ZZCD was obtained from Zhang Zhongjing Pharmacy in Henan, China. Diazepam (DZP) (H37023039) was bought from Shandong Xinyi Pharmaceutical Co. Ltd. PCPA was sourced from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Sodium pentobarbital (2018042001) was acquired from Huaxia Chemical Reagent Co., Ltd. (Sichuan, China). Enzyme-linked immunosorbent assay (ELISA) kits for various biomarkers including 5-HT, NE, DA, corticotropin-releasing hormone (CRH), ACTH, CORT, IL-1, and TNF-α were all purchased from Shanghai enzyme-linked biotechnology Co., Ltd. (Shanghai, China). For ZZCD preparation, 9 g FG and 8 g FS were soaked in 1000 mL distilled water for 1 h and then boiled for 1 h. After collecting the decoction, 800 mL of water was added and also boiled for 1 h for the second decoction. The two decoctions were combined, frozen, and then concentrated using a rotary evaporator at 65°C to prepare the ZZCD extract. Based on the conversion ratio between humans and mice, the concentrated volume of a single dose of ZZCD was calculated to be 41.8 mL. The suspension of DZP (1 mg/kg) and PCPA powder (60 mg/mL) was obtained by dissolving them in distilled water.

Male ICR mice (25–35 g) were purchased from Beijing HFK bioscience Co., Ltd. (China, Beijing, SYXK (Jing) 2024-0015) and the animals were kept in a controlled environment for 7 d. The room temperature stayed at 25 ± 2°C, the humidity at 50 ± 5%, and there was a 12 h light—dark cycle, with 12 h of light and 12 h of darkness daily. Approval for the animal experiments was bestowed by the Animal and Ethics Review Committee of Henan University of Chinese Medicine (approval number: IACUC-202410039). All male ICR mice were randomly allocated into four groups (n = 6 per group) using a computer-generated randomization sequence: control group, model group, ZZCD group, and DZP group (3 mg/kg of DZP). Except for control group, which was injected with 0.3 mL of saline, all other groups received a daily intraperitoneal injection of a PCPA suspension (300 mg/kg) once between 8:00 and 9:00 for 2 consecutive days to establish a insomnia model (Zhu et al., 2024). On the 9th day, mice injected with PCPA were observed to exhibit abnormal mental status, with rough fur and increased irritability. Between control group and model group, verification through a pentobarbital sodium sleep test found statistical differences in sleep latency and sleep duration. Subsequently, the corresponding groups were gavaged with 0.3 mL of respective samples once daily for 7 consecutive days. On the 16th day, the open field test and elevated plus maze test were conducted. On the 17th day, the pentobarbital sodium sleep test was performed and followed collecting the mice feces. On the 18th day, blood was collected from the orbital cavity, and the mice were euthanized by cervical dislocation after anesthesia with deep chloral hydrate. After euthanasia, the medulla oblongata was immediately severed at the foramen magnum, and mice’s skull was removed to fully expose the brain tissue. Half of brain was fixed for hematoxylin and eosin (H&E) and Nissl staining analysis, while the other half was collected on ice, separated, and stored in a refrigerator at −80°C.

3 d before the end of drug administration, mice underwent the open field test as follows: after 30 min of drug administration, the mice were first placed for 5 min to adapt to the environment. Afterward, mice were placed inside a 50 cm × 50 cm open field to collect behavioral parameters for 5 min. The arena was divided into a central zone (25 cm × 25 cm square in the middle) and a peripheral zone (the remaining area). Alcohol was used to eliminate the scent of previous mice before proceeding with the next experiment. The main parameters collected were the total distance moved (in cm) and the total activity time (in S). Similarly, 3 d before the end of drug administration, the mice underwent the elevated plus maze test. The elevated plus maze test is elevated 40 cm above the ground. The maze consists of two open arms and two closed arms (each arm = 30 cm, central area = 6 cm × 6 cm). The central zone in the EPM test serves both as the starting point of the experiment and an important reference area for behavioral observation. The time spent by mice in the central zone and the frequency of entries into each arm reflect their exploratory behavior and anxiety level. During the test, each mice was placed in the central zone facing an open arm and allowed to explore freely for 5 min. The number of entries and the time spent (in S) by mice in the open arms were recorded and analyzed.

