Preparation method and application of probiotic membrane vesicle and probiotic derivative component

Abstract

The invention relates to the technical field of biomedicine, in particular to a preparation method and application of probiotic membrane vesicles and probiotic derivative components. The preparation method comprises the following steps: (1) carrying out mixed culture on probiotics and a culture medium, centrifuging, and collecting bacterial liquid supernatant and bacterial precipitate; (2) filtering the bacterial liquid supernatant to obtain a cell-free fermentation supernatant; (3) filtering the bacterial liquid supernatant, and centrifuging to obtain cytoplasmic membrane vesicles; and (4) after resuspending the thallus precipitate, breaking, centrifuging, collecting a supernatant and precipitate, filtering the supernatant to obtain a lysing supernatant, dissolving the precipitate, and filtering to obtain the lysing precipitate. The invention provides a novel probiotic-sourced non-viable bacteria therapy, the membrane vesicles and the lysing supernatant which are sourced from the probiotics are used, the infection risk is avoided, substances with anti-inflammatory and anti-oxidation effects are screened, accurate treatment is carried out aiming at a specific inflammation pathway, and multi-target treatment is realized.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a preparation method and application of probiotic membrane vesicles and probiotic-derived components. Background Art

[0002] Psoriasis, also widely known as "psoriasis," is a common chronic inflammatory skin disease that often manifests as localized or widespread scaly erythema or plaques. Its pathophysiological process is closely related to abnormal activation of the immune system and persistent inflammatory responses mediated by an imbalance in the intestinal microbiome. For inflammatory skin diseases, existing technologies mainly rely on drug therapy, phototherapy, biological therapy, and lifestyle intervention, but these methods have safety issues, non-targeted nature, and single target defects. Drug therapy relies on hormones, immunosuppressants, etc., which have side effects and recurrence risks; phototherapy cycles are long and may increase the risk of skin cancer; biological therapy is expensive and may cause side effects such as autoimmune diseases; lifestyle interventions have limited effects and are difficult to adhere to.

[0003] Korean Patent KR102665403B1 discloses a pharmaceutical composition for treating or preventing allergic diseases, comprising Lactobacillus plantarum HD-02 strain, a lysate derived therefrom, a culture medium, or a mixture thereof as an active ingredient. The allergic diseases include edema, allergic reactions, rhinitis, allergic rhinitis, asthma, allergic conjunctivitis, allergic dermatitis, allergic otitis media, atopic dermatitis, contact dermatitis, urticaria, chronic spontaneous urticaria, urticaria, dry eyes, pruritus, anaphylactic shock, pollen allergy, bacterial allergy, a substance selected from fungal allergy, viral allergy, insect allergy, food allergy, drug allergy, and respiratory allergy. However, compared with the Lactobacillus plantarum ATCC BAA-793 of the present invention, the probiotic membrane vesicles and derived components prepared from Lactobacillus plantarum HD-02 are less effective in treating inflammatory skin diseases.

[0004] Chinese patent CN109498660A discloses an application of Lactobacillus plantarum CCFM8610 that can alleviate atopic dermatitis. The invention found that Lactobacillus plantarum CCFM8610 has the effect of alleviating atopic dermatitis, which is specifically reflected in: (1) significantly improving the degree of ear swelling in atopic dermatitis mice; (2) significantly improving the skin pathological symptoms of atopic dermatitis mice; (3) significantly reducing the SCORAD index of patients with mild atopic dermatitis; (4) significantly improving the DLQI score of patients with mild atopic dermatitis. Therefore, Lactobacillus plantarum CCFM8610 has great application prospects in the preparation of products for the prevention and / or treatment of atopic dermatitis. However, compared with the Lactobacillus plantarum ATCC BAA-793 of the present invention, the probiotic membrane vesicles and derived components prepared from Lactobacillus plantarum CCFM8610 are less effective in treating inflammatory skin diseases.

[0005] The literature "Wang Wanni, Li Ying, Zeng Jiahui, et al. Research progress on the prevention and treatment of atopic dermatitis with probiotics and its mechanism [J]. Acta Microbiologica Sinica, 2024, 64(5):1378-1391." reported that probiotics that can reduce type I allergic reactions mainly come from Lactobacillus and Bifidobacterium. A randomized, double-blind, placebo-controlled study using animal Bifidobacterium found that the itching symptoms of subjects in the probiotic group were significantly improved after 8 weeks. Enomoto et al.'s study found that after using Bifidobacterium breve, the atopic dermatitis score index of the probiotic group decreased. After adult atopic dermatitis patients took Lactobacillus salivarius LS01, the SCORD score of the probiotic treatment group was significantly reduced, and the Staphylococcus load in fecal microorganisms was reduced. After using Lactobacillus paracasei K71, the skin severity score of the probiotic group was reduced. A randomized controlled trial of a combination of Lactobacillus salivarius LS01 DSM 2275 and Bifidobacterium breve BR03 DSM 16604 found significant improvements in clinical scores and decreased plasma lipopolysaccharide levels in participants. Furthermore, supplementation with Lactobacillus rhamnosus HN001 during pregnancy and early infancy appears to reduce the risk of atopic dermatitis. However, this article only lists recent research advances in the use of probiotics for the prevention and treatment of atopic dermatitis and does not compare the effectiveness of different probiotics in this regard.

