Fresh mackerel bones were supplied by Zhejiang Industrial Group Co. Ltd. (Zhoushan, Zhejiang, China). The head and remaining meat were removed, and the bones were frozen at −20°C for further processing. Melatonin (MT) was purchased from Gaoxin Chemical Glass Co., Ltd. (Shanghai, China). Zebrafish dopamine (DA) and γ-aminobutyric acid (GABA) ELISA kits were purchased from Mreda Technology Co., Ltd. (Beijing, China). 2′, 7′-Dichlorofluorescein diacetate (DCFH-DA) was obtained from Solarbio Science & Technology Co., Ltd. (Beijing, China). Superoxide dismutase (SOD), catalase (CAT), and lipid peroxidation (MDA) detection kits were purchased from Yuanye Bio-Technology Co., Ltd. (Shanghai, China). TVGF and TVGF-Acp-FITC were synthesized by GL Biochem (Shanghai, China) Ltd. All other chemicals and reagents utilized in this study were purchased from local commercial establishments and were analytical grade.

Wild-type adult zebrafish (AB strain) were obtained from the Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai Ocean University (Shanghai, China). All animal experiments were based on the institutional ethical guidelines of the Shanghai Ocean University Experimentation Ethics Review Committee (SHOU-DW-2016-002). Zebrafish were maintained under standard environmental conditions: 28°C ± 0.5°C, a 14/10-h light/dark cycle, and a pH of 6.8–7.2. The night before spawning, adult zebrafish were placed in the mating tank overnight (male: female = 1: 1), with baffles separating males and females. The baffles were re-moved the next morning, allowing natural mating. Embryos were collected and incubated at 28°C ± 0.5°C in a thermostat incubator until used for exposure experiments. During this period, maintain fresh water daily and promptly remove dead embryos and debris.

At 96 h post-fertilization (hpf), zebrafish larvae were exposed to 200 lux of white light continuously for 24 h to establish a model of anxiety-related insomnia. Subsequently, larvae were randomly assigned to groups and treated with melatonin, MBP1, and TVGF for 24 h prior to transfer to 96-well plates. Including the normal control group (NC) without 24-h light exposure; anxiety-like insomnia model zebrafish control group (IC); IC larvae treated with 10⁻³ mol/L melatonin (IC + MT); IC larvae treated with low (0.03 mg/mL), medium (0.05 mg/mL), and high (0.3 mg/mL) dose of MBP1 (IC + LP, IC + MP, IC + HP); IC larvae treated with 0.2 mg/mL TVGF (IC + TVGF). All experiments were performed in triplicate and 50% of the solution in each dish was re-placed with fresh solution every 24 h during the exposure period.

Based on our previous research methodology, mackerel bones were initially defatted, chopped, and mixed with distilled water (water-to-bone ratio = 3.62:1). Papain and trypsin (1:1 enzyme ratio) were then added for hydrolysis, with a total enzyme concentration of 5,000 U/g. Hydrolysis was carried out at 62°C and pH 7.0 for 4 h under water bath oscillation (120 rpm). Following enzymatic hydrolysis, the supernatant was obtained through enzyme inactivation, centrifugation, 1% activated carbon decolorization, and filtration. MBP1 was separated using an ultrafiltration membrane with a molecular weight cutoff of less than 1 kDa (Sigma-Aldrich, USA) and lyophilized to obtain MBP1 powder (The molecular weight distribution analysis has been conducted to assess purity.) for subsequent experiments.

Zebrafish at 120 hpf were rinsed three times with culture water and individually transferred to 48-well plates (1 larva/well, n = 8/group). Prior to formal testing, larvae were gently placed in Daniovision (Noldus, Netherlands) under dark environment for 10 min to acclimate. Locomotor behavior was monitored and recorded for 1 h using the Daniovision system, with a resolution of 1,024 × 768 pixels and a frame rate of 25 frames per second (fps). Subsequently, total moving distance, swimming speed, active time and rest time were analyzed using Ethovision XT11 software.

