An extracellular vesicle of lactobacillus plantarum and use thereof

Abstract

The present application belongs to the field of microbial technology, and particularly relates to a plant lacto-plantarum extracellular vesicle and application thereof. Specifically, the plant lacto-plantarum extracellular vesicle can promote HaCaT cells to generate ceramide, up-regulate the expression of key genes in the ceramide synthesis pathway of skin, and restore the skin barrier. The plant lacto-plantarum extracellular vesicle can reduce the expression of pro-inflammatory factors in a cell inflammation model, and has good anti-inflammatory effect. The mouse skin damage model verification shows that the EVs have good skin penetration ability, can significantly reduce the skin damage score, relieve skin itching, improve the skin water content, and reduce the expression of pro-inflammatory factors and the expression of itching-related cytokines in the skin tissue.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a plant lactobacillus rhamnosus extracellular vesicle and application thereof. BACKGROUND

[0002] Skin, as the largest organ of the human body, is the first line of defense against the external environment and plays a crucial role in barrier function. The skin barrier is mainly composed of physical, chemical, and microbial barriers. The physical barrier is mainly composed of the stratum corneum and intercellular lipids. The chemical barrier is composed of the acidic environment (pH value of about 4.5-6.0), natural moisturizing factor (NMF), and lipids on the surface of the skin. The microbial barrier is composed of the microbial community on the surface of the skin, including bacteria, fungi, and viruses. These microbial communities interact to form a dynamic ecological system that can inhibit the excessive growth of harmful microorganisms and maintain the skin's microecological balance. The integrity of the skin barrier is crucial for maintaining the health of the skin. Once damaged, it can lead to dry, sensitive, and inflammatory skin, and even trigger allergic reactions or infections. Therefore, protecting the integrity of the skin barrier is key to maintaining skin health.

[0003] Ceramides are the main components of intercellular lipids in the stratum corneum of the skin, accounting for about 40%-50% of the total intercellular lipids, and play a key role in maintaining skin barrier function, with functions such as moisturizing, enhancing intercellular adhesion, and repairing the barrier.

[0004] Bacterial extracellular vesicles (BEVs) are nanoscale membrane particles secreted by gram-negative and gram-positive bacteria, with a diameter usually between 20-400 nm. BEVs have a lipid bilayer structure and contain various bioactive substances such as proteins, nucleic acids, lipids, and metabolites. These extracellular vesicles (EVs) play important roles in bacterial communication, host-microbe interactions, biofilm formation, virulence factor transmission, and antibiotic resistance gene transfer. Probiotic extracellular vesicles are extracellular vesicles secreted by probiotics, which have similar structure and function to bacterial extracellular vesicles, and can regulate the host's immune response, inhibit inflammatory response, and play an important role in intestinal health, immune regulation, and disease prevention. For example, Lactobacillus rhamnosus EVs can significantly reduce the production of pro-inflammatory factors TNF-α and IL-6. Plant lactobacillus rhamnosus extracellular vesicles are nanoscale vesicles secreted by plant lactobacillus rhamnosus. Studies have shown that EVs have shown significant potential in improving intestinal inflammation, regulating immune response, and promoting skin health.

[0005] At present, 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 application, but further optimization and expansion are still needed, especially the analysis of the efficacy components of bacterial extracellular vesicles is very scarce. At present, there is no research on Lactobacillus plantarum extracellular vesicles and their application in repairing skin barrier, and the analysis of the efficacy components of bacterial extracellular vesicles.

[0006] There is no report on how bacterial extracellular vesicles can increase the content of skin ceramides by regulating multiple genes or molecular mechanisms related to ceramide synthesis in the previously disclosed patents or patent applications. In addition, the analysis of the efficacy components of bacterial extracellular vesicles is very scarce and needs further research. Therefore, bacterial extracellular vesicles that can repair skin barrier, anti-inflammatory, relieve itching and have rapid transdermal absorption capacity have very important application value and practical significance. SUMMARY

[0007] The present application aims to overcome the shortcomings of the prior art and provide a Lactobacillus plantarum vesicle with skin barrier recovery function and anti-inflammatory and itching relief, and the analysis of the efficacy components of the Lactobacillus plantarum vesicle.

[0008] The first aspect of the present application is to provide a Lactobacillus plantarum extracellular vesicle, characterized in that the extracellular vesicle is derived from a Lactobacillus plantarum strain (L25). Lactiplantibacillus plantarum The Lactobacillus plantarum L25 is deposited with the China General Microbiological Culture Collection Center, and the deposit number is CGMCC No. 35146. The deposit address is No. 1, Yitian West Road, Beijing Institute of Microbiology, Chinese Academy of Sciences, Beijing City, Chaoyang District. The average particle size of the Lactobacillus plantarum extracellular vesicle is 130-140 nm; it is observed as a spherical shape under transmission electron microscope; preferably, the particle size of the extracellular vesicle is 132±2.24 nm.

[0009] Further, the extracellular vesicle is prepared by the following method:

[0010] (1) The strain is cultured in a liquid medium, and then the bacterial culture is centrifuged to remove the bacterial precipitate and obtain the supernatant of the bacterial culture;

[0011] (2) The supernatant is filtered using 0.45 μm and 0.22 μm bottle top filters to remove residual bacteria and cell debris;

[0012] (3) Then the sample is concentrated and eluted using a membrane bag, and then the concentrated sample is filtered using a 0.22 μm filter;

[0013] (4) The filtered sample is subjected to ultracentrifugation, the supernatant is removed, and the EVs derived from the Lactobacillus plantarum are precipitated and resuspended in PBS.

[0014] (5) The ultracentrifugation is repeated according to step (4), and finally the precipitate is resuspended in PBS, and stored at -80°C after being aliquoted or lyophilized to obtain a lyophilized powder for use.

