Sleeping peptide and differential metabolite detection method for caenorhabditis elegans

Abstract

The embodiment of the invention discloses a sleep peptide and differential metabolite detection method for caenorhabditis elegans, belongs to the technical field of organism metabolism, and aims to research sleep-awakening key metabolite of caenorhabditis elegans and a regulation mechanism of the sleep-awakening key metabolite. According to the sleep peptide for caenorhabditis elegans, the sleep peptide is an F4 monomer as shown in SEQ ID NO: 1. Compared with the prior art, the sleep and awakening process of the caenorhabditis elegans under the intervention of the goat milk-derived sleep peptide is analyzed through a metabonomics technology, the key metabolite for regulating and controlling sleep and awakening is successfully screened out, and a core metabolic pathway related to the key metabolite is disclosed. The application also constructs a metabolite-pathway-target regulation framework, fills the blank of sleep research in the aspect of metabolic regulation, provides a scientific basis for a sleep intervention strategy based on metabolic regulation, and also lays a foundation for developing metabolic markers of sleep states in the future.

Description

Technical Field

[0001] The present application relates to the field of biological metabolism technology, and in particular to a method for detecting sleep peptides and differential metabolites in Caenorhabditis elegans. Background Art

[0002] Currently, research on sleep regulation and its metabolic mechanisms primarily focuses on changes in neurotransmitters, hormones, and metabolites, while relatively few methods for systematic metabolomics analysis based on C. elegans (Caenorhabditis elegans) exist. For example, studies have found that various food-derived peptides (such as bovine casein hydrolyzed peptides and fish-derived peptides) have a certain promoting effect on sleep, primarily through pathways such as GABA, 5-HT, and melatonin. However, there are currently few reports on specific active peptides derived from goat milk and their specific metabolic regulatory effects on the sleep-wake transition.

[0003] C. elegans, as a model organism, has been widely used in nervous system research, and its sleep state is similar to that of mammals, making it highly valuable for research. Existing research focuses on the regulatory mechanisms of sleep in nematodes through the use of neurotransmitters (such as GABA, glutamate, and acetylcholine), but relatively little research has examined global metabolite changes. While initial exploration of C. elegans in metabolomics has been conducted (e.g., using LC-MS and GC-MS to analyze metabolite changes), existing studies have primarily focused on nutritional metabolism, aging, or environmental adaptation, with limited systematic metabolomics analyses specifically targeting sleep state transitions.

[0004] At present, mainstream sleep metabolomics research mainly relies on mammalian models (such as mice and rats), using LC-MS, GC-MS and other technologies to analyze brain tissue or body fluid metabolites, and analyzing sleep-related metabolites through KEGG pathways. However, the problems with these studies are high costs, long cycles, and it is difficult to conduct large-scale and high-throughput experiments on mammalian models. In contrast, C. elegans has the advantages of a short life cycle, simple genetic manipulation, and suitability for large-scale metabolomics analysis, making it suitable for the screening of sleep-regulating metabolites. A small number of studies have used metabolomics to explore sleep, but the studies mainly focus on changes in systemic metabolites, and lack precise analysis of key metabolites of sleep-wakefulness. Summary of the Invention

[0005] The main purpose of this application is to provide a method for detecting sleep peptides and differential metabolites in Caenorhabditis elegans, aiming to study the key metabolites of sleep-wakefulness in Caenorhabditis elegans and their regulatory mechanisms.

[0006] To achieve the above objectives, the technical solutions adopted in the embodiments of the present application are as follows:

[0007] In a first aspect, an embodiment of the present application provides a sleep peptide for Caenorhabditis elegans, and the sleep peptide is the F4 monomer shown in SEQ ID NO: 1.

[0008] In a second aspect, an embodiment of the present application further provides a method for detecting differential metabolites of Caenorhabditis elegans sleep-wakefulness, including the following steps:

[0009] Cultivate Caenorhabditis elegans using a target culture medium; based on the sleep peptide, establish a drug administration system for the cultivated Caenorhabditis elegans and perform synchronization treatment; divide the synchronized Caenorhabditis elegans into a sleep nematode sample group and a wakefulness nematode sample group;

[0010] Use a methanol-water mixed solvent to extract nematode metabolites from the sleep nematode sample group and the wakefulness nematode sample group respectively. After using the ultrasonic crushing method to improve the metabolite release efficiency, perform LC-MS / MS analysis using ultra-high performance liquid chromatography tandem Fourier transform mass spectrometry.

[0011] Specifically, the cultivation of Caenorhabditis elegans using the target culture medium means that after synchronization, the nematodes grow on NGM complete medium for 50 - 54 h. Use a stereomicroscope to observe the vulva structure, and select nematodes with a crescent-shaped translucent appearance as pre-sleep nematodes, and pick nematodes with a growth time of 70 - 74 h as wakefulness-stage nematodes. Collect the nematodes with a growth period of 50 - 54 h using M9 solution, wash them clean, and store them in a -80°C refrigerator as nematodes to be tested for sleep-stage omics; collect the nematodes with a growth period of 70 - 74 h using M9 solution, wash them clean, and store them in a -80°C refrigerator as nematodes to be tested for wakefulness-stage omics.

[0012] As some alternative embodiments of the present application, the target culture medium is prepared through the following steps:

[0013] Mix 17 g of agar powder, 2.5 g of tryptone, 3 g of sodium chloride, and 0.2 g of streptomycin and dissolve them in 1 L of ultrapure water to prepare a basal medium;

[0014] Mix 1 mol / L CaCl2, MgSO4, and K2HPO4-KH2PO4 buffer solution in a mixing ratio of 1:1:25, and perform autoclaving treatment together with the basal medium to obtain a sterilized culture medium; when the sterilized culture medium is naturally cooled to 85 ± 5°C in a clean environment, perform aseptic operation in a biosafety cabinet to obtain a molten culture medium; dispense the molten culture medium into pre-sterilized 60 mm culture dishes, and wait for the culture medium to solidify at room temperature to obtain the target culture medium.

[0015] As some alternative embodiments of the present application, the conditions for the autoclave treatment are 121 °C for 20 min;

[0016] The aseptic operation means: sequentially injecting 1 ml of sterilized CaCl2 solution, 1 ml of sterilized MgSO4 solution, 25 ml of phosphate buffer system with pH 6.0, and 1 ml of cholesterol ethanol solution with a concentration of 5 mg / ml sterilized through a 0.22 μm filter membrane, and fully mixing to obtain a molten medium.

[0017] As some alternative embodiments of the present application, establishing the administration system for the cultured Caenorhabditis elegans based on the sleep peptide includes:

[0018] Using the zone-streaking method to inoculate the preserved uracil auxotrophic Escherichia coli OP50 strain onto a fresh LB agar plate. After culturing at 37 °C for 24 - 48 hours, select monoclonal colonies with typical morphology and aseptically transfer them to a test tube containing 5 mL of sterile LB liquid medium; the typical morphology means presenting a small circular monoclonal colony morphology;

[0019] Subsequently, place it in a constant temperature shaking incubator for amplification culture at 37 °C and 200 rpm. After 12 hours, monitor the bacterial liquid concentration, and terminate the culture when the optical density value reaches OD600 = 0.4. The obtained live bacteria suspension can be directly used for the inoculation operation of the Caenorhabditis elegans NGM feeding plate; configure the ovine milk sleep peptide F4 monomer into a 100 μg / ml solution with pure water to obtain a sample liquid; mix the prepared Escherichia coli liquid and the sample liquid at a ratio of 9:1, and evenly coat the mixture on a 60 mm solid medium plate, and incubate at 20 °C in a laminar flow cabinet for 24 h for standby.