Following exposure experiments, zebrafish (n = 60/group) at 120 hpf were collect-ed and homogenized in an ice bath. The homogenate was then centrifuged at 3,000–4,000 rpm for 15 min at 4°C to obtain the supernatant. DA and GABA concentrations in zebrafish were quantified using commercially available ELISA kits. The concentrations of DA and GABA in the supernatant were determined according to the manufacturer’s instructions.

The levels of reactive oxygen species (ROS), malondialdehyde (MDA) concentration and antioxidant enzyme activity were measured to evaluate the experimental induced oxidative stress response of zebrafish (Guru et al., 2021). Larvae at 120 hpf after exposure were randomly transferred to 48-well plates and incubated with 20 μg/mL DCFH-DA for 1 h at 28.5°C in the dark for staining. Stained larvae were then washed three times with PBS buffer and anesthetized with 0.04% tricaine solution. Later, zebrafish were imaged in the lateral position under a fluorescence microscope (OLYMPUS IX71, Japan), and the fluorescence intensity of the zebrafish (n = 5/group) was quantified by ImageJ software to estimate the level of ROS production. The larvae (n = 120/group) at 120 hpf after exposure were collected and rinsed three times with PBS buffer, then homogenized in an ice bath as required and centrifuged (10,000–12,000 rpm, 15–20 min, 4°C) to collect the supernatant. Later, the supernatant was used to determine the levels of MDA, SOD and CAT according to the manufacturer’s instructions.

Zebrafish from each experimental group were randomly selected and fixed in the lateral position using OCT freezing embedding agent (n = 3/group). Tissue sections of 5 μm thickness were cut with a frozen slicer (Leica CM 1950, Germany) and adhered to the ZnSe windows. The windows were then dried in a desiccator (40°C, 12 h) to eliminate moisture interference for IR imaging. Subsequently, optical imaging image and infrared spectral data of the zebrafish cross-section were acquired using a Spotlight 400 system (PerkinElmer, United States). Spectral acquisition parameters were as follows: wavenumber ranged from 4,000 to 750 cm⁻¹, resolution was 4 cm⁻¹, pixel size was 6.25 μm, and each pixel was scanned 16 times. PerkinElmer spectral software (Version 10.6.4) was used to extract spectral information from the head, abdomen, and tail of the zebrafish (five for each part) randomly and evenly from the obtained chemical imaging map for processing and analysis. Finally, PeakFit software (Version 4.12) was employed to analyze the percentage of secondary structures based on the extracted spectral data.

After the exposure experiment, zebrafish (n = 20/group) were frozen with liquid nitrogen and shipped to Novogene Biotech Co., Ltd. (Beijing, China) for transcriptome sequencing analysis based on the Illumina sequencing platform. In brief, total RNA was extracted from zebrafish using the TRIzol kit (Thermo Fisher Scientific, USA), and the integrity and quality of total RNA were accurately detected and strictly controlled using the Agilent 2100 bioanalyzer. Qualified RNA samples were subjected to Illumina Hi SeqTM sequencing after mRNA enrichment, double-stranded cDNA synthesis, end repair, poly-A enrichment, fragments selection, PCR, and library quality inspection. In order to obtain clean reads, the quality and reliability of data analysis can be ensured by removing reads containing sequencing adapters, removing reads with unknown base, and removing low-quality reads (the number of bases with Q phred ≤20 bases for more than 50% of the whole read length). Later, the clean reads were aligned to the reference genome using Hisat2 v2.0.5 (the reference genome and gene model annotation files were downloaded from the genome website). Deseq2R software was used to analyze the differential expression of zebrafish in different groups and modify the p-value obtained by the original hypothesis test (padj <0.05, |log2foldchange| >1). Finally, in order to further explore the differences in functions and pathways of DEGs, used cluster Profiler 3.8.1 software for GO and KEGG enrichment analysis of DEGs.