[0006] Therefore, there is an urgent need to study a method for preparing multi-target probiotic membrane vesicles and derived components with excellent effects on inflammatory skin diseases. Summary of the Invention

[0007] The purpose of the present invention is to provide a method for preparing probiotic membrane vesicles and probiotic-derived components and their application in products for treating inflammatory diseases. The cytoplasmic membrane vesicles and active ingredients derived from Lactobacillus plantarum ATCC BAA-793 are extracted, which greatly enhances the effect of Lactobacillus plantarum in alleviating inflammatory skin diseases.

[0008] In order to achieve the above-mentioned purpose of the invention, the technical solution of the present invention is as follows: In one aspect, the present invention provides a method for preparing probiotic membrane vesicles and probiotic-derived components, comprising the following steps: (1) Mix the probiotics with the culture medium, centrifuge, and collect the supernatant and precipitate; (2) The supernatant of the bacterial solution is filtered to obtain the cell-free fermentation supernatant; (3) The supernatant of the bacterial solution was filtered and centrifuged to obtain cytoplasmic membrane vesicles; (4) After the bacterial pellet is resuspended, it is broken by high-pressure homogenization and centrifuged to collect the supernatant and precipitate. The supernatant is filtered to obtain the lysis supernatant, and the precipitate is dissolved and filtered to obtain the lysis precipitate.

[0009] Preferably, the method for preparing the probiotic membrane vesicles and derived components comprises the following steps: (1) Probiotics were mixed with culture medium, cultured anaerobically for 12-72 hours, and then centrifuged at 6000-12000g for 10-30 minutes to collect the supernatant and precipitate respectively; (2) The supernatant of the bacterial solution was filtered through a 0.22-0.25 μm microporous filter to obtain the cell-free fermentation supernatant (CFS). (3) The supernatant of the bacterial solution was filtered through a 0.45-0.48 μm microporous membrane and then ultracentrifuged (100,000-150,000 g, 80-120 min) to obtain cellular membrane vesicles (CMVs); (4) The bacterial pellet was resuspended in PBS buffer and then broken by high-pressure homogenization. The homogenized lysed bacterial solution was centrifuged at 8000-15000g for 15-45min. The supernatant and precipitate were collected. The supernatant was filtered through a 0.22-0.25μm microporous membrane to obtain the bacterial lysate supernatant (BL-S). The precipitate was dissolved in sterile PBS buffer and then filtered through a 0.22-0.25μm microporous membrane to obtain the bacterial lysate precipitate (BL-P).

[0010] Preferably, the probiotics described in step (1) are Lactobacillus plantarum ATCC BAA-793.

[0011] Preferably, the culture medium in step (1) is MRS broth culture medium.

[0012] Preferably, the method for breaking up the bacterial precipitate may also be an ultrasonic method or a lysozyme method.

[0013] As a preferred embodiment, the method for preparing the probiotic membrane vesicles and derived components comprises the following steps: (1) Lactobacillus plantarum ATCC BAA-793 was mixed with MRS broth and cultured anaerobically for 24 hours. The supernatant and precipitate were collected after centrifugation at 10,000 g for 20 minutes. (2) The cell-free fermentation supernatant was obtained by filtering the bacterial supernatant through a 0.22 μm microporous filter membrane. (3) The supernatant of the bacterial solution was filtered through a 0.45 μm microporous filter and then ultracentrifuged (150,000 g, 90 min) to obtain cytoplasmic membrane vesicles; (4) The bacterial precipitate was resuspended in PBS buffer and then broken by high-pressure homogenization. The homogenized lysed bacterial solution was centrifuged at 15,000 g for 30 min, and the supernatant and precipitate were collected. The supernatant was filtered through a 0.22 μm microporous membrane to obtain the lysed supernatant; the precipitate was dissolved in sterile PBS buffer and then filtered through a 0.22 μm microporous membrane to obtain the lysed precipitate.

[0014] On the other hand, the present invention also provides probiotic membrane vesicles and probiotic-derived components prepared by the above preparation method.

