Evaluation of Therapeutic Effects and Underlying Mechanisms of Baichuan Baile Formula in Rodent Insomnia Models

Abstract

Background/Objectives: Baichuan Baile (BCBL), a novel functional dietary formula, has been shown to exert antidepressant-like effects through modulation of the 5-HT system in our prior studies. Given the close neurobiological connections between depression and insomnia, along with its pharmacodynamic profile guided by TCM theory and nutritional assessments, BCBL is likely to possess beneficial effects against insomnia. However, this hypothesis and its underlying mechanisms require further validation. Methods: The chemical constituents of BCBL were analyzed by UPLC-Q-TOF-MS, and network pharmacology was applied to predict potential sleep-relevant targets and pathways. Subsequently, BCBL was evaluated for sedative-hypnotic effects using pentobarbital-induced hypnosis, locomotor activity, and polysomnography (EEG/EMG). Its therapeutic efficacy was further assessed in insomnia models induced by environmental stress, serotonin depletion, and rotarod-based sleep deprivation. The rotarod-induced chronic model was selected for mechanistic studies due to its sustained insomnia-like phenotype. Finally, key network-predicted targets were validated in this model through histopathology, Western blotting, and ELISA. Results: Pharmacological evaluation confirmed that BCBL significantly promoted sleep at both behavioral and EEG levels, confirming its sedative-hypnotic properties. BCBL mitigated environmental stress-triggered impairments in NREM sleep continuity and duration, and exerted protective effects against body weight loss and sleep disturbances in a serotonin depletion-induced insomnia model. In the rotarod sleep deprivation model, BCBL treatment increased spontaneous alternation rates and recognition indices, ameliorated hippocampal pathological alterations, and reduced hippocampal levels of HIF-1α, TNF-α, and IL-1β. Furthermore, BCBL elevated the p-GSK3β/GSK3β ratio and enhanced SIRT₁ expression in the hypothalamus. It also modulated the activity of key sleep–wake neurotransmitters/neuromodulators (serotonin, dopamine, adenosine, and glutamate) and key circadian rhythm regulators (BMAL₁, PER₂, and CLOCK) in this region. Conclusions: BCBL exhibits significant therapeutic efficacy against insomnia, indicating its potential as a dietary supplement for managing insomnia. Its mechanisms appear to involve anti-inflammatory effects, rebalancing of neurotransmitters/neuromodulators, and stabilization of circadian rhythm gene expression.

1. Introduction

Insomnia represents a widespread global health burden and ranks among the most prevalent sleep disorders, affecting approximately one-third of adults during their lifetime [1]. Its pathogenesis arises from complex interactions among multiple dimensions, including genetic susceptibility, environmental stress, circadian-rhythm disruption, and dysregulation of neurotransmitter systems [2,3]. Notably, a close bidirectional association exists between insomnia and psychiatric disorders, particularly depression: about 30% of patients presenting with insomnia concurrently meet diagnostic criteria for depression [4], whereas up to 70% of individuals diagnosed with major depressive disorder report clinically significant insomnia symptoms [5]. Furthermore, the clinical manifestations of both conditions often overlap, presenting with fatigue, difficulty concentrating, and irritability. These shared features stem largely from common neurobiological mechanisms, such as monoaminergic neurotransmitter dysfunction, ultimately forming an interactive “insomnia–depression comorbidity cycle” [6].

Given the profound pathological connections between insomnia and depression, sedating antidepressants, such as sedating agents like trazodone and mirtazapine, are commonly used in clinical practice to treat insomnia, especially when comorbid with depression or anxiety [7]. This approach highlights the unique value of drugs that act on shared neuroregulatory pathways in managing comorbid conditions. In recent years, novel therapies have gained increasing attention: the selective orexin-2 receptor antagonist seltorexant has met all endpoints in phase III trials for both insomnia and major depressive disorder [8]. Meanwhile, herbal therapy, recognized for its safety, multi-target effects, and tolerability, has demonstrated potential in regulating mood and improving sleep, emerging as a promising complementary or alternative strategy that continues to stimulate research interest.

Baichuan Baile (BCBL) is a functional formulation developed by integrating traditional Chinese medicine (TCM) theory, nutritional assessment, and contemporary biomedical perspectives on the pathogenesis of depression. Composed of edible herbs, Angelica dahurica, Ligusticum chuanxiong, and Schisandra chinensis, along with the food-grade additive L-menthol, this formula is designed to clear the head, resolve constraint, and tranquilize the spirit. Modern pharmacological studies have demonstrated that active fractions or monomeric compounds isolated from the constituent herbs of this formula exhibit sedative-hypnotic effects, regulate neurotransmitter systems, and alleviate neuroinflammation in multiple insomnia models [9,10,11]. In our previous study, BCBL was shown to produce antidepressant-like effects through modulation of the serotonergic (5-HT) system [12], a key pathway also involved in sleep regulation, which highlights its potential value as a herbal dietary supplement for managing insomnia.

