Elucidating the pharmacological mechanism of Yangming-Kaixin-Yizhi formula in inhibiting neuronal ferroptosis via Nrf2 in Alzheimer's disease: a study combining network pharmacology, transcriptomics, and experimental validation.

Ferroptosis is considered to be an important pathological driver of Alzheimer's disease (AD), and inhibiting neuronal ferroptosis shows a significant AD improvement effect. Yangming-Kaixin-Yizhi formula (YKY), a traditional Chinese medicine (TCM) formula, has been used for the treatment of forgetfulness for hundreds of years, but its mechanisms remain unclear. This study aimed to delineate the herb-component-target network of YKY in regulating ferroptosis for the treatment of AD, providing modern scientific basis for the traditional use of YKY. After 3×Tg-AD mice were orally treated with YKY for 11 weeks, the learning and memory ability of the mice was evaluated by Morris water maze, and hippocampal neuron loss was observed by hematoxylin-eosin (HE) staining. Transcriptomics and network pharmacology were used to analyze the underlying pharmacological mechanisms. Perls staining and immunofluorescence were used to observe brain iron deposition and hippocampal neuronal ferroptosis, respectively. The effects of YKY on iron deposition, reactive oxygen species (ROS) and lipid peroxidation in HT22 cells were detected by FerroOrange, DCFH-DA and BODIPY 581/591 C11 fluorescent probes, respectively. Western blot was used to detect the expression of Nrf2 and ferroptosis-related proteins to verify the pharmacological mechanism of YKY. YKY significantly improved the learning and memory ability and neuronal loss in 3×Tg-AD mice, and transcriptome and network pharmacology analysis suggested that its pharmacological mechanism may be related to regulation of iron metabolism, ferroptosis and Nrf2 regulated gene transcription. YKY significantly reduced intracellular iron deposition, improved cell viability, reduced ROS and lipid peroxidation levels, promoted the protein expression of Nrf2 and its downstream SLC7A11, GPX4 and FTH1, and inhibited the expression of TFR1 and NCOA4 proteins in 3×Tg-AD mice and RSL3-mediated HT22 cells. YKY may improve AD by targeting Nrf2 to inhibit neuronal ferroptosis, which provides modern scientific evidence for the use of YKY in TCM to treat disorders associated with memory loss.