[0015] Preferably, the derived components are cytoplasmic membrane vesicles, lysis supernatant, lysis precipitate and cell-free fermentation supernatant, preferably cytoplasmic membrane vesicles and lysis supernatant.

[0016] Finally, the present invention also provides the use of the probiotic membrane vesicles and derived components prepared by the above preparation method in the preparation of drugs for treating inflammatory diseases.

[0017] Preferably, the inflammatory disease is an inflammatory skin disease.

[0018] Preferably, the dosage form of the drug is powder, granules, capsules, tablets, oral liquid or pills.

[0019] Preferably, the drug further comprises pharmaceutically acceptable excipients.

[0020] Preferably, the pharmaceutically acceptable excipients include one or more of fillers, wetting agents, binders, disintegrants and lubricants.

[0021] The beneficial effects of the present invention are: (1) This invention proposes a novel probiotic-derived, non-live bacterial therapy that uses probiotic-derived membrane vesicles and lysate supernatants to avoid infection risks. Furthermore, by screening for substances with anti-inflammatory and antioxidant effects, it provides precise treatment for specific inflammatory pathways, achieving multi-target therapy. Animal experiments have confirmed that this invention can effectively alleviate psoriasis symptoms and has good biosafety, providing new ideas and methods for the treatment of inflammatory skin diseases and has broad application prospects.

[0022] (2) The present invention also reveals the different metabolite compositions of membrane vesicles and lysis supernatant derived from Lactobacillus plantarum.

[0023] (3) The present invention also reveals the material basis for the differences in efficacy of different active components of Lactobacillus plantarum: anandamide (AEA) derived from Lactobacillus plantarum exerts anti-inflammatory, antioxidant and macrophage M1 polarization inhibitory effects; 2-hydroxyisohexyl derived from Lactobacillus plantarum exerts antibacterial activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1This is a method for preparing four derivative components according to Example 1 of the present invention.

[0025] Figure 2 These are the four derivative components prepared in Example 1 of the present invention.

[0026] Figure 3 The morphology of CMVs prepared in Example 1 of the present invention (scale bar = 100 μm).

[0027] Figure 4 The particle size and particle size distribution of CMVs prepared in Example 1 of the present invention (n = 3).

[0028] Figure 5 This is the zeta potential of CMVs prepared in Example 1 of the present invention (n = 3).

[0029] Figure 6 The results of the CCK-8 test are as follows: the effects of the four derivative components prepared in Example 1 of the present invention on the cell proliferation activity of human keratinocytes HaCaT (Figure A: CCK-8 detection of the effects of the four derivative components of Lactobacillus plantarum on the proliferation activity of HaCaT cells, n = 3; Figure B: CCK-8 detection of the effects of the four derivative components of Lactobacillus plantarum on the excessive proliferation of HaCaT cells induced by TNF-α and IL-17, n = 3).

[0030] Figure 7 Representative flow cytometric images and statistical results (n = 3) of the effects of the four derivative components prepared in Example 1 of the present invention on the level of reactive oxygen species in human keratinocytes HaCaT cells induced by LPS.

[0031] Figure 8 The results of the CCK-8 test are as follows: the effects of the four derivative components prepared in Example 1 of the present invention on the proliferation of mononuclear macrophages (Figure A: CCK-8 test of the effects of the four derivative components of Lactobacillus plantarum on the proliferation activity of THP-1 macrophages, n = 3; Figure B: CCK-8 test of the effects of the four derivative components of Lactobacillus plantarum on LPS-induced THP-1 macrophage proliferation, n = 3).

[0032] Figure 9 The relative expression levels of mRNA of inflammatory factors TNF-α, IL-6 and IL-1β in mononuclear macrophages induced by LPS were detected by qPCR (n = 3).

[0033] Figure 10The results of flow cytometric analysis of the effects of CMVs and BL-S prepared in Example 1 of the present invention on LPS-induced M1 polarization of macrophages are shown (Figures A, C: flow cytometric analysis of the proportion of M1 macrophages in macrophages stimulated by LPS and treated with CMVs, and quantitative analysis, n = 3; Figures B, D: flow cytometric analysis of the proportion of M1 macrophages in macrophages stimulated by LPS and treated with BL-S, and quantitative analysis, n = 3).

[0034] Figure 11 This is the broad targeted metabolomics partial orthogonal partial least squares-discriminant analysis (OPLS-DA) score of CMVs and BL-S prepared in Example 1 of the present invention.

[0035] Figure 12 This is a cluster heat map of the metabolite composition in CMVs and BL-S prepared in Example 1 of the present invention.

[0036] Figure 13 This is a Venn diagram of the metabolite composition types in CMVs and BL-S prepared in Example 1 of the present invention.

[0037] Figure 14 The metabolite composition results of CMVs and BL-S prepared in Example 1 of the present invention are shown.

