Purple truffle exosome for improving athletic ability and intestinal flora composition of acute sleep deprivation mouse

Abstract

The invention discloses a perigord truffle exosome for improving the athletic ability and intestinal flora composition of an acute sleep deprivation mouse, and belongs to the field of biomedicine. According to the application, the perigord truffle exosome is used on a mouse acute sleep deprivation inducing model, and the application of the perigord truffle exosome in treating motor ability and intestinal injury after acute sleep deprivation is discussed. Mouse athletic ability, energy metabolism, inflammatory factors in serum, oxidative stress in liver, and Hamp in ileum tissue; e staining, expression of tight junction protein and determination of indexes such as intestinal flora and the like find that the perigord truffle exosome can effectively improve the athletic ability and the intestinal flora after acute sleep deprivation.

Description

Technical Field

[0001] The invention relates to black truffle exosomes improving the motor ability and intestinal flora composition of acute sleep deprivation mice, and belongs to the field of biomedicine. Background Art

[0002] Sleep is essential for cognitive and physical performance, and lack of sleep, known as sleep deprivation (SD), has become a major health problem worldwide. Long-term or severe sleep deprivation has multiple effects on physical and mental health, including: physical damage to the immune system, metabolic disorders, cardiovascular disease endocrine disorders, etc.; mental cognitive dysfunction, emotional problems, and risk of mental illness. Sleep deprivation is negatively correlated with the incidence of skeletal muscle morphology and motor ability by interfering with energy metabolism, immune processes, and endocrine signals. Microbial dysbiosis is an important factor in the pathogenesis of sleep disorders. Insomnia and the oxidative stress response induced by it lead to long-term energy consumption and malnutrition. At present, the main treatments for sleep deprivation include cognitive behavioral therapy, sleep hygiene education, taking melatonin and other sleep-aiding drugs, meditation and relaxation training, etc., but there are also problems such as drug dependence risks, insufficient diagnosis, and low acceptance of psychological intervention in the treatment. Therefore, it is urgent to strengthen the cognition and treatment of sleep deprivation.

[0003] Sleep deprivation can be divided into acute sleep deprivation and chronic sleep deprivation. Acute sleep deprivation usually lasts for several hours or even days; chronic sleep deprivation usually lasts for several hours a day for weeks or even months. Sleep deprivation is very common in work and daily life: sleep deprivation can occur when working night shifts, on missions, or overtime; the use of caffeinated beverages or physical or mental illnesses can also lead to sleep deprivation.

[0004] Sports events have extremely high requirements for the comprehensive qualities of athletes, especially in terms of brain alertness, physical reaction ability and cognitive decision-making ability. Athletes need to demonstrate good reaction ability and quick decision-making ability when facing sports. Studies have shown that athletes often suffer from acute sleep deprivation during training or before competition due to bad sleeping habits, high-intensity training, stress and environmental factors. Acute sleep deprivation may have a certain impact on athletes' cognitive decision-making ability and sports performance.

[0005] Black truffle (perigord truffle), also known as truffle and motherless fruit, is a wild edible fungus that grows underground. It has extremely high requirements for the growth environment and is extremely picky about sunlight, water or soil pH. Black truffles are mainly distributed in some areas of the Alps and the Himalayas, and are also distributed near Panzhihua, Sichuan Province. Black truffles are very expensive. They are known as the three major treasures in Europe and the United States along with caviar and foie gras. Black truffle exosomes are an extracellular vesicle in the development process of black truffle truffles, carrying a variety of substances. Black truffle exosomes are rich in crude protein, crude fat, crude fiber, vitamin B1, vitamin B2, vitamin C, vitamin PP, polysaccharides and other elements, and have extremely high nutritional value. It can be absorbed by cells through autocrine or paracrine pathways, and can also be absorbed by distant target tissues or organs through the circulatory system, participating in a variety of physiological and pathological processes in the body.

[0006] Black truffle exosomes are bioactive macromolecules and one of the important components of black truffles. They have no toxic side effects on biological cells. They contain a variety of nutrients such as black truffle polysaccharides. These nutrients have a variety of biological activities, such as antioxidant activity, hypoglycemic activity, anti-inflammatory activity, immunomodulation, and hypolipidemic activity. Studies have confirmed that the antioxidant capacity of truffle polysaccharides depends on the molecular weight, sulfation content, branching degree, and glycosidic bond type of polysaccharides, and its hypoglycemic activity is closely related to the molecular weight, glycosidic bond, higher structure, group, sulfation, carboxylmethylation, and acetylation of polysaccharides. Black truffles, also known as "black diamonds", are wild fungi that can be used as both medicine and food. They have a strong and unique aroma and are often used as raw materials for food and spices. Mature black truffles have high nutritional value and are rich in amino acids, vitamins, sterols, and trace elements. They can also promote human health through various ways such as enhancing immunity, benefiting the stomach, and inhibiting tumors. However, truffles of different varieties and different geographical environments have certain differences in structure and biological activity of their polysaccharide components, which affects their nutrition and biological activity.

[0007] The "gut-muscle-axis" hypothesis explores the interaction between the gut microbiota and skeletal muscle and its potential impact on muscle mass and function. This hypothesis is based on the observed correlation between changes in gut microbial composition and muscle mass, strength and endurance. Studies have shown that metabolites of gut microbes, especially short-chain fatty acids, can directly affect the metabolism and function of muscle cells, including reducing muscle atrophy, increasing muscle glycogen content and promoting fatty acid oxidation. In addition, specific intestinal bacterial species are closely related to maintaining muscle function, and exercise and nutritional interventions can affect muscle health by changing the composition and metabolic activity of gut microbes. With aging, sarcopenia, a condition in which muscle mass decreases, is associated with changes in the gut microbiota, suggesting that regulating the gut microbiota may help prevent and treat sarcopenia. The interaction between the gut microbiota and muscle may involve multiple mechanisms, including antioxidant effects, energy metabolism regulation, gene expression regulation, and hormone secretion. These findings provide new perspectives for understanding muscle physiology and pathology and provide the possibility of developing new strategies for the prevention and treatment of muscle-related diseases. Summary of the invention

[0008] Based on the "gut-muscle-axis" hypothesis, we hypothesized that FEs pretreatment helps the body establish systemic protection, that is, through a two-way communication system connecting the intestine and muscles. In this case, FEs can alleviate or even offset the decline in exercise capacity caused by acute SD. Therefore, this study has three main aspects:

[0009] (1) To verify the effect of FEs on the motor ability of acute SD mice.

