Extracellular vesicles of phytobacterium plantarum and application of extracellular vesicles

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to an extracellular vesicle of phytobacterium plantarum and application of the extracellular vesicle. Specifically, the plant lactobacillus extracellular vesicles can promote HaCaT cells to generate ceramide, up-regulate expression of key genes in a skin ceramide synthesis route and recover a skin barrier; the expression of proinflammatory factors in a cell inflammation model can be reduced, and a relatively good anti-inflammatory effect is achieved. A mouse skin injury model verifies that the EVs has good skin infiltration capacity and can remarkably reduce the skin injury score, relieve skin itch, improve the skin water content and reduce expression of proinflammatory factors in skin tissue and expression of pruritus-related cell factors.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to an extracellular vesicle of Lactobacillus plantarum and an application thereof. Background Art

[0002] As the largest organ in the human body, the skin is the first line of defense against the external environment and has a vital barrier function. The skin barrier is primarily composed of physical, chemical, and microbial barriers. The physical barrier is primarily composed of the stratum corneum and intercellular lipids. The chemical barrier is composed of the acidic environment on the skin surface (pH approximately 4.5-6.0), natural moisturizing factor (NMF), and lipids. The microbial barrier is composed of the microbial community on the skin surface, including bacteria, fungi, and viruses. These microbial communities interact to form a dynamic and balanced ecosystem that inhibits the overgrowth of harmful microorganisms and maintains the skin's microecological balance. The integrity of the skin barrier is crucial for maintaining healthy skin. Once damaged, it can lead to problems such as dryness, sensitivity, inflammation, and even trigger allergic reactions or infections. Therefore, protecting the integrity of the skin barrier is key to maintaining skin health.

[0003] Ceramide is the main component of intercellular lipids in the skin's stratum corneum, accounting for about 40% to 50% of the total intercellular lipids. It plays a key role in maintaining the skin's barrier function, and has the functions of moisturizing, enhancing intercellular adhesion and repairing the barrier.

[0004] Bacterial extracellular vesicles (BEVs) are nanoscale, membrane-bound particles secreted by both Gram-negative and Gram-positive bacteria, typically ranging in diameter from 20 to 400 nm. BEVs possess a lipid bilayer structure and contain a variety of bioactive substances, such as proteins, nucleic acids, lipids, and metabolites. These extracellular vesicles (EVs) play important roles in interbacterial communication, host-microbe interactions, biofilm formation, virulence factor transmission, and antibiotic resistance gene transfer. Probiotic EVs, secreted by probiotics, share similar structures and functions to bacterial EVs. They can modulate host immune responses, suppress inflammatory responses, and play a crucial role in intestinal health, immune regulation, and disease prevention. For example, EVs from Lactobacillus rhamnosus can significantly reduce the production of proinflammatory cytokines TNF-α and IL-6. Extracellular vesicles (EVs) are nanoscale vesicles secreted by Lactobacillus plantarum. Studies have shown that EVs have significant potential in improving intestinal inflammation, regulating immune responses, and promoting skin health.

[0005] Currently, the preparation and application of bacterial extracellular vesicles have attracted attention. The existing technology mainly focuses on the preparation of extracellular vesicles and their potential efficacy in diseases such as cervical cancer and inflammatory diseases. These technical solutions provide an important technical basis for the present invention, but still need to be further optimized and expanded, especially for the analysis of the effective components of bacterial extracellular vesicles. At present, there is no research on extracellular vesicles of Lactobacillus plantarum and their application in repairing the skin barrier, nor on the analysis of the effective components of bacterial extracellular vesicles.

[0006] Previously published patents or patent applications have yet to report on how bacterial extracellular vesicles (EVs) increase skin ceramide levels by regulating multiple genes or molecular mechanisms involved in ceramide synthesis. Furthermore, the understanding of the active ingredients in EVs is limited, requiring further research. Therefore, EVs that can simultaneously repair the skin barrier, combat inflammation, alleviate itching, and possess rapid transdermal absorption have significant application value and practical significance. Summary of the Invention

[0007] The present invention aims to overcome the shortcomings of the prior art, provide a Lactobacillus plantarum vesicle and its application that can restore skin barrier function and have anti-inflammatory and anti-itching effects, and provide an analysis of the effective components in the Lactobacillus plantarum vesicle.

[0008] The first aspect of the present invention is to provide an extracellular vesicle of Lactobacillus plantarum, characterized in that the extracellular vesicle is derived from a strain of Lactobacillus plantarum ( Lactiplantibacillus plantarum Lactobacillus plantarum L25 is deposited at the General Microbiology Center of the China Culture Collection Administration under the deposit number CGMCC No. 35146 at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The average particle size of the extracellular vesicles of Lactobacillus plantarum is 130-140 nm; they exhibit a spherical morphology under transmission electron microscopy. Preferably, the particle size of the extracellular vesicles is 132 ± 2.24 nm.

[0009] Furthermore, the extracellular vesicles are prepared by the following method: (1) The strain is cultured in a liquid culture medium, and then the bacterial culture is centrifuged to remove the bacterial precipitate and obtain the supernatant of the bacterial culture; (2) Filter the supernatant using 0.45 μm and 0.22 μm bottle-top filters to remove residual bacteria and cell debris; (3) The eluted sample was then concentrated using a membrane cassette and filtered through a 0.22 μm filter. (4) The filtered sample was ultracentrifuged, the supernatant was removed, and the EVs precipitate derived from Lactobacillus plantarum was obtained and the precipitate was resuspended in PBS.

[0010] (5) Repeat ultracentrifugation as in step (4), and finally resuspend the precipitate with PBS. Aliquot and store at -80°C for future use or freeze-dry to obtain lyophilized powder for future use.

[0011] Preferably, the culture medium in step (1) is MR liquid culture medium; the culture time is 12 to 18 hours, the centrifugal speed is 5000 to 8000 g, and the centrifugation time is 5 to 20 minutes; Preferably, the ultracentrifugation in step (4) is 12000g to 15000g, and the centrifugation time is 1 to 2 hours.