[0038] Figure 15 The metabolite traceability analysis results of CMVs and BL-S prepared in Example 1 of the present invention (volcano plot of metabolite differences and KEGG enrichment analysis of differential metabolites).

[0039] Figure 16 The statistical results of the contents of AEA, putrescine, and HICA in CMVs and BL-S prepared in Example 1 of the present invention (n = 3).

[0040] Figure 17 The relative mRNA expression levels of inflammatory factors TNF-α, IL-6, and IL-1β in inflammatory macrophages or inflammatory HaCaT cells after AEA or putrescine treatment were detected by qPCR (n = 3).

[0041] Figure 18 Figure 3 Effects of AEA or putrescine on ROS levels in LPS-induced macrophages detected by flow cytometry (n = 3).

[0042] Figure 19 To detect the effect of HICA and BL-S treatment on E. coli Results of the effects on the proliferation activity of K-12 (n = 5).

[0043] Figure 20Figures 24 and 25 show the effects of AEA or putrescine on the LPS-induced BMDM cell polarization phenotype detected by flow cytometry (Figures A, C: flow cytometric detection and quantitative analysis of the proportion of F4 / 80+CD80+ phenotype in inflammatory BMDM treated with putrescine or AEA, n = 3; Figures B, D: flow cytometric detection and quantitative analysis of the proportion of F4 / 80+CD86+ phenotype in inflammatory BMDM treated with putrescine or AEA, n = 3).

[0044] Figure 21 The purpose is to construct a mouse model of psoriasis and develop a treatment plan.

[0045] Figure 22 These are representative images of the treatment status of the dorsal skin of mice in each group on the 8th day as prepared in Example 1.

[0046] Figure 23 The skin thickness score (Figure A), erythema score (Figure B), scaling score (Figure C), and psoriasis lesion area and severity score (Figure D) of each group of mice prepared in Example 1 (n = 5).

[0047] Figure 24 Figure 2 shows the body weight changes of mice in each group (Figure A) and spleen weight on day 8 (Figure B).

[0048] Figure 25 Figure 3 H&E staining and Ki67 immunohistochemistry results of skin tissues and the levels of inflammatory factors in skin tissues of mice in each group (Figure A: H&E staining and Ki67 immunohistochemistry images of lesional skin on day 8, scale bar: 100 μm, Figure B: Quantitative analysis of epidermal thickness by H&E staining (n ≥ 4), Figure C: Quantitative analysis of Ki67 by immunohistochemistry (n ≥ 4), Figure D: Relative mRNA expression level of TNF-α in skin tissues of mice on day 8 (n = 3), Figure E: Relative mRNA expression level of IL-1β in skin tissues of mice on day 8 (n = 3)).

[0049] Figure 26 Figure 3 Immunohistochemical staining analysis results of F4 / 80 and CD206 and the results of CD86 / CD206 transcription levels in skin tissues of mice in each group (Figure A: Immunohistochemical images of F4 / 80 and CD206 in lesional skin on day 8, scale bar: 100 μm, Figure B: Quantitative analysis of F4 / 80 by immunohistochemistry (n ≥ 4), Figure C: Quantitative analysis of CD206 by immunohistochemistry (n ≥ 4), Figure D: Ratio of CD86 to CED206 mRNA expression levels in mouse skin tissues on day 8 (n = 3)).

[0050] Figure 27Representative flow cytometry plots and quantitative analysis of CD45+CD11b+F4 / 80+ macrophages (Panels A, C) and CD45+CD11b+F4 / 80+CD86+ macrophages (Panels A, D) in the spleens of mice in each group (n ≥ 4).

[0051] Figure 28 Representative H&E staining images of the heart, liver, spleen, lung, and kidney in each group on day 8. Scale bar: 100 μm. DETAILED DESCRIPTION

[0052] The following non-limiting examples are provided to enable those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way. The following is merely an illustrative description of the scope of the present invention, and those skilled in the art may make various changes and modifications to the present invention based on the disclosed content, which should also fall within the scope of the present invention.

[0053] The present invention will be further described below by way of specific examples. The various chemical reagents used in the examples of the present invention were obtained through conventional commercial channels unless otherwise specified. Unless otherwise specified, the contents described below are all by weight. Unless otherwise specified, it is understood that the experiments were conducted at room temperature.

[0054] Lactobacillus plantarum ATCC BAA-793: also known as Lactobacillus plantarum NCIM 8826 / WCFS1 / Hayward 3A, purchased from the China General Microbiological Culture Collection Center CGMCC, resource number 1511C0002100005120; the article "Effects of Lactobacillus plantarum WCFS1 on pancreatic and ileal damage in mice with acute necrotizing pancreatitis" in "Chinese Journal of Pancreas" April 2023, Volume 23, Issue 2 disclosed this bacterium.