Based on this rationale, the present study was designed to elucidate the therapeutic potential and mechanistic foundations of BCBL in treating insomnia. First, the chemical composition of BCBL was characterized, and network pharmacology analysis was conducted to predict its potential sleep-modulatory pathways and key molecular targets. Subsequently, the sedative-hypnotic efficacy of BCBL was assessed using the pentobarbital-induced hypnosis test, locomotor activity measurement, and polysomnographic analysis based on electroencephalogram/electromyogram (EEG/EMG) recordings. Next, the ability of BCBL to normalize insomnia-like behavior was evaluated in environmental stress- and serotonin depletion-induced insomnia models. A chronic insomnia model was then established through rotarod-based sleep deprivation, in which the ameliorative effects of BCBL on memory impairment were assessed using the Y-maze spontaneous alternation test and the novel object recognition test. Finally, guided by the earlier predictive insights, key molecular targets, primarily involving the suppression of neuroinflammatory responses, regulation of neurotransmitter/neuromodulator activity, and modulation of circadian rhythm factors, were experimentally validated in the rotarod-induced chronic insomnia model using molecular biology techniques.

2. Materials and Methods

2.1. Preparation and Composition Identification of BCBL

2.1.1. Extraction and Dosage Determination

The components of BCBL were procured from Tongrentang Co. Ltd. (Beijing, China). Detailed specifications are listed in

Table 1. Detailed information on the herbs in BCBL.

Chinese Name	Latin Name	Batch Number	Dosage (g)	Origin
Bai Zhi	Angelica dahurica	201116	5–20	Henan Province
Chuan Xiong	Rhizoma chuanxiong	B8031511	3–12	Sichuan Province
Wu Wei Zi	Schisandra chinensis	21041002	3–12	Liaoning Province
Bo He Nao	L-menthol	210307025	0.02–0.12	Guangdong Province

The prescribed dosages specify the therapeutic amounts of herbs and food additives to be taken by an adult each day.

. Following the method described in our previous study [12], Angelica dahurica, Ligusticum chuanxiong, and Schisandra chinensis were combined and co-extracted with water to yield a single aqueous extract. This extract was then blended with L-menthol at specific ratios and dissolved with 1% dimethyl sulfoxide to prepare the BCBL solution (water extract and L-menthol). The BCBL doses were defined as low (Low, 300 mg/kg and 1.625 mg/kg), medium (Mid, 600 mg/kg and 3.25 mg/kg), and high (High, 1200 mg/kg and 6.5 mg/kg).

2.1.2. Component Analysis of BCBL

Chemical profiling was performed using an ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) system (Waters H-Class, Milford, MA, USA) equipped with a Welch Ultimate UHPLC AQ-C18 column (2.1 × 100 mm, 1.8 µm). The mobile phase consisted of (A) water containing 0.1% formic acid and (B) acetonitrile, with the following gradient program: 0–3 min, 15% B; 3–10 min, 15–30% B; 10–20 min, 30–90% B; 20–25 min, 90% B; and 25–30 min, 10% B. The column temperature was maintained at 30 °C, and the detection wavelength was set at 254 nm. Mass spectrometry was conducted using a Sciex Triple TOF^® 4600 spectrometer coupled with Analyst TF 1.7.1 software (SCIEX, Framingham, MA, USA), acquiring data in both positive and negative electrospray ionization (ESI) modes for MS and MS/MS analysis.

2.2. Network Pharmacology Analysis

2.2.1. BCBL and Desease-Relevant Data Preparation

The active components in BCBL were identified through an integrated approach combining UPLC-Q-TOF-MS analysis (detailed in Section 2.1.2) with database screening, including the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database (https://tcmsp-e.com/tcmsp.php, accessed on 15 February 2026) and the Supervised Prediction of Drug Indications (Super-PRED) database (https://prediction.charite.de/index.php, accessed on 15 February 2026). In addition to compounds directly identified by mass spectrometry, we also utilized these databases to retrieve other potential bioactive components reported in the literature but not directly detected in the current analysis. Components with pre-calculated oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18 were retained for further analysis. Potential targets of the screened active components were obtained using the Swiss Target Prediction database (http://www.swisstargetprediction.ch/, accessed on 15 February 2026). Disease-related targets for insomnia were retrieved from the GeneCards (https://www.genecards.org/, accessed on 15 February 2026) and Therapeutic Target Database (TTD, https://db.idrblab.net/ttd/, accessed on 15 February 2026) databases. The intersection between the predicted targets of BCBL components and the insomnia disease targets was defined as the set of potential therapeutic targets of BCBL for insomnia treatment.

2.2.2. Network Establishment and Analysis

The intersection between the targets of active compounds in BCBL and insomnia genes was visualized using VENNY 2.1 (https://bioinfogp.cnb.csic.es/tools/venny/, accessed on 15 February 2026). These intersecting targets were then used to construct a protein–protein interaction (PPI) network via the STRING database (https://www.string-db.org/, accessed on 15 February 2026), with the species restricted to Homo sapiens and a high-confidence interaction score set at ≥0.400. The network was imported into Cytoscape 3.8.2 for visualization and topological analysis. Targets with a degree value ≥10 were selected to constitute the core target network. Subsequently, the core targets were uploaded to the DAVID 2021 database (https://david.ncifcrf.gov/, accessed on 15 February 2026) for Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The results were screened based on the criterion of an adjusted p-value < 0.05 for statistical significance and ranked in ascending order of p-value. The top 20 entries from the KEGG analysis and top 10 entries from the GO analysis and were selected as the key findings.