[0015] Preferably, the culture medium in step (1) is MRS liquid medium; the culture time is 12-18 h, the centrifugal speed is 5000-8000 g, and the centrifugal time is 5-20 min.

[0016] Preferably, the ultracentrifugation in step (4) is 12000 g-15000 g, and the centrifugal time is 1-2 h.

[0017] The second aspect of the present application provides a preparation method of Lactobacillus plantarum extracellular vesicles, characterized in that the method comprises the following steps:

[0018] (1) The strain is cultured in a liquid medium, and then the bacterial culture is centrifuged to remove the bacterial precipitate and obtain the supernatant of the bacterial culture;

[0019] (2) The supernatant is filtered using 0.45 μm and 0.22 μm bottle top filters to remove residual bacteria and cell debris;

[0020] (3) The sample is then concentrated and eluted using a membrane pack, and then the concentrated sample is filtered using a 0.22 μm filter;

[0021] (4) The filtered sample is subjected to ultracentrifugation, the supernatant is removed, and the EVs derived from the Lactobacillus plantarum are precipitated and resuspended in PBS.

[0022] (5) The ultracentrifugation is repeated according to step (4), and finally the precipitate is resuspended in PBS, and stored at -80°C after being aliquoted or lyophilized to obtain a lyophilized powder for use.

[0023] Preferably, the culture medium in step (1) is MRS liquid medium; the culture time is 12-18 h, the centrifugal speed is 5000-8000 g, and the centrifugal time is 5-20 min.

[0024] Preferably, the ultracentrifugation in step (4) is 12000 g-15000 g, and the centrifugal time is 1-2 h.

[0025] The third aspect of the present application provides the use of the extracellular vesicle of the Lactobacillus plantarum in the preparation of a preparation for repairing skin barrier, characterized in that the concentration of the extracellular vesicle in the preparation is 6-120 μg / mL, preferably 80-120 μg / mL, and most preferably 100 μg / mL.

[0026] The fourth aspect of the present application provides the use of the extracellular vesicle of the Lactobacillus plantarum in the preparation of a preparation for reducing skin inflammation, characterized in that the concentration of the extracellular vesicle in the preparation is 6-120 μg / mL, preferably 80-120 μg / mL, and most preferably 100 μg / mL.

[0027] The fifth aspect of the present application provides the use of the extracellular vesicle of the Lactobacillus plantarum in the preparation of a preparation for reducing skin itching, characterized in that the concentration of the extracellular vesicle in the preparation is 6-120 μg / mL, preferably 80-120 μg / mL, and most preferably 100 μg / mL.

[0028] The sixth aspect of the present application provides the use of the extracellular vesicle of the Lactobacillus plantarum in the preparation of a preparation as a skin rapid transdermal absorption carrier, characterized in that the concentration of the extracellular vesicle in the preparation is 6-120 μg / mL, preferably 80-120 μg / mL, and most preferably 100 μg / mL.

[0029] The seventh aspect of the present application provides the use of trans-11-octadecenoic acid in the preparation of a preparation for repairing skin damage.

[0030] Further, the skin damage includes skin barrier damage, inflammation, and itching.

[0031] The beneficial effects of the present application include:

[0032] The extracellular vesicle of the Lactobacillus plantarum of the present application can promote the generation of ceramide by HaCaT cells, up-regulate the expression of key genes in the ceramide synthesis pathway of the skin, and restore the skin barrier; can reduce the expression of pro-inflammatory factors in a cell inflammation model, and has good anti-inflammatory effect. Through verification of a mouse skin damage model, the EVs have good skin penetration ability, can significantly reduce the skin damage score, relieve skin itching, increase the water content of the skin, and reduce the expression of pro-inflammatory factors and the expression of itching-related cytokines in the skin tissue. In summary, the extracellular vesicle of the Lactobacillus plantarum is a multi-functional substance, has good safety, biocompatibility, and transdermal absorption capacity, and can simultaneously exert multiple functions such as repairing skin barrier, anti-inflammatory, moisturizing, anti-sensitivity, and soothing.

[0033] The microorganism of the present application is Lactobacillus plantarumLactiplantibacillus plantarum L25, which was deposited with the China General Microbiological Culture Collection Center on July 9, 2025, and has the accession number CGMCC No. 35146, and is located at No. 1, Yikhinxi Lu, Beichen, Beijing, China. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 EVs sedimentation sample images.

[0035] Figure 2 EVs transmission electron microscopy images.

[0036] Figure 3 EVs particle size.

[0037] Figure 4 Evs uptake by cells fluorescence staining images.

[0038] Figure 5 EVs improve HaCaT cell ceramide production.

[0039] Figure 6 EVs modulate HaCaT cell ceramide synthesis key enzyme gene expression.

[0040] Figure 7 EVs modulate skin barrier related gene expression in SDS-induced damaged cell model.

[0041] Figure 8 EVs modulate skin inflammation related gene expression in SDS-induced damaged cell model.

[0042] Figure 9 EVs relieve SDS-induced mouse skin damage appearance images.

[0043] Figure 10 Mouse skin lesion scores.

[0044] Figure 11 Mouse skin moisture content and trans-epidermal water loss.

[0045] Figure 12 EVs reduce inflammation gene expression in mouse skin tissue.

[0046] Figure 13 EVs reduce inflammation protein expression in mouse skin tissue.

[0047] Figure 14 EVs improve mouse skin barrier related gene expression.

[0048] Figure 15 EVs inhibit mouse skin itch related gene expression.

[0049] Figure 16 Absorption and distribution profile of EVs in mouse skin.

[0050] Figure 17 Proteomic analysis of EVs.

[0051] Figure 18 Metabolomic analysis of EVs.

[0052] Figure 19 Trans-11-octadecenoic acid modulates ceramide production in HaCaT cells.