[0012] The second aspect of the present invention is to provide a method for preparing extracellular vesicles of Lactobacillus plantarum, characterized in that the method comprises the following steps: (1) The strain is cultured in a liquid culture medium, and then the bacterial culture is centrifuged to remove the bacterial precipitate and obtain the supernatant of the bacterial culture; (2) Filter the supernatant using 0.45 μm and 0.22 μm bottle-top filters to remove residual bacteria and cell debris; (3) The eluted sample was then concentrated using a membrane cassette and filtered through a 0.22 μm filter. (4) The filtered sample was ultracentrifuged, the supernatant was removed, and the EVs precipitate derived from Lactobacillus plantarum was obtained and the precipitate was resuspended in PBS.

[0013] (5) Repeat ultracentrifugation as in step (4), and finally resuspend the precipitate with PBS, aliquot, and store at -80°C until use.

[0014] Preferably, the culture medium in step (1) is MRS liquid culture medium; the culture time is 12 to 18 hours, the centrifugal speed is 5000 to 8000 g, and the centrifugation time is 5 to 20 minutes; Preferably, the ultracentrifugation in step (4) is 12000g to 15000g, and the centrifugation time is 1 to 2 hours.

[0015] The third aspect of the present invention is to provide the use of the extracellular vesicles of Lactobacillus plantarum described in the first aspect in the preparation of a preparation for repairing the skin barrier, characterized in that the concentration of the extracellular vesicles in the preparation is 6 to 120 μg / mL, preferably 80 to 120 μg / mL; most preferably 100 μg / mL.

[0016] The fourth aspect of the present invention is to provide the use of the extracellular vesicles of Lactobacillus plantarum described in the first aspect in the preparation of a preparation for reducing skin inflammation, characterized in that the concentration of the extracellular vesicles in the preparation is 6 to 120 μg / mL, preferably 80 to 120 μg / mL; most preferably 100 μg / mL.

[0017] The fifth aspect of the present invention is to provide the use of the extracellular vesicles of Lactobacillus plantarum described in the first aspect in the preparation of a preparation for alleviating skin itching, characterized in that the concentration of the extracellular vesicles in the preparation is 6 to 120 μg / mL, preferably 80 to 120 μg / mL; most preferably 100 μg / mL.

[0018] The sixth aspect of the present invention is to provide the use of the extracellular vesicles of Lactobacillus plantarum described in the first aspect in the preparation of a skin rapid transdermal absorption carrier preparation, characterized in that the concentration of the extracellular vesicles in the preparation is 6 to 120 μg / mL, preferably 80 to 120 μg / mL; most preferably 100 μg / mL.

[0019] The seventh aspect of the present invention is to provide the use of trans-11-octadecenoic acid in preparing a preparation for repairing skin damage; Furthermore, the skin damage includes skin barrier damage, inflammation, and itching.

[0020] The beneficial effects of the present invention include: The extracellular vesicles of Lactobacillus plantarum of the present invention can promote the production of ceramide in HaCaT cells, upregulate the expression of key genes in the skin ceramide synthesis pathway, and restore the skin barrier; they can reduce the expression of proinflammatory factors in the cell inflammation model and have a good anti-inflammatory effect. It has been verified in the mouse skin injury model that EVs have good skin penetration ability, can significantly reduce the skin lesion score, relieve skin itching, increase skin water content, and reduce the expression of proinflammatory factors and itching-related cytokines in skin tissue. In summary, the extracellular vesicles of Lactobacillus plantarum are a multifunctional substance with good safety, biocompatibility and transdermal absorption ability, which can simultaneously play multiple functions such as repairing the skin barrier, anti-inflammatory, moisturizing, anti-sensitivity, and soothing.

[0021] The microorganism of the present invention is Lactobacillus plantarum ( Lactiplantibacillus plantarum ) L25, the plant lactobacillus L25 was deposited in the General Microbiology Center of the China Culture Collection Administration on July 9, 2025, with the deposit number CGMCC No. 35146, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1Figure 1. EVs precipitation sample.

[0023] Figure 2 Transmission electron microscopy images of EVs.

[0024] Figure 3 The particle size of EVs.

[0025] Figure 4 Fluorescent staining of Evs taken up by cells.

[0026] Figure 5 EVs enhance ceramide production in HaCaT cells.

[0027] Figure 6 EVs regulate the expression of key enzyme genes for ceramide synthesis in HaCaT cells.

[0028] Figure 7 EVs regulate the expression of skin barrier-related genes in an SDS-induced damaged cell model.

[0029] Figure 8 EVs regulate the expression of genes related to skin inflammation in an SDS-induced cell injury model.

[0030] Figure 9 Epigenetic image showing that EVs alleviate SDS-induced skin damage in mice.

[0031] Figure 10 Skin lesion scoring of mice.

[0032] Figure 11 Water content and transepidermal water loss in mice.

[0033] Figure 12 EVs reduce inflammatory gene expression in mouse skin tissue.

[0034] Figure 13 EVs reduce the expression of inflammatory proteins in mouse skin tissue.

[0035] Figure 14 EVs increase the expression of skin barrier-related genes in mice.

[0036] Figure 15 EVs inhibit the expression of itch-related genes in mouse skin.

[0037] Figure 16 Absorption and distribution of EVs in mouse skin.

[0038] Figure 17 Proteomic analysis of EVs.

[0039] Figure 18 Metabolomic analysis of EVs.

[0040] Figure 19 Trans-11-octadecenoic acid enhances ceramide production in HaCaT cells.

[0041] Figure 20 Trans-11-octadecenoic acid regulates the expression of skin barrier-related genes in an SDS-induced cell injury model.