The protocol for the isolation, purification and identification of peptides with hypnotic activity of MBP1 was based on our previous research (Wang et al., 2014). Briefly, the MBP1 solution (8 mg/mL) was purified by chromatography column (1.6 × 60 cm) equipped with Sephadex G-15. Ultimately, three fractions were obtained, lyophilized and their sleep-promoting activities were investigated by the light-induced sleep disturbances zebrafish model. LC-MS/MS technology was used to identify the peptide sequence by comparing and matching the database. The dominant peptide sequence TVGF was identified by mass spectrometry and synthesized by solid-phase synthesis technology. The purity and molecular weight of TVGF were determined by ONO LC-2000 (4.6 mm × 250 mm, 5 μm) HPLC and mass spectrometry (Agilent-6125B).

The 3D structure of TVGF was constructed using PyMOL software and processed with AutoDock4 software, such as hydrogenylation, charge balance, and then saved as pdbqt files. The PDB ID of the relevant receptor proteins was obtained from PDB (https://www.rcsb.org/) and then optimized by PyMOL software, including dehydration, hydrogenation, and deligandization. Autodock 4 software was used to perform semi-flexible docking of the receptor and ligand. The operation steps included setting the GridBox size, running AutoGrid molecular docking, observing the molecular con-formation of the docking and analyzing the docking data.

All experimental studies were expressed as mean ± standard deviation. The data were analyzed by ANOVA test (SPSS 21.0 statistical software). Tukey test was used to analyze the significant difference between the mean values of each parameter. Relevant data charts were generated using GraphPad Prism 8.0.2 and Microsoft Excel 2019.

Long-term light exposure can disrupt the circadian rhythm of zebrafish and make them extremely excited and active. Therefore, the impact of long-term light exposure on the locomotor behavior of zebrafish can be examined by tracking locomotor trajectories (Figure 1A) (Gong et al., 2017). The IC group exhibited more intricate and disordered movement trajectories compared to the NC group, characterized by increased movement distance, swimming speed, active time, and reduced rest time (Figures 1B,C). Treatment with MBP1 at varying concentrations effectively mitigated these behavioral changes, reducing locomotor activity and promoting rest time. The highest dose of MBP1 (IC + HP) yielded the most pronounced improvement, approaching the control group and exhibiting comparable effects to the melatonin-treated group (IC + MT). Thus, it can be preliminarily determined that the anxiety-like movement of zebrafish is alleviated under the protection of MBP1 active peptide.

Neurotransmitter imbalances are crucial factors in the development of sleep disturbances. DA in sleep-wake regulation is considered as a wakefulness-promoting ex-citatory neurotransmitter (Herrera-Solis et al., 2017), while GABA is a crucial inhibitory neurotransmitter for the initiation and maintenance of sleep in the brain (Morgan et al., 2012). As depicted in Figure 1D, exposure to prolonged light caused an imbalance in the neurotransmitter system, leading to increased DA and decreased GABA concentrations in zebrafish, ultimately causing hyperkinesis and resulting in insomnia. The effect of the IC + HP group showed a significant reversal advantage in light-induced anxiety-like insomnia, and the effect was close to that of the IC + MT group, while the effect of the IC + LP and IC + MP groups was relatively slight (Figure 1D). At higher doses of MBP1, the effect on neurotransmitter regulation was comparable to that of the positive drug group. Additionally, the changes in neurotransmitter levels in all treatment groups remained within a safe range, ensuring no other physiological damage was caused. Therefore, MBP1 can act with the neurotransmitter system to improve anxiety-related activities.

As depicted in Figures 2A–C, compared to the NC group (ROS, 100.00%; MDA, 38.68 μmol/g), the IC group exhibited significantly increased levels of ROS (142.43%) and MDA (51.88 μmol/g), visualized by enhanced green fluorescence around the yolk sac, head, and eyes. Treatment with various concentrations of MBP1 effectively attenuated these effects, reducing ROS production and MDA levels in a dose-dependent manner. Among them, the IC + HP group (ROS, 132.07%; MDA, 44.54 μmol/g) demonstrated the most significant improvement, focusing the fluorescence distribution primarily to the yolk sac, indicating its potential to regulate oxidative stress. In addition, compared with NC group (SOD, 36.02 U/g; CAT, 3.76 U/g), the activities of SOD (27.12 U/g) and CAT (2.87 U/g) in IC group were significantly reduced in the IC group. However, treatment with various doses of MBP1 reversed this trend, enhancing the expression levels of endogenous antioxidant enzymes (SOD, CAT) in a dose-dependent manner (Figures 2D,E). These findings indicate that MBP1 can alleviate anxiety-like insomnia through enhancing the activity of the antioxidant defense system.