[0010] (2) To investigate how FEs reshape intestinal microbial homeostasis and function in SD mice.

[0011] (3) To further explore the relationship between FEs and the exercise capacity, energy metabolism, and intestinal flora of acute SD mice, as well as its possible mediating mechanism between the “gut-muscle” axis.

[0012] The present invention provides a method for preparing black truffle exosomes, which are small vesicles composed of complex RNA and protein extracted from Yunnan black truffles. The protein, mRNA and miRNA components carried by the black truffle exosomes have the ability to transmit information and regulate cell functions and have rich biological activity; the method comprises the following steps:

[0013] (1) Mix black truffle and PBS buffer and homogenize;

[0014] (2) The homogenized mixture is separated by high-speed centrifugation, density gradient centrifugation or a combination of these methods to obtain black truffle-derived exosomes.

[0015] In one embodiment of the present invention, the steps specifically include:

[0016] (a) Wash the fresh truffle sample, cut it into small pieces, add pre-cooled PBS buffer and homogenize it.

[0017] (b) The treated liquid is transferred to a centrifuge tube, centrifuged for the first time, the supernatant is collected, and centrifuged for the second time. The supernatant is collected and transferred to a new centrifuge tube, and ultra-high-speed centrifugation is performed to precipitate the exosomes.

[0018] (c) Resuspension: Collect the precipitated exosomes and resuspend them in PBS buffer for storage.

[0019] In one embodiment of the present invention, in step (a), the mass of the small piece of black truffle is about 1 g.

[0020] In one embodiment of the present invention, in step (a), the volume ratio of the black truffle sample to the PBS buffer is 1:10.

[0021] In one embodiment of the present invention, in step (a), the homogenization speed is 30000-35000 rpm.

[0022] In one embodiment of the present invention, in step (b), the first centrifugation is performed at 2000-3000 rpm for 10 minutes.

[0023] In one embodiment of the present invention, in step (b), the second centrifugation is performed at 10000-12000 rpm for 30 minutes.

[0024] In one embodiment of the present invention, in step (b), the ultra-high speed centrifugation is performed at 100,000-120,000 rpm for 2 hours.

[0025] In one embodiment of the present invention, in step (c), the storage is storage in a refrigerator at -80 degrees Celsius.

[0026] The present invention provides an application of black truffle exosomes in preparing a product for preventing, improving and / or treating decreased motor ability and intestinal flora disorder caused by acute sleep deprivation.

[0027] The present invention provides an application of black truffle exosomes in preparing a product for resisting sleep deprivation damage.

[0028] In one embodiment of the present invention, the product includes but is not limited to one or more of medicines, health foods, health products, foods for special medical purposes, foods, functional foods, feeds, feed additives or pet foods; and the black truffle exosomes are derived from black truffles.

[0029] In one embodiment of the present invention, the drug can alleviate motor impairment and intestinal barrier damage caused by sleep deprivation.

[0030] In one embodiment of the present invention, the dosage form of the drug is a liquid preparation or a solid preparation.

[0031] In one embodiment of the present invention, the dosage form of the drug includes but is not limited to granules, capsules, tablets, pills or oral liquids.

[0032] In one embodiment of the present invention, the drug further comprises a pharmaceutically acceptable excipient.

[0033] In one embodiment of the present invention, the pharmaceutical excipients include: any one or more of solubilizers, emulsifiers, colorants, adhesives, disintegrants, fillers, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesive agents, integrities, pH regulators, buffers, plasticizers, defoamers, thickeners, humectants, filter aids, and release retardants;

[0034] In one embodiment of the present invention, the food is fermented food, special food, functional food.

[0035] In one embodiment of the present invention, the food includes but is not limited to cereal products, vegetable products, fruit products, fermented foods, meat products, egg products, dairy products, beverages, and snacks.

[0036] In one embodiment of the present invention, the black truffle exosomes are separated and extracted from black truffles, have a double-membrane vesicle structure, and the particle size is concentrated between 100-150nm.

[0037] In one embodiment of the present invention, the method for preparing black truffle exosomes is:

[0038] (1) Mix black truffle and PBS buffer and homogenize;

[0039] (2) The homogenized mixture is separated by high-speed centrifugation, density gradient centrifugation or a combination of these methods to obtain black truffle-derived exosomes.

[0040] The present invention also provides a product for preventing, improving and / or treating decreased motor ability and intestinal flora disorder caused by acute sleep deprivation, wherein the product contains exosomes derived from black truffles.

[0041] In one embodiment of the present invention, the product includes but is not limited to one or more of medicines, health foods, health products, foods for special medical purposes, foods, functional foods, feeds, feed additives or pet foods.

[0042] In one embodiment of the present invention, the drug prevents or treats acute kidney injury by repairing damage to renal tissue cells.

[0043] In one embodiment of the present invention, the dosage form of the drug is a liquid preparation or a solid preparation.

[0044] In one embodiment of the present invention, the dosage form of the drug includes but is not limited to granules, capsules, tablets, pills or oral liquids.

[0045] In one embodiment of the present invention, the drug further comprises a pharmaceutically acceptable excipient.

