Phospholipids from plant lactobacillus extracellular vesicles and uses thereof

Abstract

The present application belongs to the field of microbial technology, and particularly relates to a phospholipid from extracellular vesicles of plant lactobacillus and application thereof. Specifically, the extracellular vesicles of plant lactobacillus can restore skin barrier; can reduce the expression of proinflammatory factors in a cell inflammation model, and has good anti-inflammatory effect. Through metabolomic analysis, it is found that effective substances effective for skin barrier damage include phospholipid 1-palmitoyl-sn-glycero-3-phosphocholine (PC, 16:0 / 0:0); experiments prove that PC (16:0 / 0:0) can regulate the expression of skin barrier related genes in a SDS induced damage cell model; and can regulate the expression of skin inflammation related genes in the SDS induced damage cell model, which indicates that PC (16:0 / 0:0) can be used as a drug for treating skin barrier damage.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to a phospholipid derived from extracellular vesicles of Lactobacillus plantarum and its application. Background Technology

[0002] As the largest organ in the human body, the skin is the first line of defense against the external environment and plays a crucial barrier role. The skin barrier is mainly composed of the physical barrier, chemical barrier, and microbial barrier, which work together to maintain the skin's health. The physical barrier is primarily composed of the stratum corneum and intercellular lipids. The chemical barrier consists of the acidic environment of the skin surface (pH approximately 4.5-6.0), natural moisturizing factors (NMF), and lipids. The microbial barrier is composed of the microbial community on the skin surface, including bacteria, fungi, and viruses.

[0003] Ceramides are the main component of intercellular lipids in the stratum corneum of the skin, accounting for about 40% to 50% of the total intercellular lipids. They play a key role in maintaining the skin barrier function and have 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, typically ranging in diameter from 20 to 400 nm. BEVs possess a lipid bilayer structure and contain various bioactive substances, such as proteins, nucleic acids, lipids, and metabolites. Probiotic extracellular vesicles are extracellular vesicles secreted by probiotics, possessing similar structure and function to bacterial extracellular vesicles. They can regulate the host's immune response, suppress inflammatory responses, and play an important role in gut health, immune regulation, and disease prevention.

[0005] Currently, the preparation and application of bacterial extracellular vesicles have attracted attention. Existing technologies mainly focus 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 foundation for this invention, but further optimization and expansion are still needed, especially regarding the analysis of the active ingredients of bacterial extracellular vesicles, which is very lacking. Currently, there is no research on *Lactobacillus plantarum* extracellular vesicles and their application in repairing the skin barrier, nor on the analysis of the active ingredients of bacterial extracellular vesicles.

[0006] While previously published patents and patent applications have reported on vesicles used to treat inflammatory diseases, no reports have been found regarding *Lactobacillus plantarum* extracellular vesicles promoting ceramide production and thus repairing the skin barrier to alleviate skin problems. Furthermore, research on how bacterial extracellular vesicles increase skin ceramide levels by regulating multiple genes or molecular mechanisms related to ceramide synthesis is lacking. In addition, the elucidation of the active ingredients in bacterial extracellular vesicles is very limited and requires further investigation. Therefore, bacterial extracellular vesicles capable of simultaneously repairing the skin barrier, reducing inflammation, relieving itching, and possessing rapid transdermal absorption capabilities have significant application value and practical implications. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a phospholipid derived from extracellular vesicles of Lactobacillus plantarum and its application.

[0008] The first aspect of the present invention is to provide a phospholipid derived from extracellular vesicles of *Lactobacillus plantarum*, characterized in that the phospholipid is 1-palmitoyl-sn-glycerol-3-phosphocholine.

[0009] A second aspect of the present invention is to provide a formulation for treating skin lesions, characterized in that the formulation comprises the phospholipids described in the first aspect and a pharmaceutically acceptable carrier.

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

[0011] Furthermore, the phospholipid is administered at a concentration of 6 μg / mL to 15 μg / mL.

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

[0013] A third aspect of the present invention is to provide the use of the phospholipids described in the first aspect in the preparation of formulations for repairing the skin barrier.

[0014] Furthermore, the dosage concentration of the phospholipid in the formulation is 6 μg / mL to 15 μg / mL.

[0015] A fourth aspect of the present invention is to provide the use of the phospholipids described in the first aspect in the preparation of formulations that reduce skin inflammation.

[0016] Furthermore, the dosage concentration of the phospholipid in the formulation is 6 μg / mL to 15 μg / mL.

[0017] The use of the phospholipids described in the fifth aspect of the present invention in the preparation of formulations that relieve skin itching.

[0018] Furthermore, the dosage concentration of the phospholipid in the formulation is 6 μg / mL to 15 μg / mL.

