Application of roxburgh rose exosome in preparation of product for preventing and treating acute alcoholic liver injury

Abstract

The invention discloses application of roxburgh rose exosomes in preparation of products for preventing and treating acute alcoholic liver injury, relates to extraction of the roxburgh rose exosomes and new application of the roxburgh rose exosomes in prevention and treatment of the acute alcoholic liver injury, and belongs to the technical field of biology. The roxburgh rose exosome can also prevent and treat acute alcoholic liver injury. The particle size of the roxburgh rose exosome is 135.2 nm, the particle concentration is 1.3 * 10 < 11 > Particle / mL, and the total protein concentration is 2.12 mu g / mu L. A new thought is provided for research and development of drugs for preventing and treating acute alcoholic liver injury, the extraction method of the roxburgh rose exosome is simple, full development and utilization of food resources can be promoted, and a drug or a health care product for preventing and treating acute alcoholic liver injury is provided.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to new applications of prickly pear exosomes, specifically the application of prickly pear exosomes in the preparation of products for the prevention and treatment of acute alcoholic liver injury. Background Technology

[0002] Rosa Roxburghii Tratt (RRT) is a medicinal and edible plant that mainly grows in southwestern China. It is rich in functional components such as superoxide dismutase (SOD), vitamin C (VC), polysaccharides, and dietary fiber, and has good effects in anti-oxidation, anti-tumor, anti-aging and regulating intestinal flora imbalance.

[0003] Plant exosomes, in a narrow sense, refer to extracellular vesicles (EVs) of a specified size produced and secreted by plant cells. The main functions of plant exosomes are substance transport and signal transduction. They play a crucial role in plant growth, development, and immune defense. As an important line of defense against infection, plant exosomes play a vital role in the plant-pathogen interaction by transporting antimicrobial proteins and anti-infection siRNAs.

[0004] Currently, there are very few reports on research on prickly pear exosomes, especially on the prevention and treatment of acute alcoholic liver injury. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides the application of prickly pear exosomes in the preparation of products for the prevention and treatment of acute alcoholic liver injury, offering a new approach to the research and development of drugs for the prevention and treatment of acute alcoholic liver injury. The extraction method for prickly pear exosomes is simple, which can promote the full development and utilization of food resources, providing a medicine or health product for the prevention and treatment of acute alcoholic liver injury. To achieve the above objectives, this invention is implemented through the following technical solutions: The technical solution of the present invention: Application of prickly pear exosomes in the preparation of products for the prevention and treatment of acute alcoholic liver injury.

[0006] The aforementioned prickly pear exosomes had a particle size of 135.2 nm and a particle concentration of 1.3 × 10⁻⁶. 11 Particles / mL, total protein concentration was 2.12 μg / μL.

[0007] The aforementioned method for extracting prickly pear exosomes is performed according to the following steps: (1) Extraction: Take fresh prickly pear, cut it into pieces, add 1-3% of the total amount of prickly pear PBS buffer and place it in a blender to crush. Filter the juice with gauze, centrifuge the juice at 1000g for 25-35min and take the supernatant to obtain supernatant A; then centrifuge supernatant A at 3000g for 25-35min and take the supernatant to obtain supernatant B; finally, centrifuge supernatant B at 10000g for 25-35min and take the supernatant to obtain supernatant C; filter it with 70μm, 0.45μm and 0.22μm filter membranes in sequence, take the filtrate, centrifuge at 100000g for 50-70min, discard the supernatant, and resuspend the precipitate in PBS buffer. The volume ratio of the precipitate to PBS buffer is 50:0.5-1.5 to obtain the crude prickly pear exosome extract. (2) Purification: Dissolve sucrose in PBS buffer to prepare sucrose solutions with mass fractions of 8%, 30%, 45%, and 60%, and add them to centrifuge tubes in sequence. Then add crude extract of prickly pear exosomes and centrifuge at 100,000g for 50-70 minutes. The volume ratio of 8%, 30%, 45%, and 60% sucrose solutions to crude extract of prickly pear exosomes is 5-15:5-15:5-15:5-15:1. Take the interfacial band of the 30-45% sucrose solution layer to obtain prickly pear exosomes.