[0046] In one embodiment of the present invention, the pharmaceutical excipients include: any one or more of solubilizers, emulsifiers, colorants, adhesives, disintegrants, fillers, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesive agents, integrities, pH regulators, buffers, plasticizers, defoamers, thickeners, humectants, filter aids and release retardants.

[0047] In one embodiment of the present invention, the food is fermented food, special food, functional food.

[0048] In one embodiment of the present invention, the food includes but is not limited to cereal products, vegetable products, fruit products, fermented foods, meat products, egg products, dairy products, beverages, and snacks.

[0049] In one embodiment of the present invention, the black truffle exosomes are separated and extracted from black truffles, have a double-membrane vesicle structure, and the particle size is concentrated between 100-150nm.

[0050] In one embodiment of the present invention, the method for preparing black truffle exosomes is:

[0051] (1) Mix black truffles with PBS buffer and homogenize;

[0052] (2) The homogenized mixture is separated by high-speed centrifugation, density gradient centrifugation or a combination of these methods to obtain black truffle-derived exosomes.

[0053] The present invention also provides a use of black truffle exosomes in preparing a product having any one or more of the following effects:

[0054] (a) Alleviate and / or treat the decline in motor performance caused by acute sleep deprivation in individuals;

[0055] (b) alleviating and / or treating a decrease in blood glucose levels in an individual caused by acute sleep deprivation injury;

[0056] (c) improving energy metabolism, inflammation and oxidative stress levels in individuals affected by acute sleep deprivation;

[0057] (d) Alleviating and / or treating intestinal barrier damage caused by acute sleep deprivation in an individual.

[0058] In one embodiment of the present invention, the athletic ability includes but is not limited to grip strength and muscle endurance; the energy metabolism includes but is not limited to liver glycogen and muscle glycogen levels; the oxidative stress level includes but is not limited to SOD and glutathione (GSH) levels and MDA content levels in liver tissue; the inflammation level includes but is not limited to CRP levels in serum, inflammatory factor interleukin 10 levels, and TNF-α levels.

[0059] In one embodiment of the present invention, the product contains an effective dose of black truffle exosomes, and the product includes but is not limited to one or more of medicines, health foods, health products, foods for special medical purposes, foods, functional foods, dietary supplements, feeds, feed additives or pet foods; the black truffle exosomes are derived from black truffles;

[0060] In one embodiment of the present invention, the black truffle exosomes are separated and extracted from black truffles, have a double-membrane vesicle structure, and the particle size is concentrated between 100-150nm;

[0061] In one embodiment of the present invention, the method for preparing black truffle exosomes is:

[0062] (1) Mix black truffle and PBS buffer and homogenize;

[0063] (2) The homogenized mixture is separated by high-speed centrifugation, density gradient centrifugation or a combination of these methods to obtain black truffle-derived exosomes.

[0064] In one embodiment of the present invention, the drug prevents or treats acute kidney injury by repairing damage to renal tissue cells.

[0065] In one embodiment of the present invention, the dosage form of the drug is a liquid preparation or a solid preparation.

[0066] In one embodiment of the present invention, the dosage form of the drug includes but is not limited to granules, capsules, tablets, pills or oral liquids.

[0067] In one embodiment of the present invention, the drug further comprises a pharmaceutically acceptable excipient.

[0068] In one embodiment of the present invention, the pharmaceutical excipients include: any one or more of solubilizers, emulsifiers, colorants, adhesives, disintegrants, fillers, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesive agents, integrities, pH regulators, buffers, plasticizers, defoamers, thickeners, humectants, filter aids and release retardants.

[0069] In one embodiment of the present invention, the food is fermented food, special food, functional food.

[0070] In one embodiment of the present invention, the food includes but is not limited to cereal products, vegetable products, fruit products, fermented foods, meat products, egg products, dairy products, beverages, and snacks.

[0071] Beneficial Effects

[0072] 1. This invention uses animal experiments to explore the improving effect of black truffle exosomes on motor ability and intestinal biota in sleep deprivation, providing ideas for the further application of black truffle exosomes in anti-fatigue.

[0073] 2. The present invention uses black truffle exosomes on an acute sleep deprivation model induced in mice to explore the use of black truffle exosomes in the present invention for treating motor ability and intestinal damage after acute sleep deprivation. Through the determination of indicators such as mouse motor ability, energy metabolism, inflammatory factors in serum, oxidative stress in the liver, H&E staining of ileum tissue, expression of tight junction proteins, and intestinal flora, it was found that the black truffle exosomes can effectively improve motor ability and intestinal flora after acute sleep deprivation.

[0074] 3. The present invention provides a new treatment method for people who suffer from acute sleep deprivation due to bad sleeping habits, high intensity training, stress and environmental factors. That is, the use of the black truffle exosomes of the present invention can effectively improve motor ability and intestinal flora after acute sleep deprivation. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 : Effects of black truffle exosomes on the motor ability of acute sleep deprived mice; (A) is the forelimb grip strength of mice, and (B) is the motor coordination ability of mice in the rotating rod test.

[0076] Figure 2 : Effects of FEs on energy metabolism, oxidative stress balance and immune regulation of SD mice; (A)-(C) are blood glucose, liver glycogen and muscle glycogen contents, (D)-(F) are liver SOD, GSH and MDA contents, (G)-(I) are serum CRP and inflammatory factors (IL-10, TNF) contents, respectively.

[0077] Figure 3 : FEs protect the intestinal barrier by promoting the expression of tight junction proteins; (A) is the H&E staining and histopathological examination of ileum tissue, (B) is the villus height, crypt depth and the ratio of villus height to crypt depth of ileum tissue, and (C) is the mRNA expression levels of tight junction proteins ZO-1 and Occludin in ileum tissue.

[0078] Figure 4 : Effects of FEs on SD-related intestinal flora; where (A) is α diversity (Ace, Shannon, Chao index), (B) is β diversity of hierarchical clustering at the genus level, (C) is principal component analysis (PCA) at the OTU level, (D) is PCoA analysis at the genus level, (E) is the change of FEs of microbial flora at the genus level, and (F) is the relative abundance of microorganisms at the genus level.