Infrared (IR) transmission spectroscopy can simultaneously collect hundreds of pixel spectra with a high signal-to-noise ratio and low distortion, demonstrating the clear correlation between molecular structure and spectral characteristics (Chen et al., 2013). It can intuitively and completely analyze the spatial distribution and chemical composition information of biomacromolecules such as lipids and proteins in the microscopic region of biological tissues (Table 1) (Hou et al., 2020). It is observed from the IR transmission images of zebrafish cross-section that a substantial presence of proteins (red areas) occupies a significant proportion of the entire cross section (Figure 3A). Analysis of IR spectra (Figure 3B) extracted from IR transmission images revealed alterations in chemical composition following treatment with different doses of MBP1. Insufficient sleep and circadian rhythm disorder increase the risk of obesity and central obesity (Cai et al., 2018), and there is a latent correlation between lipid accumulation and tau aggregate formation (Zhao et al., 2023). In line with these findings, the characteristic lipid peaks at 2,925 cm⁻¹ (υ_as (CH2)) and 2,873 cm⁻¹ (υ_s (CH3)) were significantly elevated in the IC group compared to the NC group (Figure 3C), indicating increased lipid content in zebrafish experiencing sleep deprivation. Treatment with varying doses of MBP1 effectively reduced these lipid peaks in a dose-dependent manner, gradually approaching the NC group levels (Figure 3C), demonstrating its potential to mitigate lipid accumulation associated with insomnia.

Similarly, the characteristic peaks associated with Amide I and Amide II, indicative of protein structure, were significantly lower in the IC group compared to the NC group. However, MBP1 treatment led to a gradual restoration of these peaks towards NC levels with increasing MBP1 concentration. This trend was not observed in the melatonin (MT) treated group (Figure 3D). Further analysis of the protein region using second derivative infrared spectroscopy (SD-IR) revealed alterations in protein secondary structure within the Amide I band (Figure 3E). Compared to the NC group, the IC group exhibited increased α-helix, random coil, and β-sheet content while β-turn content decreased. This shift in protein secondary structure is consistent with the hypothesis that excessive ROS generation during sleep deprivation leads to misfolded proteins and protein aggregation (Vitaterna et al., 2019). Interestingly, the secondary structures of proteins in the IC + LP, IC + MP, and IC + HP groups were also interconverted, exhibiting a trend of α-helix, random coil, and β-sheet content decreased, β-turn content increased and gradually approached a normal level, which was particularly obvious in the IC + HP group. These results indicated that MBP1 may prevent the accumulation of excessive β-sheet protein to a certain extent, thereby preventing the aggregation of tau protein and mitigating the adverse effects of light-induced sleep disturbances on the imbalance of protein homeostasis.

RNA-Seq analysis was performed on zebrafish in the IC, IC + MT and IC + HP groups. Following quality control analysis, adapters and low-quality sequences were removed. Clean Q30 and Q20 results indicated the transcriptome sequencing was of high quality. The biological replicates of zebrafish in each group were highly correlated, and the results of subsequent transcriptome analysis were accurate and credible (Supplementary Tables S1 and S2). Differentially expressed genes (DEGs) were identified based on the criteria of |log2 FC| > 0 & padj <0.05. Volcano plot analysis revealed 192 DEGs (68 downregulated, 124 upregulated) between the IC + MT and IC groups, and 634 DEGs (543 downregulated, 91 upregulated) between the IC + HP and IC groups (Figures 4A,B). There were 114 common DEGs in the two comparison combinations, while 78 and 520 DEGs were uniquely regulated by the IC + MT and IC + HP groups, respectively (Figure 4C). To further explore the potential mechanisms by which MBP1 ameliorates light-induced sleep disturbances, |log2 FC| > 1 & padj <0.05 were used as the default criteria for difference significance, and 15 DEGs with greater significance in the two comparison combinations were selected for comparative analysis (Table 2).

