Short peptides from lacticaseibacillus plantarum extracellular vesicles and uses thereof

Abstract

The present application belongs to the field of microbial technology, and particularly relates to a short peptide from an extracellular vesicle of plant lactobacillus and application thereof. Specifically, the plant lactobacillus extracellular vesicle can restore skin barrier, can reduce the expression of proinflammatory factors in a cell inflammation model, and has good anti-inflammatory effect. Through metabolic analysis, it is found that the effective material effective for skin barrier damage includes a short peptide RFV; RFV can regulate the expression of skin barrier related genes in a SDS induced damaged cell model; and can regulate the expression of skin inflammation related genes in the SDS induced damaged cell model, which indicates that the short peptide can be used as a drug for treating skin barrier damage.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a short peptide from extracellular vesicles of Lactiplantibacillus plantarum 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. 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. The synthesis and metabolism of ceramides involve multiple enzymes, such as sphingomyelinase (acidic), serine palmitoyltransferase (SPT), glucosylceramide synthase (GCS), and ceramide synthase (CerS) family.

[0004] Bacterial extracellular vesicles (BEVs) are nanoscale membrane particles secreted by gram-negative and gram-positive bacteria, usually with a diameter of 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 transfer, and antibiotic resistance gene transfer. Probiotic extracellular vesicles are extracellular vesicles secreted by probiotics, which have similar structures and functions to bacterial extracellular vesicles, and can regulate the immune response of the host, inhibit the inflammatory response, and play an important role in intestinal health, immune regulation, and disease prevention. For example, EVs of Lactobacillus rhamnosus can significantly reduce the production of pro-inflammatory factors TNF-α and IL-6. Extracellular vesicles of Lactiplantibacillus plantarum are nanoscale vesicles secreted by Lactiplantibacillus plantarum. Studies have shown that EVs have 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 the plant lactobacillus extracellular vesicles and their application in repairing skin barrier, and the analysis of the efficacy components of bacterial extracellular vesicles.

[0006] In the previously disclosed patents or patent applications, although there have been reports of vesicles for treating inflammatory diseases, there is no report on plant lactobacillus extracellular vesicles repairing skin barrier by promoting ceramide generation and relieving skin disease problems. At the same time, there is no report on how bacterial extracellular vesicles can improve the content of skin ceramide by regulating multiple genes or molecular mechanisms related to ceramide synthesis. 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 deficiencies in the prior art and provide a short peptide from plant lactobacillus extracellular vesicles and its application.

[0008] The first aspect of the present application is to provide a short peptide from plant lactobacillus extracellular vesicles, characterized in that the short peptide is RFV.

[0009] The second aspect of the present application is to provide a preparation for treating skin damage, characterized in that the preparation comprises the short peptide of the first aspect and a pharmaceutically acceptable carrier.

[0010] Further, the preparation dosage form includes at least one of suspension, granules, capsules, powders, tablets, drop pills, suppositories, drops.

[0011] Further, the administration concentration of the short peptide is 6 μg / mL-15 μg / mL.

[0012] Further, the skin damage includes skin barrier damage, inflammation, itching.

[0013] The third aspect of the present application is to provide the use of the short peptide of the first aspect in the preparation of a preparation for repairing skin barrier.

[0014] Further, the administration concentration of the short peptide in the preparation is 6 μg / mL-15 μg / mL.

[0015] The fourth aspect of the present application provides the use of the short peptide of the first aspect in the preparation of a preparation for reducing skin inflammation.

[0016] Further, the administration concentration of the short peptide in the preparation is 6 μg / mL-15 μg / mL.

[0017] The fifth aspect of the present application provides the use of the short peptide in the preparation of a preparation for reducing skin itching.

[0018] Further, the administration concentration of the short peptide in the preparation is 6 μg / mL-15 μg / mL.

[0019] The sixth aspect of the present application provides a pharmaceutical composition, characterized in that the pharmaceutical composition comprises the short peptide of the first aspect and other active components of the plant Lactiplantibacillus extracellular vesicles.

[0020] Further, the other active components are lipid, protein or nucleic acid components.

[0021] Further, the plant Lactiplantibacillus is Lactiplantibacillus plantarum L25, which is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 35146, and the preservation address is No. 1, Beichen West Road, Yuanmingyuan District, Beijing.

[0022] The beneficial effects of the present application include:

[0023] 1) The plant Lactiplantibacillus extracellular vesicles of the present application can promote the generation of ceramides 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.