[0019] A sixth aspect of the present invention is to provide a pharmaceutical composition, characterized in that the pharmaceutical composition comprises the phospholipids described in the first aspect and other active components of extracellular vesicles of *Lactobacillus plantarum* derived from phospholipids.

[0020] Furthermore, the other active components include lipids, proteins, or nucleic acid components.

[0021] Furthermore, the Lactiplantibacillus plantarum is L25, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35146, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0022] The beneficial effects of this invention include:

[0023] 1) The extracellular vesicles of *Lactobacillus plantarum* of this invention can promote the production of ceramides by HaCaT cells, upregulate the expression of key genes in the skin ceramide synthesis pathway, and restore the skin barrier; it can also reduce the expression of pro-inflammatory factors in a cell inflammation model and has a good anti-inflammatory effect.

[0024] 2) Validated by a mouse skin injury model, EVs have good skin penetration ability, can significantly reduce skin lesion scores, relieve skin itching, increase skin moisture content, and reduce the expression of pro-inflammatory factors and itch-related cytokines in skin tissue.

[0025] 3) Metabolomics analysis revealed that the effective substances for treating skin barrier damage include phospholipid 1-palmitoyl-sn-glycerol-3-phosphocholine (PC, 16:0 / 0:0);

[0026] 4) Experiments confirmed that PC regulates the expression of skin barrier-related genes in an SDS-induced damaged cell model;

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

[0028] The microorganism of this invention is *Lactobacillus plantarum* (… Lactiplantibacillus plantarum L25, the Lactobacillus plantarum L25, was deposited on July 9, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35146. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. Attached Figure Description

[0029] Figure 1 Image of EVs precipitated sample.

[0030] Figure 2 Transmission electron microscopy image of EVs.

[0031] Figure 3 Particle size of EVs.

[0032] Figure 4 Evs fluorescence staining pattern taken up by cells, where DAPI (4′,6-diamidino-2-phenylindole) is used for staining the cell nucleus; DIO (3,3′-di-octadecyloxacarbonylcyanine perchlorate) is used for staining the cell membrane.

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

[0034] Figure 6 EVs regulate the expression of key enzyme genes involved in ceramide synthesis in HaCaT cells.

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

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

[0037] Figure 9 Epistem of EVs alleviating SDS-induced skin damage in mice.

[0038] Figure 10 Mouse skin lesion score.

[0039] Figure 11 Moisture content and transepidermal 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 increase 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 PC (16:0 / 0:0) increases ceramide production in HaCaT cells.

[0048] Figure 20 PC (16:0 / 0:0) regulates the expression of skin barrier-related genes in an SDS-induced damaged cell model.

[0049] Figure 21 Figure 21 PC (16:0 / 0:0) regulates the expression of skin inflammation-related genes in an SDS-induced damaged cell model. Detailed Implementation

[0050] 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.

[0051] Example 1: Isolation and Characterization Evaluation of EVs Derived from Lactobacillus plantarum

[0052] 1. Isolation of EVs derived from Lactobacillus plantarum

[0053] 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 1The 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.

[0054] 2. Characteristic Evaluation of EVs

[0055] (1) Morphological observation

[0056] The morphology of EVs isolated from bacterial cultures was observed using electron microscopy. EV samples were placed on a 300-mesh copper grid and stained with 2% phosphotungstic acid for 12 h. Then, images were captured using a transmission electron microscope at an accelerating voltage of 100 kV to observe the morphological characteristics of the EVs. The results are as follows: Figure 2 As shown, EVs were observed to have a spherical shape using electron microscopy.

[0057] (2) Particle size distribution determination

[0058] The particle size distribution of EVs isolated from bacterial cultures was measured using dynamic light scattering (DLS). The diameter of the EVs was determined using Zetasizer Nano S, and the results are as follows: Figure 3 As shown, the particle size of EVs is 132 ± 2.24 nm.

[0059] (3) Concentration determination

[0060] The concentration of EVs was determined using nanoparticle tracking analysis (NTA). The EV concentration was adjusted to 500 ng / mL, and 0.3 mL to 0.4 mL samples were placed in the chamber of an LM-10HS instrument. The camera focus was adjusted to ensure clear visibility of the particles, and the sample was captured by progressively decreasing the focus level to confirm no sample drift. The capture duration was set to 30 seconds to obtain data. Experimental results showed that the EV concentration could reach 3.2 × 10⁻⁶. 9 particles / mL.

[0061] Example 2: Uptake of EVs derived from Lactobacillus plantarum in HaCaT cells

[0062] 1. Cell Culture

[0063] HaCaT cells were added to DMEM high-glucose culture medium containing 10% fetal bovine serum and cultured in an incubator (37°C, 5% CO2) according to standard procedures. When the cell confluence reached 80-90%, the adherent cells were digested with trypsin and passaged, with passages or medium changes performed 2-3 times per week.