[0079] Figure 5 : FEs regulate intestinal microbial functions and metabolic pathways; (A)-(B) are linear discriminant analysis effect size (LEfSe) analysis of microorganisms at the genus level, (C) is the phylogenetic tree and species relative abundance at the genus level, (D)-(E) are microbial network interaction diagrams of the SD+FEs group, and (F) is the Mantel correlation analysis between exercise performance (as well as antioxidant capacity and energy metabolism), inflammation-related biomarkers (CRP, IL-10, TNF-α and LPS) and intestinal microbiota composition. DETAILED DESCRIPTION

[0080] The sources of raw materials involved in the following embodiments are:

[0081] Black Truffle: Obtained from Hanlu Food Store, pure, natural black truffles from Yunnan that have not been processed;

[0082] Healthy adult male C57BL / 6 mice (20 g-40 g, 6-8 weeks) were purchased from Vitallife.

[0083] The detection methods involved in the following embodiments are:

[0084] Grip strength test

[0085] In this study, the grip strength test was conducted using a Panlab dynamometer (model LE902, provided by Hong Kong Youcheng Biotechnology Co., Ltd.) to assess the forelimb grip strength of each experimental mouse. The specific operation is: gently lift the mouse's tail, guide its front paws to grasp the metal wire of the dynamometer, and then slowly pull it back while ensuring that the tail is parallel to the experimental table until the mouse releases the wire. To ensure the accuracy of the data, each mouse was tested three times in a row, and the average score was processed for subsequent statistical analysis.

[0086] Muscle endurance test

[0087] First, the mice were trained to use the rotarod. During the training, the mice were placed on an accelerating rotarod (model ZB-200, manufactured by Chengdu Taimeng Technology Co., Ltd.). The speed of the device was gradually increased from 5 rpm to 15 rpm within 5 minutes, and the mice needed to maintain balance on the rotating rod. Formal testing can only be carried out after 3 training sessions. In the formal test, the mice were placed on a rotarod with a fixed speed of 15 rpm for a duration test until the mice could no longer maintain balance and fell. The time each mouse persisted on the rotarod was recorded, and this length of time was used as a key indicator to assess their motor coordination.

[0088] Hematoxylin and eosin (HE) staining

[0089] Ileal tissue samples were collected from mice sacrificed in each group (n = 3 per group) and stored in 4% paraformaldehyde (BSA) / PBS (v / v) solution for histological evaluation. Tissue samples were dehydrated with 70%, 80%, 90%, and 100% ethanol, then embedded in paraffin and cut into 4 μm sections. Hematoxylin and eosin (H&E) staining was then performed. Finally, digital images of ileal tissues were obtained using a digital panoramic scanner (provided by Jiangsu Zhiyue Medical Technology Co., Ltd., WS-10), and detailed analysis and processing were performed using CaseViewer v2.3 software.

[0090] Detection of biological markers in serum and liver

[0091] Fresh anticoagulated whole blood samples were collected from mouse eyeballs and centrifuged at 3500 rpm for 15 min at 4°C to separate serum from blood. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of inflammatory factors including interleukin 10 (IL-10), tumor necrosis factor (TNF-α), and C-reactive protein (CRP) in serum.

[0092] Then, the oxidative stress indicators in the liver were measured, including the content of superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde (MDA). The kits used in the experiment were operated according to the instructions provided by Xiamen Huijia Biotechnology Co., Ltd. and Beijing Solebow Technology Co., Ltd.

[0093] Ileal tissue mRNA extraction and qPCR detection

[0094] Mouse ileum RNA was isolated using TRIzol reagent. Reverse transcription was performed using the SuperRT cDNA synthesis kit. Quantitative analysis was performed using SYBR Green. Data were calculated using the comparative cycle threshold method (2-ΔΔct), and β-actin was used as the normalizer gene. All primers were synthesized by Thermo Fisher Scientific and the sizes were determined. Primers were designed using Primer 5 software. The specificity of the primers was tested using the blast program, and the PCR products were detected using agarose gel electrophoresis. The specific primers used for real-time quantitative polymerase chain reaction (qPCR) are shown in Table 1.

[0095] Total RNA from ileum tissue was extracted using the TRIzol method according to the instructions. Liquid nitrogen was placed in the collected fresh ileum tissue, ground into powder, 100 mg of tissue was mixed and dissolved with 1 mL of Trizol solution, 200 μL of chloroform was added and shaken, and the supernatant was centrifuged at 4°C. The white RNA precipitate was centrifuged again and washed twice with 75% ethanol and dissolved in DEPC (diethyl pyrocarbonate) water. Total RNA was reverse transcribed into cDNA using Super ScriptⅡ reverse transcriptase (Invitrogen, Carlsbad).

[0096] Real-time quantitative PCR analysis was performed using SYBR Green Supermix dye and specific primers of the gene to be tested. The reaction system was: 5 μL SYBR Green Supermix dye, 1 μL cDNA, 0.4 μL forward primer, 0.4 μL reverse primer, 3.2 μL dd water. The amplification program set for qPCR was: preheating at 50°C for 1 min, preheating at 95°C for 2 min, maintaining at 95°C for 5 s, maintaining at 60°C for 15 s, and reacting for 40 cycles. The change in mRNA expression of the target gene relative to the housekeeping gene was calculated according to the comparative cycle threshold method (2-ΔΔct).