[0024] 2) The EVs have good skin penetration ability, can significantly reduce the skin lesion 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, as verified by a mouse skin injury model.

[0025] 3) Metabolomics analysis shows that the effective substance effective for skin barrier damage includes the short peptide RFV.

[0026] 4) Experiments have proved that RFV regulates the expression of skin barrier-related genes in a SDS-induced damaged cell model.

[0027] 5) RFV regulates the expression of skin inflammation-related genes in a SDS-induced damaged cell model.

[0028] The microorganism of the present application is Lactobacillus 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. The deposit address is No. 1, Yihuangyuan, Beichen West Road, Beijing, China. Lactiplantibacillus plantarum BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 EVs sedimentation sample figure.

[0030] Figure 2 Transmission electron microscopy of EVs.

[0031] Figure 3 Particle size of EVs.

[0032] Figure 4 Fluorescent staining of EVs being taken up by cells.

[0033] Figure 5 EVs improve ceramide production in HaCaT cells.

[0034] Figure 6 EVs modulate the expression of key enzymes in HaCaT cell ceramide synthesis.

[0035] Figure 7 EVs modulate the expression of skin barrier-related genes in SDS-induced damaged cell models.

[0036] Figure 8 EVs modulate the expression of skin inflammation-related genes in SDS-induced damaged cell models.

[0037] Figure 9 Appearance of EVs relieving SDS-induced skin damage in mice.

[0038] Figure 10 Mouse skin lesion score.

[0039] Figure 11 Moisture content and trans-epidermal water loss in mice.

[0040] Figure 12 EVs reduce the expression of inflammatory genes in mouse skin tissue.

[0041] Figure 13 EVs reduce the expression of inflammatory proteins in mouse skin tissue.

[0042] Figure 14 EVs improve the expression of skin barrier-related genes in mice.

[0043] ​Figure 15 EVs inhibit the expression of genes related to skin itching in mice.

[0044] Figure 16 Absorption and distribution of EVs in mouse skin.

[0045] Figure 17 Proteomic analysis of EVs.

[0046] Figure 18 Metabolomics analysis of EVs.

[0047] Figure 19 RFV regulates the expression of skin barrier-related genes in an SDS-induced damaged cell model.

[0048] Figure 20 RFV regulates the expression of skin inflammation-related genes in an SDS-induced damaged cell model. Detailed Implementation

[0049] The following detailed embodiments further illustrate the concept and technical effects of the present invention to fully understand its purpose, features, and effects. Unless otherwise specified, all methods described are conventional methods. Unless otherwise specified, all materials are available from publicly available commercial sources. The illustrative embodiments and descriptions of the present invention are used to explain the invention and do not constitute an undue limitation thereof. It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0050] Example 1: Isolation and Characterization Evaluation of EVs Derived from Lactobacillus plantarum

[0051] 1. Isolation of EVs derived from Lactobacillus plantarum

[0052] The *Lactobacillus plantarum* strain used was isolated from kimchi samples by the Applied Microbiology and Enzyme Engineering Laboratory of the School of Bioengineering, Tianjin University of Science and Technology. The strain was cultured in MRS liquid medium at 37°C for 14 h. The bacterial culture was then centrifuged at 6000 g for 10 min to remove the bacterial precipitate, obtaining the supernatant. The supernatant was filtered using 0.45 μm and 0.22 μm top-mounted filters to remove residual bacteria and cell debris. The sample was then concentrated and eluted using a membrane filter, followed by filtration through a 0.22 μm filter. The filtered sample was then ultracentrifuged at 140,000 g for 1.5 h to remove the supernatant, yielding the EVs precipitate derived from *Lactobacillus plantarum*. Figure 1 The precipitate was resuspended in PBS. The ultracentrifugation was repeated once with the above parameters. Finally, the precipitate was resuspended in PBS, aliquoted, and stored at -80°C for later use. The protein concentration of EVs was measured using the BCA assay.

[0053] 2. Characterization of EVs

[0054] (1) Morphological observation

[0055] 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.

[0056] (2) Particle size distribution measurement

[0057] The particle size distribution of EVs isolated from bacterial culture was measured using dynamic light scattering (DLS). The diameter of EVs was measured using a Zetasizer Nano S, and the results are shown in Figure 3 , the EVs particle size was 132 ± 2.24 nm.

[0058] (3) Concentration measurement

[0059] 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 in the sample. The capture duration was set to 30 seconds to obtain the data. The experimental results showed that the concentration of EVs was 3.2 x 10 9 particles / mL.