[0064] 2. Fluorescent labeling of EVs

[0065] 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 three times with PBS to remove unbound dye.

[0066] 3. Observation using laser confocal microscopy

[0067] When the HaCaT cell confluence reaches 80%–90%, a cell suspension is obtained using the cell passage method described above, and the cell density is adjusted to 2 × 10⁻⁶ cells / mL. 5 Cells / mL. Add 1000 μL of cell suspension to each well of a 24-well plate pre-placed with cell spreaders, and incubate for 12 h at 37°C, 5% CO2. Once the cell deposition rate meets experimental requirements, discard all old culture medium from the wells and add serum-free medium. Add DIO-labeled EVs (30 μg / mL) to the experimental groups and incubate for another 12 h, avoiding light. Then discard all cell culture medium, add fixative for 10 min, rinse three times with PBS, stain the nuclei with DAPI dye, rinse three times with PBS after 5 min, and finally add a small amount of PBS to prevent cell drying. Observe and photograph using a laser confocal microscope.

[0068] The results are as follows Figure 4 As shown, EVs labeled with DIO dye exhibit green fluorescence, while those stained with DAPI dye exhibit blue fluorescence. It is evident that EVs can penetrate into the cell periphery, indicating that EVs can be taken up by cells.

[0069] Example 3: EVs derived from Lactobacillus plantarum promote ceramide production in HaCaT cells

[0070] The cell culture method is the same as in Example 2. Once the cell confluence reaches 80%–90%, adherent cells are digested with trypsin, and the cell density is adjusted to 2 × 10⁶ cells / year. 5 Cells / mL. Add 1000 μL of cell suspension to each well of a 24-well plate and incubate for 12 h at 37°C, 5% CO2. Once the cell deposition rate meets the experimental requirements, discard the old culture medium and add serum-free medium. Three concentration gradients of EVs were set up for the experimental groups: 30 μg / mL, 12 μg / mL, and 6 μg / mL, with 75 μL of the corresponding concentration added to each well. An equal volume of Bacillus cereus fermentation supernatant was used as a positive control, and PBS as a negative control. Each group had three replicates and was incubated for 24 h. After incubation, adherent cells were scraped off using a cell scraper, centrifuged at 1000g (g is the centrifugation speed unit) for 3 min to collect the cells, and then analyzed by ELISA.

[0071] 1. Preparation before testing

[0072] (1) Remove the kit from the refrigerator 20 minutes in advance to allow it to equilibrate to room temperature;

[0073] (2) Dilute the 30-fold concentrated washing solution with distilled water 30 times and set aside.

[0074] 2. Operating Procedures

[0075] (1) Remove the required strips from the sealed bag that has been equilibrated to room temperature;

[0076] (2) Set up standard wells, blank wells and sample wells. Add 50 μL of standard at different concentrations to each standard well.

[0077] (3) Add 50 μL of the sample to be tested to the sample well, and leave the blank well empty;

[0078] (4) Set up blank wells (blank control wells without sample and enzyme-labeled reagent, all other steps are the same) and sample wells. Add 40 μL of sample diluent to the sample wells on the enzyme-labeled plate, then add 10 μL of the sample to be tested (final sample dilution is 5-fold). Add the sample to the bottom of the wells, avoiding contact with the well walls, and gently shake to mix. Seal the reaction wells with sealing film and incubate at 37°C for 30 min.

[0079] (5) Discard the liquid, pat dry on absorbent paper, and fill each well with washing solution (350 μL); let stand for 30 seconds, shake off the washing solution, pat dry on absorbent paper, and repeat this washing process 5 times.

[0080] (6) Add 50 μL of substrate enzyme-labeled reagent to each well, except for the blank wells. Incubate at 37°C in the dark for 15 min;

[0081] (7) Washing: Same as step 5;

[0082] (8) Color development: Add 50 μL of color developer A to each well first, then add 50 μL of color developer B, gently shake to mix, and develop color at 37℃ in the dark for 15 min.

[0083] (9) Add 50 μL of stop solution to each well and measure the OD value of each well at 450 nm wavelength within 15 min.

[0084] 3. Result Judgment

[0085] The OD value of each standard and specimen should be subtracted from the OD value of the blank well. Plot the standard concentration on the x-axis and the OD value on the y-axis using software, and select the best-fit curve. The concentration of the specimen can be found on the standard curve using its OD value.