[0042] Figure 21 Trans-11-octadecenoic acid regulates the expression of genes related to skin inflammation in an SDS-induced cell injury model. DETAILED DESCRIPTION

[0043] The following is a further description of the concept of the present invention and the technical effects produced in conjunction with specific embodiments, so as to fully understand the purpose, features and effects of the present invention. The methods described are all conventional methods unless otherwise specified. The materials described can be obtained from public commercial channels unless otherwise specified. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute undue limitations of the present invention. It should be noted that, unless there is a conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

[0044] Example 1 Isolation and Characterization of EVs from Lactobacillus plantarum 1. Isolation of EVs from Lactobacillus plantarum The Lactobacillus plantarum used was derived from a Lactobacillus plantarum strain screened and isolated from a kimchi sample by the Laboratory of Applied Microbiology and Enzyme Engineering, School of Bioengineering, Tianjin University of Science and Technology. The strain was cultured in MRS liquid medium at 37°C for 14 h, and then the bacterial culture was centrifuged at 6000 g for 10 min to remove the bacterial precipitate and obtain the supernatant of the bacterial culture. The supernatant was filtered using 0.45 μm and 0.22 μm bottle top filters to remove residual bacteria and cell debris. The eluted sample was then concentrated using a membrane package, and the concentrated sample was then filtered using a 0.22 μm filter. The filtered sample was ultracentrifuged at 140,000 g for 1.5 h, the supernatant was removed, and the EVs precipitate derived from Lactobacillus plantarum was obtained ( Figure 1 ) and resuspend the pellet in PBS. Repeat ultracentrifugation once using the above parameters. Finally, resuspend the pellet in PBS, aliquot, and store at -80°C until needed. Measure the protein concentration of EVs using the BCA assay.

[0045] 2. Characterization of EVs (1) Morphological observation Electron microscopy was used to observe the morphology of EVs isolated from bacterial cultures. EV samples were placed on 300-mesh copper grids and stained with 2% phosphotungstic acid for 12 h. Then, images were taken using a transmission electron microscope at an accelerating voltage of 100 kV to observe the morphological characteristics of EVs. Figure 2 As shown, EVs were observed to have a spherical shape by electron microscopy.

[0046] (2) Particle size distribution measurement Dynamic light scattering (DLS) was used to measure the size distribution of EVs isolated from bacterial cultures. The diameter of EVs was determined using a Zetasizer Nano S. Figure 3 As shown, the particle size of EVs was 132±2.24 nm.

[0047] (3) Concentration determination The concentration of EVs was determined by nanoparticle tracking analysis (NTA). The concentration of EVs was adjusted to 500 ng / mL, and 0.3 mL to 0.4 mL of sample was placed in the chamber of the LM-10HS instrument. The camera focus was adjusted to make the particles clearly visible, and the sample was captured by gradually lowering the level to confirm that the sample did not drift. The capture duration was set to 30 seconds to obtain the data. The experimental results showed that the concentration of EVs could reach 3.2×10 9 particles / mL.

[0048] Example 2: Uptake of EVs from Lactobacillus plantarum in HaCaT cells 1. Cell Culture HaCaT cells were cultured in DMEM high-glucose medium supplemented with 10% fetal bovine serum in a conventional incubator (37°C, 5% CO2). When the cells reached 80-90% confluency, adherent cells were trypsinized and passaged. The medium was changed 2-3 times per week.

[0049] 2. Fluorescent Labeling of EVs The isolated extracellular vesicles (EVs) of Lactobacillus plantarum were incubated with DIO dye at room temperature for 20 minutes in the dark to label the EVs. After incubation, the EVs were washed three times with PBS to remove unbound dye.

[0050] 3. Laser Confocal Microscopy Observation When the confluence of HaCaT cells reached 80% to 90%, the cell suspension was obtained using the above-mentioned cell passage method, and the cell density was adjusted to 2 × 10 5 / mL. The cell suspension was added to a 24-well plate with a cell slide placed in advance, and 1000 μL of cell suspension was added to each well. The cells were then cultured in an incubator (37°C, 5% CO2) for 12 hours. When the cell plating rate in the well plate met the experimental requirements, all the old culture medium in the 24-well plate was aspirated and replaced with serum-free culture medium. EVs (30 μg / mL) labeled with DIO in advance were added to the experimental group and incubated for another 12 hours, protecting the cells from light. The cell culture medium in all wells was then discarded, and the fixative was added to fix the cells for 10 minutes. The cells were then rinsed three times with PBS, and DAPI dye was added to stain the cell nuclei. After 5 minutes, the cells were rinsed three times with PBS. Finally, a small amount of PBS was added to prevent the cells from drying out. The cells were observed and photographed using a laser confocal microscope.

[0051] The results are as follows Figure 4 As shown in the figure, EVs labeled with DIO dye exhibit green fluorescence, and DAPI dye stains the cell nucleus with blue fluorescence. It can be clearly seen that EVs can penetrate into the surrounding cells, indicating that EVs can be taken up by cells.

[0052] Example 3: EVs derived from Lactobacillus plantarum promote ceramide production in HaCaT cells The cell culture method was similar to that in Example 2. When the cell confluence reached 80% to 90%, the adherent cells were digested with trypsin and the cell density was adjusted to 2 × 10 5 The cell suspension was added to a 24-well plate, with 1000 μL of cell suspension added to each well. The plate was then incubated in an incubator (37°C, 5% CO2) for 12 h. When the cell plating rate in the well plate met the experimental requirements, the old culture medium was discarded and serum-free culture medium was added. Three concentration gradients of EVs were set up in the experimental group: 30 μg / mL, 12 μg / mL, and 6 μg / mL, respectively. 75 μL of EVs of the corresponding concentration was added to each well. The same volume of Bacillus cereus fermentation supernatant was used as the positive control group, and PBS was used as the negative control group. Three replicates were set up in each group, and the cells were incubated for 24 h. After the incubation period, the adherent cells were scraped off with a cell scraper and collected by centrifugation at 1000 g for 3 min. The cells were then tested by ELISA.