[0055] Example 1 Lactobacillus plantarum ATCC BAA-793 was cultured anaerobically in MRS broth for 24 hours and then centrifuged at 10,000 g for 20 minutes to collect the supernatant and precipitate. The supernatant of the bacterial solution was filtered through a 0.22 μm microporous membrane to obtain cell-free fermentation supernatant (CFS); the supernatant of the bacterial solution was filtered through a 0.45 μm microporous membrane and then ultracentrifuged (150,000 g for 90 min) to obtain cellular membrane vesicles (CMVs); the bacterial pellet was resuspended in PBS buffer and then ruptured by high-pressure homogenization. The homogenized lysed bacterial solution was centrifuged at 15,000 g for 30 min, and the supernatant and pellet were collected. The supernatant was filtered through a 0.22 μm microporous membrane to obtain bacterial lysate supernatant (BL-S). The pellet was dissolved in sterile PBS buffer and filtered through a 0.22 μm microporous membrane to obtain bacterial lysate precipitate (BL-P). The preparation methods of the four derivative components, CMVs, BL-S, BL-P and CFS, are as follows: Figure 1 As shown, the four derived components of CMVs, BL-S, BL-P and CFS are as follows Figure 2 shown.

[0056] Test Example 1 1. Characterization of CMVs prepared in Example 1 1.1 Characterization methods Transmission electron microscopy (TEM) scanning was used to characterize the morphology of CMVs, nanoparticle tracer analysis (NTA) was used to characterize the particle size and size distribution of CMVs, and dynamic light scattering (DLS) was used to analyze the zeta potential of CMVs.

[0057] 1.2 Characterization Results TEM scanning showed that CMVs exhibited a typical spherical structure and a double lipid membrane of extracellular vesicles ( Figure 3 The average particle size of CMVs characterized by NTA was 179.8±4.1nm ( Figure 4 ); DLS analysis revealed that the zeta potential of CMVs was approximately -24.14 mV ( Figure 5 ).

[0058] Test Example 2 1. Test whether the four derivative components prepared in Example 1 can inhibit keratinocyte inflammation, proliferation and oxidative stress 1.1 Reagents Cell Counting Kit-8 (CCK-8, APExBIO Technology, K1018); Recombinant Human TNF-α (NovoProtein); Recombinant Human IL-17 / IL-17A (Solarbio); LPS (MCE, HY-D1056); 2',7'-Dichlorofluorescin diacetate DCFH-DA (Sigma-Aldrich, D6883).

[0059] 1.2 Test Method (1) CCK-8 assay was used to detect the effects of Lactobacillus plantarum derived components on the proliferation activity of (TNF-α + IL-17-induced inflammatory) human keratinocytes HaCaT: three concentration gradients were set for each derived component, and five replicates were set for each concentration group. A blank group (no cells were plated) and a control group (PBS substituted for drugs) were also set up. The concentration gradients for CMVs were 1 μg / mL, 5 μg / mL, and 20 μg / mL; the concentration gradients for BL-S were 0.001 μg / mL, 0.1 μg / mL, and 10 μg / mL; the concentration gradients for BL-P were 10 μg / mL, 50 μg / mL, and 200 μg / mL; and the concentration gradients for CFS were 4 log CFU / mL, 6 log CFU / mL, and 8 log CFU / mL. HaCaT cells with good growth status were obtained, cell suspensions were prepared, and cell counts were performed. Based on the cell count results, 5,000 cells (100 μL) were plated in 96-well plates. 100 μL of PBS was added to the perimeter of the unplated wells of a 96-well plate to minimize solvent evaporation. After cells were cultured overnight and reached a density of 50-60%, the corresponding concentration of the derivatized component was added to each well (for induction of inflammatory HaCaT cells, 50 ng / mL TNF-α and 50 ng / mL IL-17 were administered simultaneously to induce HaCaT inflammation). The cells were then cultured in a cell culture incubator. After 24 hours of culture, the culture medium was removed, the plates were washed once with PBS, and 100 μL of 10% CCK-8 reagent (10% CCK-8 in 90% DMEM basal medium) was added to each well. The plates were incubated at 37°C in the dark for 30 min-1 h, and the absorbance was measured at 450 nm using a microplate reader. The cell proliferation rate of each group relative to the control group was calculated and plotted using GraphPad Prism.