2.3. Animals

Specific-pathogen-free (SPF)-grade male Institute of Cancer Research (ICR) mice (weighing 20 ± 2 g) and SPF-grade male Sprague Dawley (SD) rats (weighing 160 ± 5 g) were purchased from HFKBiotechnology Co., Ltd. (Beijing, China). (animal production license number: SCXK 2024-0003). All procedures followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee (IACUC-DWZX-2025-571). Rats were housed individually, and mice in groups of four per cage, with both species housed in individually ventilated cages (IVC), under a 12:12 h light/dark cycle (lights on 08:00–20:00), at 22 ± 1 °C and 50 ± 10% relative humidity, with food and water available ad libitum throughout the study. During the rotarod sleep deprivation model (including drug administration), the light phase was extended to 15 h (lights on 08:00–23:00). To minimize handling stress, animals were habituated to gentle handling and mock oral gavage (empty needle) for 3 days prior to the experiment.

2.4. Reagents

All chemical and biological reagents were obtained from commercial suppliers and used in accordance with manufacturers’ protocols. Pentobarbital (No. BA-0831), diazepam (U1023403), xylene (10023418), and absolute ethanol (100092683) were sourced from China National Pharmaceutical Group Corp (Beijing, China). Dimethyl sulfoxide (DMSO, DS855) was purchased from Beijing Innochem Science & Technology Co., Ltd. (Beijing, China). P-chlorophenylalanine (PCPA, C6506) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies against 5-HT_1A (ab85615, 1:1000), CLOCK (ab3517, 1:1000), GSK3β (ab32391, 1:10,000), phospho-GSK3β (ab75814, 1:10,000), SIRT₁ (ab110304, 1:1000), β-actin (ab8227, 1:5000), BMAL₁ (14268-1-AP, 1:2000), PER₂ (GTX30117, 1:1000), ADORA₁ (DF4905, 1:1000) and HIF-1α (EPR16897, 1:5000) were purchased from Abcam (Cambridge, UK), Proteintech (Wuhan, China), GeneTex (Irvine, CA, USA), Affinity Biosciences (Cincinnati, OH, USA) and Wanlei Biotechnology (Shenyang, China), respectively. Corresponding HRP-conjugated secondary antibodies (goat anti-mouse IgG ZB-2305, 1:5000; goat anti-rabbit IgG ZB-2301, 1:5000) were obtained from Zhongshan Jinqiao Biotechnology (Beijing, China). Commercial enzyme-linked immunosorbent assay (ELISA) kits for mouse adenosine (YMS9383-A), 5-HT (JLC2655), dopamine (DA, E-EL-0046), glutamate (Glu, LB2829B), IL-1β (JL18442) and TNF-α (JL10484) were procured from Yuannuo Tiancheng (Chengdu, China), Xiguzi Biotechnology (Beijing, China), Li-Borui Biotechnology (Wuhan, China) and Jianglai Biotechnology (Shanghai, China), with all assays performed according to the manufacturers’ instructions.

2.5. Pentobarbital-Induced Hypnosis Test

Mice were randomly assigned to five experimental groups (n = 8 per group): a control group (Con), a diazepam group (DZ, 3 mg/kg), and three BCBL treatment groups receiving low (Low), medium (Mid), and high (High) doses. All drug administrations and behavioral tests were performed between 08:00 and 12:00. Diazepam and BCBL were administered orally once daily in the morning, while the control group received an equal volume of vehicle (1%DMSO, 20 mL/kg) via the same route. Thirty minutes post-administration, all animals received an intraperitoneal injection of pentobarbital (45 mg/kg, 10 mL/kg). Sleep latency was defined as the interval between pentobarbital injection and loss of the righting reflex, and sleep duration was measured from loss to spontaneous recovery of the righting reflex.

2.6. Locomotor Activity Experiment

The experimental groups and drug administration regimen followed the protocol described in Section 2.5. Thirty minutes after dosing, each mouse was individually placed in a locomotor activity chamber and allowed to move freely for 15 min. Behavior was recorded using a video tracking system (Shanghai Jiliang Software Technology Co., Ltd., Shanghai, China). The initial 5 min period served as habituation; data from the subsequent 10 min interval were analyzed for total movement distance and duration of activity.

2.7. EEG/EMG-Based Polysomnographic Analysis

To characterize the impact of BCBL on normal sleep architecture, EEG/EMG-based polysomnographic analysis was performed. Given the advantages of SD rats, including their larger body size, well-defined sleep–wake cycle, and stable sleep baseline, we selected rats for this EEG/EMG-based sleep assessment. The drug concentrations in rats were the same as those in mice. The administration volume for rats was adjusted based on body surface area using a standard interspecies conversion factor of 0.52 (mouse-to-rat) to deliver pharmacologically equivalent doses [13]. For optimal resource allocation, only the diazepam group and the BCBL dose group (n = 6) that demonstrated the most pronounced sedative-hypnotic effects (as identified in Section 2.5 and Section 2.6) were included in this analysis. Rats had been previously implanted with radiotelemetry transmitters (HD-S02, DSI Systems, St. Paul, MN, USA) for continuous recording as previously described [14]. Following baseline sleep pattern determination, rats were randomized into two groups with one receiving diazepam (3 mg/kg) and the other receiving the optimized BCBL dose, both administered orally in the morning. Sleep–wake signals were continuously recorded for 24 h, from 08:00 on the day of administration to 08:00 the following day, beginning 30 min after drug administration. During the experimental period, rats were housed in individual cages with a strictly controlled quiet environment. Sleep architecture was analyzed using NeuroScore software (v3.3.1, DSI) with the integrated rodent sleep staging module. Quantified parameters included total sleep time, rapid eye movement (REM) sleep duration, non-REM (NREM) sleep duration, total wake time, number of sleep episodes, mean episode duration, and frequency of sleep–wake transitions over the 24 h period.