[0053] 1. Preparation before testing (1) Take the kit out of the refrigerator 20 minutes in advance to equilibrate to room temperature; (2) Dilute the 30-fold concentrated washing solution 30-fold with distilled water and set aside.

[0054] 2. Operation steps (1) Take out the panels required for the test from the sealed bag that has been equilibrated to room temperature; (2) Set up standard wells, blank wells, and sample wells, and add 50 μL of standard of different concentrations to each standard well; (3) Add 50 μL of the sample to be tested to the sample well, and do not add anything to the blank well; (4) Set up blank wells (blank control wells do not contain samples and enzyme-labeled reagents, and the rest of the steps are the same) and test sample wells. First add 40 μL of sample diluent to the test sample wells on the enzyme-labeled plate, and then add 10 μL of the test sample (the final sample dilution is 5 times). Add the sample to the bottom of the well of the enzyme-labeled plate, trying not to touch the well wall, and gently shake to mix. Seal the reaction wells with a sealing film and incubate at 37°C for 30 min; (5) Discard the liquid, pat dry on absorbent paper, and fill each well with washing solution (350 μL); let it stand for 30 seconds, shake off the washing solution, pat dry on absorbent paper, and repeat this process 5 times; (6) Add 50 μL of substrate enzyme-labeled reagent to each well, except for the blank well. Incubate at 37°C in the dark for 15 min. (7) Washing: same operation as in step 5; (8) Color development: First add 50 μL of color developer A to each well, then add 50 μL of color developer B, gently shake to mix, and develop color at 37°C in the dark for 15 min; (9) Add 50 μL of stop solution to each well and measure the OD value of each well at a wavelength of 450 nm within 15 minutes.

[0055] 3. Result judgment The OD value of the blank well should be subtracted from the OD value of each standard and sample. The software plots the best-fit curve, with the standard concentration as the horizontal axis and the OD value as the vertical axis. The concentration of the sample can be found on the standard curve using the OD value of the sample.

[0056] 4. Calculation of Ceramide Content Ceramide standards were prepared into standard solutions and serially diluted to a series of known concentrations. OD values ​​at 450 nm (OD 450) were measured using the ELISA method described above. A regression equation was constructed using a standard curve. The OD 450 values ​​of each test sample were then substituted into the equation to calculate the ceramide content in each test sample. Three replicate wells were tested in each group, and the mean and standard deviation (SD) were calculated. A one-way ANOVA was performed using SPSS statistical software to compare the ceramide content of each sample group with that of the blank control group. Statistical significance was considered to be p < 0.05.

[0057] The results are as follows Figure 5 As shown in the results, EVs derived from Lactobacillus plantarum significantly promoted the production of ceramide in HaCaT cells, with a statistically significant difference compared to the blank control group (p < 0.05). Furthermore, the effect of EVs in promoting ceramide production was comparable to that of the positive control group.

[0058] Example 4: EVs derived from Lactobacillus plantarum upregulate the expression of key genes in the ceramide synthesis pathway in HaCaT cells For cell culture conditions and operation methods, please refer to Example 2. When the cell plating rate in the 6-well plate meets the experimental requirements, the old culture medium is discarded. Three concentration gradients were set in the experimental group, namely 30 μg / mL, 12 μg / mL, and 6 μg / mL EVs, and 100 μL of EVs of the corresponding concentration was added to each well. The same volume of PBS was used as a blank control group. Each group was set up with 3 replicate wells and cultured for 24 hours. After the culture was completed, the adherent cells were scraped with a cell scraper, centrifuged at 1000 g for 3 minutes, and the cells were collected for subsequent experiments.

[0059] The total RNA of the HaCaT cells collected above was extracted using the TRIZOL kit. The specific steps are as follows: 1) RNA Extraction: Add 1 mL of TRIZOL reagent to the cell sample, shake, and centrifuge (12,000 rpm, 4°C, 15 min). Collect the supernatant, add 200 μL of chloroform, vortex, and let stand at room temperature for 10 min. Centrifuge according to the above parameters and collect the supernatant. Then, add an equal volume of isopropanol to the supernatant, gently shake, and centrifuge at 14,000 rpm for 10 min. Discard the supernatant, add 75% ethanol, and centrifuge again. Resuspend the precipitate in DEPC water to obtain RNA. Store the resulting RNA at -80°C until further use.

[0060] 2) Determination of RNA concentration and quality: RNA concentration and purity were determined using Nanodrop 2000.

[0061] 3) Reverse transcription: RNA was reverse transcribed into cDNA using the TransScript® All-in-One First-Strand cDNA Synthesis SuperMix for qPCR Kit.

[0062] Prepare the reverse transcription reaction system as shown in Table 1 below: Table 1 Reverse transcription system

[0063] Perform reverse transcription reaction: Gently mix the above system and incubate at 45°C for 15 min to remove the genome and reverse transcription reaction; heat at 85°C for 5 s to inactivate TransScript® RT / RI and gDNA Remover.

[0064] 4) Real-time fluorescence quantitative PCR reaction The qRT-PCR method was used to determine the effect of EVs of Lactobacillus plantarum on the expression levels of genes related to the ceramide synthesis pathway (SPT, GCS, SMase and CerS3 genes).

[0065] Table 2 qRT-PCR amplification system

[0066] Real-time fluorescence quantitative PCR was performed using the Takala TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) kit. The amplification system and protocol are shown in Tables 2 and 3. The qPCR primers required for this experiment are shown in Table 4. First, prepare the mixed reaction system. According to Table 2, add ddH2O, upstream and downstream primers, TB Green enzyme, and ROX to the tube in that order. Vortex to mix, then briefly centrifuge. Add the DNA template and the mix solution to each tube in an 8-tube strip. Repeat five times for each sample. The entire process was performed on ice and protected from light. In all qPCR experiments, the template in the negative control was replaced with RNase-free ddH2O, and the GAPDH gene (a housekeeping gene) was used as the internal reference gene.