[0060] (2) Flow cytometry was used to detect the effects of Lactobacillus plantarum derived components on the oxidative stress level of human keratinocytes HaCaT induced by LPS: 5 concentration gradients were set for each derived component, including an unstained group, a control group (NC), and a model group (LPS). The concentration gradient of the derived components was set as follows: Figure 7 Take HaCaT cells in good growth state, make a cell suspension and count the cells. According to the cell counting results, 2×10 4 The number of cells was plated in a 12-well plate. After overnight culture, the cells adhered to the wall and the density reached over 50%. 1 μg / mL LPS was administered to the modeling group and the drug-treated group. After 6 hours of stimulation, the corresponding concentration of the derivative component was added to each group and cultured in a cell culture incubator. After 24 hours of culture, the culture medium was removed, the cells were washed once with PBS, and 10 μM DCFH-DA was added and incubated at 37°C in the dark for 15 minutes. After incubation, the cells were washed three times with PBS, digested with trypsin, and collected. After washing once with PBS, the cells were resuspended in 300-500 μL PBS and the FITC fluorescence intensity was detected by flow cytometry.

[0061] 1.3 Test Results (1) The CCK-8 test showed that the four derivative components prepared in Example 1 had no cytotoxicity after treating HaCaT cells for 24 hours. Specifically, CMVs and BL-S slightly promoted HaCaT proliferation after 24 hours of treatment, while BL-P and CFS had no significant effect on HaCaT proliferation activity after 24 hours of treatment. For the excessive proliferation of inflammatory HaCaT cells induced by TNF-α and IL-17, both CMVs and BL-S could effectively inhibit inflammation-induced HaCaT cell proliferation, among which CMVs had a significantly greater inhibitory effect. In contrast, BL-P and CFS showed opposite effects, promoting cell proliferation under inflammatory conditions ( Figure 6 ).

[0062] (2) Flow cytometry results showed that CMVs and BL-S prepared in Example 1 significantly reduced the ROS level of HaCaT cells induced by LPS, while BL-P and CFS had no significant regulatory effect ( Figure 7 ).

[0063] Test Example 3 1. Test whether the four derivative components prepared in Example 1 can inhibit macrophage inflammation and regulate the polarization phenotype of macrophages 1.1 Reagents Cell Counting Kit-8 (CCK-8, APExBIO Technology, K1018); Trizol lysis buffer (Vazyme, R401-01) LPS (MCE, HY-D1056); The reverse transcription kit and SYBR Green qPCR SuperMix were purchased from Beijing Quanshijin Company; FITC anti-mouse / human CD11b (Biolegend cat. 101206); PerCP / Cyanine5.5 anti-mouse CD80 (Biolegend cat. 104722).

[0064] 1.2 Test Method (1) CCK-8 test to detect the effect of Lactobacillus plantarum derived components on (LPS-induced) mononuclear macrophage cell proliferation: three concentration gradients were set for each derived component, and five replicates were set for each concentration group. A blank group (unplated cells) and a control group (PBS instead of drugs) were also set up. The concentration gradient of the derived components was set the same as (1) in 1.2 of Experimental Example 2. THP-1 cells with good growth status were taken, prepared into a cell suspension, and the cells were counted. According to the cell count results, 4×10 cells per well were used. 4 100 μL of cells (100 μL) were plated in a 96-well plate and 100 ng / mL PMA was added to induce differentiation of monocytes into mononuclear macrophages. 100 μL of PBS was added to the unplated wells around the 96-well plate to reduce solvent evaporation from the edges. After 48 hours of culture, THP-1 cells were differentiated into mononuclear macrophages. The corresponding concentration of the derivative component was added to each well (for induction of inflammatory mononuclear macrophages, 100 ng / mL LPS was also added to induce inflammation). The cells were cultured in a cell culture incubator. After 24 hours of culture, the culture medium was removed, the plates were washed once with PBS, and 100 μL of 10% CCK-8 reagent (10% CCK-8 in 90% DMEM basal medium) was added to each well. The cells were incubated at 37°C in the dark for 30 min-1 h, and the absorbance was measured at 450 nm using a microplate reader. The cell proliferation rate of each group relative to the control group was calculated and plotted using GraphPad Prism.