The results indicate that the core circadian clock gene per2 was upregulated in both the IC + MT and IC + HP groups compared to the IC group, suggesting that it contributed to the restoration of circadian rhythm function (Vitaterna et al., 2019). The genes involved in visual perception and retinal development, opn1mw2 was downregulated and zgc:73075 was upregulated (Sun et al., 2018). si:dkey-17e16.17 gene, which is involved in the Golgi transporter system was downregulated. Additionally, the IC + HP group could individually upregulate the visual perception and retinal light sensitivity gene arr3b (Renninger et al., 2011), and the gene sik1 regulating circadian rhythm behavior and energy metabolism was downregulated (Jagannath et al., 2013). These transcriptional changes are consistent with previous studies demonstrating the influence of circadian rhythm networks on visual function and the presence of circadian rhythms in various visual behaviors in zebrafish (Shi et al., 2019).

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were subsequently performed to explore the functional characteristics of these DEGs. Comparison of the IC and IC + MT groups revealed significant enrichment of GO terms related to lipid transport and response to stress in biological processes, while the extracellular region was the most enriched cellular component. Heme binding was the predominant molecular function enriched in this comparison (Figure 4D). GO enrichment analysis revealed that the IC + HP group was significantly enriched in proteolysis and lipid transport as compared to the IC group, corroborating the findings from the multi-molecule spectroscopy analysis. This was probably at-tributed to aberrant expression of circadian genes induced by insomnia, leading to overproduction and overaccumulation of clock proteins. The process involved selectively degrading clock proteins to regulate circadian rhythms (Tamaru and Takamatsu, 2018). The extracellular region was the most enriched cellular component in this comparison, and serine-type endopeptidase activity was the predominant molecular function (Figure 4E). The selected DEGs were subjected to KEGG analysis and enriched into typical signaling pathways (Figures 4F,G). The results revealed that the IC + MT group significantly enriched KEGG pathways related to arachidonic acid metabolism, phototransduction, and linoleic acid metabolism. Similarly, IC + HP group enriched pathways related to retinol metabolism, phototransduction, and amino sugar and nucleotide sugar metabolism.

According to LC-MS/MS identification results (Supplementary Table S3), the purity of TVGF (retention time 10.130 min, MW 422.470 Da) synthesized by solid-phase synthesis was over 98% based on HPLC and mass spectrometry analysis (Supplementary Figure S1). The binding ability of TVGF to sleep-related receptor proteins was further assessed using molecular docking binding energies (Table 3). Top-ranked IL6, HTR1A, and MAOA were selected as key targets to study the receptor-ligand interaction pattern (Figure 5). The occurrence of insomnia is associated with the change of the level of oxidative stress and inflammation in vivo, and IL6 as typical inflammatory factors with TVGF good binding energy (−6.09 kcal/mol). Residues Glu99, Lys120 and Asn144 of IL6 formed three conventional hydrogen bonds with TVGF, and Ala145 and Asn144 was bound to TVGF by Pi-Sigma and Amide-Pi Stacked, respectively (Figure 5A). HTR1A is a G-protein-coupled receptor for 5-HT that plays a role in regulating DA and 5-HT levels in the brain, thereby affecting neural activity, mood, and behavior (Xie et al., 2019). Figure 5B shows that residues Val201, Thr355 and Tyr359 of HTR1A formed three conventional hydrogen bonds with TVGF. Simultaneously, there existed alkyl interactions between TVGF and HTR1A (Val200 and Met337). Insomnia is closely related to depression, and MAOA is a key enzyme involved in the degradation of monoamine neurotransmitters and plays an important role in depression (Wang et al., 2017). As shown in Figure 5C, residues Thr205 and Asp132 of MAOA formed conventional hydrogen bonds with TVGF, while Gly110 formed carbon hydrogen bonds. Additionally, TVGF formed Amide-Pi stacked with residue TRP128 and an alkyl interaction with Val210 of MAOA. Based on these results, it could be indicated that TVGF is a potent inhibitor of anxiety-like insomnia.