[0053] Figure 20 Trans-11-octadecenoic acid modulates skin barrier-related gene expression in SDS-induced damaged cell model.

[0054] Figure 21 Trans-11-octadecenoic acid modulates skin inflammation-related gene expression in SDS-induced damaged cell model. DETAILED DESCRIPTION

[0055] The concept and the technical effects of the present application will be further described below in conjunction with specific embodiments, so as to fully understand the objects, features and effects of the present application. The methods are all conventional methods unless otherwise specified. The materials can be obtained from public commercial channels unless otherwise specified. The illustrative embodiments of the present application and the descriptions thereof are used to explain the present application, and do not constitute an improper limitation on the present application. It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0056] Example 1 Isolation and characterization of EVs derived from Lactobacillus plantarum

[0057] 1. Isolation of EVs derived from Lactobacillus plantarum

[0058] The Lactobacillus plantarum used was screened and isolated from a Lactobacillus plantarum strain in a pickled cabbage sample by the Applied Microorganisms and Enzyme Engineering Laboratory of the School of Biological Engineering, Tianjin University of Science and Technology. The strain was cultured in a 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. Then the sample was concentrated and eluted using a membrane bag, and then the concentrated sample was filtered using a 0.22 μm filter. The filtered sample was ultracentrifuged at 140000 g for 1.5 h to remove the supernatant and obtain the EVs precipitate derived from the Lactobacillus plantarum (EVs-Lp) Figure 1), and resuspended the pellet with PBS. Repeat the ultracentrifugation once with the above parameters. Finally, resuspend the pellet with PBS, aliquot and store at -80°C until use. Measure the protein concentration of EVs using BCA assay.

[0059] 2. Characterization of EVs

[0060] (1) Morphological observation

[0061] The morphology of EVs isolated from bacterial culture was observed using electron microscopy. EVs 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 acceleration voltage of 100 kV to observe the morphological characteristics of EVs. As shown in Figure 2 , EVs were observed to have a spherical shape by electron microscopy.

[0062] (2) Particle size distribution measurement

[0063] The particle size distribution of EVs isolated from bacterial culture was measured using dynamic light scattering (DLS). The diameter of EVs was determined using a Zetasizer Nano S, and the results are shown in Figure 3 , the EVs particle size was 132 ± 2.24 nm.

[0064] (3) Concentration measurement

[0065] The concentration of EVs was determined by nanoparticle tracking analysis (NTA). The concentration of EVs was adjusted to 500 ng / mL, and 0.3-0.4 mL of sample was placed in the chamber of the LM-10HS instrument. The camera focus was adjusted so that the particles were clearly visible, and the sample was captured by gradually lowering the level, confirming that there was no 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 x 10 9 particles / mL.

[0066] Example 2: Uptake of EVs derived from Lactobacillus plantarum in HaCaT cells

[0067] 1. Cell culture

[0068] HaCaT cells were added to DMEM high glucose culture medium containing 10% fetal bovine serum and placed in an incubator (37°C, 5% CO2) for routine culture. When the cell confluence reached 80-90%, the adherent cells were digested with trypsin and passaged, and the medium was changed 2-3 times per week.

[0069] 2. Fluorescent labeling of EVs

[0070] The isolated plantamylactobacillus extracellular vesicles (EVs) were incubated with DIO dye at room temperature for 20 min in the dark to label the EVs. After incubation, the EVs were washed with PBS 3 times to remove unbound dye.

[0071] 3. Laser confocal microscope observation

[0072] When the HaCaT cells reached 80% to 90% confluence, the cell suspension was obtained by the above cell passage method, and the cell density was adjusted to 2 x 10 5 The cell suspension was added to the 24-well plate in which the cell slides were previously placed, 1000 μL of cell suspension was added to each well, and the plate was further incubated in the incubator (37°C, 5% CO2) for 12 h. When the cell plating rate in the plate met the test requirements, all the old culture medium in the 24-well plate was first aspirated, and serum-free medium was added. The experimental group was added with EVs labeled with DIO in advance (30 μg / mL), and incubated for another 12 h, with attention to avoid light. Then the cell culture medium in all wells was discarded, the cells were fixed with fixing solution for 10 min, and then washed with PBS 3 times. DAPI dye was added to stain the cell nucleus, and after 5 min, the cells were washed with PBS 3 times. Finally, a small amount of PBS was added to prevent the cells from drying, and a laser confocal microscope was used for observation and photography.

[0073] The results, as shown in Figure 4 , the EVs labeled with DIO dye showed green fluorescence, and the cell nucleus stained with DAPI dye showed blue fluorescence. It could be clearly seen that the EVs could penetrate into the cells around, indicating that the EVs could be taken up by the cells.

[0074] Example 3: EVs derived from plantamylactobacillus promote the generation of ceramide by HaCaT cells

[0075] The cell culture method was as 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 x 10 5 The cell suspension was added to the 24-well plate, 1000 μL of cell suspension was added to each well, and the plate was further incubated in the incubator (37°C, 5% CO2) for 12 h. When the cell plating rate in the plate met the test requirements, the old culture medium was discarded, and serum-free medium was added. Three concentration gradients were set in the experimental group, which were 30 μg / mL, 12 μg / mL, and 6 μg / mL of EVs, and 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. Each group had 3 replicate wells, and the culture was continued for 24 h. After the culture was completed, the adherent cells were scraped with a cell scraper, and the cells were collected by centrifugation at 1000 g for 3 min. Then, ELISA detection was performed.

[0076] 1. Pre-test preparation

[0077] (1) Take the kit out of the refrigerator 20 min in advance to balance to room temperature;

[0078] (2) The 30-fold concentrated washing solution is diluted 30 times with distilled water and is ready for use.