[0008] Specifically, the aforementioned method for extracting prickly pear exosomes is carried out according to the following steps: (1) Extraction: Take fresh prickly pear, cut it into pieces, add 2% of the total amount of prickly pear PBS buffer and place it in a blender to crush. Filter the juice with gauze, take the juice and centrifuge at 1000g for 30min to get the supernatant, get supernatant A; take supernatant A and centrifuge at 3000g for 30min to get the supernatant B; finally take supernatant B and centrifuge at 10000g for 30min to get the supernatant C; filter it with 70μm, 0.45μm and 0.22μm filter membranes in sequence, take the filtrate, centrifuge at 100000g for 60min, discard the supernatant, add PBS buffer to resuspend the precipitate, the volume ratio of the precipitate to PBS buffer is 50:1, and the crude extract of prickly pear exosomes is obtained. (2) Purification: Dissolve sucrose in PBS buffer to prepare sucrose solutions with mass fractions of 8%, 30%, 45%, and 60%, and add them to centrifuge tubes in sequence. Then add crude extract of prickly pear exosomes and centrifuge at 100,000g for 60 min. The volume ratio of 8%, 30%, 45%, 60% sucrose solution and crude extract of prickly pear exosomes is 10:10:10:10:1. Take the interfacial band of the 30-45% sucrose solution layer to obtain prickly pear exosomes.

[0009] The aforementioned products include pharmaceuticals or health products.

[0010] The aforementioned preparation method involves combining prickly pear exosomes with acceptable excipients from the drug, processing them according to conventional methods, and producing corresponding pharmaceutical preparations or health products.

[0011] The dosage forms of the aforementioned products include tablets, capsules, granules, decoctions, or pills.

[0012] A product for the prevention and treatment of acute alcoholic liver injury, wherein the raw material of the product contains the aforementioned prickly pear exosomes.

[0013] A product for the prevention and treatment of acute alcoholic liver injury, the product being mainly prepared from the aforementioned prickly pear exosomes.

[0014] The beneficial effects of this invention are 1. This invention provides the application of prickly pear exosomes in the preparation of products for the prevention and treatment of acute alcoholic liver injury. Experiments have demonstrated that prickly pear exosomes (hereinafter referred to as RRTENs) have the efficacy of preventing and treating acute alcoholic liver injury.

[0015] 2. Prickly pear exosomes can significantly reduce the levels of inflammatory cytokines IL-6, IL-1β, and IL-18 in liver tissue. They can also alleviate alcohol-induced oxidative stress imbalance in liver tissue by reducing the production of oxidative damage products and increasing the levels of antioxidant enzymes. Prickly pear exosomes can also improve alcohol-induced inflammatory responses by inhibiting the TLR4 / MyD88 / NF-κB signaling pathway. Attached Figure Description

[0016] Figure 1 Purification of prickly pear exosomes using sucrose gradient centrifugation; Figure 2 : BCA standard curve of prickly pear exosomes; Figure 3 Transmission electron microscopy image of prickly pear exosomes; Figure 4 Particle size distribution of prickly pear exosomes; Figure 5 : Positive ion mode (TIC) of total ion chromatogram of prickly pear exosomes (sample information in the upper right corner, P represents positive ion mode); Figure 6 : Negative ion mode (TIC) of total ion chromatogram of prickly pear exosomes (sample information in the upper right corner, N represents negative ion mode); Figure 7 The structural formulas of the top 20 components by peak area; Figure 8 : Expression level of aspartate aminotransferase (AST) in mouse liver; Figure 9 : Expression level of alanine aminotransferase (ALT) in mouse liver; Figure 10 HE staining of mouse liver tissue (200×); Figure 11 : Expression level of interleukin IL-1β in mouse liver; Figure 12 : Expression level of interleukin IL-6 in mouse liver; Figure 13 : Expression level of interleukin IL-18 in mouse liver; Figure 14 : MDA expression level in mouse liver; Figure 15 : Expression level of reactive oxygen species (ROS) in mouse liver; Figure 16 : Expression level of superoxide dismutase (SOD) in mouse liver; Figure 17 WB banding of the NF-κB / TLR4 / MyD88 signaling pathway in mouse liver; Figure 18 In vivo imaging of mice after oral administration of prickly pear exosomes at 0, 0.5, 1, 2, 6, 12, and 24 hours. Figure 19 Distribution of prickly pear exosomes in mice at 0, 0.5, 1, 2, 6, 12, and 24 hours after oral administration. Detailed Implementation

[0017] Example 1: Method for extracting exosomes from prickly pear (1) Extraction: Take fresh prickly pear, cut it into pieces, add 2% of the total amount of prickly pear PBS buffer and place it in a blender to crush. Filter the juice with gauze, take the juice and centrifuge at 1000g for 30min to get the supernatant, get supernatant A; take supernatant A and centrifuge at 3000g for 30min to get the supernatant B; finally take supernatant B and centrifuge at 10000g for 30min to get the supernatant C; filter it with 70μm, 0.45μm and 0.22μm filter membranes in sequence, take the filtrate, centrifuge at 100000g for 60min, discard the supernatant, add PBS buffer to resuspend the precipitate, the volume ratio of the precipitate to PBS buffer is 50:1, and the crude extract of prickly pear exosomes is obtained. (2) Purification: Dissolve sucrose in PBS buffer to prepare sucrose solutions with mass fractions of 8%, 30%, 45%, and 60%, and add them to centrifuge tubes in sequence. Then add crude extract of prickly pear exosomes and centrifuge at 100,000g for 60 min. The volume ratio of 8%, 30%, 45%, 60% sucrose solution and crude extract of prickly pear exosomes is 10:10:10:10:1. Take the interfacial band of the 30-45% sucrose solution layer to obtain prickly pear exosomes.