[0065] (2) qPCR detection of the effects of Lactobacillus plantarum derived components on LPS-induced monocyte-macrophage inflammation levels (TNF-α, IL-6, and IL-1β mRNA): Three concentration gradients were set for each derived component, and a control group (NC) and a model group (LPS) were set. The concentration gradient of the derived components was set the same as (1) in 2.2. THP-1 cells with good growth status were taken, prepared into a cell suspension, and the cells were counted. According to the cell counting results, 5×10 cells per well were added. 5100 ng / mL PMA was added to induce monocyte differentiation into mononuclear macrophages. After 48 hours of culture, THP-1 cells were differentiated into mononuclear macrophages. 100 ng / mL LPS and the corresponding concentrations of the derivative components were administered simultaneously to the model and treatment groups and cultured in a cell culture incubator. After 24 hours of culture, the culture medium was removed, the cells were washed once with PBS, and 500 μL of Trizol lysis buffer was added to each well to lyse the cells on ice for 30 minutes. 100 μL of chloroform was added, the cells were thoroughly mixed, and the mixture was incubated on ice for 15 minutes. The cells were centrifuged at 12,000 rpm for 15 minutes, and the top layer of clear liquid was transferred to a new sterile EP tube. An equal volume of isopropanol was added to the mixture, the mixture was incubated on ice for 15 minutes, and the cells were centrifuged at 12,000 rpm for 15 minutes. The supernatant was removed and the pellet was washed twice with 75% ethanol. The RNA pellet was air-dried and dissolved in sterile, enzyme-free water, and the RNA concentration was measured using a Nanodrop instrument. RNA was reverse transcribed into cDNA using a reverse transcription kit, and qPCR was then performed using the SYBR system to detect TNF-α, IL-6, and IL-1β mRNA levels. The primer sequences used are shown in Table 1.

[0066] Table 1

[0067] (3) Flow cytometry was used to detect the effects of Lactobacillus plantarum derived components on LPS-induced macrophage M1 polarization: 5 concentration gradients of CMV-S, CMV-L and BL-S were set, and the unstained group, control group (NC) and model group (LPS) were set. The concentration gradients of CMVs, CMV- and BL-S were set as in (1) in 2.2. iBMDM cells with good growth status were taken, prepared into cell suspension and the cells were counted. According to the cell counting results, 1.5×10 cells were added to each well. 5 The number of cells was plated in a 12-well plate. After overnight culture, the cells adhered to the wall and the density reached 50-60%, 1 μg / mL LPS and the corresponding concentration of derivative components were given to the model group and the drug group at the same time, and continued to be cultured in the cell culture incubator. After 24 hours of culture, the culture medium was removed, washed with PBS, and the cells were collected by trypsin digestion. 100 μL Cell Staining Buffer, 1 μL FITC anti-mouse / human CD11b and 1 μL PerCP / Cyanine5.5 anti-mouse CD80 were added to each well and incubated on ice in the dark for 15 minutes. After the incubation, the cells were centrifuged at 1500 rpm for 5 minutes to collect the cells, washed with PBS once, and resuspended in 300-500 μL PBS. The proportion of CD11b+CD80+ double-positive cells was detected by flow cytometry.

[0068] 1.3 Test Results (1) CCK-8 test showed that in the PMA-induced THP-1 macrophage model, the four derivative components prepared in Example 1 had no significant effect on cell activity. Further studies found that in the LPS-induced inflammatory macrophage proliferation model, CMVs could significantly inhibit the increase in proliferation level, while BL-S only showed a significant proliferation inhibitory effect at low concentrations, and BL-P and CFS had no significant regulatory effect ( Figure 8 ).

[0069] (2) qPCR results showed that the mRNA expression levels of pro-inflammatory factors TNF-α, IL-6, and IL-1β in the CMVs prepared in Example 1 and BL-S treatment groups were significantly reduced, while the expression levels of the above factors in the BL-P and CFS treatment groups showed no statistical difference or a slight upward trend ( Figure 9 ).

[0070] (3) Flow cytometry results showed that among the anti-inflammatory phenotype Lactobacillus plantarum derived components (CMVs and BL-S), only CMV-S significantly reduced the proportion of CD11b+CD80+ double positive cells in LPS-stimulated iBMDM ( Figure 10 ).

[0071] Test Example 4 1. Exploring the material basis for the efficacy of the derivative components prepared in Example 1 1.1 Reagents Anandamide (AEA, MCE, HY-10863); Putrescine (Sigma-Aldrich); LPS (MCE, HY-D1056); Recombinant Human TNF-α(NovoProtein); Trizol lysis buffer (Vazyme, R401-01); The reverse transcription kit and SYBR Green qPCR SuperMix were purchased from Beijing Quanshijin Company; 2',7'-Dichlorofluorescin diacetate DCFH-DA (Sigma-Aldrich, D6883); anti-mouse-F4 / 80-APC (Biolegend); anti-mouse-CD80-FITC (Biolegend); anti-mouse-CD86-PE (Biolegend).