[0079] 2. Operation steps

[0080] (1) Take the required board from the sealed bag which has been balanced to room temperature for the test;

[0081] (2) Set the standard hole, blank hole and sample hole, and add 50 μL of different concentrations of standard to each standard hole;

[0082] (3) Add 50 μL of the sample to be tested to the sample hole, and do not add anything to the blank hole;

[0083] (4) Set the blank hole (the blank control hole does not add sample and enzyme labeled reagent, and the rest of the operation is the same) and the sample hole to be tested. Add 40 μL of sample diluent to the sample hole to be tested on the enzyme labeled coating plate, and then add 10 μL of sample to be tested (the final dilution of the sample is 5 times). Add the sample to the bottom of the enzyme labeled plate hole, try not to touch the hole wall, and gently shake to mix. Seal the reaction hole with a sealing film, and incubate at 37°C for 30 min;

[0084] (5) Discard the liquid, dry it on a paper towel, and add full washing solution (350 μL) to each hole. Rest for 30 seconds, shake off the washing solution, dry it on a paper towel, and repeat the plate washing 5 times;

[0085] (6) Add 50 μL of substrate enzyme labeled reagent to each hole, except the blank hole. Incubate at 37°C in the dark for 15 min;

[0086] (7) Washing: the operation is the same as 5;

[0087] (8) Color development: add 50 μL of color developing agent A to each hole first, and then add 50 μL of color developing agent B, mix gently, and develop color at 37°C in the dark for 15 min;

[0088] (9) Add 50 μL of termination solution to each hole, and measure the OD value of each hole at 450 nm wavelength within 15 min.

[0089] 3. Result judgment

[0090] The OD value of each standard and sample should be subtracted from the OD value of the blank hole. Take the standard concentration as the horizontal coordinate and the OD value as the vertical coordinate, and draw and select the best fitting curve by software. The concentration of the sample can be found on the standard curve through the OD value of the sample.

[0091] 4. Ceramide content calculation

[0092] The standard sample of ceramide was configured into a standard solution, and was diluted into a series of solutions with known concentrations in proportion step by step. The OD value (OD 450) at 450 nm was measured by the above-mentioned ELISA method, and the regression equation of the standard curve was fitted. The OD 450 value of each test sample was brought into the equation, and the ceramide content result in the test sample was calculated. Each group was tested in triplicate, and the average value and standard deviation (SD) were calculated. The ceramide content of each sample group measured in the above-mentioned test was subjected to one-way ANOVA analysis with the blank control group by using SPSS statistical software, and p < 0.05 was considered to have statistical significance.

[0093] The results are shown in Table 1. Figure 5 As shown in Table 1, the EVs derived from Lactobacillus plantarum significantly promoted the generation of ceramide in HaCaT cells, and the difference was statistically significant compared with the blank group (p < 0.05). At the same time, the effect of EVs on promoting the generation of ceramide was equivalent to that of the positive control group.

[0094] Example 4: EVs derived from Lactobacillus plantarum up-regulate the expression of key genes in the ceramide synthesis pathway of HaCaT cells

[0095] The cell culture conditions and operation mode are described in Example 2. When the cell plating rate in the 6-well plate meets the test requirements, the old culture solution is aspirated. Three concentration gradients are set in the experimental group, which are 30 μg / mL, 12 μg / mL and 6 μg / mL of EVs, respectively. 100 μL of EVs with the corresponding concentration is added to each well. The same volume of PBS is used as the blank control group. Each group has 3 replicate wells, and the culture is carried out for 24 h. After the culture is completed, the adherent cells are scraped with a cell scraper, centrifuged at 1000 g for 3 min, and the cells are collected for subsequent experiments.

[0096] The total RNA of the above-mentioned collected HaCaT cells is extracted by using the TRIZOL kit, and the specific steps are as follows:

[0097] 1) Extraction of RNA: 1 mL of TRIZOL reagent is added to the above-mentioned cell sample, and after shaking, it is centrifuged (12000 rpm, 4℃, 15 min). The supernatant is collected, 200 uL of chloroform is added, and after vortexing at room temperature for 10 min, it is centrifuged according to the above-mentioned parameters, and the supernatant is collected. Then, an equal volume of isopropanol is added to the above-mentioned supernatant, and it is shaken gently. It is centrifuged at 14000 rpm for 10 min, and the supernatant is discarded. 75 % ethanol is added and centrifuged again, and then the precipitate is resuspended with DEPC water to obtain RNA. The obtained RNA is stored at -80℃ for standby use.

[0098] 2) RNA concentration and quality determination: RNA concentration and purity were determined using Nanodrop 2000.

[0099] 3) Reverse transcription: RNA was reverse transcribed into cDNA using TransScript® All-in-One First-Strand cDNA Synthesis SuperMix for qPCR kit.

[0100] The reverse transcription reaction system was prepared according to the following Table 1:

[0101] Table 1 Reverse transcription system

[0102]

[0103] The reverse transcription reaction was performed: the above system was mixed gently, incubated at 45°C for 15 min, and the genomic DNA and reverse transcription reaction were removed; TransScript® RT / RI and gDNA Remover were inactivated by heating at 85°C for 5 s.

[0104] 4) Real-time fluorescent quantitative PCR reaction

[0105] The method of qRT-PCR was used to determine the effect of L. plantarum EVs on the expression of genes related to the ceramide synthesis pathway (SPT, GCS, SMase, and CerS3 genes).

[0106] Table 2 qRT-PCR amplification system

[0107]

[0108] Real-time fluorescent quantitative PCR was performed using Takala kit TB Green® Premix Ex Taq™ II (Tli RNaseH Plus), and the amplification system and amplification procedure are shown in Tables 2 and 3. The qPCR primers used in this experiment are shown in Table 4. First, the mixed reaction system Mix was prepared, and ddH2O, upstream and downstream primers, TB Green enzyme and ROX were added in the tube according to Table 2, and then mixed and centrifuged briefly. In the 8-tube tube, DNA template and mix solution were added, and each sample was repeated 5 times. The whole process was carried out on ice and in the dark. In all qPCR experiments, RNase-free ddH2O was used instead of template in the negative control, and GAPDH gene (housekeeping gene) was selected as the internal reference gene.