[0018] Example 2: Method for extracting exosomes from prickly pear (1) Extraction: Take fresh prickly pear, cut it into pieces, add 1% of the total amount of prickly pear PBS buffer and place it in a blender to crush. Filter the juice with gauze, centrifuge the juice at 1000g for 35min and take the supernatant to obtain supernatant A; then centrifuge supernatant A at 3000g for 35min and take the supernatant B; finally centrifuge supernatant B at 10000g for 25min and take the supernatant C; filter it with 70μm, 0.45μm and 0.22μm filter membranes in sequence, take the filtrate, centrifuge at 100000g for 70min, discard the supernatant, and resuspend the precipitate with PBS buffer. The volume ratio of the precipitate to PBS buffer is 50:0.5, which is the crude extract of prickly pear exosomes. (2) Purification: Dissolve sucrose in PBS buffer to prepare sucrose solutions with mass fractions of 8%, 30%, 45%, and 60%, and add them to centrifuge tubes in sequence. Then add crude extract of prickly pear exosomes and centrifuge at 100,000g for 50 min. The volume ratio of 8%, 30%, 45%, 60% sucrose solution and crude extract of prickly pear exosomes is 5:5:15:15:1. Take the interfacial band of the 30-45% sucrose solution layer to obtain prickly pear exosomes.

[0019] Example 3: Method for extracting exosomes from prickly pear (1) Extraction: Take fresh prickly pear, cut it into pieces, add 3% of the total amount of prickly pear PBS buffer and place it in a blender to crush. Filter the juice with gauze, centrifuge the juice at 1000g for 25min and take the supernatant to obtain supernatant A; then centrifuge supernatant A at 3000g for 25min and take the supernatant B; finally centrifuge supernatant B at 10000g for 35min and take the supernatant C; filter it with 70μm, 0.45μm and 0.22μm filter membranes in sequence, take the filtrate, centrifuge at 100000g for 50min, discard the supernatant, and resuspend the precipitate in PBS buffer. The volume ratio of the precipitate to PBS buffer is 50:1.5, which is the crude extract of prickly pear exosomes. (2) Purification: Dissolve sucrose in PBS buffer to prepare sucrose solutions with mass fractions of 8%, 30%, 45%, and 60%, and add them to centrifuge tubes in sequence. Then add crude extract of prickly pear exosomes and centrifuge at 100,000g for 70 min. The volume ratio of 8%, 30%, 45%, 60% sucrose solution and crude extract of prickly pear exosomes is 15:15:5:5:1. Take the interfacial band of the 30-45% sucrose solution layer to obtain prickly pear exosomes.

[0020] Example 4: Method for extracting exosomes from prickly pear (1) Extraction: Take fresh prickly pear, cut it into pieces, add 2% of the total amount of prickly pear PBS buffer and place it in a blender to crush. Filter the juice with gauze, centrifuge the juice at 1000g for 28min and take the supernatant to obtain supernatant A; then centrifuge supernatant A at 3000g for 33min and take the supernatant to obtain supernatant B; finally centrifuge supernatant B at 10000g for 26min and take the supernatant to obtain supernatant C; filter it with 70μm, 0.45μm and 0.22μm filter membranes in sequence, take the filtrate, centrifuge at 100000g for 55min, discard the supernatant, and resuspend the precipitate in PBS buffer. The volume ratio of the precipitate to PBS buffer is 50:0.8, which is the crude extract of prickly pear exosomes. (2) Purification: Dissolve sucrose in PBS buffer to prepare sucrose solutions with mass fractions of 8%, 30%, 45%, and 60%, and add them to centrifuge tubes in sequence. Then add crude extract of prickly pear exosomes and centrifuge at 100,000g for 50-70 minutes. The volume ratio of 8%, 30%, 45%, 60% sucrose solution and crude extract of prickly pear exosomes is 10:5:5:10:1. Take the interfacial band of the 30-45% sucrose solution layer to obtain prickly pear exosomes.

