ObjectiveThis study aims to explore the effect and mechanism of Hei Xiaoyaosan in preventing Alzheimer's disease （AD） by regulating the PTEN-induced kinase 1 （PINK1）/Parkin signaling pathway and intervening in mitophagy.MethodsFifty 4-month-old male APP/PS1 mice were randomly divided into a model group， the donepezil hydrochloride group （0.65 mg·kg^-1）， and the high-， medium-， and low-dose Hei Xiaoyaosan groups （22.10， 11.05， and 5.53 g·kg^-1）. Ten additional male C57BL/6J mice of the same age served as the blank control group. Following a 90-day treatment period， learning and memory functions were evaluated using the Y-maze test. Additionally， hippocampal morphology was examined through hematoxylin-eosin （HE） staining. Fluoro-Jade B （FJB） staining was employed to examine neuronal damage and the number of damaged cells. Transmission electron microscopy （TEM） was utilized to observe mitochondrial damage. Immunofluorescence staining was employed to assess the expression of autophagy-related proteins microtubule-associated protein 1 light chain 3 （LC3） and selective autophagy adaptor protein 1 （p62） in the hippocampus. Western blot analysis was performed to quantify the protein expression of PINK1， Parkin， phosphorylated Parkin （p-Parkin）， LC3， and p62. Real-time quantitative polymerase chain reaction （Real-time PCR） was utilized to evaluate the mRNA expression levels of PINK1 and Parkin.ResultsCompared with the blank group， the model group exhibited a decreased alternation rate in the Y-maze test （P<0.01）， disordered hippocampal cell arrangement， enlarged intercellular space， and severe nuclear pyknosis. There was increased neuronal damage and number of damaged cells （P<0.01）， severe mitochondrial swelling and rupture， reduced protein and mRNA expression levels of PINK1 and p-Parkin （P<0.01）， increased immunofluorescence intensity of p62 （P<0.01）， and decreased fluorescence signal intensity of LC3 （P<0.01）. Compared with the model group， the donepezil hydrochloride group and the high- and medium-dose Hei Xiaoyaosan groups showed an increased alternation rate （P<0.05， P<0.01）， alleviated hippocampal neuronal damage， and reduced numbers of damaged cells （P<0.01）. There was a narrowed intercellular arrangement， improved nuclear morphology， and alleviated mitochondrial injury. The donepezil hydrochloride group and the high-dose Hei Xiaoyaosan group exhibited reduced p62 fluorescence intensity （P<0.05， P<0.01）， increased LC3 fluorescence intensity （P<0.05， P<0.01）， elevated protein expression of PINK1 and p-Parkin （P<0.05， P<0.01）， increased LC3 expression （P<0.05， P<0.01）， and decreased p62 expression （P<0.05， P<0.01）. The donepezil hydrochloride group and the high- and medium-dose Hei Xiaoyaosan groups showed increased mRNA expression levels of PINK1 （P<0.05， P<0.01）， and the donepezil hydrochloride group and the high-dose Hei Xiaoyaosan group exhibited elevated mRNA levels of Parkin （P<0.05）.ConclusionHei Xiaoyaosan can improve learning and memory abilities and ameliorate pathological damage in APP/PS1 mice. The mechanism may be related to the regulation of the PINK1/Parkin signaling pathway， enhancement of mitophagy， and repair of damaged neurons to prevent and treat AD.

Alzheimer's disease;Hei Xiaoyaosan;PTEN-induced kinase 1 （PINK1）/Parkin signaling pathway;mitophagy