[0086] 4. Calculation of ceramide content

[0087] Ceramide standards were prepared into standard solutions and serially diluted to a series of solutions of known concentrations. The OD value (OD 450) at 450 nm was measured using the ELISA method described above. A regression equation for a standard curve was constructed by fitting the equation, and the OD 450 value of each test sample was substituted into the equation to calculate the ceramide content in that sample. Each group was tested in triplicate, and the mean and standard deviation (SD) were calculated. One-way ANOVA was performed using SPSS statistical software to compare the ceramide content of each sample group with the blank control group. A p-value < 0.05 was considered statistically significant.

[0088] The results are as follows Figure 5 As shown, EVs derived from *Lactobacillus plantarum* significantly promoted ceramide production in HaCaT cells, with a statistically significant difference compared to the control group (p < 0.05). Furthermore, the effect of EVs in promoting ceramide production was comparable to that of the positive control group.

[0089] Example 4: EVs derived from *Lactobacillus plantarum* upregulate the expression of key genes in the ceramide synthesis pathway in HaCaT cells.

[0090] The cell culture conditions and procedures are described in Example 2. Once the cell deposition rate in the 6-well plate met the experimental requirements, the old culture medium was discarded. Three concentration gradients of EVs were set up for the experimental groups: 30 μg / mL, 12 μg / mL, and 6 μg / mL. 100 μL of the corresponding concentration of EVs was added to each well. An equal volume of PBS was used as a blank control group. Each group had three replicates, and the cells were cultured for 24 h. After culture, adherent cells were scraped off using a cell scraper, centrifuged at 1000 g for 3 min, and the cells were collected for subsequent experiments.

[0091] Total RNA was extracted from the collected HaCaT cells using the TRIZOL kit, and the specific steps are as follows:

[0092] 1) RNA extraction: Add 1 mL of TRIZOL reagent to the cell sample, vortex, and centrifuge (12000 rpm, 4℃, 15 min). Collect the supernatant, add 200 μL of chloroform, vortex, incubate at room temperature for 10 min, and centrifuge again using the above parameters. Collect the supernatant. Then add an equal volume of isopropanol to the supernatant, gently mix, centrifuge at 14000 rpm for 10 min, discard the supernatant, add 75% ethanol, centrifuge again, and then resuspend the precipitate in DEPC water to obtain RNA. Store the obtained RNA at -80℃ for later use.

[0093] 2) Determination of RNA concentration and quality: RNA concentration and purity were determined using Nanodrop 2000.

[0094] 3) Reverse transcription: RNA was reverse transcribed into cDNA using the TransScript® All-in-One First-Strand cDNA Synthesis SuperMix for qPCR kit.

[0095] Prepare the reverse transcription reaction system as shown in Table 1 below:

[0096] Table 1 Reverse transcription system

[0097]

[0098] Perform reverse transcription: Gently mix the above system, incubate at 45°C for 15 min to remove the genome and reverse transcription reaction; heat at 85°C for 5 s to inactivate TransScript®RT / RI and gDNA Remover.

[0099] (TransScript® RT / RI: This kit contains reverse transcriptase (RT) and RNase inhibitor (RI). RT is used to reverse transcribe RNA template into cDNA; RI inhibits residual RNase in the sample, protecting RNA from degradation. They are usually provided as a single enzyme or mixture, hence the name TransScript® RT / RI. gDNA Remover: This is a reagent for removing genomic DNA (usually an endonuclease / deoxyribonuclease, similar to DNase I). Its function is to digest residual genomic DNA in the RNA sample, preventing amplification in subsequent qPCR / RT-PCR and thus avoiding interference with results.)

[0100] 4) Real-time quantitative PCR reaction

[0101] The effects of EVs from *Lactobacillus plantarum* on the expression levels of genes related to the ceramide synthesis pathway (SPT, GCS, SMase, and CerS3) were determined using qRT-PCR.

[0102] Table 2 qRT-PCR amplification system

[0103]

[0104] Real-time quantitative PCR was performed using the Takala TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) kit. The amplification system and procedure are shown in Tables 2 and 3. The qPCR primers required for this experiment are shown in Table 4. First, the mixed reaction system (Mix) was prepared. According to Table 2, ddH2O, forward and reverse primers, TB Green enzyme, and ROX were added sequentially to the tubes. After vortexing and brief centrifugation, DNA template and mix solution were added to each of the 8-tube strips. Each sample was repeated 5 times. The entire process was performed on ice and protected from light. In all qPCR experiments, RNase-free ddH2O was used instead of template in the negative control, and the GAPDH gene (housekeeping gene) was selected as the internal control gene.

[0105] Table 3 qRT-PCR amplification program

[0106]

[0107] Exploit 2 -ΔΔCT The relative expression levels of the above-mentioned related genes were statistically analyzed, with GAPDH as an internal reference. The relative expression levels of the target gene mRNA were determined according to 2... －ΔΔCt calculate.