[0021] Example 5: Method for extracting exosomes from prickly pear (1) Extraction: Take fresh prickly pear, cut it into pieces, add 2.5% of the total amount of prickly pear PBS buffer and place it in a blender to crush. Filter the juice with gauze, take the juice and centrifuge at 1000g for 32min to get the supernatant, get supernatant A; take supernatant A and centrifuge at 3000g for 29min to get the supernatant B; finally take supernatant B and centrifuge at 10000g for 33min to get the supernatant C; filter it with 70μm, 0.45μm and 0.22μm filter membranes in sequence, take the filtrate, centrifuge at 100000g for 65min, discard the supernatant, add PBS buffer to resuspend the precipitate, the volume ratio of the precipitate to PBS buffer is 50:1.3, and the crude extract of prickly pear exosomes is obtained. (2) Purification: Dissolve sucrose in PBS buffer to prepare sucrose solutions with mass fractions of 8%, 30%, 45%, and 60%, and add them to centrifuge tubes in sequence. Then add crude extract of prickly pear exosomes and centrifuge at 100,000g for 50-70 minutes. The volume ratio of 8%, 30%, 45%, 60% sucrose solution and crude extract of prickly pear exosomes is 5:15:10:10:1. Take the interfacial band of the 30-45% sucrose solution layer to obtain prickly pear exosomes.

[0022] Example 6: Preparation of tablets Take 100g of prickly pear exosomes from Example 1, freeze-dry, add an appropriate amount of conventional excipients, mix well, make into granules, dry, compress into tablets, and make 1000 tablets (each tablet contains 100mg of prickly pear exosomes) to obtain tablets.

[0023] Treatment of symptoms: Prevention and treatment of acute alcoholic liver injury.

[0024] Instructions for use: Oral administration.

[0025] Dosage and administration: 1-2 tablets each time, twice a day.

[0026] Example 7: Preparation of Capsules Take 100g of prickly pear exosomes from Example 1, freeze-dry them, add a certain amount of sucrose powder and dextrin, granulate by dry method, sieve, fill into capsules, and make 1000 capsules (each capsule contains 100mg of prickly pear exosomes), thus obtaining the capsule formulation.

[0027] Treatment of symptoms: Prevention and treatment of acute alcoholic liver injury.

[0028] Instructions for use: Oral administration.

[0029] Dosage and administration: 1-2 capsules each time, twice a day.

[0030] Example 8: Preparation of Granules Take 100g of prickly pear exosomes from Example 1, freeze-dry them, add a certain amount of sucrose powder and dextrin, and dry-process them into granules. Package them in 5-10g / packet (each packet contains 100mg of prickly pear exosomes) to obtain granules.

[0031] Treatment of symptoms: Prevention and treatment of acute alcoholic liver injury.

[0032] Instructions for use: Oral administration.

[0033] Dosage and administration: 1-2 packets each time, twice a day.

[0034] Example 9: Preparation of decoction paste Take 100g of prickly pear exosomes from Example 1, add a certain amount of refined honey, and mix to prepare a decoction.

[0035] Treatment of symptoms: Prevention and treatment of acute alcoholic liver injury.

[0036] Instructions for use: Take orally or dissolved in boiling water.

[0037] Dosage and administration: 10-20g each time (each g contains 5mg of prickly pear exosomes), twice a day.

[0038] Example 10: Preparation of pills Take 100g of prickly pear exosomes from Example 1, freeze-dry them, and make them into pills with conventional excipients to make 1000 pills (each pill contains 100mg of prickly pear exosomes), thus obtaining the pills.

[0039] Treatment of symptoms: Prevention and treatment of acute alcoholic liver injury.

[0040] Instructions for use: Oral administration.

[0041] Dosage and administration: 1-2 pills each time, twice a day.

[0042] The inventors conducted numerous experiments. The following is a study on the specific implementation methods of preparing prickly pear exosomes for the prevention and treatment of acute alcoholic liver injury as described in this invention: 1. Extraction and purification of prickly pear exosomes Fresh prickly pear was cut into chunks, and the mixture was blended with an appropriate amount of PBS using a high-speed blender. The juice was filtered through gauze. The mixture was centrifuged at 1000g for 30 min, and the supernatant was collected. Then, it was centrifuged at 3000g for 30 min, and the supernatant was collected again. The mixture was then filtered sequentially at 70μm, 0.45μm, and 0.22μm. After centrifugation at 10000g for 60 min, the supernatant was discarded, and the precipitate was resuspended in an appropriate amount of PBS to obtain the crude prickly pear exosome extract. The crude extract was centrifuged at 10000g for 60 min in a sucrose gradient (8%, 30%, 45%, 60%). The volume ratio of the 8%, 30%, 45%, and 60% sucrose solutions to the crude prickly pear exosome extract was 10:10:10:10:1. The 30%-45% interfacial band was collected to obtain the prickly pear exosomes (see...). Figure 1 The total protein concentration of prickly pear exosomes was quantified using a BCA protein assay kit. The results are shown in Table 1 and 2. Figure 2 .