2.8. Environmental Stress-Triggered Rat Insomnia Model

The protocol for implanting radiotelemetry transmitters, conducting baseline tests, and administering drugs was the same as that in Section 2.7, except that a model group (Mod, receiving 1% DMSO, n = 6) was added to the experimental design. Thirty minutes post-administration, rats were exposed to environmental stress conditions induced by the cage-change method [15], during which sleep signals were continuously recorded for a 6 h period (from 08:00 to 14:00).

2.9. Serotonin Depletion-Induced Insomnia Model

Serotonin depletion-induced insomnia was established in mice by intraperitoneal injection of para-chlorophenylalanine (PCPA, 300 mg/kg) once daily for 3 consecutive days [16]. In the preliminary experiment, mice were divided into a control group and a model group (n = 8). The model group received PCPA (10 mL/kg), while the control group was administered an equivalent volume of physiological saline. Modeling efficacy was validated on day 4 using the pentobarbital-induced hypnosis test, as evidenced by significantly prolonged sleep latency and reduced total sleep duration in the model group. Additionally, changes in body weight also served as an important reference index for model evaluation. Based on the pilot results, a new cohort of mice was acclimatized for seven days and then randomly assigned to six groups (n = 8 per group): control (Con, 1%DMSO), model (Mod, 1%DMSO), diazepam (DZ, 3 mg/kg), and low- (Low), medium- (Mid), and high-dose (High) BCBL groups. From day 1 onward, all groups received their respective treatments (20 mL/kg) once daily by oral gavage at 08:00. Starting on day 5, the modeling procedure was initiated: the control group received saline intraperitoneally 30 min after the daily gavage, while the remaining groups were injected with PCPA at the same time. Drug administration continued throughout the modeling period. The experimental timeline is summarized in Figure 6A. On the day after the final PCPA injection, the pentobarbital-induced hypnosis test was performed again to evaluate the therapeutic effects of BCBL.

2.10. Rotarod Sleep Deprivation-Based Insomnia Model

Chronic insomnia model was established using a sleep deprivation device that employs sustained horizontal rotational interference. Mice were randomly allocated to six groups (n = 8 per group): control (Con, 1%DMSO), model (Mod, 1%DMSO), diazepam (DZ, 3 mg/kg), and low- (Low), medium- (Mid), and high-dose (High) BCBL groups. All mice were acclimated to the rotarod sleep deprivation apparatus. With the exception of the control group, all other groups underwent sleep deprivation according to the following schedule: on day 1, deprivation was conducted for 4 h (8:00–12:00); on day 2, for 4 h (12:00–16:00); and on day 3, for 8 h (8:00–16:00). Subsequently, a continuous 14-day sleep deprivation protocol was implemented, consisting of 14 h of daily deprivation (8:00–22:00). The drug intervention was initiated on day 11 and continued thereafter with a single evening administration (22:00–23:00) via gavage (20 mL/kg) each day. The experimental timeline is illustrated in Figure 7A. The deprivation protocol operated intermittently at 6 rotations/minute, with a 10 s rotation followed by a 20 s stationary interval. The Y-maze spontaneous alternation test and novel object recognition test were performed between 08:00 and 12:00 on the day after the final administration.

2.11. Y-Maze Spontaneous Alternation Test

Mice were individually placed into one arm of the Y-maze for a 5 min free exploration session, with their behavior recorded throughout by a video tracking system (Shanghai Xinruan Information Technology Co., Ltd., Shanghai, China). The spontaneous alternation rate was calculated as the number of consecutive entries into three different arms divided by (total arm entries − 2) × 100% [17].

2.12. Novel Object Recognition Test

The novel object recognition test was performed according to a previously established protocol [18], with mouse behavior recorded using the video tracking system described in Section 2.11. The familiar objects were two identical cylindrical wooden blocks (3 cm in diameter, 7 cm in height, and red in color). The novel object matched the familiar objects in size, shape, and material, differing solely in color (green). Object exploration was defined specifically as time spent sniffing or touching the object with the nose; climbing on or resting against the object was excluded. The recognition index was calculated as the time spent exploring the novel object divided by the total exploration time directed toward both objects.

2.13. Sample Collection and Preparation

Immediately after behavioral tests, mice subjected to the rotarod sleep deprivation model were euthanized for sample collection. Serum was collected for ELISA. Brains were processed accordingly: samples for histopathology were fixed; those for ELISA and Western blot were dissected on ice. Based on the 6th edition of the Mouse Brain in Stereotaxic Coordinates, the hippocampus was exposed by retracting the cortex, freed from white matter and subiculum using curved micro-forceps, and bilaterally pooled per animal. The hypothalamus was dissected ventrally, bounded by the optic chiasm, mammillary bodies, and hypothalamic fissures. Both tissues were snap-frozen in liquid nitrogen and stored at −80 °C. Tissues were subsequently thawed, lysed, and centrifuged, and the supernatant was aliquoted and stored at −80 °C for later analysis.

