ObjectiveBased on transcriptomics to explore the mechanism of Hei Xiaoyaosan regulating the phosphatidylinositol 3-kinase（PI3K）/protein kinase B（Akt） signaling pathway to improve the learning and memory ability of insomnia rats with liver depression syndrome.MethodsSixty male 8-week-old SD rats were selected and randomly assigned to six groups： a blank group， a model group， and three groups receiving low， medium， and high doses of Hei Xiaoyaosan（3.82， 7.65， and 15.30 g·kg^-1， respectively）， along with an eszopiclone group（0.09 mg·kg^-1）， with ten animals in each group. All groups， except for the blank group， underwent chronic restraint， tail clamping stimulation， and intraperitoneal injection of p-chlorophenylalanine（PCPA） to induce insomnia associated with liver depression syndrome. Each treatment group received intragastric administration according to the specified dosage， once daily， for a duration of 14 consecutive days. The sodium pentobarbital synergistic sleep test， open field test， and Morris water maze test were used to test the sleep amount， depressive-like behavior， and learning and memory abilities of rats. Additionally， enzyme-linked immunosorbent assay（ELISA） was utilized to quantify levels of 5-hydroxytryptamine（5-HT）， γ-aminobutyric acid（GABA）， brain-derived neurotrophic factor（BDNF）， glial cell line-derived neurotrophic factor（GDNF）， and nitric oxide（NO） in hippocampal tissue. Hematoxylin-eosin（HE） staining was performed to examine pathological changes in the hippocampal tissue， while in situ terminal labeling（TUNEL） was used to evaluate apoptosis in hippocampal neurons. Transcriptomic sequencing technology was employed to identify differentially expressed genes in hippocampal tissue between the model group and the blank group， as well as between the medium-dose of Hei Xiaoyaosan group and the model group. Gene Ontology（GO） and Kyoto Encyclopedia of Genes and Genomes（KEGG） enrichment analysis was performed on the intersection genes obtained by the two groups. Subsequently， the enriched key genes and signaling pathways were analyzed and verified. Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） was utilized to assess the mRNA expression of the phosphatase and tensin homologous gene（PTEN）， Bcl2-like protein 11（BCL2L11）， and mitogen-activated protein kinase 1（MAPK1） in hippocampal tissue. Additionally， Western blot analysis was employed to evaluate the protein levels of phosphatidylinositol 3-kinase（PI3K）， phosphorylated PI3K（p-PI3K）， protein kinase B（Akt）， phosphorylated Akt（p-Akt）， B-cell lymphoma-2（Bcl-2）， Bcl-2-related X protein（Bax）， and cleaved Caspase-3 in the same tissue.ResultsIn comparison to the blank group， the model group exhibited a reduction in body weight（P<0.01）， an increase in sleep latency（P<0.01）， a decrease in sleep duration（P<0.01）， and a reduction in both the number of grid crossings and instances of standing upright（P<0.01）. Additionally， the escape latency was prolonged（P<0.01）， while the frequency of platform crossings decreased（P<0.01）. Furthermore， the levels of 5-HT， GABA， NO， BDNF， and GDNF in hippocampal tissue were lower（P<0.01）. Histological examination revealed a disorganized cell arrangement in the CA1 region of the hippocampus， characterized by irregular cell shapes， a reduced cell count， deeply stained and pyknotic nuclei， increased vacuolar degeneration， and an elevated apoptosis rate（P<0.01）. Compared with the model group， the high and medium dose groups of Hei Xiaoyaosan， along with the eszopiclone group， increased the body weight of insomnia rats with liver depression syndrome（P<0.01）. These treatments also shortened sleep latency（P<0.01） and extended sleep duration（P<0.05， P<0.01）. Additionally， there was an increase in the number of grid crossings and instances of standing upright（P<0.05， P<0.01）. They also reduced escape latency（P<0.01） and increased the frequency of platform crossings（P<0.05， P<0.01）. Furthermore， there was an increase in the levels of 5-HT， GABA， NO， BDNF， and GDNF in the hippocampus（P<0.01， P<0.05）. These interventions also ameliorated pathological damage in the hippocampal CA1 area and reduced the apoptosis of hippocampal neurons（P<0.01）. Transcriptomic sequencing results indicate that Hei Xiaoyaosan may exert a therapeutic effect by regulating the PI3K/Akt signaling pathway through key mRNAs， including PTEN， BCL2L11， and MAPK1. The roles of these key mRNAs and proteins within the PI3K/Akt signaling pathway were further validated. In comparison to the blank group， the expression levels of PTEN， BCL2L11， and MAPK1 mRNA in the hippocampal tissue of rats in the model group were increased（P<0.01）. The protein expression levels of p-PI3K， p-Akt， and Bcl-2 were decreased（P<0.01）. Additionally， the protein expression levels of PTEN， Bax， and cleaved Caspase-3 were increased（P<0.01）. Compared to the model group， the high-dose and medium-dose groups of Hei Xiaoyaosan， along with the eszopiclone group， down-regulated the expression of PTEN， BCL2L11， and MAPK1 mRNAs（P<0.01）. Additionally， they up-regulated the expression of p-PI3K， p-Akt， and Bcl-2 proteins（P<0.01）， while also down-regulating the protein expression of PTEN， Bax， and cleaved Caspase-3（P<0.05， P<0.01）.ConclusionHei Xiaoyaosan may exert an anti-apoptotic effect by down-regulating the expression of key genes， including PTEN， BCL2L11， and MAPK1， while also modulating the PI3K/Akt signaling pathway. This mechanism may enhance the learning and memory capabilities of insomnia rats with liver depression syndrome.

Hei Xiaoyaosan;insomnia with liver depression syndrome;learning and memory abilities;transcriptomic sequencing;differential genes;phosphatidylinositol 3-kinase（PI3K）/protein kinase B（Akt） signaling pathway