[0043] Table 1. Results of particle size, particle concentration, and total protein concentration of prickly pear exosomes. 2. Characterization of prickly pear exosomes Place a copper mesh on filter paper, add 10 μL of diluted prickly pear exosomes to the copper mesh, let stand for 10 min, add 1 drop of PBS, let it dry slightly, then add 4% paraformaldehyde solution and air dry. Observe the morphology of the prickly pear exosomes using a transmission electron microscope. Figure 3 The particle size and distribution of *Rhizopus rosacea* vesicles were determined using a nanoparticle size analyzer and a nanoparticle tracking analyzer. Figure 4 ).

[0044] The results showed that the morphology of *Rhizopus rosaceus* exosomes observed by transmission electron microscopy was uniform, with a distinct double-membrane structure on the outside. They exhibited saucer-like or hemispherical structures of varying sizes, consistent with the microscopic identification characteristics of exosomes. The average particle size of the obtained *Rhizopus rosaceus* exosomes was 135.2 nm, and the particle concentration was 1.3 × 10⁻⁶. 11 Particles / mL, total protein concentration 2.12 μg / μL.

[0045] UHPLC-Q-Exactive HF Analysis of Prickly Pear Exosome Components Take an appropriate amount of well-mixed prickly pear exosomes and place them in a 2 mL centrifuge tube. Add an appropriate amount of pure methanol and vortex for 10 min to mix the sample. Centrifuge the mixed liquid sample at 12000 rpm for 10 min at 4℃. Filter the supernatant through a 0.22 μm microporous membrane and add 100 μg / mL internal standard solution (2-chlorophenylalanine) to make its concentration 1 mg / L. Then, perform analysis.

[0046] Chromatographic conditions: column temperature 30 ℃; flow rate 0.3 mL / min; mobile phase A: 0.1% formic acid aqueous solution, B: pure acetonitrile; injection volume 2 μL, autosampler temperature 4℃.

[0047] Table 2 Mobile phase conditions Positive mode: Heater temperature 325 °C; Sheath flow rate: 45 arb; Auxiliary flow rate: 15 arb; Purge flow rate: 1 arb; Electrospray voltage: 3.5 kV; Capillary temperature: 330 °C; S-Lens RF Level: 55%.

[0048] Negative mode: heater temperature 325 °C; sheath flow rate: 45 arb; auxiliary flow rate: 15 arb; purge flow rate: 1 arb; electrospray voltage: 3.5 kV; capillary temperature: 330 °C; S-Lens RF Level: 55%.

[0049] Scanning modes: Full scan (m / z 100~1500) and data-dependent secondary mass spectrometry (dd-MS2, TopN = 5); Resolution: 120,000 (primary mass spectrometry) & 60,000 (secondary mass spectrometry). Collision mode: High-energy collision dissociation (HCD).