2.14. Hematoxylin and Eosin Staining and Morphological Observation

Mouse brains were fixed in 4% paraformaldehyde at 4 °C for 48 h, then dehydrated through graded ethanol, cleared in xylene, and embedded in paraffin. Coronal sections (4 µm) of the hippocampus were cut consecutively, mounted onto slides, and baked at 60 °C for 2 h. Sections were deparaffinized, rehydrated, stained with Harris hematoxylin for 5 min and eosin Y for 1 min, differentiated in 1% acid alcohol, blued in tap water, and mounted with neutral balsam. Hippocampal subregions, specifically the cornu ammonis 1(CA1), cornu ammonis 3 (CA3), and dentate gyrus (DG), were examined under a light microscope (Nikon Eclipse C1, Nikon, Tokyo, Japan). Overall structural integrity and lesion extent were assessed at 10× magnification; neuronal morphological features, such as pyknosis, nuclear membrane integrity, and cytoplasmic vacuolation, were observed at 40× magnification.

2.15. ELISA Test

Levels of adenosine, 5-HT, Glu, DA, IL-1β, and TNF-α were tested by ELISA. Specifically, IL-1β and TNFα were measured in mouse hippocampal tissue; 5-HT and adenosine were measured in hypothalamic tissue; dopamine and glutamate were assessed in both hypothalamic tissue and serum. Sample preparation and detection followed the manufacturers’ instructions. Standard curves were generated using Curve Expert software (https://www.curveexpert.net/, accessed on 15 February 2026), and sample concentrations were calculated using regression equations.

2.16. Western Blotting Analysis

Protein expression levels of HIF-1α, 5-HT_1A, ADORA₁, GSK3β, p-GSK3β, SIRT₁, BMAL₁, CLOCK, and PER₂ were determined by Western blotting. Hippocampal tissue was used for the detection of HIF-1α, while hypothalamic tissue was employed for the measurement of all other proteins. Brain tissues were homogenized, and total protein was extracted and quantified using a BCA assay. Subsequent steps including gel electrophoresis, membrane transfer, and blocking were performed according to standard protocols. Membranes (Millipore, Bedford, MA, USA) were incubated with primary antibodies at 4 °C overnight, followed by incubation with species-matched horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h. Protein bands were visualized using a chemiluminescence imaging system (ProteinSimple, San Jose, CA, USA), and band intensities were quantified with Image J software (https://imagej.net/software/fiji/, accessed on 15 February 2026). Protein expression was quantified by normalizing target band intensity to β-actin, with the cumulative grayscale values of all visible bands in the same lane used for proteins showing multiple bands to represent total expression.

2.17. Statistical Analysis

Statistical analyses were performed using GraphPad Prism (version 8.0.2). All of the data were presented as mean ± SEM (standard error of mean). Student’s t-test was used to evaluate differences between two groups in the preliminary experiment on serotonin depletion-induced insomnia. The data from EEG/EMG recordings and longitudinal body weight measurements were analyzed by two-way analysis of variance (ANOVA), and the remaining datasets were evaluated using one-way ANOVA. Post hoc comparisons were conducted using Tukey’s multiple comparisons test, with the significance level set at p ≤ 0.05.

3. Results

3.1. Chemical Compounds of BCBL

A total of 75 chemical constituents were identified in BCBL by UPLC-Q-TOF-MS analysis. The representative base peak ion (BPI) chromatogram is presented in Figure 1. Detailed information for the major detected compounds, including their mass-to-charge ratio (m/z), retention time, molecular formula, and tentative identification is provided in Supplementary Data S1.

3.2. Network Pharmacology Analysis of BCBL in Insomnia Treatment

Network pharmacology analysis was performed to elucidate the potential anti-insomnia mechanisms of BCBL. A total of 108 overlapping targets were identified between BCBL-related targets and known insomnia-associated targets (Figure 2A). PPI network analysis revealed several highly connected hub targets, including GSK3β, SIRT₁, HIF-1α, TNF, and ADORA1 (Figure 2B), suggesting their potential role as key mediators of BCBL’s bioactivity. Functional enrichment analysis based on KEGG and GO databases showed that these overlapping targets were significantly enriched in pathways related to neurotransmitter regulation, such as serotonin, dopamine, and glutamate signaling, and biological processes associated with circadian rhythm (Figure 2C,D). Collectively, these findings suggest that BCBL may exert its anti-insomnia effects through a multi-target mechanism, primarily involving the modulation of inflammation-related factors, neurotransmitters and neuromodulators, as well as rhythm processes.

3.3. Effects of BCBL on Pentobarbital-Induced Hypnosis and Locomotor Activity in Mice

In the pentobarbital-induced hypnosis test, both high-dose BCBL and diazepam significantly shortened sleep latency (Figure 3B) and extended sleep duration (Figure 3C) compared with the control group. Consistent with these findings, locomotor activity was markedly suppressed by high-dose BCBL and diazepam, as evidenced by reductions in total movement distance (Figure 3E) and activity time (Figure 3F). Collectively, these results confirm the sedative-hypnotic properties of BCBL. Given its pronounced efficacy, the high-dose BCBL was selected as the optimal dosage for subsequent EEG/EMG-based polysomnographic analysis.