[0097] Table 1 Specific primers used in q-PCR

[0098]

[0099] 16S rRNA analysis of intestinal microbiota

[0100] Microbial DNA was rapidly extracted from the colonic contents of mice in a sterile environment using a genomic DNA test kit (Omega Bio-Tek). However, 16S rRNA gene sequencing (V3-V4 region) detected only bacteria, which were amplified from the entire genome using a barcoded 341F / 806R primer pair (341F 5'-CCTAYGGGRBGCASCAG-3' and 806R, 5'-GGACTACHVGGGTWTCTAAT-3'). PCR conditions were set at 95°C for 5 minutes, followed by 95°C / 30s, 60°C / 30s, 75°C / 30s, and a final extension at 75°C for 10 minutes. All amplifications were purified using a DNA Gel / PCR Purification Mini prep kit (Biomiga, BW-DC3511-01, China), quantified using a QFEsnt-iT PicoGreen dsDNA As-say kit (LifeTechnologies, Carlsgood, USA), and then sequenced on an Illumina NovaSeq platform. Paired-end sequences were stitched based on direct overlap relationships of PE (Paired-end) reads, and bases with read quality values ​​≤ 20 were filtered using Flash (version 1.2.11). Subsequently, quality control was performed by filtering unqualified sequences with quality values ​​≤ 20. Bacteria with sequence similarity ≥ 0.97 were exclusively assigned an operational taxonomic unit (OTU) and clustered using USEARCH11-uparse based on the Unite database. .

[0101] Statistical data analysis

[0102] To ensure the accuracy and reliability of the data, all results in this study were expressed as mean ± standard error. To explore the differences among the groups, one-way analysis of variance was performed using SPSS24.0 software (SPSS, Chicago, IL, USA), and Dunnett's multiple comparison test was used to further determine the significant differences between the groups. In the statistical results, *p<0.05 and **p<0.01 indicated significant and extremely significant differences, respectively, compared with the Con group; and #p<0.05 and ##p<0.01 also indicated significant and extremely significant differences, respectively, compared with the SD group. In order to intuitively display the data and conduct in-depth analysis, we used the ggpubr and ggplot2 packages in R software (v4.1.0) and GraphPad Prism 8.1.0 to generate statistical analysis and boxplots. In addition, we used the Spearman correlation coefficient to perform correlation analysis on each factor to explore the degree of association between them.

[0103] At the same time, in order to more comprehensively understand the interactions and relationships between various factors, we used Cytoscape (v3.8.0) to build a network model. This model not only helps us intuitively understand the complex relationships between various factors, but also provides strong support for subsequent research.

[0104] Example 1: Extraction of black truffle exosomes

[0105] The specific steps are as follows:

[0106] 1. Preparation of black truffle exosomes

[0107] (1) Sample preparation:

[0108] Wash the fresh black truffle samples and cut them into small pieces weighing about 1 gram.

[0109] (2) Homogenization:

[0110] The black truffle sample obtained in step (1) is added to a pre-cooled PBS buffer solution (pH: 7.35-7.45), the mass volume ratio of the added black truffle sample to the PBS buffer solution is about 1:10, and the mixture is homogenized using a homogenizer.

[0111] (3) Centrifugation:

[0112] The homogenized liquid was transferred to a centrifuge tube and centrifuged at 3000 rpm for 10 minutes to remove cell debris. The supernatant was collected and centrifuged again at 10,000 rpm for 30 minutes to remove large particles. The supernatant from the previous centrifugation was collected again and transferred to a new centrifuge tube and ultracentrifuged at 100,000 rpm for 2 hours to precipitate exosomes.

[0113] (4) Resuspension:

[0114] The exosomes precipitated in step (3) were collected and resuspended in PBS buffer (pH 7.35-7.45), and stored at -20°C until subsequent experimental analysis.

[0115] 2. Characterization of black truffle exosomes

[0116] After the preparation is completed, the black truffle exosomes prepared in step 1 are subjected to NTA and TEM detection. The nanoparticle tracking detection data of black truffle exosomes are shown in Table 2, and the nano-projection electron microscopy detection image is shown in Figure 1 .

[0117] Table 2: NTA test results of black truffle exosomes

[0118]

[0119] The results showed that the black truffle exosomes extracted by ultracentrifugation had a clear double-membrane vesicle structure, with a particle size of approximately 100-150nm and an average particle size of 120.3nm, which met the typical identification characteristics of exosomes.

[0120] Example 2: Effects of black truffle exosomes on acute sleep deprivation in mice

[0121] Thirty-two healthy adult male C57BL / 6 mice (Wei Tong Li Hua) weighing 20g-40g were acclimated for one week under specific pathogen-free (SPF) conditions at the Experimental Animal Center of Jiangnan University (Wuxi, China). The experimental protocol was approved by the Ethics Committee of the Experimental Animal Center of Jiangnan University. The black truffle exosomes prepared in Example 1 were used to intervene in acute sleep deprivation of mice.

[0122] The specific steps are as follows:

[0123] In animal experiments, a dose of 20 mg of black truffle exosomes per kilogram of body weight is equivalent to an estimated human dose of about 120 mg per day, which is within the acceptable range of human equivalent doses.

[0124] The experimental mice were randomly divided into four groups (8 mice in each group). From the 1st to the 7th day of the experiment, the mice in each group were free to eat ordinary maintenance feed (ordinary feed purchased from Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd.) and drink water freely.