[0020] As some alternative embodiments of the present application, the synchronization treatment includes:

[0021] Rinse the young adult population with pre-cooled M9 buffer and collect it into a 1.5 mL sterile centrifuge tube to obtain a nematode suspension; freshly prepare a NaOH / NaClO mixed lysis system and mix it with the nematode suspension in an equal volume. After vortexing for 30 seconds, centrifuge at 3000 × g for 60 seconds to remove adult tissue debris. Retain the precipitate, wash it with M9 buffer and centrifuge (3000 × g, 1 min / time), repeat twice to obtain the bottom precipitate; disperse the bottom precipitate with sterile water, quantitatively spot 2 μL on an NGM plate covered with an OP50 bacterial lawn, and incubate at 25 °C for 48 hours to obtain a developmentally synchronized L4 larval population; the NaOH / NaClO mixed lysis system includes primary water: 1 mol / L NaOH: 10% NaClO with a volume ratio of 1:1:1.

[0022] In some alternative embodiments of the present application, the synchronized Caenorhabditis elegans is divided into a group of sleeping nematode samples and a group of waking nematode samples, including:

[0023] Caenorhabditis elegans cultured under a constant temperature condition of 25 ± 0.5 °C for 48 - 52 h is confirmed by a stereomicroscope that more than 95% of the Caenorhabditis elegans individuals enter the L4 developmental stage. Subsequently, low-temperature induction at 4 °C for 10 min is used to promote the sedimentation of the worm body, and agarose gradient purification is carried out using M9 buffer containing 0.01% TritonX-100. A high-purity worm body precipitate is obtained by centrifugation at 3000×g for 3 min; after sampling, the survival rate is verified to be > 98% by trypan blue staining method, and the worm body concentration is adjusted to 300 ± 50 individuals / mL. Finally, a cryopreservation solution containing 15% DMSO is added for programmed cooling preservation to obtain nematodes in the sleeping stage.

[0024] Caenorhabditis elegans cultured under a constant temperature condition of 25 ± 0.5 °C for 68 - 72 h is processed in the same way as the nematodes in the sleeping stage to obtain nematodes in the waking stage.

[0025] In some alternative embodiments of the present application, the nematode metabolites in the group of sleeping nematode samples and the group of waking nematode samples are respectively extracted using a methanol-water mixed solvent, and an ultrasonic disruption method is used to improve the metabolite release efficiency, including:

[0026] A well-cultured nematode population is collected into a 15 ml centrifuge tube, centrifuged at 3000 rpm and 4 °C for 2 min. After discarding the supernatant, it is washed 3 times with M9 buffer pre-cooled to 5 °C to remove residual bacteria, and the residual liquid is removed after the last washing.

[0027] The nematode sample is quickly placed in liquid nitrogen and frozen for 5 min to lyse the cells and improve the metabolite release efficiency. After taking it out, 1 ml of a methanol-water mixed solvent with a volume ratio of 1:1 pre-cooled to 5 °C is added, and ultrasonic disruption is carried out under ice bath conditions to ensure sufficient lysis; after disruption, it is centrifuged at 12000 rpm and 4 °C for 10 min, and the supernatant is collected into a new 1.5 ml centrifuge tube, and the protein and particulate matter can be removed by filtration through a 0.22 μm filter membrane to obtain a clear metabolite extract.

[0028] In some alternative embodiments of the present application, the LC-MS / MS analysis is carried out using ultra-high performance liquid chromatography tandem Fourier transform mass spectrometry, including:

[0029] The metabolite extract is subjected to LC-MS / MS analysis using ultra-high performance liquid chromatography tandem Fourier transform mass spectrometry;

[0030] The liquid phase conditions are: Waters ACQUITY HSS T3 C18 chromatographic column (100 mm × 2.1 mm, 1.8 μm), mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is 0.1% formic acid acetonitrile, flow rate is 0.3 mL / min, gradient elution program is: 0.0 min, 95% of phase A, 5% of phase B; 2.0 min, 80% of phase A, 20% of phase B; 6.0 min, 50% of phase A, 50% of phase B; 10.0 min, 5% of phase A, 95% of phase B; 12.0 min, 5% of phase A, 95% of phase B; 14.0 min, 95% of phase A, 5% of phase B; end at 15.0 min, 95% of phase A, 5% of phase B; column temperature is set at 40 °C, injection volume is 5 μL, detection mode is tandem mass spectrometry, electrospray ionization source, positive and negative ion mode scanning;

[0031] Chromatographic conditions are as follows: 5 μL of the sample is separated by an HSS T3 100 mm × 2.1 mm i.d., 1.8 μm chromatographic column and then enters the mass spectrometry detection; mobile phase A is 95% water + 5% acetonitrile; mobile phase B is 47.5% acetonitrile + 47.5% isopropanol + 5% water; flow rate is 0.40 mL / min, column temperature is 40 °C;

[0032] Mass spectrometry conditions are as follows: The sample mass spectrometry signal acquisition adopts positive and negative ion scanning mode, and the mass scanning range is 70 - 1050 m / z; the sheath gas flow rate is: 50 psi, the auxiliary gas flow rate is 13 psi, the auxiliary gas heating temperature is 425 °C, the positive mode ion spray voltage is set at 3500 V, the negative mode ion spray voltage is set at -3500 V, the ion transfer tube temperature is 325 °C, and the normalized collision energy is 20 - 40 - 60 V cyclic collision energy. The resolution of the first-stage mass spectrometry is 60000, the resolution of the second-stage mass spectrometry is 7500, and data is collected in DDA mode.

[0033] Compared with the prior art, this application analyzes the sleep and wakefulness processes of Caenorhabditis elegans under the intervention of goat milk-derived sleep peptide through metabolomics technology, successfully screens out the key metabolites that regulate sleep and wakefulness, and reveals the core metabolic pathways involved. These pathways include linoleic acid metabolism, sulfur metabolism, histidine metabolism related to sleep, and purine metabolism, riboflavin metabolism, pantothenic acid and CoA biosynthesis related to wakefulness, etc. Purine metabolism is determined as the core pathway for sleep regulation, and key metabolites such as uric acid, GMP, IMP, and AMP are identified. This application not only provides metabolic evidence for the mechanism of action of sleep peptide, but also constructs a regulatory framework of metabolite - pathway - target, filling the gap in metabolic regulation in sleep research, providing a scientific basis for sleep intervention strategies based on metabolic regulation, and also laying a foundation for the future development of metabolic markers for sleep status. Description of the Drawings

[0034] Figure 1PCA result diagrams, OPLS-DA result diagrams, and permutation test analysis result diagrams of the falling asleep process and waking up process of the blank group and the drug administration group involved in the embodiments of the present application; wherein: Figure a1, Figure a2, and Figure a3 are respectively the PCA result diagram, OPLS-DA result diagram, and permutation test analysis diagram of the falling asleep and waking up processes of the blank group; Figure b1, Figure b2, and Figure b3 are respectively the PCA result diagram, OPLS-DA result diagram, and permutation test analysis diagram of the falling asleep process of the blank group and the F4 sample group; Figure c1, Figure c2, and Figure c3 are respectively the PCA result diagram, OPLS-DA result diagram, and permutation test analysis diagram of the waking up process of the blank group and the F4 sample group; Figure d1, Figure d2, and Figure d3 are respectively the PCA result diagram, OPLS-DA result diagram, and permutation test analysis diagram of the falling asleep and waking up processes of the F4 sample group;