3.4. Effects of BCBL on Sleep in Rats Under Standard Housing Conditions

Twenty-four-hour sleep monitoring revealed that, compared with baseline recordings, both diazepam and high-dose BCBL significantly increased total sleep time and NREM sleep duration, while decreasing wake time (Figure 4B). Furthermore, these treatments prolonged the average duration of NREM sleep episodes (Figure 4D) and reduced the frequency of transitions between wakefulness and NREM sleep (Figure 4E). Although a trend toward reduced NREM sleep fragmentation was observed in both groups, this effect did not reach statistical significance (Figure 4C). Collectively, these findings indicate that BCBL enhances total sleep time primarily by improving the continuity and duration of NREM sleep. Under standard conditions, rats spend approximately 75–85% of their total sleep time during the light phase (08:00–20:00). The sleep-promoting effect of BCBL was particularly evident during this period, with a statistically significant increase in sleep time relative to baseline specifically in the intervals of 8:00–12:00 and 16:00–20:00 (Figure 4F).

3.5. BCBL Attenuates Environmental Stress-Induced Insomnia

After six hours of EEG/EMG-based polysomnographic analysis, compared to baseline sleep, the model group showed a significant reduction in total sleep time and NREM sleep time (Figure 5B), along with an increase in the number of sleep fragments (Figure 5C) and a decrease in their duration (Figure 5D). The number of transitions between wakefulness and NREM sleep also increased (Figure 5E), but these changes were not statistically significant. In comparison to the model group, both diazepam and high-dose BCBL significantly increased total sleep time and NREM sleep duration while reducing wake time. Additionally, both groups reduced the total number of sleep fragments and NREM sleep fragments, and significantly prolonged the average duration of NREM sleep episodes. Regarding the reduction in the number of awakenings, the diazepam group showed a statistically significant difference compared to the model group, whereas the high-dose BCBL group exhibited a decreasing trend that did not reach statistical significance. These results indicate that BCBL mitigates environmental stress-triggered impairments in NREM sleep continuity and duration, with its regulated effects being most pronounced within 1 h after administration (Figure 5F).

3.6. BCBL Improves Sleep Disturbance and Exerts Protective Effects on Body Weight in Insomnia Induced by Serotonin Depletion

In the preliminary experiment, intraperitoneal PCPA administration induced serotonin depletion and established a valid insomnia model, as demonstrated by significantly prolonged sleep latency (Figure 6B), shortened sleep duration (Figure 6C), and suppressed weight gain (Figure 6E) in the model group compared to the control group. Following pharmacological intervention in the replicated model, both the diazepam and high-dose BCBL groups showed significantly reduced sleep latency (Figure 6F), extended sleep duration (Figure 6G), and mitigated weight loss (Figure 6H) compared to the model group. Although the low- and medium-dose BCBL groups prolonged sleep duration relative to the model group, their effects remained significantly inferior to the high-dose BCBL group in sleep latency and weight recovery. These results indicate that BCBL exerts protective effects on body weight and sleep disturbances in PCPA-induced insomnia.

3.7. BCBL Alleviates Memory Impairment and Promotes Weight Gain in Rotarod Sleep Deprivation-Induced Insomnia Mice

Compared with the control group, model mice exhibited attenuated weight gain (Figure 7B,C) and significant reductions in both spontaneous alternation rate (Figure 7F) and recognition index (Figure 7I). High-dose BCBL treatment significantly reversed these insomnia-induced deficits, enhancing weight gain and restoring cognitive performance in both behavioral tests. In contrast, diazepam administration only ameliorated weight gain impairment but did not significantly improve memory-related metrics. These results suggest that BCBL, unlike diazepam, confers protective effects against body weight loss and effectively mitigates insomnia-associated memory impairment.

3.8. BCBL Alleviates Hippocampal Neuron Damage in Rotarod Sleep Deprivation-Induced Insomnia Mice

Compared with the control group, the model group exhibited loosely arranged hippocampal neurons (Figure 8 (B2,B3)), with evident structural disorganization in the CA1 (cornu ammonis 1), CA3, and DG (dentate gyrus) subfields. These regions showed widened intercellular spaces, along with increased neuronal condensation and hyperchromasia (indicated by black arrows). In contrast, the high-dose BCBL group displayed hippocampal morphology closely resembling that of the control group. Neuronal architecture in CA1, CA3, and DG appeared compact and well-organized, with clearly delineated molecular, pyramidal (or granular), and polymorphic layers. Pyramidal and granular cells were orderly arranged, exhibiting abundant cytoplasm and large, round nuclei (Figure 8 (F2–F4)). The diazepam group still showed notable neuronal condensation and deep staining in CA1 and DG (Figure 8 (C2,C4)).

3.9. BCBL Inhibits Hippocampal Levels of Hypoxia and Inflammation-Related Factors in Rotarod Sleep Deprivation-Induced Insomnia Mice

Hippocampal expression of HIF-1α, TNF-α, and IL-1β was significantly elevated in model mice compared to controls (Figure 9B–D). In contrast to diazepam, which failed to alter these increases, high-dose BCBL treatment significantly downregulated all three proteins, suggesting a specific role in mitigating neuroinflammation and the hypoxic response.