This study investigated the ability of TVGF to mitigate anxiety-like insomnia in zebrafish. To track the distribution of TVGF in zebrafish, a fluorescently labeled analog TVGF-Acp-FITC was synthesized, which emits green fluorescence. Fluorescence dis-tribution maps (Figure 6A) revealed the presence of TVGF-Acp-FITC in various parts of the zebrafish following digestion and catabolism. Yolk sacs of zebrafish treated with TVGF concentrations ranging from 0.05 to 0.30 mg/mL exhibited significant green fluorescence. The head and eyes of zebrafish in 0.20–0.30 mg/mL TVGF group also emitted weak fluorescence. Based on these findings, a concentration of 0.20 mg/mL TVGF was selected for further behavioral analysis. Behavioral experiments demonstrated that TVGF treatment significantly reduced abnormal behavior indicators in zebrafish compared to the IC group (Figure 6B). Specific behavioral changes included decreased moving distance, slower swimming speeds, reduced active time, and increased rest time. These findings suggest that TVGF exhibits beneficial sedative effects by effectively transporting into zebrafish via digestion and catabolism. The observed reduction in abnormal behaviors in zebrafish treated with TVGF highlights its potential as a therapeutic agent for mitigating anxiety-like insomnia.

RNA-seq analysis was performed on zebrafish in the IC, IC + MT and IC + TVGF groups to elucidate the molecular mechanism of synthetic peptides to improve anxiety-like insomnia by analyzing the transcriptome. Detailed quality control analyses are presented in Supplementary Tables S4 and S5. The comparison between the IC + MT and IC groups identified a total of 1993 DEGs (1,052 downregulated and 941 upregulated). Similarly, the IC + TVGF group revealed 3,131 DEGs (1,589 downregulated and 1,542 upregulated) that exhibited differential expression compared to the IC group (Figures 7A,B). A further comparative analysis revealed 906 common DEGs between the two comparisons, while 1,087 DEGs were uniquely regulated by the IC + MT group and 2,225 DEGs were uniquely regulated by the IC + TVGF group (Figure 7C). Similarly, 15 DEGs of greater significance in the two comparison combinations were also screened for analysis (Table 4).

It was observed that several previously reported core circadian genes, including cry1bb, cry1ba and per2, were also upregulated and per1b was downregulated in both groups. Furthermore, upregulation of genes associated with visual perception systems such as opn1mw1, gnb3b, arr3b and gnat2 was observed. Similarly, it was also observed that the TVGF group could individually downregulate the circadian rhythm function gene sik1 (Jagannath et al., 2013), the core gene pnp5a related to purine metabolism (Keller et al., 2022), and the gene fkbp5 (Ke et al., 2018) related to anxiety disorders mediating the recovery of stress response.

Subsequently, GO and KEGG enrichment analyses were performed. In comparison between the IC and IC + MT groups, the organonitrogen compound catabolic process was the most enriched GO term in biological processes, while the proteasome core complex dominated in cellular composition, and threonine-type endopeptidase activity was mainly enriched in molecular function. In contrast, the IC + TVGF group exhibited a distinct enrichment profile, with the purine nucleotide metabolic process being the most affected biological process. The cell junction was predominantly affected by cell composition, and molecular function was significantly enriched in calcium ion binding (Figures 7D,E) This enrichment in calcium ion binding is crucial for biosynthesis, metabolic regulation, and maintaining calcium homeostasis within mitochondria (Friedman and Nunnari, 2014). KEGG enrichment analysis also identified the most affected the pathways in melatonin and TVGF treatment compared to the IC group. The IC + MT group demonstrated enrichment in the Proteasome, Spliceosome, and Ribosome regulatory pathways, while the IC + TVGF group showed enrichment Spliceosome and Proteasome regulatory pathways (Figures 7F,G).