Abstract: BACKGROUND: Modern medicine often uses benzodiazepines for the treatment of insomnia complicated by anxiety; however, long-term use can lead to drug dependence and adverse reactions. Traditional Chinese medicine, based on syndrome differentiation and holistic treatment, demonstrates definite efficacy and high safety, offering multi-pathway and multi-target comprehensive effects, thereby providing new ideas and directions for the treatment of this condition.
OBJECTIVE: To investigate the effects of Yuping Shen’an Granules on hippocampal neuronal autophagy in a mouse model of insomnia complicated by anxiety via the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway.
METHODS: The components of Yuping Shen’an Granules were identified using ultra-performance liquid chromatography-mass spectrometry, and key pathways were predicted through network pharmacology. Ninety male C57BL/6J mice were randomly divided into nine groups (n=10 per group): normal group, model group, low-, medium-, and high-dose Yuping Shen’an groups [3.125, 6.25, and 12.5 g/(kg·d)], eszopiclone group [0.06 mg/(kg·d)], verification experiment-model group, verification experiment-model + PI3K inhibitor [10 mg/(kg·d)] group, and verification experiment-Yuping Shen’an [12.5 g/(kg·d)] + PI3K inhibitor [10 mg/(kg·d)] group. All groups except for the normal group underwent 2 weeks of chronic unpredictable mild stress and 4 days of intraperitoneal injection of p-chlorophenylalanine to establish a mouse model of insomnia complicated by anxiety. Behavioral assessments were conducted using the open field test and pentobarbital sodium-induced sleep test. Pathological changes in the hippocampus were observed via hematoxylin-eosin staining and Nissl staining. Serotonin, γ-aminobutyric acid, dopamine, and norepinephrine levels were measured using enzyme-linked immunosorbent assay. LC3B expression was detected through immunofluorescence staining. The Western blot assay was employed to measure the expression of proteins associated with the PI3K/Akt/mTOR pathway and autophagy-related proteins (P62, Beclin-1, LC3B), as well as the serotonin 1A receptor. The number of autophagosomes was quantified using a transmission electron microscope.  
RESULTS AND CONCLUSION: Ultra-performance liquid chromatography-mass spectrometry identified 2 276 components, with 35 major components screened. Network pharmacology analysis indicated the PI3K/Akt/mTOR pathway as a potential target. Compared with the normal group, the model group exhibited increased exploratory behavior, prolonged sleep latency (P < 0.01), significant hippocampal neuronal damage, decreased levels of serotonin and γ-aminobutyric acid, and increased levels of dopamine and norepinephrine (P < 0.01). Additionally, there was upregulation of LC3B fluorescence and protein expression (P < 0.01), downregulation of phosphorylated PI3K/Akt/mTOR, P62, and the serotonin 1A receptor, and upregulation of Beclin-1 and LC3B (P < 0.05), along with an increased number of autophagosomes. Compared with the model group, the verification experiment-model + PI3K inhibitor group exhibited aggravated damage, while the high-dose Yuping Shen’an group and the verification experiment-Yuping Shen’an + PI3K inhibitor group significantly reversed these changes. They improved behavioral indicators, alleviated neuronal damage, increased levels of serotonin and γ-aminobutyric acid, and decreased levels of dopamine and norepinephrine (P < 0.05). Additionally, they modulated the expression of autophagy-related proteins (P < 0.05) and reduced the number of autophagosomes. These findings indicate that Yuping Shen’an Granules improve symptoms of insomnia complicated by anxiety by activating the PI3K/Akt/mTOR pathway, regulating neurotransmitter balance, and inhibiting excessive autophagy in hippocampal neurons.

Key words: Yuping Shen’an Granules, insomnia with anxiety, network pharmacology, phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), mammalian target of rapamycin (mTOR), autophagy