[0035] Figure 2 Metabolite volcano diagrams of the falling asleep process and waking up process of the blank group and the drug administration group involved in the embodiments of the present application; a represents the differential metabolite situation of the blank group in comparing falling asleep and waking up, b represents the differential metabolite situation of the sample group after F4 administration compared with the blank group during the falling asleep process, c represents the differential metabolite situation of the sample group after F4 administration compared with the blank group during the waking up process, and d represents the differential metabolite situation of the sample group after F4 administration in comparing the falling asleep process and the waking up process;

[0036] Figure 3 Venn diagrams of differential metabolites of the blank group and the sample group during the falling asleep and waking up processes involved in the embodiments of the present application;

[0037] Figure 4 Cluster heat maps involved in the embodiments of the present application; a represents the heat map (Heatmap) of differential metabolites of the blank group and the sample group during the falling asleep process, and b represents the heat map (Heatmap) of differential metabolites of the blank group and the sample group during the waking up process;

[0038] Figure 5 Pathway enrichment analysis diagrams involved in the embodiments of the present application; a represents the metabolic pathway analysis diagram of activating the falling asleep of Caenorhabditis elegans after F4 monomer administration, b represents the metabolic pathway analysis diagram of activating the waking up of Caenorhabditis elegans after F4 monomer administration, and c represents the pathways significantly enriched during the process from falling asleep to waking up. Detailed implementation manners

[0039] It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0040] According to the elaboration of the background technology, several defects existing in the prior art can be clearly identified:

[0041] (1) Lack of systematic research: Currently, research on sleep-promoting food-derived peptides mainly focuses on bovine casein peptides, while there is relatively little research on specific sleep peptides derived from goat milk. The molecular mechanism of action and the regulatory mode of the sleep-wake metabolism pathway have not been clearly elucidated.

[0042] (2) Limitations of sleep metabolomics research: Current sleep metabolism research mainly relies on mammalian models such as mice or rats. These models are costly and have long experimental cycles, which is not conducive to high-throughput screening. It is difficult for these models to comprehensively analyze the metabolite changes of specific bioactive peptides during the sleep-wake transition.

[0043] (3) One-sidedness of nematode sleep research: Currently, nematode sleep research mainly focuses on neurotransmitters (such as GABA, glutamate, acetylcholine, etc.), while there is relatively little research on its global metabolic changes. Existing metabolomics research is mostly used in fields such as lifespan, aging, and nutritional metabolism, and the metabolomics analysis for the sleep-wake transition has not been perfected.

[0044] (4) Insufficiency of key metabolite screening methods: Currently, there is little research on systematically mining key metabolites for the sleep-to-wake transition using a method combining metabolomics and network pharmacology. The functional analysis and pathway verification of metabolites are still scattered, and a complete mechanism chain has not been constructed.

[0045] Based on this, an embodiment of the present application is directed to a sleep peptide for Caenorhabditis elegans and a detection method for differential metabolites of Caenorhabditis elegans during sleep-wake transition.

[0046] Among them, for the sleep peptide for Caenorhabditis elegans, the sleep peptide is the F4 monomer with the amino acid sequence shown in SEQ ID NO: 1, and its amino acid sequence is TQTPVVVPPFLQPEIM.

[0047] Among them, the detection method for differential metabolites of Caenorhabditis elegans during sleep-wake transition includes the following steps:

[0048] Step 1: Culture Caenorhabditis elegans using a target culture medium; based on the sleep peptide, establish a drug administration system for the cultured Caenorhabditis elegans and perform synchronization treatment; divide the synchronized Caenorhabditis elegans into a sleep nematode sample group and a wake nematode sample group.

[0049] The target culture medium is prepared through the following steps: Dissolve 17 g of agar powder, 2.5 g of tryptone, 3 g of sodium chloride, and 0.2 g of streptomycin in 1 L of ultrapure water to prepare a basal culture medium; Mix 1 mol / L CaCl2, MgSO4, and K2HPO4-KH2PO4 buffer solution according to a mixing ratio of 1:1:25, and perform high-pressure steam sterilization treatment together with the basal culture medium to obtain a sterilized culture medium; When the sterilized culture medium is placed in a clean environment and naturally cooled to 85 ± 5 °C, perform aseptic operation in a biosafety cabinet to obtain a molten culture medium; Aliquot the molten culture medium into pre-sterilized 60 mm petri dishes, and after the culture medium solidifies at room temperature, obtain the target culture medium.

[0050] The conditions of the high-pressure steam sterilization treatment are 121 °C for 20 min; The aseptic operation means: Sequentially inject 1 ml of sterilized CaCl2 solution, 1 ml of sterilized MgSO4 solution, 25 ml of phosphate buffer system with pH 6.0, and 1 ml of 5 mg / ml cholesterol ethanol solution sterilized through a 0.22 μm filter membrane, and mix well to obtain a molten culture medium.

[0051] Based on the sleep peptide, establish the dosing system for the cultured Caenorhabditis elegans, including: Inoculate the preserved uracil auxotrophic Escherichia coli OP50 strain onto a fresh LB agar plate by the zone streaking method, and after incubating at 37 °C for 24 - 48 hours, select a monoclonal colony with typical morphology and transfer it aseptically to a test tube containing 5 mL of sterile LB liquid medium; Then place it in a constant temperature shaking incubator and perform amplification culture under the conditions of 37 °C and 200 rpm. After 12 hours, monitor the bacterial liquid concentration, and terminate the culture when the optical density value reaches OD600 = 0.4. The live bacterial suspension obtained at this time can be directly used for the inoculation operation of the Caenorhabditis elegans NGM feeding plate; Configure the sheep milk sleep peptide F4 monomer into a 100 μg / ml solution with pure water to obtain a sample liquid; Mix the prepared Escherichia coli liquid and the sample liquid in a ratio of 9:1, and after mixing, evenly coat it on a 60 mm solid culture medium plate, and incubate at 20 °C in a laminar flow hood for 24 h for standby.

[0052] Among them, the NGM culture plate is obtained through the following steps: accurately weigh 17 g of agar powder, 2.5 g of tryptone, 3 g of sodium chloride, and 0.2 g of streptomycin, and dissolve them in 1 L of ultrapure water; then place the mixed solution together with 1 mol / L of CaCl2, MgSO4, and K2HPO4-KH2PO4 buffer solution in an autoclave and sterilize at 121 °C for 20 min. After sterilization, wait for the solution to cool naturally to 80-90 °C, and then add 1 ml of CaCl2, 1 ml of MgSO4, 25 ml of K2HPO4-KH2PO4 buffer solution, and 1 ml of 5 mg / ml cholesterol ethanol solution filtered through a 0.22 μm filter membrane in sequence in a laminar flow hood, and mix well. Finally, transfer the uniformly mixed culture medium solution to a pre-sterilized petri dish, wait for it to solidify, and store it refrigerated to obtain a solid nematode growth medium (NGM).

[0053] Among them, the Escherichia coli solution is obtained through the following steps: Strain culture and coating: Streak the strain of uracil-deficient Escherichia coli (E. coli) OP50 on a solid medium plate and culture for 1-2 days. Pick a single colony and transfer it to a sterilized 100 mL LB (Luria-Bertani) liquid medium. E. coli OP50 is cultured in a shaker at 37 °C and 200 r / min for 12 h. When OD600 = 0.4, it can be inoculated onto the NGM plate and cultured on the plate for 1 day. Observe the growth of the bacterial solution. If no contaminants are seen, it indicates that the bacterial solution preparation is successful and can be used to feed nematodes.