[0067] Table 3 qRT-PCR amplification procedure

[0068] Exploit 2 -ΔΔCT The relative expression levels of the above-mentioned related genes were statistically analyzed by GAPDH as the internal reference. The relative expression levels of the target gene mRNA were calculated based on 2 －ΔΔCt calculate.

[0069] The results are as follows Figure 6 As shown in the results, EVs derived from Lactobacillus plantarum can significantly promote the expression of key enzyme genes (SPT, CerS3, SMase and GCS) in the ceramide synthesis pathway (p < 0.05), indicating that EVs can promote the synthesis of ceramide in HaCaT cells.

[0070] Table 4 Primers

[0071] Example 5: EVs derived from Lactobacillus plantarum promote the expression of genes related to skin barrier repair To evaluate the effect of EVs derived from Lactobacillus plantarum on the expression of skin barrier-related genes, a skin barrier damage model was established by treating HaCaT cells with 1% sodium dodecyl sulfate (SDS). EVs derived from Lactobacillus plantarum were isolated using the method of Example 1. The cell culture method was similar to that of Example 2. When the cell confluence reached 80% to 90%, the cell density was adjusted to 5 × 10 5 Cells were plated at a concentration of 100 μg / mL and added to a 6-well plate. 2 mL of cell suspension was added to each well and cultured in an incubator (37°C, 5% CO2) for 12 hours. When the cell plating rate met the experimental requirements, the cells were divided into three groups: an experimental group (SDS+EVs), a model group (SDS), and a blank group (DMEM). Each group received the corresponding intervention, with three replicates per well. The old culture medium in the 6-well plate was discarded, and serum-free culture medium was added to all wells. 1% SDS was added to all wells except the blank group, and the cells were cultured for an additional 12 hours. After the 12-hour incubation, EVs were added to the experimental group. Three concentration gradients of EVs were set: 30 μg / mL, 12 μg / mL, and 6 μg / mL. 100 μL of the corresponding concentration of EVs was added to each well, and the cells were incubated for an additional 24 hours. The cell culture medium in all wells was then discarded, and 1 mL of PBS was added. The adherent cells were scraped off with a cell scraper and collected by centrifugation at 1000 g for 3 minutes. Total RNA from HaCaT cells was extracted and transcribed into cDNA according to the method in Example 4. The qRT-PCR method was used to determine the effect of EVs from Lactobacillus plantarum on the expression levels of skin barrier repair-related genes keratin 10, filaggrin (FLG), and desmoglein 1 (DSG1). The primers used in this experiment are shown in Table 5.

[0072] The results are as follows Figure 7 As shown in the results, compared with the model group, the expression of skin barrier repair-related genes such as keratin 10 (KRT10), filaggrin (FLG) and desmoglein 1 (DSG1) were significantly upregulated after treatment with different concentrations (6 μg / mL, 12 μg / mL and 30 μg / mL) of Lactobacillus plantarum EVs (p < 0.05), indicating that EVs can promote the expression of skin barrier-related genes, thereby accelerating the repair of the skin barrier.

[0073] Table 5 Primers

[0074] Example 6: EVs derived from Lactobacillus plantarum downregulate the expression of inflammation-related genes in HaCaT cells Cell culture conditions and experimental procedures were the same as in Example 5. Total RNA from HaCaT cells was extracted and transcribed into cDNA according to the method in Example 4. qRT-PCR was used to determine the effect of Lactobacillus plantarum EVs on the expression levels of inflammation-related genes tumor necrosis factor-α, interleukin-33, and thymic stromal lymphopoietin in HaCaT cells. The primers used in this experiment are shown in Table 6.

[0075] The results are as follows Figure 8 As shown in the data, compared with the model group, the mRNA levels of TNF-α, IL-33 and TSLP encoding genes were significantly reduced after treatment with different concentrations (6 μg / mL, 12 μg / mL and 30 μg / mL) of Lactobacillus plantarum EVs, and showed a concentration-dependent manner.

[0076] Table 6 Primers

[0077] Example 7: Effects of EVs derived from Lactobacillus plantarum on a mouse skin barrier damage model A mouse skin barrier damage model was established, and EVs derived from Lactobacillus plantarum were used for intervention. The number of scratching times and skin lesion scores of the mice were calculated, and the skin barrier-related indicators of the mouse back, including water content, transepidermal water loss, and expression of barrier repair-related factor genes in the skin barrier tissue, were measured. The gene and protein levels of pro-inflammatory cytokines in the skin tissue were measured, and the therapeutic effect of EVs derived from Lactobacillus plantarum on skin barrier damage was evaluated.

[0078] 1. Establishment and intervention of mouse skin lesion model The mice were first adaptively raised for 1 week. Female Balb / c mice, weighing 20±2 g, were used. The hair on the abdomen and back of the mice was removed with electric clippers (the hair removal area was 2 cm×3 cm). 150 μL of 5% sodium dodecyl sulfate (SDS) was applied continuously for the first 7 days. Then, 150 μL of 4% SDS was applied every three days for 24 days to establish the model.

[0079] The experiment was divided into a blank group (Control), a model group (SDS), a positive group (MFC), and an EVs group (four concentration gradients: 6 μg / mL, 12 μg / mL, 30 μg / mL, and 100 μg / mL), with 8 mice in each group. The positive group received mometasone furoate ointment, while the blank group received saline. Intervention began on the eighth day of the experiment. Each of the blank, positive, and EVs groups received a single application of 100 μL of saline, mometasone furoate ointment, and EVs of varying concentrations to the dorsal lesions daily for 24 consecutive days.

[0080] 2. Number of mouse scratches On the 10th, 15th and 20th days of administration, the mice were placed in an environment where no one would disturb them, and filmed for 10 minutes using a camera to record the number of times the mice scratched.