[0108] The results are as follows Figure 6 As shown, EVs derived from Lactobacillus plantarum significantly promoted the expression of key enzyme genes (SPT, CerS3, SMase, and GCS) in the ceramide synthesis pathway (p < 0.05), indicating that EVs can promote ceramide synthesis in HaCaT cells.

[0109] Table 4 Primers

[0110]

[0111] Example 5: EVs derived from Lactobacillus plantarum promote the expression of genes related to skin barrier repair.

[0112] To evaluate the effect of *Lactobacillus plantarum* EVs on the expression of skin barrier-related genes, a skin barrier damage model was established by treating HaCaT cells with 1% sodium dodecyl sulfate (SDS). *Lactobacillus plantarum* EVs were isolated using the method described in Example 1, and the cell culture method was the same as described in Example 2 above. When the cell confluence reached 80%–90%, the cell density was adjusted to 5 × 10⁶ cells / year. 5Cells were added at a density of 1 / mL to 6-well plates, with 2 mL of cell suspension added to each well. The plates were then incubated at 37°C (5% CO2) for 12 h. Once the cell deposition rate met experimental requirements, the cells were divided into three groups: experimental group (SDS+EVs), model group (SDS), and control group (DMEM). Each group underwent corresponding interventions, with three replicates per group. The old culture medium in the 6-well plates was first discarded, and serum-free medium was added to all wells. Except for the control group wells, 1% SDS was added to all other wells, and the plates were incubated for another 12 h. After 12 h of incubation, EVs were added to the experimental group, with three concentration gradients of 30 μg / mL, 12 μg / mL, and 6 μg / mL EVs. 100 μL of the corresponding concentration of EVs was added to each well, and the plates were incubated for another 24 h. The cell culture medium in all wells was then discarded, and 1 mL of PBS was added. Adherent cells were scraped off using a cell scraper and collected by centrifugation at 1000 g for 3 min. Total RNA was extracted from HaCaT cells and transcribed into cDNA according to the method in Example 4. The effects of EVs of Lactobacillus plantarum on the expression levels of skin barrier repair-related genes keratin 10, filaggrin (FLG) and desmosome core glycoprotein 1 (DSG1) were determined by qRT-PCR. The primers used in this experiment are shown in Table 5.

[0113] 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.

[0114] Table 5 Primers

[0115]

[0116] Example 6: EVs derived from Lactobacillus plantarum downregulate the expression of inflammation-related genes in HaCaT cells

[0117] 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.

[0118] The results are as follows Figure 8As shown, compared with the model group constructed in Example 5, the mRNA levels of the encoding genes of Lactobacillus plantarum EVs at different concentrations (6 μg / mL, 12 μg / mL and 30 μg / mL) were significantly reduced after treatment, and the reduction was concentration-dependent.

[0119] Table 6 Primers

[0120]

[0121] Example 7: Effects of EVs derived from Lactobacillus plantarum on a mouse model of skin barrier damage

[0122] 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.

[0123] 1. Establishment and intervention of mouse skin lesion model

[0124] Mice were first acclimatized for one week using Balb / c mice, female, 20±2 g. Hair on the abdomen and back of the mice was removed using electric clippers (hair removal area 2 cm × 3 cm). For the first 7 days, 150 μL of 5% sodium dodecyl sulfate (SDS) was applied continuously. Then, 150 μL of 4% SDS was applied every three days to establish the model, and the application was continued for 24 days.

[0125] The experiment consisted of four groups: a control group (Control), a model group (SDS), a positive control group (MFC), and an EVs group (with four concentration gradients: 6 μg / mL, 12 μg / mL, 30 μg / mL, and 100 μg / mL), with eight mice in each group. The positive control group received mometasone furoate ointment, while the control group received saline. Intervention began on day 8. The control, positive, and EVs groups applied 100 μL of saline, mometasone furoate ointment, and different concentration gradients of EVs to the skin lesions on the back once daily for 24 consecutive days.

[0126] 2. Number of scratches by mice

[0127] On days 10, 15, and 20 after administration, the mice were placed in an undisturbed environment and filmed for 10 minutes to record the number of times the mice scratched.

[0128] Table 7 Number of scratches (times / 10 min)

[0129]

[0130] Note: Compared with the control group, ### p < 0.001; compared with the model group, *** p < 0.001

[0131] The results are shown in Table 7. Compared with the blank group, the number of scratches in the model group was significantly increased (p < 0.01). Compared with the model group, the intervention of *Lactobacillus plantarum* EVs significantly reduced the number of scratches in mice (p < 0.01), and the effect was concentration-dependent, with 100 μg / mL of EVs showing the most significant effect.