[0109] Table 3 qRT-PCR amplification procedure

[0110]

[0111] Utilizing 2 -ΔΔCT The relative expression of the above related genes was statistically analyzed and analyzed, GAPDH as internal reference, the relative expression of mRNA of target gene was calculated according to 2 －ΔΔCt .

[0112] The results are shown in Figure 6 , 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.

[0113] Table 4 primer

[0114]

[0115] Example 5: EVs derived from Lactobacillus plantarum promote the expression of skin barrier repair related genes

[0116] In order to evaluate the expression of skin barrier related genes promoted by EVs derived from Lactobacillus plantarum, skin epithelial cell line (HaCaT) cells were treated with 1% sodium dodecyl sulfate (SDS) to establish a skin barrier damage model. EVs derived from Lactobacillus plantarum were isolated by the method of Example 1, and the cell culture method refers to the method of Example 2 described above. When the cell confluence reached 80%-90%, the cell density was adjusted to 5×10 5 6-well plates, 2 mL of cell suspension was added to each well, and the plates were incubated in an incubator (37℃, 5% CO2) for 12 h. When the cell plating rate in the plate met the test requirements, the cells were divided into three groups: experimental group (SDS+EVs), model group (SDS) and blank group (DMEM), and the corresponding intervention was carried out, and 3 replicate wells were set in each group. The old culture solution in the 6-well plate was aspirated, and serum-free medium was added to all wells, except for the blank group wells, and 1% SDS was added to all other wells, and then incubated for 12 h. After 12 h of incubation, EVs were added to the experimental group for intervention, and three concentration gradients of EVs were set, 30 μg / mL, 12 μg / mL and 6 μg / mL of EVs, 100 μL of corresponding concentration of EVs was added to each well, and incubated for 24 h. Then discard the cell culture solution in all wells, add 1 mL of PBS, scrape the adherent cells with a cell scraper, and collect the cells by centrifugation at 1000 g for 3 min. Total RNA of HaCaT cells was extracted and transcribed into cDNA according to the method of Example 4, and the effect of EVs derived from Lactobacillus plantarum on the expression level of skin barrier repair related genes keratin 10, filaggrin (FLG) and desmoglein 1 (DSG1) was determined by qRT-PCR method, the primers used in this experiment are shown in Table 5.

[0117] Results as shown in Figure 7 Compared with the model group, the expression of skin barrier repair-related genes such as keratin 10 (KRT10), filaggrin (FLG), and desmoglein 1 (DSG1) was significantly up-regulated after treatment with different concentrations (6 μg / mL, 12 μg / mL, and 30 μg / mL) of L. plantarum EVs (p < 0.05), indicating that EVs can promote the expression of skin barrier-related genes and thus accelerate the repair of the skin barrier.

[0118] Table 5 Primers

[0119]

[0120] Example 6: EVs derived from L. plantarum down-regulate the expression of inflammation-related genes in HaCaT cells

[0121] The cell culture conditions and experimental operation methods were the same as in Example 5. Total RNA was extracted from HaCaT cells and transcribed into cDNA according to the method of Example 4. The effect of EVs derived from L. plantarum on the expression levels of inflammation-related genes TNF-α, IL-33, and TSLP in HaCaT cells was determined by qRT-PCR. The primers used in this experiment are shown in Table 6.

[0122] Results as shown in Figure 8 Compared with the model group, the mRNA levels of TNF-α, IL-33, and TSLP coding genes were significantly reduced after treatment with different concentrations (6 μg / mL, 12 μg / mL, and 30 μg / mL) of L. plantarum EVs, and showed a concentration-dependent manner.

[0123] Table 6 Primers

[0124]

[0125] Example 7: Effect of EVs derived from L. plantarum on a mouse skin barrier damage model

[0126] A mouse skin barrier damage model was established, and EVs derived from L. plantarum were used for intervention. The number of scratches and the skin lesion score of mice were calculated, and the skin barrier-related indicators of the back skin of mice, including the water content, trans-epidermal water loss, and the expression of barrier repair-related factor genes in the skin barrier tissue, were determined. The gene and protein levels of pro-inflammatory cytokines in the skin tissue were measured to evaluate the therapeutic effect of EVs derived from L. plantarum on skin barrier damage.

[0127] 1. Establishment and intervention of mouse skin lesion model

[0128] First, the mice were acclimated for 1 week. Balb / c mice, female, 20±2 g, were used. The abdominal and dorsal hair of the mice was removed using an electric clipper (hair removal area 2 cm x 3 cm). For the first 7 days, 150 μL of 5% sodium dodecyl sulfate (SDS) was applied continuously. Thereafter, 150 μL of 4% SDS was used every three days for modeling, and the application was continued for 24 days.

[0129] The experiment was divided into a blank group (Control), a model group (SDS), a positive group (MFC), and an EVs group (four concentration gradients were set, 6 μg / mL, 12 μg / mL, 30 μg / mL, and 100 μg / mL, respectively), with 8 mice in each group. The positive group was intervened with mometasone furoate ointment, and the blank group was intervened with normal saline. Intervention began on the 8th day of the experiment. The blank group, the positive group, and the EVs group were each applied with 100 μL of normal saline, mometasone furoate ointment, and EVs of different concentration gradients, respectively, once a day on the back skin lesions, and the application was continued for 24 days.