3.10. BCBL Regulates the Levels of Neurotransmitters and Neuromodulators in Rotarod Sleep Deprivation-Induced Insomnia Mice

Compared with the control group, the model group exhibited downregulated hypothalamic 5-HT_1A receptor expression (Figure 10B) and upregulated ADORA₁ receptor levels (Figure 10C), accompanied by significantly decreased concentrations of 5-HT (Figure 10D) and DA in the hypothalamus (Figure 10G) as well as serum DA (Figure 10F). In contrast, hypothalamic adenosine (Figure 10E) and Glu levels in both serum (Figure 10H) and hypothalamus (Figure 10I) were markedly elevated. High-dose BCBL treatment significantly counteracted these changes by upregulating 5-HT_1A expression and increasing 5-HT and DA levels, while downregulating ADORA₁ receptors and reducing adenosine and Glu concentrations. Medium-dose BCBL only significantly reduced hypothalamic ADORA₁ and adenosine levels, whereas diazepam showed no significant effects on any of these neurochemical parameters.

3.11. BCBL Reverses the Abnormal Expression of Circadian Rhythm-Related Genes in Rotarod Sleep Deprivation-Induced Insomnia Mice

In comparison to the control group, the model group demonstrated a significant reduction in the p-GSK3β/GSK3β ratio (Figure 11B), as well as in the expression levels of BMAL₁ (Figure 11C), SIRT₁ (Figure 11D), and CLOCK (Figure 11F), while showing an increase in PER₂ expression (Figure 11E) in hypothalamic tissue. High-dose BCBL treatment significantly reversed these alterations by upregulating the p-GSK3β/GSK3β ratio, SIRT₁, CLOCK, and BMAL₁, while downregulating PER₂ expression. In contrast, diazepam administration only induced a significant increase in SIRT₁ expression.

4. Discussion

Insomnia is a common clinical disorder with high comorbidity with depression, a connection underpinned by shared neurobiological mechanisms, notably those involving the serotonergic (5-HT) system. Our prior work demonstrated that Baichuan Baile (BCBL), a novel functional dietary formula derived from traditional Chinese medicine (TCM), modulates the 5-HT pathway and exerts antidepressant effects. Building on this mechanistic and clinical linkage, the present study investigates the therapeutic potential of BCBL for insomnia, aiming to expand its application as a multi-target sleep-modulating agent.

Initial behavioral characterization confirmed that BCBL possesses clear sedative-hypnotic properties, as evidenced by shortened sleep latency, prolonged sleep duration, and reduced locomotor activity. Importantly, polysomnographic recordings revealed that BCBL not only increased total sleep time by extending non-rapid eye movement (NREM) sleep but also improved sleep continuity, reflected by longer mean NREM episode durations and fewer state transitions. This profile, which contrasts with the sleep-fragmenting effects of certain conventional hypnotics [19], suggests that BCBL may offer qualitative advantages in sleep consolidation.

Given the multifaceted etiology of insomnia, we employed three complementary rodent models to systematically evaluate BCBL: environmental stress-induced, para-chlorophenylalanine (PCPA)-induced (serotonin depletion), and rotarod-based chronic sleep deprivation. BCBL consistently ameliorated core sleep disturbances across all paradigms. Notably, in the chronic sleep deprivation model, it significantly attenuated memory deficits and normalized changes in body weight, demonstrating its ability to address both core symptoms and common comorbidities. Owing to its unique capacity to sustain prolonged and stable insomnia-like symptoms, thus more effectively mimicking the persistent pathology of chronic insomnia, the rotarod paradigm was selected for subsequent in-depth mechanistic investigation.

Reflecting the multi-component and multi-target nature of traditional Chinese medicine, we adopted an integrated strategy combining network pharmacology and experimental validation to systematically explore the mechanisms through which BCBL alleviates insomnia. To this end, network pharmacology analysis revealed that BCBL’s anti-insomnia effects are strongly linked to key targets such as GSK3β, SIRT₁, TNF, and ADORA1, and to the modulation of neurotransmitter signaling (e.g., serotonin, dopamine, and glutamate) and circadian rhythm regulation. These findings thereby provide a clear roadmap for further research into the action mechanisms of BCBL.

Insomnia has been linked to cerebral energy depletion and a hypoxic microenvironment, which contribute to the stabilization and accumulation of HIF-1α [20,21]. Although not traditionally classified as an inflammatory mediator, HIF-1α plays a key role in driving the expression of pro-inflammatory cytokines such as IL-1β and TNF-α [22]. In the present study, BCBL significantly reduced hippocampal levels of HIF-1α and its downstream pro-inflammatory factors. Moreover, H&E staining suggested a preservation of neuronal structural integrity and layered organization. These results preliminarily indicate that BCBL may attenuate insomnia-associated hippocampal pathology by suppressing the HIF-1α-mediated neuroinflammatory pathway.

Nevertheless, it should be noted that the necessity of this pathway has not been directly validated through specific interventions targeting HIF-1α signaling, such as genetic knockout or pharmacological antagonism. Consequently, whether BCBL exerts its effects unequivocally via this pathway remains to be further substantiated. Moreover, although H&E staining offers insight into tissue morphology, it does not allow quantitative assessment of critical cellular functional parameters, including neuronal viability and synaptic plasticity. Future investigations should integrate more refined methodologies, such as immunofluorescence and electron microscopy, to provide a comprehensive evaluation of these aspects.