[0081] Table 7 Number of scratching (times / 10 min)

[0082] Note: Compared with the blank group, ### p < 0.001; compared with the model group, *** p < 0.001 The results are shown in Table 7. Compared with the blank group, the number of scratching in the model group increased significantly (p < 0.01). Compared with the model group, Lactobacillus plantarum EVs intervention significantly reduced the number of scratching in mice (p < 0.01), and the effect was concentration-dependent, with 100 μg / mL EVs having the most significant effect.

[0083] 3. Epigenetic Changes of Local Skin Lesions in Mice Before the intervention and on the last day of the intervention, the apparent changes of the back skin of each group of mice were recorded using a camera ( Figure 9 Obvious scabs, dry skin, and desquamation were observed on the back skin of mice in the model group. Compared with the model group, the skin condition of mice in the positive drug group was better, and the scabs on the back skin were smaller. The skin condition of mice in the EVs group was good, and the dryness and desquamation of the skin were effectively alleviated. The intervention effect of 100 μg / mL EVs was better than that of the positive group.

[0084] 4. Scoring of Skin Lesion Severity in Mice Before and on the last day of intervention, the severity of skin lesions on the back of mice was scored on a scale of 0 (no symptoms), 1 (mild symptoms), 2 (moderate symptoms), and 3 (severe symptoms) (Table 8).

[0085] Table 8 Scoring criteria for skin lesion severity

[0086] The rating results are shown as Figure 10 As shown in the figure, the skin lesion score of the model group increased; compared with the model group, the score of the positive group and the EVs group decreased, and the differences were significant (p < 0.05).

[0087] 5. Determination of Mouse Skin Moisture Content and Transepidermal Water Loss On day 24 after administration, three different areas of the mouse modeling area were randomly selected for testing using a skin tester. The water content of the stratum corneum and the transepidermal water loss at these areas were recorded. Finally, the average of the water content of the stratum corneum and the transepidermal water loss at these three areas was calculated to assess the hydration status and barrier function of the skin.

[0088] like Figure 11 As shown in the results, compared with the model group, treatment with different concentrations of Lactobacillus plantarum EVs (6 μg / mL, 12 μg / mL, 30 μg / mL, and 100 μg / mL) significantly increased skin moisture content (p < 0.05) and significantly reduced transepidermal water loss (p < 0.05). This result indicates that Lactobacillus plantarum EVs can effectively restore skin moisture and improve skin barrier function.

[0089] 6. Determination of Gene Expression Levels of Pro-inflammatory Cytokines in Mouse Skin Tissue The dorsal tissues of mice in each group were homogenized, and total RNA was extracted according to the method of Example 4. The RNA was reverse transcribed into cDNA, and the mRNA expressions of related pro-inflammatory cytokines TNF-α, IL-4, IL-31 and TSLP were detected by qRT-PCR. GAPDH was used as an internal reference, and 2 -△△Ct The relative expression levels of each gene were calculated using the primers used in this experiment as shown in Table 9.

[0090] The experimental results are as follows Figure 12 As shown in the results, compared with the model group, treatment with Lactobacillus plantarum EVs at different concentrations (6 μg / mL, 12 μg / mL, 30 μg / mL, and 100 μg / mL) significantly reduced the mRNA levels of genes encoding TNF-α, IL-31, IL-4, and TSLP (p < 0.05), and this reduction was clearly concentration-dependent. This suggests that Lactobacillus plantarum EVs can inhibit the gene expression of inflammation-related cytokines and chemokines in a concentration-dependent manner, thereby exerting a significant anti-inflammatory effect.

[0091] Table 9 Primers

[0092] 7. Determination of protein expression levels of pro-inflammatory cytokines in mouse skin tissue The dorsal tissues of mice in each group were homogenized and tested using ELISA kits.

[0093] (1) ELISA test a. Preparation before testing: Taking the TNF-α kit as an example 1) Take the kit out of the refrigerator 20 minutes in advance to equilibrate to room temperature.

[0094] 2) Dilute the 20× concentrated washing solution to 1× working solution with double distilled water.

[0095] b. Steps: 1) Remove the required strips from the sealed bag that has been equilibrated to room temperature. Place the unused strips and desiccant back into the aluminum foil bag, press the self-sealing strip, seal the bag, and return it to 4°C.

[0096] 2) Set up standard wells, blank wells, and sample wells. Add 50 μL of standard solution of varying concentrations to each well.

[0097] 3) Add 50 μL of the sample to be tested to the sample well; do not add anything to the blank well.

[0098] 4) Add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each well, except the blank wells, of the standard wells and sample wells. Seal the wells with a sealing film and incubate at 37°C in a thermostat or water bath for 60 min.

[0099] 5) Discard the liquid, pat dry with absorbent paper, and fill each well with washing solution (350 μL). Let it stand for 1 minute, then discard the washing solution and pat dry with absorbent paper. Repeat this process 5 times.

[0100] 6) Add 50 μL of substrate A and substrate B to each well and incubate at 37°C in the dark for 15 min.

[0101] 7) Add 50 μL of stop solution to each well and measure the OD value of each well at a wavelength of 450 nm within 15 minutes (within 3 minutes).

[0102] c. Result judgment: The OD value of the blank well should be subtracted from the OD value of each standard and sample. The software plots the best-fit curve, with the standard concentration as the horizontal axis and the OD value as the vertical axis. The concentration of the sample can be found on the standard curve using the OD value of the sample.

[0103] (2) Calculation of TNF-α, IL-1β and IL-6 content TNF-α, IL-1β, and IL-6 standards were prepared into standard solutions and serially diluted to a series of known concentrations. OD values ​​at 450 nm were measured using the ELISA method described above. A regression equation was constructed using the standard curves, and the OD450 values ​​of each test sample were substituted into the equation to calculate the TNF-α, IL-1β, and IL-6 content in each sample. Three replicate wells were tested in each group, and the mean and standard deviation (SD) were calculated. One-way analysis of variance was performed using SPSS statistical software to compare the TNF-α, IL-1β, and IL-6 content of each sample group with that of the blank control group. P < 0.05 was considered statistically significant.