[0050] Compound Discoverer 3.3 was used for retention time correction, peak identification, and peak extraction to obtain the total ion chromatogram of prickly pear exosomes. Figure 5 and Figure 6Based on the secondary mass spectrometry information, the substances were identified using the Thermo mzCloud online database and the Thermo mzValut local database. The qualitative results of metabolites from the prickly pear exosome liquid sample (positive ion mode) are shown in Table 3. The structural formulas of the top 20 peak areas are shown in [Table 3]. Figure 7 : Table 3. Qualitative results of sample metabolites Serial Number English name Chinese name Molecular formula reserve Peak area Peak area 1 L-Ethionine L-ethionine <![CDATA[C6H 13 NO2S]]> 0.779 753986020.2 2.44% 2 Phenyl sulfoxide Phenyl sulfoxide <![CDATA[C 12 H 10 OS]]> 0.793 5622098169 18.17% 3 D-(−)-Quinic acid D-(−)-quinic acid <![CDATA[C7H 12 O6]]> 0.828 315308717.6 1.02% 4 Nicotinic acid niacin <![CDATA[C6H5NO2]]> 0.865 48263774.31 0.16% 5 7-Fluoro-3-methyl-1-benzothiophene 1,1-dioxide 7-Fluoro-3-methyl-1-benzothiophene 1,1-dioxide <![CDATA[C9H7FO2S]]> 0.874 567016959.4 1.83% 6 D-(+)-Pipecolinic acid D-piperacic acid <![CDATA[C6H 11 NO2]]> 0.877 273445512.3 0.88% 7 Methylenesuccinate Itaconic acid <![CDATA[C5H4O4]]> 1.09 447106341.6 1.45% 8 (Ammoniomethyl)phosphonate (aminomethyl)phosphonates <![CDATA[CH5NO3P]]> 1.091 187117466.3 0.61% 9 Erythorbic acid D-isoascorbic acid <![CDATA[C6H8O6]]> 1.093 2771003134 8.95% 10 Anilic Acid 2,5-Dihydroxy-p-benzoquinone <![CDATA[C6H4O4]]> 1.094 845393509.7 2.73% 11 Adenine adenine <![CDATA[C5H5N5]]> 1.111 119762388.8 0.39% 12 L-Leucine L-Leucine <![CDATA[C6H 13 NO2]]> 1.308 117949032.8 0.38% 13 2-Methyl-4-isothiazolin-3-one Methylisothiazolinone <![CDATA[C4H5NOS]]> 1.598 21134240.78 0.07% 14 L-Phenylalanine L-phenylalanine <![CDATA[C9H 11 NO2]]> 2.311 66253720.92 0.21% 15 4-Methoxybenzaldehyde p-Methoxybenzaldehyde <![CDATA[C8H8O2]]> 2.419 39633826.29 0.13% 16 2-Hydroxyquinoline 2-Hydroxyquinoline <![CDATA[C9H7NO]]> 2.622 35366487.41 0.11% 17 Cinnamaldehyde Cinnamaldehyde <![CDATA[C9H8O]]> 4.508 8274355.978 0.03% 18 4-Chlorophenylacetylene 4-Chlorophenylacetylene <![CDATA[C8H5Cl]]> 4.574 246538711 0.80% 19 Indole Indole <![CDATA[C8H7N]]> 4.583 35770498.71 0.12% 20 trans-3-Indoleacrylic acid Indole-3-acrylic acid <![CDATA[C 11 H9NO2]]> 4.629 187595778.1 0.61% 21 2-Naphthylamine 2-Naphthylamine <![CDATA[C 10 H9N]]> 4.633 10410548.15 0.03% 22 Caffeic acid caffeic acid <![CDATA[C9H8O4]]> 4.757 27783468.34 0.09% 23 2,4-Xylidine 2,4-Dimethylaniline <![CDATA[C8H 11 N]]> 4.786 183993193.4 0.60% 24 Coumarin Coumarin <![CDATA[C9H6O2]]> 5.148 5309275.337 0.02% 25 Phthaldialdehyde Phthalate <![CDATA[C8H6O2]]> 5.2 8421129.102 0.03% 26 5-Chloro-2-methyl-4-isothiazolin-3-one Isothiazolinone <![CDATA[C4H4ClNOS]]> 5.291 2692255.673 0.01% 27 Protocatechualdehyde Protocatechuic aldehyde <![CDATA[C7H6O3]]> 5.39 313252127 1.01% 28 Benzoic acid benzoic acid <![CDATA[C7H6O2]]> 5.391 65828370.44 0.21% 29 p-Coumaric acid p-hydroxycinnamic acid <![CDATA[C9H8O3]]> 5.408 87557233.47 0.28% 30 Isoferulic acid Isoflavonic acid <![CDATA[C 10 H 10 O4]]> 5.674 52313488.57 0.17% 31 3',4'-Dimethoxyacetophenone 3,4-Dimethoxyacetophenone <![CDATA[C 10 H 12 O3]]> 5.84 5602179.372 0.02% 32 2,6-Dimethyl-γ-pyrone 2,6-Dimethyl-4-pyranone <![CDATA[C7H8O2]]> 6.093 4786805.761 0.02% 33 7-Methoxy-4-methylcoumarin 7-Methoxy-4-methylcoumarin <![CDATA[C 11 H 10 O3]]> 6.16 3343811.796 0.01% 34 10-HDA 10-Hydroxy-2-decenoic acid <![CDATA[C 10 H 18 O3]]> 6.334 2738546.624 0.01% 35 Ethyl nicotinate Ethyl nicotinic acid <![CDATA[C8H9NO2]]> 6.624 7363697.903 0.02% 36 Dibenzylamine Dibenzylamine <![CDATA[C 14 H 15 N]]> 6.743 9463329.922 0.03% 37 Ligustilide ligustilide <![CDATA[C 12 H 14 O2]]> 6.803 115898765.6 0.38% 38 Perillene Perillaene <![CDATA[C 10 H 14 O]]> 6.876 15216692.82 0.05% 39 Heptanophenone Phenylopeptone <![CDATA[C 13 H 18 O]]> 7.054 38083029.22 0.12% 40 2-Adamantanone 2-Adamantane <![CDATA[C 10 H 14 O]]> 7.062 6722831.848 0.02% 41 2,4-Dimethylbenzaldehyde 2,4-Dimethylbenzaldehyde <![CDATA[C9H 10 O]]> 7.179 4040630.726 0.01% 42 Ferulaldehyde Coniferaldehyde <![CDATA[C 10 H 10 O3]]> 7.237 7684132.87 0.03% 43 4-Ethylbenzaldehyde 4-Ethylbenzaldehyde <![CDATA[C9H 10 O]]> 7.732 5150707.024 0.02% 44 Triethyl phosphate Triethyl phosphate <![CDATA[C6H 15 O4P]]> 7.81 15849093.07 0.05% 45 Valpromide valproamide <![CDATA[C8H 17 NO]]> 9.663 2095537.364 0.01% 46 5-Hydroxy-1-tetralone 5-Hydroxy-tetrahydronaphthone <![CDATA[C 10 H 10 O2]]> 10.171 3354263.724 0.01% 47 2,2,6,6-Tetramethyl-1-piperidinol (TEMPO) 2,2,6,6-Tetramethylpiperidine oxide <![CDATA[C9H 19 NO]]> 10.561 8493536.986 0.03% 48 Decanamide Decylamine <![CDATA[C 10 H 21 NO]]> 11.36 3546016.047 0.01% 49 6,7-Dihydroxy-4-methylcoumarin 4-Methylesculin <![CDATA[C 10 H8O4]]> 11.403 3182155.625 0.01% 4. Study on the protective effect and mechanism of prickly pear exosomes (RRTENs) against alcoholic liver injury in mice. 4.1 Laboratory Animals C57BL / 6J mice (10 weeks old), male, were purchased from Henan Scibes Biotechnology Co., Ltd. Approved by the Experimental Animal Ethics Review Committee of Guizhou University of Traditional Chinese Medicine (20250611007).