[0132] 3. Phenotypic changes in local skin lesions in mice

[0133] Before and on the last day of the intervention, the apparent changes in the skin on the backs of mice in each group were recorded using a camera. Figure 9 The model group mice showed obvious crusting, dryness, and desquamation on their backs. Compared with the model group, the positive drug group mice had better skin condition and less crusting on their backs. The EVs group mice had good skin condition on their backs, and the dryness and desquamation were effectively relieved. The intervention effect of 100 μg / mL EVs was better than that of the positive group.

[0134] 4. Severity score of mouse skin lesions

[0135] The severity of skin lesions on the backs of mice was scored before 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).

[0136] Table 8. Scoring Criteria for Skin Lesion Severity

[0137]

[0138] The rating results show that... Figure 10 As shown, the skin lesion score of the model group increased; while compared with the model group, the scores of the positive group and the EVs group decreased, and the differences were significant (p < 0.05).

[0139] 5. Determination of skin moisture content and transepidermal water loss in mice

[0140] On day 24 after drug administration, three different sites in the mouse model area were randomly selected using a skin testing device for testing. The stratum corneum water content and transepidermal water loss at these sites were recorded. Finally, the average stratum corneum water content and transepidermal water loss at these three sites were calculated to assess the skin's hydration status and barrier function.

[0141] like Figure 11As shown, compared with the model group, treatment with different concentrations (6 μg / mL, 12 μg / mL, 30 μg / mL and 100 μg / mL) of *Lactobacillus plantarum* EVs significantly increased skin moisture content (p < 0.05) while significantly decreasing transepidermal water loss (p < 0.05). This result indicates that *Lactobacillus plantarum* EVs can effectively restore skin moisture and improve skin barrier function.

[0142] 6. Determination of gene expression levels of pro-inflammatory cytokines in mouse skin tissue

[0143] The dorsal tissues of mice in each group were homogenized, and total RNA was extracted according to the method in Example 4. The RNA was reverse transcribed into cDNA, and the mRNA expression of relevant pro-inflammatory cytokines TNF-α, IL-4, IL-31, and TSLP was detected by qRT-PCR. GAPDH was used as an internal control. -△△Ct The relative expression levels of each gene were calculated using a method. The primers used in this experiment are shown in Table 9.

[0144] Experimental results are as follows Figure 12 As shown, compared with the model group, treatment with different concentrations (6 μg / mL, 12 μg / mL, 30 μg / mL, and 100 μg / mL) of *Lactobacillus plantarum* EVs significantly reduced the mRNA levels of TNF-α, IL-31, IL-4, and TSLP encoding genes (p < 0.05), with the reductions in TNF-α, IL-31, and IL-4 exhibiting a clear concentration-dependent effect. This indicates that *Lactobacillus plantarum* EVs can inhibit the gene expression of inflammation-related cytokines and chemokines in a concentration-dependent manner, thereby exerting a significant anti-inflammatory effect.

[0145] Table 9 Primers

[0146]

[0147] 7. Determination of protein expression levels of pro-inflammatory cytokines in mouse skin tissue

[0148] The back tissues of mice in each group were homogenized and detected using an ELISA kit.

[0149] (1) ELISA detection

[0150] a. Preparation before testing: Taking the TNF-α kit as an example

[0151] 1) Remove the kit from the refrigerator 20 minutes in advance to allow it to equilibrate to room temperature.

[0152] 2) Dilute the 20× concentrated washing solution with double-distilled water to make a 1× working solution.

[0153] b. Operating steps:

[0154] 1) Remove the required strips and desiccant from the sealed bag that has been equilibrated to room temperature. Put the unused strips and desiccant back into the aluminum foil bag, press the self-sealing strip firmly, seal the bag, and return it to 4°C.

[0155] 2) Set up standard wells, blank wells, and sample wells. Add 50 μL of standard at different concentrations to each standard well.

[0156] 3) Add 50 μL of the sample to be tested to the sample well; do not add anything to the blank well.

[0157] 4) Except for the blank wells, add 100 μL of horseradish peroxidase (HRP) labeled detection antibody to each standard well and sample well, seal the reaction wells with sealing film, and incubate at 37°C in an incubator or water bath for 60 min.

[0158] 5) Discard the liquid, pat dry on absorbent paper, fill each well with washing solution (350 μL); let stand for 1 min, shake off the washing solution, pat dry on absorbent paper, and repeat the washing process 5 times.

[0159] 6) Add 50 μL of substrate A and 50 μL of substrate B to each well and incubate at 37°C in the dark for 15 min.

[0160] 7) Add 50 μL of stop solution to each well. Within 15 min, measure the OD value of each well at a wavelength of 450 nm (within 3 min).

[0161] c. Result Interpretation:

[0162] The OD value of each standard and specimen should be subtracted from the OD value of the blank well. Plot the standard concentration on the x-axis and the OD value on the y-axis using software, and select the best-fit curve. The concentration of the specimen can be found on the standard curve using its OD value.