[0060] Example 2: Uptake of EVs derived from Lactobacillus plantarum in HaCaT cells

[0061] 1. Cell culture

[0062] 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 trypsinized and passaged, and the medium was changed 2-3 times per week.

[0063] 2. Fluorescent labeling of EVs

[0064] The isolated Lactobacillus plantarum 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 3 times with PBS to remove unbound dye.

[0065] 3. Laser confocal microscope observation

[0066] When the confluence of HaCaT cells reached 80%~90%, the cell suspension was obtained by the above cell passage method, and the cell density was adjusted to 2x10 5 individuals / mL. The cell suspension was added to the 24-well plate in which the cell crawling sheet was previously placed, 1000 μL of cell suspension was added to each well, and it was continued to be cultured in the incubator (37°C, 5% CO2) for 12 h. When the cell plating rate in the well plate met the test requirements, all the old culture medium in the 24-well plate was first aspirated and discarded, and serum-free medium was added. The experimental group was added with EVs labeled in advance with DIO (30 μg / mL), and incubated for 12 h, paying 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 the cells were washed with PBS for 3 times, DAPI dye was added to stain the cell nucleus, and after 5 min, the cells were washed with PBS for 3 times, and finally a small amount of PBS was added to prevent the cells from drying. Laser confocal microscope was used for observation and photography.

[0067] The results are shown in Figure 4 , the EVs labeled with DIO dye showed green fluorescence, and the cell nucleus stained with DAPI dye showed blue fluorescence, and it could be clearly seen that EVs could penetrate into the surrounding cells, indicating that EVs could be taken up by cells.

[0068] Example 3: EVs derived from plant Lactobacillus promote the generation of ceramide by HaCaT cells

[0069] The cell culture method is referred to Example 2. When the cell confluence reached 80%~90%, the adherent cells were digested with trypsin, and the cell density was adjusted to 2x10 5 individuals / mL. The cell suspension was added to the 24-well plate, 1000 μL of cell suspension was added to each well, and it was continued to be cultured in the incubator (37°C, 5% CO2) for 12 h. When the cell plating rate in the well plate met the test requirements, the old culture medium was discarded, and serum-free medium was added. The experimental group was set at 3 concentration gradients, 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 was cultured for 24 h. After the culture was completed, the adherent cells were scraped with a cell scraper, the cells were collected by centrifugation at 1000 g for 3 min, and then ELISA detection was performed.

[0070] 1. Preparation before detection

[0071] (1) Take the kit out of the refrigerator 20 min in advance to balance to room temperature;

[0072] (2) The 30-fold concentrated washing solution is diluted 30 times with distilled water and used as needed.

[0073] 2. Operation steps

[0074] (1) Take out the required board from the sealed bag that has been equilibrated to room temperature;

[0075] (2) Set up standard holes, blank holes and sample holes, and add 50 μL of standard of different concentrations to each standard hole;

[0076] (3) Add 50 μL of sample to be tested to the sample hole, and add nothing to the blank hole;

[0077] (4) Set up a blank hole (the blank control hole does not add sample and enzyme-labeled reagent, and the rest of the steps are the same) and a sample hole. Add 40 μL of sample diluent to the sample hole 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;

[0078] (5) Discard the liquid, dry it on a blotting paper, and add full washing solution (350 μL) to each hole. Rest for 30 seconds, shake off the washing solution, dry it on a blotting paper, and repeat the plate washing 5 times;

[0079] (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;

[0080] (7) Washing: the operation is the same as in 5;

[0081] (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, gently shake to mix, and develop color at 37°C in the dark for 15 min;

[0082] (9) Add 50 μL of termination solution to each hole, and measure the OD value of each hole at 450 nm within 15 min.

[0083] 3. Result judgment

[0084] 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.

[0085] 4. Ceramide content calculation

[0086] The standard sample of ceramide was configured into a standard solution, and was diluted into a series of solutions with known concentrations in proportion and step by step. The OD value (OD 450) at 450 nm was measured by the ELISA method described above, 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 experiment was subjected to one-way ANOVA with the blank control group by using SPSS statistical software, and p < 0.05 was considered to have statistically significant difference.

[0087] The results are shown in Figure 5 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 comparable to that of the positive control group.

[0088] Example 4: EVs derived from Lactobacillus plantarum up-regulate the expression of key genes in the ceramide synthesis pathway of HaCaT cells

[0089] 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. The experimental group is set at three concentration gradients, i.e. 30 μg / mL, 12 μg / mL and 6 μg / mL of EVs, and 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 three 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.