[0104] like Figure 13As shown in the results, compared with the model group, treatment with Lactobacillus plantarum EVs at different concentrations (6 μg / mL, 12 μg / mL, 30 μg / mL, and 100 μg / mL) significantly reduced the protein levels of TNF-α, IL-1β, and IL-6 (p < 0.05). This result indicates that Lactobacillus plantarum EVs can inhibit the protein expression of inflammation-related cytokines in a concentration-dependent manner, thereby effectively exerting an anti-inflammatory effect.

[0105] 8. Determination of Gene Expression Levels of Skin Barrier Repair-related Cytokines in Mouse Skin Tissue The dorsal tissues of mice in each group were homogenized, total RNA was extracted, and reverse transcribed into cDNA. The mRNA expressions of skin barrier-related cytokines filaggrin, keratin 10, and ceramide synthase 3 were detected by qRT-PCR, with GAPDH as the internal reference. -△△Ct The relative expression levels of each gene were calculated using the primers used in this experiment. Table 10 shows the primers used in this experiment.

[0106] like Figure 14 As shown, compared with the model group, treatment with Lactobacillus plantarum EVs at different concentrations (6 μg / mL, 12 μg / mL, 30 μg / mL, and 100 μg / mL) significantly upregulated the mRNA levels of skin barrier-related genes FLG, KRT10, and CerS3 (p < 0.05), and this upregulation was clearly concentration-dependent. This suggests that Lactobacillus plantarum EVs can effectively repair the skin barrier function of mice with SDS-induced skin damage and promote skin barrier repair.

[0107] Table 10 Primers

[0108] 9. Determination of pruritus-related cytokine gene expression levels in mouse skin tissue The dorsal tissues of mice in each group were homogenized, total RNA was extracted, and reverse transcribed into cDNA. The mRNA expressions of pruritus-related cytokines IL-31RA and OSMA were detected by qRT-PCR, with GAPDH as the internal reference. -△△Ct The relative expression levels of each gene were calculated using the primers used in this experiment. Table 11 shows the primers used in this experiment.

[0109] like Figure 15As shown in the results, compared with the model group, treatment with Lactobacillus plantarum EVs at different concentrations (6 μg / mL, 12 μg / mL, 30 μg / mL, and 100 μg / mL) significantly reduced the expression levels of the itch-related genes OSMA and IL-31RA (p < 0.05). This result indicates that Lactobacillus plantarum EVs can significantly inhibit the pruritus response induced by DNFB in mice with skin damage, effectively alleviating the symptoms of pruritus.

[0110] Table 11 Primers

[0111] Example 8: Absorption and distribution of EVs derived from Lactobacillus plantarum in mouse skin A mouse back skin smear experiment was conducted to observe the uptake and distribution of extracellular vesicles (EVs) derived from Lactobacillus plantarum in skin tissue, thereby evaluating their transdermal absorption properties. Labeled Lactobacillus plantarum EVs were evenly applied to the back skin of mice. In vivo imaging was then used to monitor the penetration, uptake, and distribution of EVs within the skin in real time. By analyzing the imaging data, the transdermal ability of Lactobacillus plantarum EVs and their absorption efficiency in skin tissue were systematically evaluated.

[0112] 1. Mouse Handling Before the experiment began, mice in the experimental group (EVs + DIO) and the control group (DIO) underwent hair removal. Hair removal should be performed one day in advance to ensure a clean skin surface and no hair that would interfere with subsequent experimental procedures. The hair removal method was similar to that described in Example 7. During the procedure, damage to the mouse skin was avoided to ensure the accuracy and reliability of the experimental results.

[0113] 2. Fluorescent Labeling of EVs The labeling method was completed with reference to Example 2.

[0114] 3. In vivo efficacy observation The labeled EVs were evenly applied to the dorsal skin of mice, using 100 μL per mouse. Care was taken to avoid light during the procedure to prevent quenching of the fluorescence signal. Two hours after application, the mice were gently placed in the darkroom platform of the in vivo imaging system. The field of view was adjusted and a background image was captured to ensure imaging accuracy. Subsequently, the fluorescence signal in the mice was captured under darkfield conditions to record the distribution of EVs in the skin tissue in detail.

[0115] like Figure 16As shown in the figure, EVs labeled with DIO dye exhibit green fluorescence. Experimental results show that EVs penetrated the skin on the back of mice just 2 hours after application. This phenomenon indicates that EVs derived from Lactobacillus plantarum have good transdermal absorption and are rapidly taken up and distributed by mouse skin.

[0116] Example 9: Proteomic analysis of EVs derived from Lactobacillus plantarum The extraction method for EVs from Lactobacillus plantarum was similar to that in Example 1. The protein concentration of the EV samples was determined using BCA reagent to ensure that the protein concentration of each sample was greater than 200 μg / mL. The EV samples were then lysed and the proteins in the samples were identified using liquid chromatography-mass spectrometry (LC-MS / MS). The mass spectrometry data were analyzed using mass spectrometry data analysis software (such as MaxQuant or Proteome Discoverer) to identify the protein species in the vesicles. Bioinformatics tools (such as Gene Ontology and KEGG pathway analysis) were used to perform functional annotation and pathway enrichment analysis on the identified proteins.

[0117] The results showed that proteomic analysis of EVs derived from Lactobacillus plantarum identified a total of 1537 proteins. Figure 17 As shown in the figure, KEGG pathway analysis showed that proteins related to metabolic function dominated the total proteins, accounting for about 61.76% of the total proteins.