[0054] The synchronization treatment includes: rinsing the young adult population with pre-cooled M9 buffer solution and collecting it into a 1.5 mL sterile centrifuge tube to obtain a nematode suspension; freshly prepare a NaOH / NaClO mixed lysis system and mix it with the nematode suspension in an equal volume. After vortexing for 30 seconds, centrifuge at 3000×g for 60 seconds to remove adult tissue debris. Retain the precipitate and wash it twice with M9 buffer solution (3000×g, 1 min / time) to finally obtain high-purity eggs; take the embryo suspension enriched at the bottom, quantitatively spot 2 μL on the NGM plate covered with the OP50 bacterial lawn, and incubate at 25 °C for 48 hours to obtain a population of developmentally synchronized L4-stage larvae; the NaOH / NaClO mixed lysis system includes primary water: 1 mol / L NaOH: 10% NaClO with a volume ratio of 1:1:1.

[0055] The synchronized Caenorhabditis elegans is divided into a sleeping nematode sample group and a waking nematode sample group, including: Caenorhabditis elegans cultured under constant temperature conditions of 25±0.5°C for 48-52h, and it is confirmed through a stereomicroscope that more than 95% of the Caenorhabditis elegans individuals enter the L4 developmental stage. Subsequently, 10 min of low-temperature induction at 4°C is used to promote the sedimentation of the worms, and agarose gradient purification is carried out using M9 buffer containing 0.01% TritonX-100. A high-purity worm precipitate is obtained by centrifugation at 3000×g for 3 min; after sampling, the survival rate is verified to be >98% through trypan blue staining, and the worm concentration is adjusted to 300±50 individuals / mL. Finally, a cryopreservation solution containing 15% DMSO is added for programmed cooling preservation to obtain nematodes in the sleeping stage; Caenorhabditis elegans cultured under constant temperature conditions of 25±0.5°C for 68-72h is processed in the same way as the nematodes in the sleeping stage to obtain nematodes in the waking stage.

[0056] Step 2: Use a methanol-water mixed solvent to extract nematode metabolites from the sleeping nematode sample group and the waking nematode sample group respectively. After using the ultrasonic disruption method to improve the metabolite release efficiency; use ultra-high performance liquid chromatography tandem Fourier transform mass spectrometry for LC-MS / MS analysis.

[0057] The use of a methanol-water mixed solvent to extract nematode metabolites from the sleeping nematode sample group and the waking nematode sample group respectively, and using the ultrasonic disruption method to improve the metabolite release efficiency, includes: Collecting a well-cultured nematode population into a 15 ml centrifuge tube, centrifuging at 3000 rpm and 4°C for 2 min. After discarding the supernatant, washing 3 times with M9 buffer pre-cooled to 5°C to remove residual bacteria, and removing the residual liquid after the last wash; quickly placing the nematode sample in liquid nitrogen and freezing for 5 min to lyse the cells and improve the metabolite release efficiency. After taking it out, add 1 ml of methanol-water mixed solvent with a volume ratio of 1:1 pre-cooled to 5°C, and perform ultrasonic disruption under ice bath conditions to ensure sufficient lysis; after disruption, centrifuge at 12000 rpm and 4°C for 10 min, collect the supernatant into a new 1.5 ml centrifuge tube, and the protein and particulate matter can be removed by filtering through a 0.22 μm filter membrane to obtain a clear metabolite extract.

[0058] The use of ultra-high performance liquid chromatography tandem Fourier transform mass spectrometry for LC-MS / MS analysis includes: Using ultra-high performance liquid chromatography tandem Fourier transform mass spectrometry to perform LC-MS / MS analysis on the metabolite extract;

[0059] The liquid phase conditions are: Waters ACQUITY HSS T3 C18 chromatographic column (100 mm × 2.1 mm, 1.8 μm), mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is 0.1% formic acid acetonitrile, flow rate is 0.3 mL / min, gradient elution program is: 0.0 min, 95% of phase A, 5% of phase B; 2.0 min, 80% of phase A, 20% of phase B; 6.0 min, 50% of phase A, 50% of phase B; 10.0 min, 5% of phase A, 95% of phase B; 12.0 min, 5% of phase A, 95% of phase B; 14.0 min, 95% of phase A, 5% of phase B; end at 15.0 min, 95% of phase A, 5% of phase B; column temperature is set at 40 °C, injection volume is 5 μL, detection mode is tandem mass spectrometry, electrospray ionization source, positive and negative ion mode scanning;

[0060] The chromatographic conditions are as follows: 5 μL of the sample is separated by an HSS T3 100 mm × 2.1 mm i.d., 1.8 μm chromatographic column and then enters the mass spectrometry detection; mobile phase A is 95% water + 5% acetonitrile; mobile phase B is 47.5% acetonitrile + 47.5% isopropanol + 5% water; the flow rate is 0.40 mL / min, and the column temperature is 40 °C;

[0061] The mass spectrometry conditions are as follows: the sample mass spectrometry signal acquisition adopts positive and negative ion scanning mode, and the mass scanning range is 70 - 1050 m / z; the sheath gas flow rate is: 50 psi, the auxiliary gas flow rate is 13 psi, the auxiliary gas heating temperature is 425 °C, the positive mode ion spray voltage is set at 3500 V, the negative mode ion spray voltage is set at -3500 V, the ion transfer tube temperature is 325 °C, and the normalized collision energy is 20 - 40 - 60 V cyclic collision energy. The resolution of the first-stage mass spectrometry is 60000, and the resolution of the second-stage mass spectrometry is 7500. The data is collected in DDA mode.

[0062] It can be seen that compared with the prior art, in this application, through metabolomics technology, the sleep and awakening processes of Caenorhabditis elegans under the intervention of caprine milk-derived sleep peptide are analyzed, key metabolites regulating sleep and awakening are successfully screened out, and the core metabolic pathways involved are revealed. These pathways include linoleic acid metabolism, sulfur metabolism, histidine metabolism related to sleep, and purine metabolism, riboflavin metabolism, pantothenic acid and CoA biosynthesis related to awakening, etc. Purine metabolism is identified as the core pathway of sleep regulation, and key metabolites such as uric acid, GMP, IMP, and AMP are identified. This application not only provides metabolic evidence for the mechanism of action of sleep peptide, but also constructs a regulatory framework of metabolite - pathway - target, fills the gap in sleep research in terms of metabolic regulation, provides a scientific basis for sleep intervention strategies based on metabolic regulation, and also lays a foundation for the future development of metabolic markers of sleep state.

[0063] The following is a more detailed description of the technical solution of the present application in combination with specific embodiments, so as to facilitate those skilled in the art to understand the technical solution of the present application.