[0125] The specific groups are as follows:

[0126] Normally fed control group (Con group): From day 8 to day 14, mice were gavaged with normal saline at a dose of 0.1 mL / 10 g body weight every day and slept normally;

[0127] Model group (SD group): On days 8 to 12, mice were gavaged with normal saline at a dose of 0.1 mL / 10 g body weight every day and slept normally; on days 13 to 14, mice were sleep deprived for 48 h and gavaged with normal saline at a dose of 0.1 mL / 10 g body weight every day;

[0128] Caffeine-fed group (Pos group, caffeine was gavaged at a daily dose of 10 mg / kg body weight, purity ≥98%, purchased from Shanghai Chunyou Biological Co., Ltd., dissolved in physiological saline for later use): On days 8 to 12, mice were gavaged with caffeine solution at a dose of 0.1 mL / 10 g body weight (10 mg / kg) every day and slept normally; on days 13 to 14, mice were subjected to 48 h of sleep deprivation and gavaged with caffeine solution at a dose of 0.1 mL / 10 g body weight (10 mg / kg) every day;

[0129] The group fed with black truffle exosomes (SD+FEs group, the daily gavage dose of black truffle exosomes was 20 mg / kg body weight, dissolved in normal saline for later use): on days 8 to 12, the mice were gavaged with black truffle exosome solution (FELNs solution, black truffle exosomes were dissolved in normal saline according to the dosage requirements) at a dose of 0.1 mL / 10 g body weight (20 mg / kg) every day, and slept normally; on days 13 to 14, they were subjected to 48 hours of sleep deprivation and gavage with FELNs solution at a dose of 0.1 mL / 10 g body weight (20 mg / kg) every day.

[0130] The 48-hour sleep deprivation is to construct an acute SD model, as follows:

[0131] Mice were placed in an automatic sleep deprivation system (YAN-239, Shanghai Yuyan Scientific Instrument Co., Ltd.), and mice were free to take water and food. A stainless steel rod was firmly fixed in the center of each cage. The mice in the group that needed sleep deprivation were placed in the SD state for 4 hours before the experiment. Subsequently, a rotating metal rod with timed movement (5 rpm) was used to smoothly sweep across the bottom of the cage, thereby preventing the mice from falling asleep during the 48-hour SD period described above.

[0132] After the experiment, each mouse in each group was subjected to open field and Morris water maze tests, and finally all mice were killed and tissue samples were collected.

[0133] Example 3: Experimental results

[0134] The effects of the motor ability, body energy metabolism, inflammation level and oxidative stress level, and bacterial species of the intestinal flora of each group of mice after the experiment in Example 2 were tested, as follows:

[0135] 1. FEs improve the motor ability of acute SD mice

[0136] In order to evaluate the effect of black truffle exosomes on the motor ability of SD mice and other groups of mice, forelimb grip strength test and rotarod test were performed on SD mice and other groups of mice after the experiment in Example 2.

[0137] The results show:

[0138] Normally fed control group (Con group): The grip strength of mice was 124.24N, and the muscle endurance was 1010.6s;

[0139] Acute sleep deprivation model group (SD group): The grip strength of mice was 67.86N, and the muscle endurance was 445.2s;

[0140] The group fed with caffeine (Pos group): the grip strength of mice was 102.53N, and the muscle endurance was 796.5s;

[0141] The group fed with black truffle exosomes (SD+FEs group): the grip strength of mice was 106.67N, and the muscle endurance was 911.25s;

[0142] It can be seen that black truffle exosomes significantly improved the grip strength of mice ( Figure 1 A), indicating that black truffle exosomes enhanced muscle strength compared with the model group SD mice.

[0143] At the same time, in the rotarod test, the muscle endurance of SD+ black truffle exosome mice was significantly improved ( Figure 1 B in the figure). These behavioral results confirm that black truffle exosomes significantly improve the motor ability of SD mice. Since motor ability is related to the metabolic utilization of energy substrates in the body, we further evaluated the glucose metabolism, oxidative stress and inflammation levels of mice below.

[0144] 2. Black truffle exosomes FEs improve the body's energy metabolism, inflammation and oxidative stress levels

[0145] For individuals exposed to SD, intervention with black truffle exosomes can improve their energy metabolism, inflammation level and oxidative stress level. After the experiment, fresh anticoagulated whole blood samples were collected from the eyeballs of each group of mice after the experiment in Example 2, and centrifuged at 3500rpm for 15 minutes at 4°C to separate serum from the blood; blood glucose, liver glycogen in the liver, and muscle glycogen in muscle tissue were tested; SOD level, GSH level, MDA content in liver tissue, and CRP and inflammatory factors IL-10 and TNF-α levels in serum were tested.

[0146] The results show:

[0147] In the control group (Con group) fed normally, the blood glucose, liver glycogen and muscle glycogen levels of mice were 7.2mmol / L, 9.24mg / g and 0.35mg / g, respectively; the SOD, GSH levels and MDA content in liver tissue were 34.41nmol / mg prot, 228.7μg / mg prot and 0.25nmol / mg prot, respectively; the CRP and inflammatory factors IL-10 and TNF-α levels in serum were 166.11μg / L, 319.63pg / mL and 6.18ng / mL, respectively;

[0148] Model group (acute sleep deprivation model SD group): The blood glucose, liver glycogen and muscle glycogen levels of mice were 4.4mmol / L, 3.96mg / g and 0.25mg / g, respectively; the SOD, GSH levels and MDA content in liver tissue were 15.17nmol / mgprot, 173.86μg / mgprot and 0.74nmol / mgprot, respectively; the CRP and inflammatory factors IL-10 and TNF-α levels in serum were 219.19μg / L, 537.54pg / mL and 9.12ng / mL, respectively;

[0149] The group fed with caffeine (Pos group): the blood glucose, liver glycogen and muscle glycogen levels of mice were 5.84mmol / L, 6.37mg / g and 0.36mg / g respectively; the SOD, GSH levels and MDA content in liver tissue were 35.38nmol / mg prot, 231μg / mgprot and 0.44nmol / mg prot respectively; the CRP and inflammatory factors IL-10 and TNF-α levels in serum were 171.81μg / L, 410.39pg / mL and 7.12ng / mL respectively;

[0150] The group fed with black truffle exosomes (SD+FEs group): the blood glucose, liver glycogen and muscle glycogen levels of mice were 6.33mmol / L, 6.79mg / g and 0.4mg / g, respectively; the SOD, GSH levels and MDA content in liver tissue were 31.27nmol / mgprot, 227.62μg / mg prot and 0.51nmol / mg prot, respectively; the CRP and inflammatory factors IL-10 and TNF-α levels in serum were 184.9μg / L, 405.77pg / mL and 7.4ng / mL, respectively;

[0151] It can be seen that Figure 2 As shown in Figure 2, pretreatment with black truffle exosomes significantly increased blood glucose and liver glycogen and muscle glycogen levels in SD mice ( Figure 2AC). At the same time, black truffle exosomes maintained the oxidative stress balance in the SD mice of the model group by increasing the levels of SOD and GSH in liver tissue and reducing the content of MDA ( Figure 2 DF). In addition, SD significantly increased the inflammatory level of mice, while pretreatment with black truffle exosomes significantly reduced the levels of CRP and inflammatory factors (IL-10 and TNF-α) in serum ( Figure 2 in GI).