[0090] The total RNA of the collected HaCaT cells is extracted by using the TRIZOL kit, and the specific steps are as follows:

[0091] 1) Extraction of RNA: 1 mL of TRIZOL reagent is added to the above 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 parameters, and the supernatant is collected. Then an equal volume of isopropanol is added to the above supernatant, shaken gently, centrifuged at 14000 rpm for 10 min, the supernatant is discarded, 75 % ethanol is added and centrifuged again, then the precipitate is resuspended with DEPC water to obtain RNA. The obtained RNA is stored at -80℃ for standby use.

[0092] 2) Determination of RNA concentration and quality: The RNA concentration and purity are determined by using Nanodrop 2000.

[0093] 3) Reverse transcription: Reverse transcription of RNA into cDNA with TransScript® All-in-One First-Strand cDNA Synthesis SuperMix for qPCR kit.

[0094] The reverse transcription reaction system was prepared according to the following Table 1:

[0095] Table 1 Reverse transcription system

[0096]

[0097] 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.

[0098] 4) Real-time fluorescent quantitative PCR reaction

[0099] Using the method of qRT-PCR, the effect of L. plantarum EVs on the expression of genes related to the ceramide synthesis pathway (SPT, GCS, SMase and CerS3 genes) was determined.

[0100] Table 2 qRT-PCR amplification system

[0101]

[0102] Real-time fluorescent quantitative PCR was performed using the 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.

[0103] Table 3 qRT-PCR amplification procedure

[0104]

[0105] Using 2 -ΔΔCTThe relative expression of the above related genes was statistically analyzed and analyzed, GAPDH was used as an internal reference, and the relative expression of the mRNA of the target gene was calculated according to 2 －ΔΔCt Calculation.

[0106] The results are shown in Figure 6 Fig. 2, and the EVs derived from L. 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 the EVs can promote the synthesis of ceramide in HaCaT cells.

[0107] Table 4 primers

[0108]

[0109] Example 5: EVs derived from L. plantarum promote the expression of skin barrier repair related genes

[0110] In order to evaluate the expression of skin barrier related genes promoted by EVs derived from L. plantarum, a skin barrier damage model was established by treating skin epithelial cell line (HaCaT) cells with 1% sodium dodecyl sulfate (SDS). The EVs derived from L. 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 5x10 5 6-well plates, 2 mL of cell suspension was added to each well, and the plates were further incubated in an incubator (37°C, 5% CO2) for 12 h. When the cell plating rate in the plate met the test requirements, the cells were divided into three groups: the experimental group (SDS+EVs), the model group (SDS) and the blank group (DMEM), and the corresponding intervention was performed, 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, i.e. 30 μg / mL, 12 μg / mL and 6 μg / mL of EVs, 100 μL of EVs of the corresponding concentration was added to each well, and the incubation was continued for 24 h. Then the cell culture solution in all wells was discarded, 1 mL of PBS was added, and the adherent cells were scraped with a cell scraper, and the cells were collected by centrifugation at 1000 g for 3 min. The 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 L. 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, and the primers used in this experiment are shown in Table 5.

[0111] The results are as follows Figure 7 As shown, compared with the model group, treatment with different concentrations (6 μg / mL, 12 μg / mL and 30 μg / mL) of *Lactobacillus plantarum* EVs significantly upregulated the expression of skin barrier repair-related genes such as keratin 10 (KRT10), filaggrin (FLG) and desmosome core glycoprotein 1 (DSG1) (p < 0.05), indicating that EVs can promote the expression of skin barrier-related genes, thereby accelerating skin barrier repair.

[0112] Table 5 Primers

[0113]

[0114] Example 6: EVs derived from Lactobacillus plantarum downregulate the expression of inflammation-related genes in HaCaT cells

[0115] The cell culture conditions and experimental procedures were the same as in Example 5. Total RNA was extracted from HaCaT cells and transcribed into cDNA according to the method in Example 4. The effects 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 were determined using qRT-PCR. The primers used in this experiment are shown in Table 6.

[0116] The results are as follows Figure 8 As shown, compared with the model group, treatment with different concentrations (6 μg / mL, 12 μg / mL and 30 μg / mL) of *Lactobacillus plantarum* EVs significantly reduced the mRNA levels of the genes encoding TNF-α, IL-33 and TSLP, and this reduction was concentration-dependent.

