Alzheimer's disease (AD) is a clinically common degenerative disease of the central nervous system, characterized by progressive memory loss and overall cognitive impairment (Khan S et al., 2020). With the acceleration of the global aging process, AD patients are increasing year by year, and AD has gradually become a global public health problem, and more and more AD patients have brought burdens to families and society. Although breakthroughs have been made in the development and research of therapeutic drugs for Alzheimer's disease, it is still necessary to update therapeutic concepts and search for potential treatments due to limitations such as time Windows and applicable populations (Breijyeh Z and Karaman R, 2020).

The etiology of AD involves a variety of pathophysiological factors. Although the Aβ cascade is the dominant pathogenesis hypothesis of AD, the reasons for accelerating Aβ deposition remain to be explored. Recent studies have suggested that excessive brain iron may promote the production of Aβ by affecting the hydrolysis of amyloid precursor protein, and brain iron overload has gradually been updated as one of the pathological features of AD. Combined with the published literature and our previous findings, we found that cognitive dysfunction in APP/PS1 mice was associated with age-dependent iron accumulation. We mixed the standard compounds of icariin, astragaloside IV, and puerarin, and the mixture was named YHG as a therapeutic agent for APP/PS1 mice. We found that YHG inhibited brain iron overload, down-regulated Aβ expression, and reduced senile plaque deposition in APP/PS1 mice (Xian-hui et al., 2015a; Dong et al., 2015). However, the specific downstream mechanisms are still unclear.

With the deepening of research, a new cell death mode, ferroptosis, has been proposed (Dixon SJ et al., 2012). Ferroptosis is a cell death caused by uncontrolled iron-dependent lipid peroxidation (Villalón-García I et al., 2023). Iron overload is a biochemical feature of ferroptosis (Derry PJ et al., 2020; Ayton S et al., 2020), and morphological changes similar to ferroptosis have been reported in the hippocampus of APP/PS1 mice (Tan Q et al., 2021; Jakaria M et al., 2021). Gene enrichment analysis showed that the differential genes related to ferroptosis were highly enriched in AD subgroups, and more and more research data suggested that ferroptosis was involved in the progression of AD (Li LB et al., 2019; Bao WD et al., 2021).The core link of ferroptosis is the uncontrolled lipid peroxidation process, which involves the abnormal expression of the SystemXc-GPX4-ACSL4-LPCAT3 signal axis (Floros KV et al., 2021; Wang H et al., 2022). SystemXc consists of 7 member 11 of the solute carrier family (SLC7A11) and 3 member 2 of the solute carrier family (SLC3A2) and is expressed as a heterodimer on the cell membrane (Liu J et al., 2020). Inhibition of SLC7A11 and SLC3A2 in AD has been shown to activate iron death and inhibit furin protein, promote amyloidogenic β- and γ-secretase processing of amyloid precursor protein, and accelerate Aβ deposition (Huat TJ et al., 2019).The primary function of SystemXc is to provide a substrate for GPX4 generation (Lee N et al., 2021). GPX4 is the only protein found to inhibit peroxidation in mammals, and it limits the ferroptosis cascade by converting toxic hydroperoxides to nontoxic lipoalcohols (Seibt TM et al., 2019). GPX4 is considered to be a protein resistant to ferroptosis, and its silencing can aggravate neuronal damage and lead to cognitive impairment (Lv Y et al., 2022). Long-chain acyl lipid CoA synthetase 4 (ACSL4) and lysophosphatidyltransferase 3 (LPCAT3) are the main catalytic enzymes in the process of iron death-lipid peroxidation (Liu J et al., 2022; Kagan VE et al., 2017). They play a synergistic role with the iron-participating Fenton (Liu M et al., 2022) reaction and "ignite" the lipid peroxidation cascade with PUFA in the phospholipid of the cell membrane as the substrate. Produce MDA, LPO, and other lipid peroxides, accelerate cell damage. Regulating the expression of the SystemXc-GPX4-ACSL4-LPCAT3 signaling axis is one of the strategies to inhibit ferroptosis-lipid peroxidation in AD.

In this study, APP/PS1 mice were used as AD animal model, YHG composed of Icariin, Astragaloside A and Puerarin was used as the treatment drug, and ferroptosis inhibitor DFX was used as the positive control drug to observe the effect of YHG on the behaviour of APP/PS1 mice. Iron content and lipid peroxidation and antioxidant levels in brain tissue and serum were detected by biochemical assay. The effect of YHG on the ultrastructure of hippocampus in APP/PS1 mice was evaluated by TEM. The expressions of SLC7A11, SLC3A2, GPX4, ACSL4, and LPCAT3 were detected by immunofluorescence, Western Blot, and real-time qPCR. To explore the mechanism of YHG improving cognitive impairment in APP/PS1 mice.