[0152] The above results indicate that preventive treatment with black truffle exosomes can regulate energy metabolism, intracellular redox balance, and systemic inflammation levels, which are essential for the homeostasis of the internal environment of SD mice.

[0153] 3. Black truffle exosomes FEs protect the intestinal barrier by promoting the expression of tight junction proteins

[0154] In order to evaluate the effect of FEs on the intestinal barrier, we performed H&E staining and qPCR on the ileum tissues of mice after the experiment in Example 2. After the experiment, the mice were killed, and the ileum tissues were taken for staining and qPCR.

[0155] The results show that Figure 3 ):

[0156] In the control group (Con group) fed normally, the villus length, crypt depth, and villus / crypt ratio of mice were 209.03, 106.94, and 1.98, respectively; the mRNA expression levels of ZO-1 and Occludin proteins in ileum tissue were 0.59 and 0.67, respectively;

[0157] Model group (SD group): the villus length, crypt depth, and villus / crypt ratio of mice were 145.87, 135.52, and 1.25, respectively; the mRNA expression levels of ZO-1 and Occludin proteins in ileum tissue were 0.26 and 0.37, respectively;

[0158] In the group fed with caffeine (Pos group), the villus length, crypt depth, and villus / crypt ratio of mice were 207.6, 94.32, and 1.96, respectively; the mRNA expression levels of ZO-1 and Occludin proteins in ileum tissue were 0.63 and 0.72, respectively;

[0159] The group fed with black truffle exosomes (SD+FEs group): the villus length, crypt depth, and villus / crypt ratio of mice were 198, 102.83, and 1.88, respectively; the mRNA expression levels of ZO-1 and Occludin proteins in ileum tissue were 0.72 and 0.8, respectively;

[0160] It can be seen that Figure 3As shown in the figure, the ileal mucosa of the Con group was intact and smooth, the epithelial cells were arranged in an orderly manner, and there was no inflammatory cell infiltration. In contrast, the model group SD group showed severe epithelial defects, crypt damage, and extensive inflammatory cell infiltration. The ileal mucosal damage in the SD+FEs group was milder, the inflammatory cell infiltration was reduced, and the crypt glands were more complete.

[0161] In addition, qPCR results showed that FEs significantly increased the expression of tight junction proteins ZO-1 and Occludin in ileum tissue, indicating that FEs may enhance intestinal barrier function by activating ZO-1 and Occludin proteins. FEs have a regulatory effect on the intestinal flora of acute SD mice.

[0162] 4. Black truffle exosomes FEs regulate bacterial species in the intestinal flora of mice

[0163] In order to evaluate the effect of FEs on the composition of intestinal flora, after the experiment, the intestinal flora community was analyzed by 16s rRNA amplicon sequencing of the colon contents obtained from each group of mice Con, model group SD and SD+FEs group after the experiment in Example 2.

[0164] The results show:

[0165] (1) Compared with the control group (Con group) fed with normal diet and the model group (SD group) fed with normal saline, FEs increased the α diversity (Ace and Chao index) of the microbiota ( Figure 4 A).

[0166] (2) Diversity results showed that the intestinal microbiota of the group fed with black truffle exosomes (SD+FEs group) may show microbial community characteristics comparable to those of the control group (Con group) fed with normal saline. Cluster forest analysis showed that there were significant differences between the Con and SD+FEs groups compared with the model group (SD group) fed with normal saline ( Figure 4 B).

[0167] (3) The Adonis test was used to evaluate the diversity of bacterial community structure. The experimental results in the principal component analysis (PCA) score diagram revealed that there was a significant difference in the OTU level between the control group (Con group) fed with normal feeding and the model group (SD group) fed with saline ( Figure 4 C).

[0168] (4) This study also conducted enterotype analysis at the genus level of the dominant microbial community. Figure 4 D) in order to group different samples with similar dominant microbial community structures according to the relative abundance of microbial groups.

[0169] (5) Through the above cluster analysis, the microbial groups with significant differences between the control group (Con group) fed with normal feeding, the group fed with black truffle exosomes (SD+FEs group) and the model group (SD group) fed with normal saline were identified as Lactobacillus enteric microbiota. From the results, it can be seen that black truffle exosomes optimized the intestinal flora structure of SD mice and made it different from other groups ( Figure 4 E).

[0170] (6) At the genus level, black truffle exosomes significantly increased the abundance of Alistipes, Bacteroides, and Muribaculum, while inhibiting harmful bacteria such as Ruminococcus and Lachnoclostridium ( Figure 4 F).

[0171] 5. Black truffle exosomes FEs regulate intestinal microbial functions and metabolic pathways

[0172] Example 2 After the experiment, the colon contents obtained from Con, model group SD and SD+FEs group were sequenced, and linear discriminant effect size (LEfSe) analysis was performed on Con, SD and SD+FEs groups from the phylum to the genus level ( Figure 5 AB in ); further study the role of key microbial groups and explore their interrelationships within the community ( Figure 5 AB in).

[0173] We performed a linear discriminant analysis (LDA) and found that the main representative microbial community branches of the SD+FEs group were: Rikenellaceae (LDA value = 4.85) and Bacteroides_acidifaciens (LDA value = 4.53).

