Regulatory effects of Yuping Shen’an Granules on neuronal autophagy in a mouse model of insomnia

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 mice with insomnia and anxiety via the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway. METHODS: Ultra-high performance liquid chromatography-tandem mass spectrometry was used to identify the components of Yuping Shen’an Granules, and network pharmacology was used to predict key pathways. Ninety male C57BL/6J mice were randomly divided into normal group, model group, low-, medium-, and high-dose Yuping Shen’an Granules groups [3.125, 6.25, 12.5 g/(kg·d)], eszopiclone group [0.06 mg/(kg·d)], recovery-model group, recovery-model + PI3K inhibitor group [10 mg/(kg·d)], recovery-Yuping Shen’an [12.5 g/(kg·d)] + inhibitor group [10 mg/(kg·d)], with 10 mice per group. Except for the normal group, all groups were exposed to chronic unpredictable mild stress for 14 days and intraperitoneal injection of para-chlorophenylalanine for 4 days to establish a mouse model of insomnia with anxiety. Behavioral assessments included open field and pentobarbital sodium sleep tests; hematoxylin-eosin and Nissl staining were used to observe hippocampal pathological changes; enzyme-linked immunosorbent assay was used to detect levels of 5-hydroxytryptamine, γ-aminobutyric acid, dopamine, and norepinephrine; immunofluorescence was used to detect microtubule-associated protein light chain 3B expression; western blotting was used to detect PI3K/AKT/mTOR pathway-related proteins, autophagy-related proteins (P62, Beclin-1, microtubule-associated protein light chain 3B), and 5-hydroxytryptamine receptor 1A protein expression; transmission electron microscopy was used to observe the number of autophagosomes. RESULTS AND CONCLUSION: ① Ultra-high performance liquid chromatography-tandem mass spectrometry identified 2,276 components of Yuping Shen’an Granules, and 35 main components were screened out; ② Network pharmacology analysis showed that the PI3K/AKT/mTOR pathway was a potential target; ③ Compared with the normal group, the model group showed increased exploratory behavior, prolonged sleep latency (P < 0.01), significant hippocampal neuronal damage, decreased levels of 5-hydroxytryptamine and γ-aminobutyric acid, increased levels of dopamine and norepinephrine (P < 0.01), upregulated microtubule-associated protein light chain 3B fluorescence and protein expression (P < 0.01), downregulated phosphorylated PI3K/AKT/mTOR, P62, and 5-hydroxytryptamine receptor 1A expression, upregulated Beclin-1 and microtubule-associated protein light chain 3B expression (P < 0.05), and increased number of autophagosomes; ④ Compared with the model group, the recovery-model + inhibitor group showed aggravated damage, while the high-dose Yuping Shen’an Granules group and recovery-Yuping Shen’an + inhibitor group significantly reversed the above changes, improving behavioral indicators, alleviating neuronal damage, increasing 5-hydroxytryptamine and γ-aminobutyric acid levels, decreasing dopamine and norepinephrine levels (P < 0.05), and reversing autophagy-related protein expression (P < 0.05), with reduced autophagosomes. These results indicate that Yuping Shen’an Granules improve insomnia with anxiety symptoms by activating the PI3K/AKT/mTOR pathway, regulating neurotransmitter balance, and inhibiting excessive autophagy in hippocampal neurons.

1. Introduction

Insomnia is a complex sleep disorder characterized by prolonged sleep onset latency, difficulty maintaining sleep, and early awakening, accompanied by daytime functional impairment such as fatigue and cognitive decline [1-2]. Notably, insomnia frequently coexists with anxiety disorders, with epidemiological studies reporting a comorbidity rate of 50%-60%, forming a complex pathophysiological association [3-4]. National epidemiological surveys indicate that the prevalence of insomnia among Chinese adults is 20%-30%, and exceeds 35% in the elderly, representing a significant public health challenge [5]. The economic burden of insomnia can exceed 1% of a country's gross domestic product, with 70%-80% of costs attributed to indirect losses from reduced productivity and increased accident risk [6]. Current first-line pharmacotherapy primarily relies on benzodiazepine receptor agonists, which, although effective in the short term, are associated with long-term risks of drug dependence, tolerance, and cognitive impairment [7].

Traditional Chinese medicine (TCM) offers advantages of multi-target action and low toxicity for treating insomnia with anxiety [8]. Professor Xie Daojun, a renowned TCM expert in Anhui Province, developed Yuping Shen’an Granules based on 30 years of clinical experience. This hospital preparation (Anhui Drug Administration Preparation No. [2024] 0320) consists of eight herbs: Bupleurum chinense, Paeonia lactiflora, Curcuma aromatica, Aesculus chinensis, Moutan cortex, Albizia julibrissin, Akebia quinata, and Ostrea gigas. Previous studies have demonstrated its significant clinical efficacy. This study aims to investigate the regulatory effects of Yuping Shen’an Granules on hippocampal neuronal autophagy in a mouse model of insomnia, focusing on the PI3K/AKT/mTOR signaling pathway.