[0117] Table 6 Primers

[0118]

[0119] Example 7: Effects of EVs derived from Lactobacillus plantarum on a mouse model of skin barrier damage

[0120] A mouse skin barrier injury model was established, and EVs derived from *Lactobacillus plantarum* were used for intervention. The number of scratches and skin lesion scores of mice were calculated, and skin barrier-related indicators on the back of mice, including water content, transepidermal water loss, and the 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 to evaluate the therapeutic effect of EVs derived from *Lactobacillus plantarum* on skin barrier injury.

[0121] 1. Establishment and intervention of mouse skin lesion model

[0122] 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.

[0123] 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.

[0124] 2. Scratching frequency of mice

[0125] 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.

[0126] Table 7. Scratching frequency (times / 10 min)

[0127]

[0128] Note: Compared with the blank group, ### p < 0.001; compared with the model group, *** p < 0.001

[0129] 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.

[0130] 3. Apparent changes in local skin lesions of mice

[0131] 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.

[0132] 4. Mouse lesion severity score

[0133] 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).

[0134] Table 8. Lesion severity score criteria

[0135]

[0136] The results of the scoring showed that, as shown in Figure 10 the model group, the lesion score value increased; while relative to the model group, the score values of the positive group and the EVs group were reduced, and there was a significant difference (p < 0.05).

[0137] 5. Measurement of skin moisture content and transepidermal water loss in mice

[0138] On the 24th day after administration, the skin test instrument 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.

[0139] As shown in Figure 11 compared with the model group, after treatment with different concentrations (6 μg / mL, 12 μg / mL, 30 μg / mL and 100 μg / mL) of L. plantarum EVs, the skin moisture content was significantly increased (p < 0.05), while the transepidermal water loss was significantly reduced (p < 0.05). This result showed that L. plantarum EVs could effectively restore skin moisture and improve skin barrier function.

[0140] 6. Measurement of gene expression levels of pro-inflammatory cytokines in mouse skin tissue

[0141] The back tissues of the mice in each group were homogenized, total RNA was extracted by the method of Example 4, reverse transcribed into cDNA, and qRT-PCR was used to detect the mRNA expression of related pro-inflammatory cytokines TNF-α, IL-4, IL-31 and TSLP, with GAPDH as an internal reference. 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.

[0142] 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 exerting a significant anti-inflammatory effect.

[0143] Table 9 Primers

[0144]

[0145] 7. Protein expression level determination of pro-inflammatory cytokines in mouse skin tissue

[0146] The back tissues of mice in each group were homogenized and detected by ELISA kit.

[0147] (1) ELISA detection

[0148] a. Preparation before detection: take TNF-a kit as an example

[0149] 1) Take the kit out of the refrigerator 20 min in advance and equilibrate to room temperature.

[0150] 2) Dilute the 20x concentrated washing solution with double distilled water to 1x working solution.

[0151] b. Operation steps:

[0152] 1) Take the required strips from the sealed bag which has been equilibrated to room temperature. Unused strips and desiccant should be put back into the aluminum foil bag, compacted and sealed, and stored at 4°C.

[0153] 2) Set up standard wells, blank wells and sample wells. Add different concentrations of standards to each standard well, 50 μL.

[0154] 3) Add 50 μL of sample to be tested to the sample well; no addition to the blank well.

[0155] 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 sealing film, and incubate in a 37°C incubator or water bath for 60 min.

[0156] 5) Discard the liquid, pat dry on a paper towel, and add enough washing solution (350 μL) to each well; stand for 1 min, shake off the washing solution, pat dry on a paper towel, and repeat the plate washing for 5 times.

[0157] 6) Add 50 μL of substrate A and B to each well, and incubate at 37°C for 15 min in the dark.

[0158] 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).

[0159] c. Result determination:

[0160] 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.

[0161] (2) TNF-a, IL-1β and IL-6 content calculation

[0162] 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 variance analysis using SPSS statistical software, and p < 0.05 was considered to be statistically significant.

[0163] 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.

[0164] 8. Determination of the gene expression level of skin barrier repair-related cytokines in the skin tissue of mice

[0165] 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 the 2 -△△Ct method. The primers used in this experiment are shown in Table 10.

[0166] 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.

[0167] Table 10 Primers

[0168]

[0169] 9. Measurement of the expression levels of skin itch-related cytokine genes in mouse skin tissue

[0170] 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 itch-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.

[0171] 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.