[0064] Example 1

[0065] 1 Culture method of Caenorhabditis elegans

[0066] 1.1 Preparation of nematode growth medium

[0067] First, accurately weigh 17 g of agar powder, 2.5 g of tryptone, 3 g of sodium chloride, and 0.2 g of streptomycin, and mix and dissolve them in 1 L of ultrapure water to prepare a basal medium. The prepared basal medium and the pre-prepared 1 mol / L CaCl2, MgSO4, and K2HPO4-KH2PO4 buffer solutions are jointly subjected to high-pressure steam sterilization (121 °C, 20 min). After the sterilization procedure is completed, when the medium is naturally cooled to 85 ± 5 °C in a clean environment, aseptic operation is carried out in a biosafety cabinet: 1 ml of sterilized CaCl2 solution, 1 ml of sterilized MgSO4 solution, 25 ml of phosphate buffer system (pH 6.0), and 1 ml of cholesterol ethanol solution (5 mg / ml) sterilized through a 0.22 μm filter membrane are injected in sequence. The molten medium is fully mixed and dispensed into pre-sterilized 60 mm petri dishes. After the medium solidifies at room temperature, it is transferred to a 4 °C refrigerated environment for storage and standby. During the whole operation process, attention should be paid to the aseptic operation specification, and all added reagents need to be sterilized in advance.

[0068] 1.2 Nematode drug administration route

[0069] The preserved uracil auxotrophic Escherichia coli OP50 strain is inoculated onto a fresh LB agar plate by the zone-streaking method. After culturing at 37 °C for 24 - 48 hours, select typical monoclonal colonies and transfer them to a test tube containing 5 mL of sterile LB liquid medium under aseptic operation. Subsequently, it is placed in a constant temperature shaking incubator (37 °C, 200 rpm) for amplification culture. After 12 hours, the bacterial liquid concentration is monitored, and the culture is terminated when the optical density value reaches OD600 = 0.4. The live bacterial suspension obtained at this time can be directly used for the inoculation operation of the nematode NGM feeding plate. The ovine milk sleep peptide F4 monomer is configured into a 100 μg / ml solution with pure water. The prepared Escherichia coli liquid is configured and mixed at a concentration of (Escherichia coli: sample liquid = 9:1) for standby. The prepared sample is evenly coated on a 60 mm solid medium plate and cultured in a laminar flow hood for 24 h for standby.

[0070] 1.3 Synchronization of nematodes

[0071] Rinse the young adult population with pre-chilled M9 buffer and collect it into a 1.5 mL sterile centrifuge tube. Prepare a freshly mixed NaOH / NaClO lysis system (V deionized water:V 1 mol / L NaOH:V 10%

[0072] NaClO = 1:1:1), mix it with the nematode suspension in equal volume, vortex for 30 seconds, then centrifuge at 3000×g for 60 seconds to remove adult tissue debris. Retain the precipitate and wash it twice with M9 buffer (3000×g, 1 min each time). Finally, obtain high-purity eggs. Take the embryo suspension enriched at the bottom, quantitatively spot 2 μL on an NGM plate covered with an OP50 bacterial lawn, and incubate at 25 °C for 48 hours to obtain a synchronized L4 larval population.

[0073] 1.4 Collection and sampling of nematodes to be tested

[0074] For nematodes cultured for 48 - 52 h under a constant temperature condition of 25 ± 0.5 °C, first confirm that more than 95% of the individuals have entered the L4 developmental stage (the gonad extends to half of the body length and the gonadal primordium is formed) through a stereomicroscope (Nikon SMZ25). Subsequently, induce sedimentation of the worms by low-temperature induction at 4 °C for 10 min, and perform agarose gradient purification using M9 buffer containing 0.01% TritonX-100. Obtain a high-purity worm precipitate by centrifugation at 3000×g for 3 min. After sampling, verify the survival rate > 98% by trypan blue staining, and adjust the worm concentration to 300 ± 50 individuals / mL (microscopic counting method). Finally, add a cryopreservation solution containing 15% DMSO and perform programmed cooling for preservation as nematodes in the sleeping stage to be tested. Similarly, collect nematodes at 68 - 72 h as nematodes in the waking stage to be tested.

[0075] 2 Extraction of nematode metabolites

[0076] Collect the well-cultured nematode population into a 15 ml centrifuge tube, centrifuge at 3000 rpm and 4 °C for 2 min. After discarding the supernatant, wash it 3 times with pre-chilled M9 buffer to remove residual bacteria, and try to remove the residual liquid as much as possible after the last wash. Subsequently, quickly place the nematode sample in liquid nitrogen and freeze it for 5 min to lyse the cells and improve the metabolite release efficiency. After taking it out, add 1 ml of pre-chilled methanol-water (1:1, v / v) mixed solvent, and perform ultrasonic fragmentation under ice bath conditions (200 W, working for 5 s, interval of 5 s, lasting for 10 min) to ensure sufficient lysis. After fragmentation, centrifuge at 12,000 rpm and 4 °C for 10 min, collect the supernatant into a new 1.5 ml centrifuge tube, and filter it through a 0.22 μm filter membrane to remove proteins and particulate matter to obtain a clear metabolite extract. The obtained extract can be directly used for LC-MS or GC-MS metabolomics analysis. If not detected immediately, store it at -80 °C and avoid repeated freezing and thawing.

[0077] 3 Nematode Metabolomics Detection Method

[0078] Perform LC-MS / MS analysis (HPLC-QExactive-Orbitrap) on the samples using the Thermo Fisher Scientific's ultra-high performance liquid chromatography tandem Fourier transform mass spectrometry UHPLC-QExactive HF-X system.

[0079] Liquid phase conditions: Waters ACQUITY HSS T3 C18 chromatographic column (100mm×2.1mm, 1.8μm), mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is 0.1% formic acid acetonitrile, flow rate is 0.3mL / min, gradient elution program is: 0.0min, 95% of phase A, 5% of phase B; 2.0min, 80% of phase A, 20% of phase B; 6.0min, 50% of phase A, 50% of phase B; 10.0min, 5% of phase A, 95% of phase B; 12.0min, 5% of phase A, 95% of phase B; 14.0min, 95% of phase A, 5% of phase B; end at 15.0min, 95% of phase A, 5% of phase B. The column temperature is set at 40°C, the injection volume is 5μL, the detection mode is tandem mass spectrometry (MS / MS), electrospray ionization source (ESI), and positive and negative ion mode scanning.

[0080] Chromatographic conditions: After 5μL of the sample is separated by the HSS T3 chromatographic column (100mm×2.1mm i.d., 1.8μm), it enters the mass spectrometry detection. Mobile phase A is 95% water + 5% acetonitrile (containing 0.1% formic acid), and mobile phase B is 47.5% acetonitrile + 47.5% isopropanol + 5% water (containing 0.1% formic acid). The flow rate is 0.40mL / min, and the column temperature is 40°C.

[0081] Mass spectrometry conditions: The sample mass spectrometry signal acquisition uses positive and negative ion scanning mode, and the mass scanning range is 70 - 1050m / z. The sheath gas flow rate is: 50psi, the auxiliary gas flow rate is 13psi, the auxiliary gas heating temperature is 425°C, the positive mode ion spray voltage is set at 3500V, the negative mode ion spray voltage is set at -3500V, the ion transfer tube temperature is 325°C, and the normalized collision energy is 20 - 40 - 60V cyclic collision energy. The resolution of the first-level mass spectrometry is 60000, the resolution of the second-level mass spectrometry is 7500, and data is collected using the DDA mode.

[0082] 4 Result Analysis

[0083] 4.1 Data Quality Inspection

[0084] Metabolomics is used to identify the relationships between physiological and pathological processes and to reveal the differential expression of metabolites in animal models. In this work, the blank group consisted of nematodes fed normally, the sample group consisted of nematodes fed with F4 monomers, CK54 was the group of nematodes fed normally and in the sleeping state, F454 was the group of nematodes fed with F4 monomers and in the sleeping state, CK72 was the group of nematodes fed normally and in the waking state, and F472 was the group of nematodes fed with F4 monomers and in the waking state. The experimental idea was to compare the metabolism in vivo of nematodes in the blank group with those in the sample group during the falling asleep and waking up processes, to find metabolites with significant differences during the falling asleep and waking up processes of nematodes based on the action of F4 monomers, and to summarize the mechanism of action of this monomer.