[0174] To confirm the evolutionary relationship of the intestinal flora, a phylogenetic tree was generated at the genus level and the relative abundance of species at the genus level for each sample ( Figure 5 C in the figure). The results showed that FEs optimized the composition of intestinal flora, making it different from that of the SD group and the Con group. By establishing a network interaction diagram of the black truffle exosome SD+FEs group microorganisms, it was found that Actinobacteriota, Bacteroidota, Deferribacterota, Firmicutes, and Verrucomicrobiotas had a strong correlation ( Figure 5 DE in ).

[0175] According to Mantel correlation analysis, strong correlations were observed between inflammatory factors, exercise performance, oxidative stress, and energy metabolism ( Figure 5At the same time, systemic inflammation is also closely related to the composition of intestinal flora.

[0176] In conclusion, our findings suggest a reciprocal relationship between the gut-muscle axis and that the microbiota may represent a viable therapeutic target for enhancing motility and gut barrier function through administration of FEs.

[0177] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. Application of black truffle exosomes in the preparation of products to resist sleep deprivation damage.

2. The use according to claim 1, characterized in that: The product contains an effective dose of black truffle exosomes, and the product includes but is not limited to one or more of medicines, health foods, health products, foods for special medical purposes, foods, functional foods, dietary supplements, feeds, feed additives or pet foods; the black truffle exosomes are derived from black truffles.

3. The use according to claim 1 or 2, characterized in that: The drug can alleviate motor impairment and intestinal barrier damage caused by sleep deprivation; Preferably, the dosage form of the drug is a liquid preparation or a solid preparation; Preferably, the dosage form of the drug includes but is not limited to granules, capsules, tablets, pills or oral liquids; Preferably, the drug further comprises a pharmaceutically acceptable excipient; Preferably, the pharmaceutical excipients include: any one or more of solubilizers, emulsifiers, colorants, adhesives, disintegrants, fillers, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesive agents, integrities, pH regulators, buffers, plasticizers, defoamers, thickeners, humectants, filter aids and release retardants; Preferably, the food includes, but is not limited to, cereal products, vegetable products, fruit products, fermented foods, meat products, egg products, dairy products, drinks, and snacks; Preferably, the food also includes special dietary food; Preferably, the health care product further contains acceptable excipients.

4. The use according to any one of claims 1 to 3, characterized in that: The black truffle exosomes are separated and extracted from black truffles, have a double-membrane vesicle structure, and the particle size is concentrated between 100-150nm; Preferably, the method for preparing the black truffle exosomes is: (1) Mix black truffle and PBS buffer and homogenize; (2) The homogenized mixture is separated by high-speed centrifugation, density gradient centrifugation or a combination of these methods to obtain black truffle-derived exosomes.

5. Application of black truffle exosomes in the preparation of products having any one or more of the following effects: (a) Alleviate and / or treat the decline in motor performance caused by acute sleep deprivation in individuals; (b) alleviating and / or treating a decrease in blood glucose levels in an individual caused by acute sleep deprivation injury; (c) improving energy metabolism, inflammation and oxidative stress levels in individuals affected by acute sleep deprivation; (d) Alleviating and / or treating intestinal barrier damage caused by acute sleep deprivation in an individual.

6. The use according to claim 5, characterized in that: The athletic ability includes but is not limited to grip strength and muscle endurance; the energy metabolism includes but is not limited to liver glycogen and muscle glycogen levels; the oxidative stress level includes but is not limited to SOD, glutathione GSH levels and MDA content levels in liver tissue; the inflammation level includes but is not limited to CRP levels in serum, inflammatory factor interleukin 10 levels, and TNF-α levels.

7. The use according to claim 5 or 6, characterized in that: The product contains an effective dose of black truffle exosomes, and the product includes but is not limited to one or more of medicines, health foods, health products, foods for special medical purposes, foods, functional foods, dietary supplements, feeds, feed additives or pet foods; the black truffle exosomes are derived from black truffles; Preferably, the black truffle exosomes are separated and extracted from black truffles, have a double-membrane vesicle structure, and the particle size is concentrated between 100-150nm; Preferably, the method for preparing the black truffle exosomes is: (1) Mix black truffle and PBS buffer and homogenize; (2) The homogenized mixture is separated by high-speed centrifugation, density gradient centrifugation or a combination of these methods to obtain black truffle-derived exosomes.

8. A product for preventing, improving and / or treating decreased motor ability and intestinal flora disorder caused by acute sleep deprivation, characterized in that: The product contains an effective dose of black truffle exosomes.

9. The product according to claim 8, characterized in that The product includes, but is not limited to, one or more of a drug, a health food, a health product, a food for special medical purposes, a food, a functional food, a feed, a feed additive or a pet food; Preferably, the drug can alleviate motor impairment and intestinal barrier damage caused by sleep deprivation; Preferably, the dosage form of the drug is a liquid preparation or a solid preparation; Preferably, the dosage form of the drug includes but is not limited to granules, capsules, tablets, pills or oral liquids; Preferably, the drug further comprises a pharmaceutically acceptable excipient; Preferably, the pharmaceutical excipients include: any one or more of solubilizers, emulsifiers, colorants, adhesives, disintegrants, fillers, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesive agents, integrities, pH regulators, buffers, plasticizers, defoamers, thickeners, humectants, filter aids and release retardants; Preferably, the food is fermented food, special food or functional food.

10. The product according to claim 8 or 9, characterized in that The black truffle exosomes are separated and extracted from black truffles, have a double-membrane vesicle structure, and the particle size is concentrated between 100-150nm; Preferably, the method for preparing the black truffle exosomes is: (1) Mix black truffle and PBS buffer and homogenize; (2) The homogenized mixture is separated into black pine by one or more of high-speed centrifugation and density gradient centrifugation. Exosomes from exposure sources.

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