It is widely acknowledged that the pathophysiology of insomnia is characterized by hyperarousal, insufficient sleep drive, and the resulting daytime dysfunction, all of which are closely associated with dysregulation of neurotransmitter/neuromodulator systems [23,24]. Adenosine accumulates during wakefulness as a sleep-pressure marker [25], while 5-HT supports daytime stability and facilitates sleep transition via 5-HT_1A receptors [26]. Dysregulated excitatory systems, such as impaired dopamine signaling and overactive glutamate transmission, further contribute to hyperarousal [27]. In this study, BCBL treatment in an insomnia model elevated levels of 5-HT, 5-HT_1A receptors, and dopamine, and reduced glutamate and adenosine. These shifts suggest a moderation of hyperarousal and improved sleep–wake regulation. Behaviorally, BCBL-treated mice showed a trend toward better performance in memory tests (Y-maze and novel object recognition). The findings imply that BCBL may modulate the activity of neurotransmitters/neuromodulators, providing preliminary support for its potential role in alleviating insomnia and related daytime dysfunction.

Evidence indicates that long-term insomnia or shift work can disrupt normal circadian rhythms by interfering with the synchronization of the body to natural light–dark cycles and disturbing regular sleep–wake patterns [28]. The generation and remodeling of circadian rhythms involve a multi-level, complex process, the core mechanism of which relies on an autonomous transcriptional–translational feedback loop formed by core clock genes and their protein products.

In mammals, this system is governed by the central pacemaker located in the suprachiasmatic nucleus of the hypothalamus. The molecular basis involves the heterodimerization of BMAL₁ and CLOCK proteins, which bind to E-box elements in the promoter regions of downstream rhythm genes (such as PER₂-PER₂ and PER₂-Cry₁) to drive their transcription. Subsequently, the accumulated PER₂ and CRY₁ proteins form complexes, enter the nucleus, and inhibit the transcriptional activity of CLOCK-BMAL₁, thereby forming a negative feedback oscillatory loop with a period of approximately 24 h [29]. Furthermore, the kinase GSK3β fine-tunes the circadian period by phosphorylating core clock proteins (e.g., promoting BMAL₁ degradation and regulating PER₂ stability) [30], while the deacetylase SIRT1 influences the amplitude and phase of rhythm gene transcription by modulating the acetylation status of CLOCK and the activity of BMAL₁ [31].

The insomnia model induced by rotarod sleep deprivation simulates chronic insomnia and circadian rhythm disruption (i.e., reversed day–night cycles). In this study, we observed that following BCBL intervention, the expression levels of circadian rhythm factors in mice tended to align with those of the control group. Specifically, this was reflected in elevated expression of BMAL₁/CLOCK and reduced PER₂ levels, accompanied by inhibition of GSK3β activity (via increased p-GSK3β/GSK3β ratio) and upregulation of SIRT₁. At a single time point, these changes may suggest that BCBL possesses regulatory potential for the molecular network of the circadian clock; these changes may suggest that BCBL possesses regulatory potential for the molecular network of the circadian clock.

This study provides the first systematic investigation into the effects of BCBL on insomnia. Through integrated behavioral, network, and molecular analyses, we demonstrate that BCBL alleviates chronic insomnia by coordinately modulating neuroinflammation, neurotransmitter systems, and circadian rhythms. This multi-target action supports its potential as an integrated therapy for sleep disorders. Clinically, chronic insomnia frequently co-occurs with depression, memory deficit, and low-grade inflammation, reflecting a complex pathophysiology. BCBL’s ability to simultaneously regulate these overlapping pathways suggests it may be better suited than single-target hypnotics for treating such multifactorial insomnia. This work offers preliminary mechanistic evidence for this approach.

In addition to the limitations mentioned above, several aspects of this study could be further refined in future work. First, all experiments were conducted using male mice, and the potential impact of sex differences on the outcomes remains unexamined. Second, while diazepam served as a positive control to benchmark pharmacological efficacy, its mechanism primarily via gamma-aminobutyric acid type A receptor modulation differs fundamentally from the multi-target network predicted for BCBL. Future mechanistic studies should therefore utilize pathway-specific controls for more precise comparison. Third, molecular indicators were assessed at a single time point, lacking dynamic profiling, and a systematic dose–response relationship remains to be established, which may affect mechanistic interpretation. Fourth, pharmacokinetic properties (e.g., half-life, tissue distribution) were not analyzed, limiting insight into in vivo processes. Furthermore, the specific active components of BCBL responsible for its effects, along with their metabolic pathways and relative contributions, require further elucidation.

5. Conclusions

In summary, this study demonstrates that BCBL, a TCM-inspired functional formula, alleviates insomnia-like behaviors through a multi-system mechanism encompassing anti-inflammatory actions, neurotransmitters/neuromodulator rebalancing, and circadian stabilization. These findings highlight the potential of multi-target herbal formulations in treating complex sleep disorders, especially those with overlapping neuropsychiatric features. Addressing the noted limitations in future work will strengthen the translational rationale for BCBL and contribute to the development of integrative, mechanism-based therapies for insomnia.