[0085] As Figure 1 shown, they are the PCA result diagrams, OPLS-DA result diagrams, and permutation test analysis result diagrams of the falling asleep and waking up processes of the blank group and the drug administration group; where:

[0086] Figure a1, Figure a2, and Figure a3 are respectively the PCA result diagram, OPLS-DA result diagram, and permutation test analysis diagram of the falling asleep and waking up processes of the blank group;

[0087] Figure b1, Figure b2, and Figure b3 are respectively the PCA result diagram, OPLS-DA result diagram, and permutation test analysis diagram of the falling asleep process of the blank group and the F4 sample group;

[0088] Figure c1, Figure c2, and Figure c3 are respectively the PCA result diagram, OPLS-DA result diagram, and permutation test analysis diagram of the waking up process of the blank group and the F4 sample group;

[0089] Figure d1, Figure d2, and Figure d3 are respectively the PCA result diagram, OPLS-DA result diagram, and permutation test analysis diagram of the falling asleep and waking up processes of the F4 sample group.

[0090] Based on the PCA result diagrams shown in Figure a1, Figure b1, Figure c1, and Figure d1, it can be seen that the samples of the sample group and the blank group are well aggregated in the positive and negative ion modes, which indicates that the instrumental method is stable. Therefore, the samples of the control group and the F4 drug administration group are better separated, and the individual samples within each group do not overlap, which means there are significant differences between the blank group and the sample group.

[0091] Based on the OPLS-DA result diagrams shown in Figure a2, Figure b2, Figure c2, and Figure d2, it can be seen that using supervised OPLS-DA in the positive and negative ion modes with stronger discrimination ability, the nematode samples of all groups are better aggregated, and the F4 sample group is completely separated from the blank group, with significant overall differences.

[0092] Based on the permutation test analysis diagrams shown in Figure a3, Figure b3, Figure c3, and Figure d3, it can be seen that when comparing the four groups, R2 is greater than 0 and Q2 is less than 0, indicating that the model is effective and can be analyzed.

[0093] 4.2 Component Distribution

[0094] In this application, a volcano plot is used to display the significance and fold change of differential metabolites. The horizontal axis of the volcano plot represents the log2 fold change (log2FC) of metabolites, and the vertical axis represents the negative logarithm P-value (-log10 P-value) of the significance level. In the figure, each point represents a metabolite, and its position and color are used to distinguish the change situation of metabolites. According to the preset threshold criteria, the screening conditions for significantly differential metabolites are: |log2FC| > X and P-value < Y (or FDR < Z). In the volcano plot, metabolites that meet the screening conditions are marked with different colors (e.g., red represents up-regulated metabolites, and blue represents down-regulated metabolites), while non-significant metabolites are marked in gray. In addition, the red / blue area above the horizontal axis represents the metabolites that are significantly up-regulated or down-regulated in metabolomics analysis. In this experiment, a comparison between the blank group and the drug-administered group during the falling asleep and waking up processes was designed to summarize the situation of differential metabolites.

[0095] As Figure 2 shown, it is the volcano plot of metabolites during the falling asleep and waking up processes of the blank group and the drug-administered group; Figure 2 Among them, a represents the situation of differential metabolites in the comparison of falling asleep and waking up in the blank group, b represents the situation of differential metabolites in the comparison of the sample group after F4 administration and the blank group during the falling asleep process, c represents the situation of differential metabolites in the comparison of the sample group after F4 administration and the blank group during the waking up process, and d represents the situation of differential metabolites in the comparison of the sample group after F4 administration during the falling asleep and waking up processes.

[0096] It can be seen that a total of 1,451 metabolites were detected in the comparison of sleep and wakefulness in the blank group, among which 117 were differential metabolites, 101 were significantly up-regulated metabolites, and 16 were significantly down-regulated metabolites, indicating that there were significant metabolic changes during the process of nematode falling asleep to waking up in the blank group. A total of 916 metabolites were detected in the comparison of the sample group after F4 administration and the blank group during the sleep process, among which 450 were differential metabolites, 384 were significantly up-regulated metabolites, and 66 were significantly down-regulated metabolites, indicating that there were significant metabolic changes during the sleep process of nematodes after administration. A total of 906 metabolites were detected in the comparison of the sample group after F4 administration and the blank group during the wakefulness process, among which 280 were differential metabolites, 158 were significantly up-regulated metabolites, and 122 were significantly down-regulated metabolites, indicating that there were significant metabolic changes during the wakefulness process of nematodes after administration. A total of 856 metabolites were detected in the comparison of sleep and wakefulness after F4 administration, among which 19 were differential metabolites, 3 were significantly up-regulated metabolites, and 16 were significantly down-regulated metabolites, indicating that there were no significant metabolic changes during the process of nematodes falling asleep to waking up with the same sleep time after administration. These differential metabolites may be related to the biological process of GABA sleep regulation, providing a basis for subsequent biological function research.

[0097] 4.3 Metabolite differences

[0098] This application aims to analyze the metabolite differences between the blank group and the drug group during the sleep process and the wakefulness process to identify the specific and common metabolites under different treatment conditions. The blank group was cultured normally, while the drug group was fed with F4 monomer. Samples were taken once during the sleep process and once during the wakefulness process for LC-MS analysis.

[0099] As Figure 3 shown, it is the Venn diagram of differential metabolites of the blank group and the sample group during the sleep and wakefulness processes; by comparing the metabolite data of the four groups, this application hopes to find the specific metabolites related to this stimulus and the differential metabolites during the sleep process and the wakefulness process under the action of F4.

[0100] It can be seen that the sleep and wakefulness process of the blank group (CK54VSCK72) contains 117 differential metabolites, the sleep and wakefulness process of the drug group (F454VSF472) contains 20 differential metabolites, the comparison between the blank group and the drug group during the sleep process (CK54VSF454) contains 207 metabolites, and the comparison between the blank group and the drug group during the wakefulness process (CK72VSF472) contains 224 differential metabolites. After summarization, among them, under the influence of F4, the differential metabolites during the sleep process of nematodes contain 77, and the differential metabolites during the wakefulness process contain 46, as shown in Table 1 - Table 2. Pathway enrichment analysis was performed on the differential metabolites in these two parts.

[0101] Table 1: Differential metabolites in the awakening process of Caenorhabditis elegans under the action of F4 identified by LC-MS

[0102]

[0103]

[0104]

[0105] Table 2: Differential metabolites in the falling asleep process of Caenorhabditis elegans under the action of F4 identified by LC-MS

[0106]

[0107]

[0108]

[0109] 4.4 Cluster analysis

[0110] In this application, the metabolite differences between the blank group and the drug group of nematodes during sleep and awakening processes were compared through metabolomics analysis. Metabolite data of nematode samples were obtained using LC-MS. Under the influence of F4, there were 77 differential metabolites in the sleep process of nematodes and 46 differential metabolites in the awakening process. After standardization processing, a cluster heat map as shown in Figure 4 was generated. Among them, Figure 4 a represents the heat map of differential metabolites in the falling asleep process of the blank group and the sample group (Heatmap), and b represents the heat map of differential metabolites in the awakening process of the blank group and the sample group (Heatmap). It can be seen from Figure 4 that there are significant differences in the metabolite expression patterns of nematodes during sleep and awakening processes after F4 administration.