1 d before the end of drug administration, a pentobarbital sodium sleep experiment was conducted. 1 h after gavage, mice were intraperitoneally injected with pentobarbital sodium (48 mg/kg) and then placed supine on a cushion. Sleep latency (in S) and sleep duration (in min) were recorded. Sleep was defined as the loss of righting reflex, and awakening as its recovery. If uncertain, mice were placed back on their backs; if they flipped over within 1 min, the first flip time was recorded as the recovery time; otherwise, the second flip time was used.

After 7 d of drug administration, all ICR mice were fasted for 12 h. Blood samples were collected via orbital bleeding and centrifuged using a high-speed centrifuge at 4°C (1,000 g, 20 min) to obtain serum. Using the serum samples, ELISA kits were used to detect the levels of CRH, ACTH, CORT, IL-1, and TNF-α, respectively. After euthanasia, hippocampal tissues of mice were promptly dissected and stored at −80°C. Subsequently, hippocampal tissues were thoroughly homogenized using a tissue homogenizer with phosphate-buffered saline at −4°C. After that, homogenates were centrifuged at 5000 g for 5 min, and the supernatants were collected. Following the instructions provided, ELISA kits were utilized to measure the levels of GABA and GLU. After the mice are euthanized, hypothalamic tissue is quickly isolated and stored at −80°C. Consequently, hypothalamic tissue was homogenized using a tissue grinder at a ratio of 1:9 with PBS at −4°C. After 5,000 g × 10 min centrifugation, supernatant is collected and the levels of monoamine neurotransmitters 5-HT, DA, and NE are measured using ELISA kits. All ELISA measurements were performed in duplicate, with an intra-assay coefficient of variation (CV) < 10%, ensuring the reliability of the results.

Using 16S rDNA high-throughput sequencing, gut microbiota analysis of animal is conducted as follows: mice feces were subjected to total DNA extraction for microbial communities using the FastPure stool DNA isolation kit (MJYH, Shanghai, China). PCR amplification of full-length 16S rDNA gene was performed using primers 27F (5’-AGRGTTYGATYMTGGCTCAG-3′) and 1492R (5’-RGYTACCTTGTTACGACTT-3′) with barcodes, following an initial denaturation at 95°C for 3 min, 27 cycles of denaturation at 95°C for 30 S, annealing at 60°C for 30 S, and extension at 72°C for 30 S, followed by a final extension at 72°C for 10 min, and storage at 4°C (Weisburg et al., 1991). PCR products were detected by 2% agarose gel electrophoresis and then purified using AMPure PB beads (Pacific Biosciences, CA, United States). After that, PacBio raw reads were processed using the SMRTLink analysis software (version 11.0) to obtain high-quality Hifi reads with a minimum of three full passes and 99% sequence accuracy. Data from individual samples were distinguished based on barcode sequences, filtered by length, and sequences ranging from 1,000–1800 bp (for bacteria) were retained. Usearch 11 was used to cluster sequences into operational taxonomic units (OTUs) at a 97% similarity threshold and to remove chimeras (Edgar, 2013). To minimize the impact of sequencing depth on subsequent α- and β-diversity analyses, the sequence count for all samples was rarefied to 6,000. RDP classifier was used to annotate the OTU taxonomic classification by aligning against NT_16s (v20230830) with a confidence threshold of 70%, and the community composition of each sample was tallied at different taxonomic levels (Wang et al., 2007). Finally, PICRUSt2 software (version 2.2.0) was employed for 16S functional prediction analysis (Douglas et al., 2020).

Using H&E staining and Nissl staining, pathological examination of hypothalamic tissue was conducted. Brain tissues fixed in paraformaldehyde were retrieved and rinsed with PBS, and subsequently dehydrated with ethanol in a graded series. After embedding with wax, tissues were cut to continuous sections with a thickness of 5 μm and subjected to staining, dehydration, clearing, and mounting. This was achieved by treating the sections with increasing concentrations of ethanol solutions and xylene, ultimately leading to their mounting. The sections were stained separately with eosin and toluidine blue to observe the pathological changes in hippocampus and Nissl bodies in neuronal cells.

The statistical analysis was carried out utilizing SPSS 21.0 software, and the results were presented in the form of mean ±SD. The t-test and one-way ANOVA were employed to examine group differences (p value <0.05 was considered statistically significant). Graphs were generated using GraphPad Prism 9.0.