[0118] Example 10: Metabolomic Analysis of EVs from Lactobacillus plantarum The extraction method for EVs from Lactobacillus plantarum was similar to that in Example 1. An appropriate amount of metabolite extraction reagent was then added to the EV sample to be tested. After thorough vortexing and mixing, the sample was immediately snap-frozen in liquid nitrogen and then ultrasonically disrupted to release the EV contents. After refrigerated centrifugation, the supernatant was collected and transferred to a liquid chromatography-mass spectrometry (LC-MS) vial. Metabolomics analysis of the sample was performed using a liquid chromatography-tandem mass spectrometry (LC-MS / MS) system.

[0119] Relative abundance is a dimensionless value that represents the ratio of a specific ion signal intensity to the total ion signal intensity. Figure 18 As shown, lipids and their derivatives were the most abundant in the metabolome. Further analysis revealed that among lipids and their derivatives, trans-11-octadecenoic acid (VA) had a mean relative abundance of 102,377.9927, a high content, and possesses anti-inflammatory and barrier repair potential, making it a key active component in EVs.

[0120] Example 11: Analysis and Verification of Trans-11-Octadecenoic Acid, a Key Active Ingredient in EVs from Lactobacillus plantarum High-purity trans-11-octadecenoic acid (VA) was obtained through in vitro synthesis. HaCaT cells were treated with VA at concentrations of 6 μg / mL and 15 μg / mL. Simple cell culture medium and vitamin C served as blank and positive controls, respectively. Ceramide production, gene expression of barrier repair-related factors, and inflammatory factors were measured in the cells, as described in Examples 3, 4, 5, and 6, respectively.

[0121] like Figure 19 As shown in the results, VA intervention significantly increased the ceramide production in HaCaT cells. Figure 20 As shown in the results of the SDS-induced damaged cell model, VA intervention significantly upregulated the gene expression levels of protein factors closely related to skin barrier function, including filaggrin, ceramide synthase 3, and keratin 10. Figure 21 As shown in Figure 3, VA treatment also significantly reduced the expression levels of inflammatory factors such as IL-25, IL-33, and TSLP. In summary, trans-11-octadecenoic acid is an effector molecule that plays a key role in EVs.

[0122] The embodiments described above are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work shall fall within the scope of protection of the present invention.

Claims

1. An extracellular vesicle of Lactobacillus plantarum, characterized in that The extracellular vesicles are derived from a strain of Lactobacillus plantarum ( Lactiplantibacillus plantarum ) L25, the L25 is deposited in the General Microbiology Center of the China Culture Collection Administration Committee with a deposit number of CGMCC No. 35146. The average particle size of the extracellular vesicles of Lactobacillus plantarum is 100-200 nm; and a spherical morphology is observed under a transmission electron microscope.

2. The extracellular vesicle according to claim 1, characterized in that The extracellular vesicles are prepared by the following method: (1) The strain is cultured in a liquid culture medium, and then the bacterial culture is centrifuged to remove the bacterial precipitate and obtain the supernatant of the bacterial culture; (2) Filter the supernatant using 0.45 μm and 0.22 μm filters to remove residual bacteria and cell debris; (3) The eluted sample was then concentrated using a membrane cassette and filtered through a 0.22 μm filter. (4) The filtered sample was ultracentrifuged, the supernatant was removed, and the EVs precipitate derived from Lactobacillus plantarum was obtained and the precipitate was resuspended in PBS; (5) Repeat ultracentrifugation as in step (4), and finally resuspend the precipitate with PBS. Aliquot and store at -80°C or freeze-dry to obtain lyophilized powder for future use.

3. A method for preparing extracellular vesicles of Lactobacillus plantarum, characterized in that: The method comprises the following steps: (1) The strain is cultured in a liquid culture medium, and then the bacterial culture is centrifuged to remove the bacterial precipitate and obtain the supernatant of the bacterial culture; (2) Filter the supernatant using 0.45 μm and 0.22 μm bottle-top filters to remove residual bacteria and cell debris; (3) The eluted sample was then concentrated using a membrane cassette and filtered through a 0.22 μm filter. (4) The filtered sample was ultracentrifuged, the supernatant was removed, and the EVs precipitate derived from Lactobacillus plantarum was obtained and the precipitate was resuspended in PBS; (5) Repeat ultracentrifugation as in step (4), and finally resuspend the precipitate with PBS, aliquot, and store at -80°C until use.

4. The method according to claim 3, characterized in that The culture medium in step (1) is MRS liquid culture medium; the culture time is 12 to 18 hours, the centrifugal speed is 5000 to 8000 g, and the centrifugation time is 5 to 20 minutes; the ultracentrifugation in step (4) is 120,000 to 150,000 g, and the centrifugation time is 1 to 2 hours.

5. Use of the extracellular vesicles of Lactobacillus plantarum according to claim 1 or 2 in preparing a preparation for repairing skin barrier, characterized in that: The concentration of the extracellular vesicles in the preparation is 6 to 120 μg / mL.

6. Use of the extracellular vesicles of Lactobacillus plantarum according to claim 1 or 2 in preparing a preparation for alleviating skin inflammation, characterized in that: The concentration of the extracellular vesicles in the preparation is 6 to 120 μg / mL.

7. Use of the extracellular vesicles of Lactobacillus plantarum according to claim 1 or 2 in preparing a preparation for alleviating skin itching, characterized in that: The concentration of the extracellular vesicles in the preparation is 6 to 120 μg / mL.

8. Use of the extracellular vesicles of Lactobacillus plantarum according to claim 1 or 2 in the preparation of a skin rapid transdermal absorption carrier preparation, characterized in that: The concentration of the extracellular vesicles in the preparation is 6 to 120 μg / mL.

9. The use according to any one of claims 5 to 8, characterized in that: The concentration of the extracellular vesicles in the preparation is 80-120 μg / mL.

10. Use of trans-11-octadecenoic acid in preparing a preparation for repairing skin damage, characterized in that: The skin damage includes skin barrier damage, inflammation, and itching.

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