[0130] 2. Scratching frequency of mice

[0131] On the 10th, 15th, and 20th days of administration, the mice were placed in an undisturbed environment, and a camera was used to take pictures for 10 min. The scratching frequency of the mice was recorded.

[0132] Table 7. Scratching frequency (times / 10 min)

[0133]

[0134] Note: compared with the blank group, ### p < 0.001; compared with the model group, *** p < 0.001

[0135] The results are shown in Table 7. Compared with the blank group, the scratching frequency of the model group was significantly increased (p < 0.01); compared with the model group, the intervention of plant lactobacillus EVs significantly reduced the scratching frequency of the mice (p < 0.01), and it was concentration-dependent, with the effect of 100 μg / mL EVs being the most significant.

[0136] 3. Apparent changes in the local skin lesions of mice

[0137] Before intervention and on the last day of intervention, the apparent changes in the back skin of the mice in each group were recorded using a camera. Figure 9 Compared with the model group, the skin condition of the mice in the positive drug group was better, and the back skin scabbing was smaller. The skin condition of the mice in the EVs group was good, and the dry skin and desquamation were effectively relieved. The intervention effect of 100 μg / mL EVs was better than that of the positive group.

[0138] 4. Mouse lesion severity score

[0139] The severity of the lesions on the back of the mice was scored before the intervention and on the last day of the intervention. The scoring criteria were 0 (no symptoms), 1 (mild symptoms), 2 (moderate symptoms), and 3 (severe symptoms) (Table 8).

[0140] Table 8. Lesion severity score criteria

[0141]

[0142] The results of the scoring showed that, as shown in Figure 10 the model group, the lesion score increased; while compared with the model group, the score of the positive group and the EVs group decreased, and there was a significant difference (p < 0.05).

[0143] 5. Measurement of skin moisture content and transepidermal water loss in mice

[0144] On the 24th day after administration, the skin tester was used to detect 3 different parts of the mouse modeling area at random, and the skin stratum corneum water content and transepidermal water loss of these parts were recorded. Finally, the average value of the stratum corneum water content and transepidermal water loss of the three parts was calculated to evaluate the hydration state and barrier function of the skin.

[0145] As shown in Figure 11 compared with the model group, the skin moisture content was significantly increased (p < 0.05) and the transepidermal water loss was significantly reduced (p < 0.05) after treatment with different concentrations (6 μg / mL, 12 μg / mL, 30 μg / mL and 100 μg / mL) of L. plantarum EVs. This result showed that L. plantarum EVs could effectively restore skin moisture and improve skin barrier function.

[0146] 6. Measurement of gene expression levels of pro-inflammatory cytokines in mouse skin tissue

[0147] The back tissues of the mice in each group were homogenized, total RNA was extracted by the method of Example 4, and reverse transcribed into cDNA. The mRNA expression of related pro-inflammatory cytokines TNF-α, IL-4, IL-31 and TSLP was detected by qRT-PCR, GAPDH was used as an internal reference, and the relative expression level of each gene was calculated by the 2 -△△Ct method. The primers used in this experiment are shown in Table 9.

[0148] The experimental results are shown in Figure 12As shown, compared with the model group, the mRNA levels of TNF-a, IL-31, IL-4 and TSLP coding genes were significantly reduced (p < 0.05) after treatment with different concentrations (6 μg / mL, 12 μg / mL, 30 μg / mL and 100 μg / mL) of L. plantarum EVs, and this reduction showed a clear concentration-dependent manner. This indicates that L. plantarum EVs can inhibit the gene expression of inflammatory-related cytokines and chemokines in a concentration-dependent manner, thereby playing a significant anti-inflammatory role.

[0149] Table 9 Primers

[0150]

[0151] 7. Protein expression level determination of pro-inflammatory cytokines in mouse skin tissue

[0152] The back tissues of mice in each group were homogenized and detected by ELISA kit.

[0153] (1) ELISA detection

[0154] a. Preparation before detection: take TNF-a kit as an example

[0155] 1) Take the kit out of the refrigerator 20 min in advance and equilibrate to room temperature.

[0156] 2) Dilute the 20x concentrated washing solution with double distilled water into 1x working solution.

[0157] b. Operation steps:

[0158] 1) Take the required plate strips from the sealed bag which has been equilibrated to room temperature, and put the unused plate strips and desiccant back into the aluminum foil bag, press the self-sealing strip, seal the bag, and put it back into 4°C.

[0159] 2) Set up standard wells, blank wells and sample wells. Add different concentrations of standard 50 μL to each standard well.

[0160] 3) Add 50 μL of sample to be tested to the sample well; the blank well is not added.

[0161] 4) Add 100 μL of horseradish peroxidase (HRP) labeled detection antibody to each well of the standard well and sample well except the blank well, seal the reaction well with a plate membrane, and incubate in a 37°C incubator or water bath for 60 min.

[0162] 5) Discard the liquid, pat dry on the blotting paper, and add enough washing solution (350 μL) to each well; stand for 1 min, shake off the washing solution, pat dry with blotting paper, and repeat the plate washing for 5 times.

[0163] 6) Add 50 μL of substrate A and B to each well, and incubate at 37°C for 15 min in the dark.

[0164] 7) Add 50 μL of stop solution to each well, and measure the OD value of each well at 450 nm within 15 min (within 3 min).

[0165] c. Result judgment:

[0166] The OD value of each standard and sample should be subtracted from the OD value of the blank well. With the standard concentration as the horizontal coordinate and the OD value as the vertical coordinate, the software draws and selects the best fitting curve. The concentration of the sample can be determined on the standard curve by the OD value of the sample.