[0111] In the heat map, the blank group samples are clustered together, and the drug-administered group samples are also clustered into one category, indicating that there are obvious distinctions in the metabolic characteristics between the two groups. Further analysis found that after F4 administration, during the sleep process of nematodes, a large class of neurotransmitters, fatty acids, hormones, etc. were metabolically up-regulated, including 6-hydroxy melatonin, D-arginine, glutamate, GABA, etc. This indicates that F4 may affect the sleep regulation mechanism through different pathways (such as neurotransmitter, hormone regulation, fatty acid metabolism, etc.), while the blank group maintains normal metabolism.

[0112] Further analyzing the differential metabolites during the awakening process, the present application found that metabolites such as TBP were upregulated, and neurotransmitters such as D-phenylalanine and Giminabant (CB1 receptor antagonist) were downregulated, suggesting that F4 may regulate the wakefulness process through different mechanisms, including aspects such as energy metabolism, neurotransmitter conduction, antioxidant effects, and cell signaling pathways. They may participate in or regulate physiological functions related to wakefulness, such as neural activity, metabolic balance, and immune response.

[0113] 4.5 Pathway analysis

[0114] Performing pathway enrichment analysis on these differential metabolites, the results are as Figure 5 shown, where Figure 5 in, a represents the metabolic pathway analysis diagram of activating the sleep of Caenorhabditis elegans after administering F4 monomer, b represents the metabolic pathway analysis diagram of activating the awakening of Caenorhabditis elegans after administering F4 monomer, and c represents the pathways significantly enriched during the sleep-to-awakening process.

[0115] As Figure 5 can be seen, the present application found that after administering F4, during the sleep process of Caenorhabditis elegans, sulfur metabolism, histidine metabolism, and linoleic acid metabolism were enriched. The upregulation of sulfur metabolism may enhance neuroprotective effects or affect the synthesis and activity of neurotransmitters, thereby promoting neural homeostasis during sleep. The upregulation of linoleic acid metabolism may affect the sensitivity of the nervous system through the regulatory effects of substances such as prostaglandins, and thus play a regulatory role during sleep. The upregulation of histidine metabolism indicates an increase in histamine synthesis. Histamine is a key raw material synthesized by nerve cells, and the regulation of cell repair during sleep increases. The enrichment results of these metabolic pathways indicate that the regulation of the sleep process of C. elegans by F4 may be achieved by regulating the metabolic pathways of the nervous system (such as lipid metabolism, amino acid metabolism, and sulfur metabolism), thereby affecting the regulation of the sleep-wake cycle. In particular, the metabolism of linoleic acid and histidine may regulate the activity of the nervous system by affecting neurotransmitters and the structural functions of cell membranes, and thus regulate the sleep process of C. elegans.

[0116] As Figure 5It can be seen that during the awakening process, the regulatory effect of F4 on the awakening process of nematodes is mainly reflected in a series of metabolite changes and the enrichment of related metabolic pathways. These results reveal that F4 regulates the awakening process of nematodes by affecting multiple key metabolic pathways (such as riboflavin metabolism, purine metabolism, pantothenic acid and coenzyme A synthesis, fatty acid degradation, etc.). By enhancing riboflavin metabolism, coenzyme A synthesis, and fatty acid degradation, F4 may help nematodes provide sufficient energy to support the awakening process, especially when the energy demand of cells increases. By regulating purine metabolism and the synthesis of deoxyinosine, F4 may promote cell repair and regeneration during the awakening process. By promoting the synthesis of energy intermediates such as adenylate and inosinate, F4 may help activate neurons and synthesize neurotransmitters during the awakening process, ensuring that the nervous system can effectively transition from the sleep state to the waking state. The downregulation of uric acid may be related to reducing oxidative stress and protecting the nervous system from excessive stimulation, helping nematodes maintain cellular homeostasis and nerve function during the awakening process.

[0117] In summary, F4 promotes the rapid and effective transition of nematodes from the sleep state to the waking state through the regulation of multiple metabolic pathways, including energy metabolism, cell repair, and nerve activity regulation. These metabolite changes and pathway enrichments reflect the important role of F4 in the awakening process, especially in aspects such as energy supply, cell metabolism, and neurotransmitter regulation.

[0118] Through pathway analysis, 12 key metabolites in the sleep and awakening processes of nematodes after F4 administration were summarized, as shown in Tables 3 - 4:

[0119] Table 3: Metabolites in the key metabolic pathways during the sleep induction process detected by LC-MS after F4 administration

[0120] NO. Metabolites Ret.Time(min) M / Z Formula VIP P FC Trend HMDB Scanmode 2 Sulfate 1.419 96.96033 H2O4S 1.06003 0.00067621 1872.88816 ↑ HMDB0001448 + 39 Linoleic Acid 5.873 279.23223 C18H32O2 1.06142 0.00151562 12.9678439 ↑ HMDB0000673 - 40 L-Histidine 0.767 154.06224 C6H9N3O2 1.03614 0.00013577 12.7077040 ↑ HMDB0000177 -

[0121] Table 4: Metabolites in the key metabolic pathways during the awakening process detected by LC-MS after F4 administration

[0122]

[0123] In summary, this application constructs a high-resolution mass spectrometry technology for determining key metabolites in the sleep and awakening processes of Caenorhabditis elegans; further analyzes the metabolic pathways related to sleep regulation, and successfully identifies the key metabolites of caprine sleep peptide in regulating nematode sleep and the key metabolites during the awakening process; in addition, this application also constructs a metabolic molecular regulatory mechanism for caprine sleep peptide to intervene in insomnia. Through an efficient, rapid, and low-cost method, this application deeply explores the metabolites in the sleep and awakening processes of nematodes, reveals the role of caprine sleep peptide as a key metabolite in regulating nematode sleep and awakening, and reports for the first time a sleep mechanism other than GABA, melatonin, and serotonin.

[0124] The above are only the preferred embodiments of the present application and are not intended to limit the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application shall be included within the protection scope of the present application.

Claims

1. A sleep peptide for Caenorhabditis elegans, characterized in that, The sleep peptide is the F4 monomer shown in SEQ ID NO:

1.

2. A method for detecting differential metabolites in the sleep-wake cycle of Caenorhabditis elegans, characterized in that, The method includes the following steps: Culturing Caenorhabditis elegans with a target culture medium; establishing a drug administration system for the cultured Caenorhabditis elegans based on the sleep peptide and performing synchronization treatment; dividing the synchronized Caenorhabditis elegans into a sleep nematode sample group and a waking nematode sample group; Using a methanol-water mixed solvent to extract nematode metabolites from the sleep nematode sample group and the waking nematode sample group respectively. After using the ultrasonic disruption method to improve the metabolite release efficiency, LC-MS / MS analysis is performed using ultra-high performance liquid chromatography tandem Fourier transform mass spectrometry.

3. The detection method according to claim 2, wherein The target culture medium is prepared through the following steps: Mixing 17 g of agar powder, 2.5 g of tryptone, 3 g of sodium chloride and 0.2 g of streptomycin and dissolving them in 1 L of ultrapure water to prepare a basal medium; Mixing 1 mol / L CaCl2, MgSO4 and K2HPO4-KH2PO4 buffer solution in a mixing ratio of 1:1:25, and performing high-pressure steam sterilization treatment together with the basal medium to obtain a sterilized culture medium; when the sterilized culture medium is placed in a clean environment and naturally cooled to 85 ± 5 °C, aseptic operation is carried out in a biosafety cabinet to obtain a molten culture medium; the molten culture medium is dispensed into pre-sterilized 60 mm petri dishes, and after the culture medium solidifies at room temperature, the target culture medium is obtained.

