OBJECTIVE   To explore whether inhaling Bingchangsan (BCS) can modulate the Nrf2/HO-1 signaling pathway to improve ferroptosis and cognitive dysfunction in Alzheimer's Disease (AD) mouse models.

  METHODS   30 APP/PS1 mice were randomly divided into three groups: a model group (APP/PS1 group), a low-dose Bingchangsan group (BCS-L group), and a high-dose Bingchangsan group (BCS-H group). 10 age-matched wild-type (WT) mice were used as a control group. The WT group and the APP/PS1 group were treated with nebulized pure water, while the BCS-L group received 0.5μL of Bingchangsan inhalation solution per day, and the BCS-H group received 1μL, both administered via nebulization for 10 min daily for 2 weeks. 2 weeks post-treatment, spatial cognitive abilities of the mice were assessed using the Morris water maze test. Following the water maze experiment, the mice were euthanized, and their brain tissues were collected for Aβ_1-42 immunofluorescence, P-tau immunohistochemistry, and Nissl staining. Hippocampal tissues were extracted for Western blot and qPCR analysis to measure the protein and mRNA expression levels of Keap1, Nrf2, HO-1, GPX4, SCL7A11, TFRC, DMT1, FTL, and FTH1. Additionally, brain tissues were used for glutathione (GSH) content measurement using a GSH assay kit.

  RESULTS   In the BCS groups, the latency period for escape shortened, and the time spent in the quadrant with the platform, as well as the number of times crossing the platform, increased. Compared to the APP/PS1 group, the expression levels of Aβ_1-42 and P-tau in the hippocampal CA1 region and cortex of mice in the BCS groups were significantly reduced (P < 0.05, P < 0.01). Nissl staining revealed a denser arrangement of neurons with more Nissl bodies in the BCS groups. In the hippocampal tissue, compared to untreated APP/PS1 mice, significant increases in the protein and mRNA levels of Nrf2, HO-1, SLC7A11, GPX4, and FTL were observed in all BCS groups (P < 0.05, P < 0.01). While the BCS-L group showed increased levels of FTH1 protein and mRNA, these changes were not statistically significant. Furthermore, the expression levels of TFRC, DMT1, and Keap1 were significantly downregulated in the BCS groups (P < 0.05, P < 0.01). Additionally, treatment with BCS effectively restored the glutathione (GSH) content in the brain (P < 0.01).

  CONCLUSION   Inhalation of BCS can improve cognitive dysfunction in APP/PS1 mice. BCS activates the Nrf2/HO-1 pathway, increases the expression of GPX4, and enhances Nrf2 transcriptional activity by inhibiting Keap1. This leads to an upregulation of SLC7A11 and restoration of GSH levels, effectively countering ferroptosis. Additionally, BCS effectively inhibits TFRC and DMT1, while upregulating FTL and FTH1 expression, thereby maintaining intracellular iron homeostasis. This contributes to mitigating the impact of ferroptosis on APP/PS1 mice, subsequently enhancing their cognitive functions.