[0167] (2) TNF-a, IL-1β and IL-6 content calculation

[0168] The standard solution of TNF-a, IL-1β and IL-6 was prepared, and a series of solutions with known concentrations were diluted by proportioning. The OD value at 450 nm was measured by the above ELISA method, and the regression equation of the standard curve was fitted. The OD450 value of each test sample was brought into the equation, and the TNF-a, IL-1β and IL-6 content in the test sample was calculated. Each group was tested in triplicate, and the average value and standard deviation (SD) were calculated. The TNF-a, IL-1β and IL-6 contents of each sample group measured by the above experiment and the blank control group were analyzed by single factor analysis of variance, and p < 0.05 was considered to be statistically significant.

[0169] As shown in Figure 13 compared with the model group, the protein levels of TNF-a, IL-1β and IL-6 were significantly reduced after treatment with different concentrations (6 μg / mL, 12 μg / mL, 30 μg / mL and 100 μg / mL) of L. plantarum EVs (p < 0.05). This result showed that L. plantarum EVs could inhibit the protein expression of inflammatory-related cytokines in a concentration-dependent manner, thereby effectively exerting anti-inflammatory effect.

[0170] 8. Determination of the gene expression level of skin barrier repair-related cytokines in the skin tissue of mice

[0171] The back tissues of each group of mice were homogenized, total RNA was extracted, and cDNA was reverse transcribed. The mRNA expression of skin barrier-related cytokines filaggrin, keratin 10 and ceramide synthase 3 was detected by qRT-PCR, GAPDH was used as an internal reference, and the relative expression level of each gene was calculated by 2 -△△Ct method. The primers used in this experiment are shown in Table 10.

[0172] As shown in Figure 14 As shown in Figure 8, compared with the model group, the mRNA levels of skin barrier-related genes FLG, KRT10 and CerS3 were significantly up-regulated (p < 0.05) after treatment with different concentrations (6 μg / mL, 12 μg / mL, 30 μg / mL and 100 μg / mL) of L. plantarum EVs, and this up-regulation showed a clear concentration-dependent effect. This indicates that L. plantarum EVs can effectively repair the skin barrier function of skin-damaged mice induced by SDS and promote the repair of the skin barrier.

[0173] Table 10 Primers

[0174]

[0175] 9. Measurement of the expression levels of skin itching-related cytokine genes in mouse skin tissue

[0176] The back tissues of the mice in each group were homogenized, total RNA was extracted, and reverse-transcribed into cDNA. The mRNA expression of skin itching-related cytokines IL-31RA and OSMA was detected by qRT-PCR, GAPDH was used as an internal reference, and the relative expression levels of each gene were calculated using the 2 -△△Ct method. The primers used in this experiment are shown in Table 11.

[0177] As shown in Figure 15 As shown in Figure 8, compared with the model group, the mRNA levels of skin barrier-related genes FLG, KRT10 and CerS3 were significantly up-regulated (p < 0.05) after treatment with different concentrations (6 μg / mL, 12 μg / mL, 30 μg / mL and 100 μg / mL) of L. plantarum EVs, and this up-regulation showed a clear concentration-dependent effect. This indicates that L. plantarum EVs can effectively repair the skin barrier function of skin-damaged mice induced by SDS and promote the repair of the skin barrier.

[0178] Table 11 Primers

[0179]

[0180] Example 8: Absorption and distribution of EVs derived from L. plantarum in mouse skin

[0181] Through the mouse back skin smearing experiment, the uptake and distribution of extracellular vesicles (EVs) derived from L. plantarum in skin tissue were observed, and the transdermal absorption characteristics were evaluated. In the experiment, the labeled L. plantarum EVs were evenly smeared on the surface of the mouse back skin, and then the penetration, uptake and distribution dynamics of EVs in the skin were monitored in real time using live imaging technology. By analyzing the imaging data, the transdermal ability of L. plantarum EVs and their absorption efficiency in skin tissue were systematically evaluated.

[0182] 1. Mouse treatment

[0183] Before the experiment, the experimental group (EVs + DIO) and the blank group (DIO) mice were depilated. The depilation operation was performed one day in advance to ensure that the skin surface was clean and free of hair interference with subsequent experimental operations. The depilation method referred to the established Example 7, and the operation process avoided damage to the mouse skin to ensure the accuracy and reliability of the experimental results.

[0184] 2. Fluorescent labeling of EVs

[0185] The labeling method was completed according to Example 2.

[0186] 3. In vivo efficacy observation

[0187] The labeled EVs were evenly applied to the skin of the mouse back, with a dosage of 100 μL per mouse. During the operation, attention should be paid to avoid light to prevent quenching of the fluorescent signal. 2 h after application, the mouse was gently placed in the dark box platform of the in vivo imaging system, the field of view was adjusted, and the background image was taken to ensure the accuracy of the imaging. Subsequently, the fluorescent signal in the mouse was photographed under dark field conditions, and the distribution of EVs in the skin tissue was recorded in detail.

[0188] As shown in Figure 16 , the EVs labeled with DIO dye showed green fluorescence. The experimental results showed that the EVs could penetrate into the inside of the mouse back skin only 2 h after application. This phenomenon indicated that the EVs derived from P. luminescens had good transdermal absorption capacity and could be quickly taken up and distributed by the mouse skin.

[0189] Example 9: Proteomic analysis of EVs derived from P. luminescens

[0190] The extraction method of EVs from P. luminescens was referred to Example 1. The protein concentration of the EVs sample was determined using BCA reagent to ensure that the protein concentration of each sample was higher than 200 μg / mL. Subsequently, the EVs sample was subjected to lysis treatment, and the proteins in the sample were identified using liquid chromatography-mass spectrometry (LC-MS / MS) technology. The mass spectrometry data were analyzed by mass spectrometry data analysis software (such as MaxQuant or Proteome Discoverer), the protein types in the vesicles were identified, and the identified proteins were functionally annotated and pathway enrichment analyzed using bioinformatics tools (such as Gene Ontology, KEGG pathway analysis).