[0163] (2) Calculation of TNF-α, IL-1β and IL-6 levels

[0164] Standard solutions of TNF-α, IL-1β, and IL-6 were prepared and serially diluted to a series of known concentrations. The OD values ​​at 450 nm were measured using the ELISA method described above. A regression equation for a standard curve was constructed by fitting the equation, and the OD450 values ​​of each test sample were substituted into the equation to calculate the TNF-α, IL-1β, and IL-6 content in the test sample. Each group was tested in triplicate, and the mean and standard deviation (SD) were calculated. One-way ANOVA was performed using SPSS statistical software to compare the TNF-α, IL-1β, and IL-6 content of each sample group with the blank control group. A p-value < 0.05 was considered statistically significant.

[0165] like Figure 13 As shown, compared with the model group, treatment with different concentrations (6 μg / mL, 12 μg / mL, 30 μg / mL and 100 μg / mL) of *Lactobacillus plantarum* EVs significantly reduced the protein levels of TNF-α, IL-1β and IL-6 (p < 0.05). This result indicates that *Lactobacillus plantarum* EVs can inhibit the protein expression of inflammation-related cytokines in a concentration-dependent manner, thereby effectively exerting an anti-inflammatory effect.

[0166] 8. Determination of gene expression levels of skin barrier repair-related cytokines in mouse skin tissue

[0167] The dorsal tissues of mice in each group were homogenized, total RNA was extracted, and reverse transcribed into cDNA. The mRNA expression of skin barrier-related cytokines filaggrin, keratin 10, and ceramide synthase 3 was detected by qRT-PCR, with GAPDH used as an internal control. -△△Ct The relative expression levels of each gene were calculated using the method shown in Table 10.

[0168] like Figure 14 As shown, compared with the model group, treatment with different concentrations (6 μg / mL, 12 μg / mL, 30 μg / mL and 100 μg / mL) of *Lactobacillus plantarum* EVs significantly upregulated the mRNA levels of skin barrier-related genes FLG, KRT10 and CerS3 (p < 0.05), and this upregulation showed a clear concentration-dependent effect. This indicates that *Lactobacillus plantarum* EVs can effectively repair the skin barrier function of mice with SDS-induced skin damage and promote skin barrier repair.

[0169] Table 10 Primers

[0170]

[0171] 9. Determination of gene expression levels of pruritus-related cytokines in mouse skin tissue

[0172] The dorsal tissues of mice in each group were homogenized, total RNA was extracted, and reverse transcribed into cDNA. The mRNA expression of pruritus-related cytokines IL-31RA and OSMA was detected by qRT-PCR, with GAPDH as an internal control. -△△Ct The relative expression levels of each gene were calculated using the method shown in Table 11.

[0173] like Figure 15As shown, compared with the model group, treatment with different concentrations (6 μg / mL, 12 μg / mL, 30 μg / mL and 100 μg / mL) of *Lactobacillus plantarum* EVs significantly reduced the expression levels of pruritus-related genes OSMA and IL-31RA (p < 0.05). This result indicates that *Lactobacillus plantarum* EVs can significantly inhibit the pruritus response induced by DNFB in mice with damaged skin and effectively alleviate pruritus symptoms.

[0174] Table 11 Primers

[0175]

[0176] Example 8: Study on the absorption and distribution of EVs derived from Lactobacillus plantarum in mouse skin

[0177] This study used a mouse dorsal skin application assay to observe the uptake and distribution of extracellular vesicles (EVs) derived from *Lactobacillus plantarum* in skin tissue, thereby evaluating their transdermal absorption characteristics. In the experiment, labeled *Lactobacillus plantarum* EVs were uniformly applied to the dorsal skin surface of mice. In vivo imaging technology was then used to monitor the penetration, uptake, and distribution dynamics of EVs within the skin in real time. Analysis of the imaging data systematically evaluated the transdermal ability of *Lactobacillus plantarum* EVs and their absorption efficiency in skin tissue.

[0178] 1. Mouse treatment

[0179] Before the experiment, both the experimental group (EVs+DIO) and the control group (DIO) mice underwent hair removal. Hair removal was performed one day in advance to ensure the skin surface was clean and free of hair that could interfere with subsequent experimental procedures. The hair removal method followed the established Example 7, taking care to avoid damaging the mouse skin during the procedure to ensure the accuracy and reliability of the experimental results.

[0180] 2. Fluorescent labeling of EVs

[0181] The marking method is completed according to Example 2.