4. The detection method according to claim 3, wherein The conditions for the high-pressure steam sterilization treatment are treatment at 121 °C for 20 min; The aseptic operation refers to: sequentially injecting 1 ml of sterilized CaCl2 solution, 1 ml of sterilized MgSO4 solution, 25 ml of phosphate buffer system with pH 6.0, and 1 ml of 5 mg / ml cholesterol ethanol solution sterilized by a 0.22 μm filter membrane, and mixing well to obtain a molten culture medium.

5. The detection method according to claim 2, wherein Establishing the drug administration system for the cultured Caenorhabditis elegans based on the sleep peptide includes: Inoculating the preserved uracil auxotrophic Escherichia coli OP50 strain onto an LB agar plate by the zone streaking method. After culturing at 37 °C for 24-48 hours, selecting a monoclonal colony with typical morphology and transferring it to a test tube containing 5 mL of sterile LB liquid medium under aseptic operation; Subsequently, placing it in a constant temperature shaking incubator for amplification culture at 37 °C and 200 rpm. After 12 hours, monitoring the bacterial liquid concentration, and terminating the culture when the optical density value reaches OD600 = 0.

4. The obtained live bacterial suspension can be directly used for the inoculation operation of the Caenorhabditis elegans NGM feeding plate; configuring the sheep milk sleep peptide F4 monomer into a 100 μg / ml solution with pure water to obtain a sample liquid; mixing the prepared Escherichia coli liquid and the sample liquid in a ratio of 9:1, and evenly coating the mixture on a 60 mm solid culture medium plate, and culturing at 20 °C for 24 h in a laminar flow hood for standby.

6. The detection method according to claim 2, wherein Performing the synchronization treatment includes: Rinse the young adult population with pre-cooled M9 buffer and collect it into a 1.5 mL sterile centrifuge tube to obtain a nematode suspension. Immediately prepare a NaOH / NaClO mixed lysis system and mix it with the nematode suspension in an equal volume. After vortexing for 30 seconds, centrifuge at 3000×g for 60 seconds to remove adult tissue debris. Retain the precipitate, wash it with M9 buffer and centrifuge, repeating twice to obtain the bottom precipitate. Disperse the bottom precipitate with sterile water, quantitatively spot 2 μL onto an NGM plate covered with an OP50 lawn, and incubate at 25 °C for 48 hours to obtain a population of developmentally synchronized L4-stage larvae. The NaOH / NaClO mixed lysis system includes primary water: 1 mol / L NaOH: 10% NaClO with a volume ratio of 1:1:

1.

7. The detection method according to claim 2, wherein Dividing the synchronized Caenorhabditis elegans into a sleeping nematode sample group and a waking nematode sample group includes: For Caenorhabditis elegans cultured under constant temperature conditions of 25 ± 0.5 °C for 48 - 52 h, confirm that more than 95% of the Caenorhabditis elegans individuals enter the L4 developmental stage through a stereomicroscope. Subsequently, induce sedimentation of the worms by low-temperature induction at 4 °C for 10 min, and perform agarose gradient purification using M9 buffer containing 0.01% TritonX-100. Centrifuge at 3000×g for 3 min to obtain a high-purity worm precipitate. After sampling, verify that the survival rate > 98% by trypan blue staining, and adjust the worm concentration to 300 ± 50 individuals / mL. Finally, add a cryopreservation solution containing 15% DMSO and perform programmed cooling preservation to obtain nematodes in the sleeping stage. After treating Caenorhabditis elegans cultured under constant temperature conditions of 25 ± 0.5 °C for 68 - 72 h in the same manner as the nematodes in the sleeping stage, waking-stage nematodes are obtained.

8. The detection method according to claim 2, characterized in that, Using a methanol-water mixed solvent to extract nematode metabolites from the sleeping nematode sample group and the waking nematode sample group respectively, and adopting an ultrasonic disruption method to improve the metabolite release efficiency, includes: Collect a well-cultured nematode population into a 15 ml centrifuge tube, centrifuge at 3000 rpm and 4 °C for 2 min. After discarding the supernatant, wash it 3 times with M9 buffer pre-cooled to 5 °C to remove residual bacteria, and remove the residual liquid after the last wash. Quickly place the nematode sample in liquid nitrogen and freeze it for 5 min to lyse the cells and improve the metabolite release efficiency. After taking it out, add 1 ml of methanol-water mixed solvent pre-cooled to 5 °C with a volume ratio of 1:1, and perform ultrasonic disruption under ice bath conditions to ensure sufficient lysis. After disruption, centrifuge at 12000 rpm and 4 °C for 10 min, collect the supernatant into a new 1.5 ml centrifuge tube, and filter through a 0.22 μm filter membrane to remove proteins and particulate matter to obtain a clear metabolite extract.

9. The detection method according to claim 2, wherein Using ultra-high performance liquid chromatography tandem Fourier transform mass spectrometry for LC-MS / MS analysis, includes: Perform LC-MS / MS analysis on the metabolite extract using ultra-high performance liquid chromatography tandem Fourier transform mass spectrometry.

10. The detection method according to claim 9, characterized in that The liquid phase conditions, chromatographic conditions, and mass spectrometry conditions for the LC-MS / MS analysis are as follows: The liquid phase conditions were as follows: Waters ACQUITY HSS T3 C18 chromatographic column (100 mm × 2.1 mm, 1.8 μm), mobile phase A was 0.1% formic acid aqueous solution, mobile phase B was 0.1% formic acid acetonitrile, the flow rate was 0.3 mL / min, and the gradient elution program was: at 0.0 min, 95% of phase A and 5% of phase B; At 2.0 min, phase A is 80% and phase B is 20%. 6.0 min, phase A 50%, phase B 50%; 10.0 min, phase A 5%, phase B 95%; 12.0 min, phase A 5%, phase B 95%; 14.0 min, phase A 95%, phase B 5%; The reaction was completed after 15.0 min, with phase A at 95% and phase B at 5%. The column temperature was set at 40°C, the injection volume was 5 μL, and the detection mode was tandem mass spectrometry with an electrospray ionization source and positive and negative ion mode scanning. Chromatographic conditions were as follows: 5 μL of sample was separated on an HSS T3 100 mm × 2.1 mm ID, 1.8 μm column before mass spectrometry detection; mobile phase A was 95% water + 5% acetonitrile; mobile phase B was 47.5% acetonitrile + 47.5% isopropanol + 5% water; flow rate was 0.40 mL / min, and column temperature was 40°C. The mass spectrometry conditions were as follows: the sample mass spectrometry signal was acquired in positive and negative ion scanning mode with a mass scanning range of 70-1050 m / z; the sheath gas flow rate was 50 psi, the auxiliary gas flow rate was 13 psi, the auxiliary gas heating temperature was 425°C, the positive mode ion spray voltage was set to 3500 V, the negative mode ion spray voltage was set to -3500 V, the ion transfer tube temperature was 325°C, and the normalized collision energy was 20-40-60 V cyclic collision energy; the primary mass spectrometry resolution was 60,000, the secondary mass spectrometry resolution was 7500, and the data were collected in DDA mode.

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