[0172] Table 11 Primers

[0173]

[0174] Example 8: Absorption and distribution of EVs derived from L. plantarum in mouse skin

[0175] The uptake and distribution of EVs derived from L. plantarum in skin tissue were observed by applying the EVs to the back skin of mice, and the transdermal absorption characteristics of the EVs were evaluated. In the experiment, labeled L. plantarum EVs were evenly applied to the surface of the back skin of mice, and then the penetration, uptake and distribution of the EVs in the skin were monitored in real time using live imaging technology. The transdermal ability of L. plantarum EVs and their absorption efficiency in skin tissue were systematically evaluated by analyzing the imaging data.

[0176] 1. Mouse treatment

[0177] 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.

[0178] 2. Fluorescent labeling of EVs

[0179] The labeling method was completed according to Example 2.

[0180] 3. In vivo efficacy observation

[0181] 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.

[0182] 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.

[0183] Example 9: Proteomic analysis of EVs derived from P. luminescens

[0184] The extraction method of EVs from P. luminescens referred to Example 1. The protein concentration of the EVs sample was determined using BCA reagent, and the protein concentration of each sample was ensured to be 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).

[0185] The results showed that proteomic analysis of EVs derived from P. luminescens identified 1537 proteins. As shown in Figure 17As shown, KEGG pathway analysis indicates that proteins related to metabolic function account for the majority of total protein, approximately 39.43%.

[0186] Example 10: Metabolomics analysis of EVs derived from Lactobacillus plantarum

[0187] The extraction method for *Lactobacillus plantarum* EVs was as described in Example 1. Then, an appropriate amount of metabolite extraction reagent was added to the EV sample to be tested. After thorough shaking and mixing, the sample was immediately flash-frozen in liquid nitrogen, followed by ultrasonic disruption to release the substances contained within the EVs. After freeze-centrifugation, the supernatant was collected and transferred to a dedicated liquid chromatography-mass spectrometry (LC-MS) sample vial. Metabolomics analysis of the sample was performed using a liquid chromatography-tandem mass spectrometry (LC-MS / MS) system.

[0188] like Figure 18 As shown, in non-targeted metabolomics analysis, arginine-phenylalanine-valine (Arg-Phe-Val) (RFV) ranked among the top 20 in relative abundance among total metabolites, with a significantly higher relative abundance than other metabolites. Further analysis indicated that Arg-Phe-Val possesses anti-inflammatory and barrier repair potential and is a key active ingredient in EVs.

[0189] Example 11: Analysis and verification of key active components arginine-phenylalanine-valine in EVs derived from Lactobacillus plantarum

[0190] High-purity arginine-phenylalanine-valine (Arg-Phe-Val) was obtained through in vitro synthesis. HaCaT cells were treated with Arg-Phe-Val at concentrations of 6 μg / mL and 15 μg / mL. In the experiment, simple cell culture medium and SDS were used as the blank control group and model group, respectively. Then, the gene expression of barrier repair-related factors and inflammatory factors in the cells was measured according to Examples 4, 5, and 6.

[0191] like Figure 19 As shown, the results of the skin injury cell model indicate that Arg-Phe-Val intervention significantly upregulated the expression levels of protein factor genes closely related to skin barrier function, including filaggrin and ceramide synthase 3. Figure 20 As shown, Arg-Phe-Val treatment also significantly reduced the expression levels of inflammatory factors such as IL-25 and TSLP. In conclusion, arginine-phenylalanine-valine is a key effector molecule in EVs.

[0192] 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. Use of a short peptide RFV from an extracellular vesicle of Lactobacillus plantarum cells for the preparation of a preparation for repairing the skin barrier, characterized in that, The concentration of the short peptide in the preparation is 6-15 μg / mL.

2. Use of a short peptide RFV from an extracellular vesicle of Lactobacillus plantarum cells for the manufacture of a preparation for reducing inflammation of the skin, characterized in that, The concentration of the short peptide in the preparation is 6-15 μg / mL.

3. Use of a short peptide RFV from an extracellular vesicle of Lactobacillus plantarum cells for the manufacture of a preparation for reducing skin itch, characterized in that, The concentration of the short peptide in the preparation is 6-15 μg / mL.

4. The use according to any one of claims 1 to 3, characterized in that, The preparation further comprises a pharmaceutically acceptable carrier.

5. The use according to any one of claims 1 to 3, characterized in that, The preparation dosage form comprises at least one of a suspension, a granule, a capsule, a powder, a tablet, a dripping pill, a suppository, and a drop.

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