[0182] 3. Observation of in vivo efficacy

[0183] The labeled EVs were evenly applied to the skin on the back of mice, with 100 μL used per mouse. During the procedure, care was taken to avoid light exposure to prevent quenching of the fluorescence signal. Two hours after application, the mice were gently placed in the dark chamber platform of the in vivo imaging system. The field of view was adjusted, and a background image was taken to ensure imaging accuracy. Subsequently, fluorescence signals within the mice were captured under dark-field conditions, and the distribution of EVs in the skin tissue was recorded in detail.

[0184] like Figure 16As shown, EVs labeled with DIO dye exhibited green fluorescence. Experimental results showed that EVs could penetrate into the skin on the back of mice within just 2 hours after application. This phenomenon indicates that EVs derived from *Lactobacillus plantarum* have excellent transdermal absorption capabilities and can be rapidly absorbed and distributed through the mouse skin.

[0185] Example 9: Proteomic analysis of EVs derived from Lactobacillus plantarum

[0186] The extraction method for *Lactobacillus plantarum* EVs was as described in Example 1. The protein concentration of the EV samples was determined using BCA reagent, ensuring that the protein concentration of each sample was higher than 200 μg / mL. Subsequently, the EV samples were lysed, and the proteins in the samples were identified using liquid chromatography-mass spectrometry (LC-MS / MS). The mass spectrometry data was analyzed using mass spectrometry data analysis software (such as MaxQuant or Proteome Discoverer) to identify the types of proteins in the vesicles. Bioinformatics tools (such as Gene Ontology and KEGG pathway analysis) were used to perform functional annotation and pathway enrichment analysis on the identified proteins.

[0187] The results showed that proteomic analysis of EVs derived from *Lactobacillus plantarum* identified a total of 1537 proteins. For example... Figure 17 As shown, KEGG pathway analysis indicates that proteins related to metabolic function account for the majority of total protein, approximately 61.76%.

[0188] Example 10: Lipidomics analysis of EVs derived from Lactobacillus plantarum

[0189] 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.

[0190] like Figure 18 As shown, in the non-targeted metabolomics analysis, PC (16:0 / 0:0) (1-palmitoyl-sn-glycerol-3-phosphorylcholine) in vesicles had a high Fragmentation Score, ranking 5th. This compound has anti-inflammatory and barrier repair potential and is a key active ingredient in extracellular vesicles (EVs).

[0191] Example 11: Analysis and validation of PC (16:0 / 0:0) (1-hexadecanoyl-sn-glycero-3-phosphocholine), a key active ingredient in EVs derived from *Lactobacillus plantarum*.

[0192] High-purity PC (16:0 / 0:0) was obtained through in vitro synthesis. HaCaT cells were treated with PC (16:0 / 0:0) concentrations of 6 μg / mL and 15 μg / mL. Simple cell culture medium and vitamin C were used as the blank control and positive control, respectively. Then, according to Examples 3, 4, 5, and 6, the levels of ceramide production, barrier repair-related factors, and the gene expression of inflammatory factors in the cells were measured.

[0193] like Figure 19 As shown, the results indicate that PC (16:0 / 0:0) intervention significantly increased ceramide production in HaCaT cells. Figure 20 As shown, the results of the skin injury cell model indicated that PC (16:0 / 0:0) intervention significantly upregulated the expression levels of protein factor genes closely related to skin barrier function, including filaggrin, ceramide synthase 3, and keratin 10. Figure 21 As shown, PC (16:0 / 0:0) treatment also significantly reduced the expression levels of inflammatory factors such as IL-25, IL-33, and TSLP. In conclusion, PC (16:0 / 0:0) is an effector molecule that plays a key role in EVs.

[0194] The embodiments described above are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

Claims

1. The application of a phospholipid, 1-palmitoyl-sn-glycerol-3-phosphocholine, in the preparation of a formulation for repairing the skin barrier, characterized in that, The dosage concentration of the phospholipid in the formulation is 6 μg / mL to 15 μg / mL.

2. The use of a phospholipid, 1-palmitoyl-sn-glycerol-3-phosphocholine, in the preparation of an agent to alleviate skin inflammation caused by skin barrier damage, characterized in that... The dosage concentration of the phospholipid in the formulation is 6 μg / mL to 15 μg / mL.

3. The use of a phospholipid, 1-palmitoyl-sn-glycerol-3-phosphocholine, in the preparation of an agent to relieve pruritus caused by skin barrier damage, characterized in that... The dosage concentration of the phospholipid in the formulation is 6 μg / mL to 15 μg / mL.

4. The application according to any one of claims 1 to 3, characterized in that, The formulation also includes a pharmaceutically acceptable carrier.

5. The application according to any one of claims 1 to 3, characterized in that, The dosage forms mentioned include at least one of suspensions, granules, capsules, powders, tablets, pills, suppositories, and drops.

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