, and reduced expression of mitochondrial functional proteins ATP5A and ATP5D. All of these changes were reversed by YXQN treatment. The transmission electron microscopy (TEM) analysis revealed typical ferroptotic mitochondrial alterations in the hippocampal neurons of 2-VO group, which was largely improved by YXQN. Transcriptomic analysis revealed that the ferroptosis-related gene Dpp4 was upregulated in the 2-VO group and downregulated following YXQN administration. In vitro experiments confirmed that YXQN inhibited Erastin-induced ferroptosis in HT22 cells. Furthermore, Dpp4 knockdown significantly attenuated Erastin-induced downregulation of GPX4, while Dpp4 overexpression abolished YXQN-mediated upregulation of GPX4 expression in HT22 cells. Molecular docking analysis showed that Ursolic Acid (UA), an active compound in YXQN, strongly binds to Dpp4, and UA also inhibited Erastin-induced Dpp4 expression in HT22 cells.

CONCLUSION

This study demonstrated that YXQN ameliorated cognitive dysfunction and hippocampal neuronal degeneration in 2-VO rats through inhibition of ferroptosis, with UA identified as a critical component mediating the inhibition of Dpp4. This study provided scientific evidence for the application of YXQN in VaD prevention and treatment.