ObjectiveTo observe the effect of Hei Xiaoyaosan on improving the learning and memory abilities of Alzheimer's disease （AD） model mice and explore the anti-AD mechanism of Hei Xiaoyaosan by regulating gut microbiota.MethodsTen four-month-old male C57BL/6J mice were selected as a blank group， and 60 male amyloid precursor protein/presenilin 1 （APP/PS1） double transgenic mice of the same age were randomly divided into a model group， low-dose， medium-dose， and high-dose groups of Hei Xiaoyaosan （5.53， 11.05， 22.10 g·kg^-1）， a group of Bifidobacterium bifidum tetrapartum tablets （0.585 g·kg^-1）， and a group of donepezil hydrochloride （6.5 mg·kg^-1）， with 10 mice in each group. The continuous drug intervention was performed for 90 d. New object recognition experiments were performed to observe the learning and memory abilities of each group. Morphological pathological changes in the Cornu Ammonis 1 （CA1） area of the hippocampus were analysed by hematoxylin-eosin （HE） staining， and the changes in gut microbiota in the feces of the mice were detected by 16S rRNA sequencing. Ultrastructural changes in the blood-brain barrier were observed by electron transmission endomicroscopy （TEM）， and Western blot was employed to detect the expression of related proteins such as Claudin-1， Occludin， and zonula occludens-1 （ZO-1）.ResultsCompared with that in the blank group， the new object recognition index was significantly decreased in the model group （P<0.01）. The number of neuronal cells was reduced， and the arrangement was disordered. The cell nuclei were pyknosed. The cell structures were damaged to varying degrees. The vascular endothelial cell membranes were extensively dissolved. The tight junction gaps were enlarged， with the membranes dissolved， and the junction proteins were reduced. A small part of the vascular basement membrane was dissolved and discontinuous. The astrocytic endfeet matrix and mitochondria were edematous. The expression of Claudin-1， Occludin， and ZO-1 was significantly decreased （P<0.01）. Compared with that in the model group， after the intervention with Hei Xiaoyaosan， the new object recognition index was significantly increased （P<0.05， P<0.01）. The number of neuronal cells increased. The arrangement was neater. The pyknosis of cell nuclei was reduced. The cell bodies were clear， and the structures were relatively complete. The endothelial cell membranes were partially dissolved. The tight junction gaps were slightly enlarged. The junction proteins were relatively abundant. The dissolution of the vascular basement membranes was alleviated， and the swelling of the astrocytic endfeet and mitochondria was reduced. The expression of Claudin-1， Occludin， and ZO-1 was significantly increased （P<0.05， P<0.01）. The results of the gut microbiota analysis showed that Hei Xiaoyaosan could regulate the structure and composition of the gut microbiota in APP/PS1 mice. Compared with the blank control group， the model group showed a significant decrease in the abundance of Firmicutes， Lactobacillus， and Bifidobacterium （P<0.05， P<0.01） and a significant increase in the abundance of Bacteroidetes and unidentified_S24-7 （P<0.01）. Compared with the model group， the Hei Xiaoyaosan groups showed a significant increase in the abundance of Firmicutes， Actinobacteriota， Lactobacillus， and Bifidobacterium （P<0.05， P<0.01） and a significant decrease in the abundance of Bacteroidetes and unidentified_S24-7 （P<0.05， P<0.01）.ConclusionHei Xiaoyaosan can improve cognitive dysfunction and pathological damage in AD mice. Its mechanism may be related to adjusting the abundance and structure of the gut microbiota， reducing the permeability of the blood-brain barrier， and thus improving hippocampal neuronal damage.

Hei Xiaoyaosan;Alzheimer's disease;gut microbiota;blood-brain barrier