[0191] The results showed that proteomic analysis of EVs derived from P. luminescens identified 1537 proteins. As shown in Figure 17As shown, KEGG pathway analysis showed that proteins related to metabolic functions accounted for a major proportion of total proteins, about 61.76% of the total protein.

[0192] Example 10: Metabolomics analysis of EVs derived from Lactobacillus plantarum

[0193] The extraction method of EVs of Lactobacillus plantarum refers to Example 1. Then, an appropriate amount of metabolite extraction reagent was added to the EVs sample to be tested, and after thorough shaking and mixing, immediate liquid nitrogen freezing treatment was performed, followed by ultrasonic fragmentation technology to release the contents in the EVs. After freezing centrifugation, the supernatant was collected and transferred to a liquid chromatography-mass spectrometry (LC-MS) special sample bottle. The sample was subjected to metabolomics analysis using a liquid chromatography-tandem mass spectrometry (LC-MS / MS) system.

[0194] Relative abundance is a dimensionless number representing the proportion of the intensity of a specific ion signal in all ion signals. As shown in Figure 18 As shown, lipids and their derivatives had the highest content in the metabolome. Further analysis showed that among lipids and their derivatives, the average relative abundance of trans-11-octadecenoic acid (VA) was 102377.9927, which had a high content and had anti-inflammatory and barrier repair potential, and was a key active ingredient in EVs.

[0195] Example 11: Analysis and verification of key active ingredient trans-11-octadecenoic acid in EVs derived from Lactobacillus plantarum

[0196] High-purity trans-11-octadecenoic acid (VA) was obtained by in vitro synthesis technology, and HaCaT cells were intervened using VA concentrations of 6 μg / mL and 15 μg / mL. In the experiment, the simple cell culture medium and vitamin C were used as blank control group and positive control group, respectively. Then, the ceramide production, barrier repair-related factors and gene expression of inflammatory factors in the cells were determined according to Examples 3, 4, 5 and 6, respectively.

[0197] As shown in Figure 19 The results showed that VA intervention significantly increased the ceramide production of HaCaT cells. As shown in Figure 20 The results of the SDS-induced damaged cell model showed that VA intervention significantly up-regulated the gene expression levels of protein factors closely related to skin barrier function, including filaggrin, ceramide synthase 3 and keratin 10, etc. As shown in Figure 21 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.

[0198] The above described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present application.

Claims

1. An extracellular vesicle of Lactobacillus plantarum, characterized in that, The extracellular vesicle is from a plant lactobacillus (L25) Lactiplantibacillus plantarum The L25 is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 35146, the average particle size of the extracellular vesicle of the plant lactobacillus is 100-200 nm, and the spherical morphology is observed under a transmission electron microscope; and the extracellular vesicle is prepared by the following method: (1) the strain is cultured in a liquid medium, and then the bacterial culture is centrifuged to remove the bacterial body precipitate and obtain the supernatant of the bacterial culture; (2) the supernatant is filtered using 0.45 μm and 0.22 μm filters to remove residual bacteria and cell fragments; (3) the sample is then concentrated and eluted using a membrane pack, and then the concentrated sample is filtered using a 0.22 μm filter; (4) the filtered sample is subjected to ultracentrifugation to remove the supernatant, obtain the EVs precipitate derived from Lactiplantibacillus plantarum, and resuspend the precipitate in PBS; (5) repeat the ultracentrifugation according to step (4), finally resuspend the precipitate in PBS, and store after aliquoting at -80℃ or obtain a freeze-dried powder by freeze-drying for standby use.

2. A method for preparing an extracellular vesicle of Lactobacillus plantarum, characterized by, The method comprises the following steps: (1) the strain is cultured in a liquid medium, and then the bacterial culture is centrifuged to remove the bacterial body precipitate and obtain the supernatant of the bacterial culture; (2) the supernatant is filtered using 0.45 μm and 0.22 μm filters to remove residual bacteria and cell fragments; (3) the sample is then concentrated and eluted using a membrane pack, and then the concentrated sample is filtered using a 0.22 μm filter; (4) the filtered sample is subjected to ultracentrifugation to remove the supernatant, obtain the EVs precipitate derived from Lactiplantibacillus plantarum, and resuspend the precipitate in PBS; (5) repeat the ultracentrifugation according to step (4), finally resuspend the precipitate in PBS, and store after aliquoting at -80℃ or obtain a freeze-dried powder by freeze-drying for standby use. Among them, the plant lactobacillus ( Lactiplantibacillus plantarum The strain is L25, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35146.

3. The method of claim 2, wherein, The medium in step (1) is MRS liquid medium; the culture time is 12-18 h, the centrifugal speed is 5000-8000 g, and the centrifugal time is 5-20 min; the ultracentrifugation in step (4) is 120000 g-150000 g, and the centrifugal time is 1-2 h.

4. Use of the extracellular vesicles of Lactobacillus plantarum of claim 1 for the preparation of a formulation for repairing the skin barrier, characterized in that, The concentration of extracellular vesicles in the preparation is 6-120 μg / mL.

5. Use of the extracellular vesicles of Lactobacillus plantarum of claim 1 for the preparation of a preparation for reducing inflammation of the skin, characterized in that, The concentration of extracellular vesicles in the preparation is 6-120 μg / mL.

6. Use of the extracellular vesicles of Lactobacillus plantarum of claim 1 in the manufacture of a preparation for reducing skin itch, characterized in that, The concentration of extracellular vesicles in the preparation is 6-120 μg / mL.

7. Use of the extracellular vesicles of Lactobacillus plantarum of claim 1 in the preparation of a formulation as a carrier for the rapid transdermal absorption of the skin, characterized in that, The concentration of extracellular vesicles in the preparation is 6-120 μg / mL. The concentration of extracellular vesicles in the preparation is 6-120 μg / mL.

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