[0051] 4.2 Establishment of an in vivo model of acute alcoholic liver injury Thirty mice were randomly divided into six groups: control group (saline), alcohol group (56℃ Red Star Erguotou liquor), positive control group (biphenyl diester 28 mg / mL), low-dose treatment group (RRTENs-L 1 mg / mL), medium-dose treatment group (RRTENs-M 10 mg / mL), and high-dose treatment group (RRTENs-H 100 mg / mL). The positive control group and alcohol group were administered 0.1 mL / 10 g of 56℃ liquor by gavage every morning to induce an alcoholic liver injury model. The low, medium, and high-dose treatment groups were administered 0.1 mL / 10 g of liquor by gavage every afternoon for intervention treatment. The control group was administered the same dose of saline by gavage daily.

[0052] Determination of in vivo dosage of prickly pear exosomes: 100 mL of prickly pear exosomes were extracted from 5 kg of fresh prickly pear. The daily dosage of dried prickly pear for adults is 3-10 g. In this study, the safe dosage of fresh prickly pear was selected as 5 g per day for adults. Based on the conversion factor between mouse and human body weight (mouse (20 g): human (60 kg) = 1:9) and the yield of prickly pear exosomes, the maximum daily effective concentration for mice was calculated to be 100 mg / kg.

[0053] 4.3 Liver function index analysis Alcohol stimulates an increase in AST and ALT levels in the liver; treatment with biphenyl diester and RRTENs reduced this effect of alcohol (see...). Figure 8 , Figure 9 ).

[0054] 4.4 HE staining This study used HE staining to observe the morphology of mouse livers. Compared with the control group, the results showed... Figure 10The model group showed moderate abnormalities in liver tissue structure, with widespread severe edema of hepatocytes, loose cytoplasm, vacuolar degeneration of some cells, and dilation of hepatic sinusoids. A small number of inflammatory cells were visible within the lobules. In contrast, the liver tissue structure in the biphenyl diester and RRTENs treatment groups was basically normal, with a small number of hepatocytes showing mild edema and loose cytoplasm. No obvious inflammatory cell infiltration was observed in the tissue, and the degree of damage was significantly improved compared to the model group.

[0055] 4.5 Detection of inflammatory factors in liver tissue Figures 11-13 As can be seen, the levels of inflammatory cytokines IL-6, IL-1β, and IL-18 in liver tissue were detected by ELISA. The results showed that the levels of these cytokines were significantly upregulated in the model group compared to the control group. However, RRTENs treatment significantly reduced their levels.

[0056] 4.6 Detection of oxidative factors MDA, ROS, and SOD in liver tissue The pathogenesis of alcohol-induced liver injury is complex, but acetaldehyde and reactive oxygen species (ROS), the oxidative metabolites of ethanol, play important roles in the development and progression of the disease. Increased ROS induces oxidative stress. (See...) Figures 14-16 Compared to the control group, alcohol-induced liver injury significantly increased ROS levels in rat liver, while RRTENs dose-dependently reduced ROS levels in rat liver. Regarding the effects of RRTENs on SOD and MDA levels in the liver, compared to the control group, the model group showed significantly lower SOD levels in the liver, while RRTENs treatment alleviated liver damage to varying degrees. In particular, high-dose RRTENs treatment significantly increased SOD levels in the liver. Conversely, alcohol-induced liver injury significantly upregulated MDA compared to the control group. RRTENs-treated rats showed a clear trend of improvement compared to the model group. These results indicate that RRTENs can alleviate alcohol-induced oxidative stress imbalance in liver tissue by reducing the production of oxidative damage products and increasing the levels of antioxidant enzymes.