In the open field test, anxiety-related behavior was assessed by measuring the total distance moved and the time spent in the central zone. Mice in the model group exhibited significant decreases in both parameters relative to the control group (Figures 1A,B, p < 0.001). Mice receiving ZZCD treatment demonstrated a markedly higher overall distance covered and a considerably longer time period in the central region in comparison to the model group. Subsequently, elevated plus maze test was conducted on mice using the number of entries and the time spent in open arms as assessment indicators. Mice in the model group exhibited a significant reduction in both the frequency of open arm entries and the duration spent in open arms compared to the control group (Figures 1C,D, p < 0.05). Administration of ZZCD significantly enhanced open arm exploration, as evidenced by increased entry frequency and duration. The experimental results indicate that ZZCD can alleviate tension and anxiety-like behaviors in insomnia mice. Compared to control group, observations on sleep latency revealed a significant increase in model group (Figures 1E,F, p < 0.001). Both the ZZCD group and the DZP group displayed a notable decrease in sleep latency in comparison to the model group (p < 0.001). Sleep duration was significantly reduced in the model group relative to the control group (p < 0.01). Meanwhile, both ZZCD group and DZP group showed an increase in sleep duration (p < 0.05). ICR mice exhibited distinct locomotor trajectories across behavioral tests (Figure 1G).

The levels of CRH, ACTH, and CORT in the serum were assessed using commercially available ELISA kits. The model group showed elevated serum levels of CRH, ACTH, and CORT relative to the control group (Figures 2A–C, p < 0.01). Administration of ZZCD led to a decrease in serum concentrations of CRH, ACTH, and CORT relative to the model group (p < 0.05). Simultaneously, DZP administration significantly lowered serum levels of ACTH and CORT (p < 0.0001). These findings imply that ZZCD might have an impact on the sleep condition of insomniac mice through regulating the HPA axis.

To determine the secretion levels of GABA and GLU, we measured their contents in hippocampus using ELISA kits. Relative to the control group, the model group showed notably lower levels of GABA in the hippocampus (Figures 2D,E, p < 0.0001), accompanied by an increase in GLU content (p < 0.0001). In view of this, ZZCD improves sleep quality in mice with insomnia, potentially by adjusting the secretion of GABA and GLU. By assessing the amounts of 5-HT, DA, and NE in the hypothalamus, this research intended to delve into the influence of ZZCD on neurotransmitters in insomniac mice. The model group showed elevated DA and NE levels (Figures 2G,H, p < 0.05), along with a reduction in 5-HT (Figure 2F, p < 0.01), relative to the control group. In comparison with the model group, both ZZCD and DZP markedly enhanced 5-HT content (p < 0.0001) and reduced DA levels (p < 0.05). These results imply a potential mechanism whereby ZZCD improves sleep quality through serotonergic activation and dopaminergic inhibition in a murine model of insomnia.

To examine the inflammatory profile associated with insomnia, we evaluated serum TNF-α and IL-1 concentrations. As shown in Figures 2I,J, TNF-α and IL-1 levels were significantly higher in the model group than in the control group (p < 0.01). Treatment with ZZCD resulted in a significant decrease in TNF-α and IL-1 levels (p < 0.001). Thus, we can conclude that ZZCD improves sleep in mice by modulating the secretion of TNF-α and IL-1.

As shown in Figure 7, H&E staining and Nissl staining indicate that hippocampal neurons in control group mice are tightly and neatly arranged, with regular morphology and clearly visible Nissl bodies. In contrast, hippocampal neurons in model group mice exhibit disordered arrangement, reduced neuron number, and a large number of darkly stained neurons. Additionally, nuclear pycnosis, cytoplasmic dissolution, enlarged intercellular spaces, disordered arrangement, and decreased Nissl bodies are observed in model group. Compared with model group, both ZZCD group and DZP group show an increase in number of hippocampal neurons, with neatly arranged cells. Occasionally, darkly stained nuclei and relatively intact cell membranes are seen, indicating improvements in cell morphology and structure, along with an increase in Nissl bodies. These findings suggest that ZZCD has a certain repair effect on hippocampal neurons and Nissl bodies in insomnia mice induced by PCPA. Model group exhibited an increased number of inflammatory cells in colon, damaged villi, slight exfoliation of mucosal epithelial cells, loose arrangement of intestinal glands in lamina propria, and a thinned basement membrane. Compared to model group, ZZCD treatment significantly reduced colonic inflammatory cell infiltration, restored villi to levels comparable with the normal group, and induced structural improvements including compactly arranged lamina propria intestinal glands and a markedly thickened basement membrane.