[0057] 4.7 Western blot detection of liver tissue proteins Alcohol stimulates a cascading inflammatory response, ultimately activating the NF-κB signaling pathway. (See...) Figure 17 Compared with the control group, the expression of TLR4, MyD88, phosphorylated NF-κB, and phosphorylated IkBα proteins was significantly increased in the liver of alcohol-injured rats, while RRTENs intervention significantly reduced the expression of these proteins. These results suggest that RRTENs may improve alcohol-induced inflammatory responses by inhibiting the TLR4 / MyD88 / NF-κB signaling pathway.

[0058] 4.8 In vivo imaging Cy7 was selected as the fluorescent dye to observe the in vivo distribution of RRTENs+Cy7 and free Cy7 after gavage. (See...) Figure 18 and Figure 19 The results showed that RRTENs+Cy7 remained in the liver for a longer time than free Cy7 dye over 24 hours.

Claims

1. Application of prickly pear exosomes in the preparation of products for the prevention and treatment of acute alcoholic liver injury.

2. The application according to claim 1, characterized in that: The prickly pear exosomes had a particle size of 135.2 nm and a particle concentration of 1.3 × 10⁻⁶. 11 Particles / mL, total protein concentration was 2.12 μg / μL.

3. The application according to claim 1 or 2, characterized in that: The extraction method for prickly pear exosomes is performed according to the following steps: (1) Extraction: Take fresh prickly pear, cut it into pieces, add 1-3% of the total amount of prickly pear PBS buffer and place it in a blender to crush. Filter the juice with gauze, centrifuge the juice at 1000g for 25-35min and take the supernatant to obtain supernatant A; then centrifuge supernatant A at 3000g for 25-35min and take the supernatant to obtain supernatant B; finally, centrifuge supernatant B at 10000g for 25-35min and take the supernatant to obtain supernatant C; filter it with 70μm, 0.45μm and 0.22μm filter membranes in sequence, take the filtrate, centrifuge at 100000g for 50-70min, discard the supernatant, and resuspend the precipitate in PBS buffer. The volume ratio of the precipitate to PBS buffer is 50:0.5-1.5 to obtain the crude prickly pear exosome extract. (2) Purification: Dissolve sucrose in PBS buffer to prepare sucrose solutions with mass fractions of 8%, 30%, 45%, and 60%, and add them to centrifuge tubes in sequence. Then add crude extract of prickly pear exosomes and centrifuge at 100,000g for 50-70 minutes. The volume ratio of 8%, 30%, 45%, and 60% sucrose solutions to crude extract of prickly pear exosomes is 5-15:5-15:5-15:5-15:

1. Take the interfacial band of the 30-45% sucrose solution layer to obtain prickly pear exosomes.

4. The application according to claim 3, characterized in that: The extraction method for prickly pear exosomes is performed according to the following steps: (1) Extraction: Take fresh prickly pear, cut it into pieces, add 2% of the total amount of prickly pear PBS buffer and place it in a blender to crush. Filter the juice with gauze, take the juice and centrifuge at 1000g for 30min to get the supernatant, get supernatant A; take supernatant A and centrifuge at 3000g for 30min to get the supernatant B; finally take supernatant B and centrifuge at 10000g for 30min to get the supernatant C; filter it with 70μm, 0.45μm and 0.22μm filter membranes in sequence, take the filtrate, centrifuge at 100000g for 60min, discard the supernatant, add PBS buffer to resuspend the precipitate, the volume ratio of the precipitate to PBS buffer is 50:1, and the crude extract of prickly pear exosomes is obtained. (2) Purification: Dissolve sucrose in PBS buffer to prepare sucrose solutions with mass fractions of 8%, 30%, 45%, and 60%, and add them to centrifuge tubes in sequence. Then add crude extract of prickly pear exosomes and centrifuge at 100,000g for 60 min. The volume ratio of 8%, 30%, 45%, 60% sucrose solution and crude extract of prickly pear exosomes is 10:10:10:10:

1. Take the interfacial band of the 30-45% sucrose solution layer to obtain prickly pear exosomes.

5. The application according to any one of claims 1-4, characterized in that: The products mentioned include pharmaceuticals or health products.

6. The application according to claim 5, characterized in that: The preparation method of the product is to combine prickly pear exosomes with acceptable excipients in the drug, process them according to conventional methods, and make them into corresponding drug preparations or health products.

7. The application according to claim 6, characterized in that: The dosage forms of the product include tablets, capsules, granules, decoctions, or pills.

8. A product for the prevention and treatment of acute alcoholic liver injury, characterized in that: The raw materials of the product contain prickly pear exosomes as described in claim 1 or 2.

9. A product for the prevention and treatment of acute alcoholic liver injury, characterized in that: The product is mainly prepared from prickly pear exosomes as described in claim 1 or 2.

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