[0072] 1.2 Research Methods Non-targeted metabolomics sequencing was performed on two derivative components, CMVs and BL-S, which have anti-inflammatory phenotypes, to analyze the material composition and enriched metabolites of the two derivative components and verify the effects of the enriched metabolites on cellular inflammation, oxidative stress and macrophage polarization. The details are as follows: (1) qPCR detection of the effects of AEA and putrescine on the inflammatory levels (TNF-α, IL-6, and IL-1β mRNA) of LPS-induced iBMDM cells and LPS + TNF-α-induced HaCaT cells: a control group (NC) and a model group (LPS / PC) were set up. iBMDM or HaCaT cells with good growth status were taken, prepared into cell suspensions, and the cells were counted. According to the cell counting results, 1.5×10 cells were added to each well. 5 iBMDM cells or 5×10 4 HaCaT cells were plated in a 12-well plate. After overnight culture, the cells adhered to the wall and the density reached 60%. 1 μg / mL LPS or 1 μg / mL LPS + 50 ng / mL TNF-α and AEA or putrescine was given to the modeling group and the drug-treated group at the same time, and the cells were cultured in a cell culture incubator. After 24 hours of culture, the culture medium was removed, the cells were washed once with PBS, and RNA was extracted using the Trizol method. After reverse transcription, qPCR was performed to detect the mRNA levels of TNF-α, IL-6, and IL-1β. The method was the same as described in 1.2 (2) of Experimental Example 2. The primer sequences used are shown in Table 2.

[0073] Table 2

[0074] (2) Flow cytometry was used to detect the effects of AEA and putrescine on the oxidative stress level of RAW264.7 cells induced by LPS: an unstained group, a control group (NC), and a model group (LPS) were set up. RAW264.7 cells with good growth status were taken, cell suspensions were prepared, and cell counts were performed. According to the cell counting results, 5×10 cells were added to each well. 5 The number of cells was plated in a 12-well plate. After overnight culture, the cells adhered to the wall and the density reached more than 60%. 1 μg / mL LPS and AEA or putrescine were administered to the modeling group and the drug-treated group, and cultured in a cell culture incubator. After 24 hours of culture, the culture medium was removed, the cells were washed once with PBS, and 10 μM DCFH-DA was added and incubated at 37°C in the dark for 15 minutes. After incubation, the cells were washed three times with PBS, digested with trypsin, and collected. After washing once with PBS, the cells were resuspended in 300-500 μL PBS and the FITC fluorescence intensity was detected by flow cytometry.

[0075] (3) Detection of the effects of HICA and BL-S on E. coliEffect of K-12 proliferation activity: E. coli K-12 cells were evenly plated in a 96-well plate at a density of 5%, with 5 replicates per group. PBS was added to the NC group, and the corresponding concentrations of HICA or BL-S were added to the drug group. After 24 h of culture, the OD value was measured using a microplate reader. 600nm .

[0076] (4) Flow cytometry was used to detect the effects of AEA and putrescine on the polarization phenotype of BMDM cells induced by LPS: an unstained group, a control group (NC) and a model group (LPS) were set up. 1×10 bone marrow cells were extracted from the leg bones of mice. 6 Cells were plated at 1 / 4 cells / mL in 12-well plates and induced for 7 days in DMEM high-glucose medium supplemented with 10% FBS, 1% P / S, and 30% L929 culture supernatant. Fresh induction medium was replaced every two days. AEA or putrescine was pre-administered within 1-7 days of induction. On the last day, all groups except the NC group were given 1 μg / mL LPS to induce M1 polarization and continued to culture in a cell culture incubator. After 24 hours of culture, cells were washed once with PBS and harvested by trypsinization. After washing with PBS, 100 μL of cell staining buffer was added to each tube, followed by 1 μL of anti-mouse-F4 / 80-APC, 1 μL of anti-mouse-CD80-FITC, and 1 μL of anti-mouse-CD86-PE, respectively. After mixing, cells were stained at 4°C in the dark for 15 minutes. After staining, cells were harvested by centrifugation and resuspended in 300-500 μL of PBS for analysis by flow cytometry.

[0077] 1.3. Exploration Results (1) Non-targeted metabolomics revealed different metabolite composition patterns and screening of differential metabolites between CMVs and BL-S: Non-targeted metabolomics principal component analysis showed that there were significant intergroup differences between CMVs and BL-S samples ( Figure 11 The cluster heat map shows that the biological replication of the samples is good and the data credibility is high. It also reveals that the two groups of samples have different metabolite composition patterns, and there are significant differences in metabolite composition between BL-S and CMVs ( Figure 12 ). Non-targeted metabolomics detected a total of 705 metabolites in CMVs and 1001 metabolites in BL-S, of which 558 metabolites were common in both ( Figure 13). Further analysis of the material composition showed that the three most abundant metabolites in CMVs were heterocyclic compounds, hormones and hormone-related compounds, and organic acids and their derivatives, while the three most abundant metabolites in BL-S were organic acids and their derivatives, amino acids and their metabolites, and nucleotides and their metabolites. Interestingly, the organic acid with the highest content in the category of organic acids and their derivatives in CMVs was AEA, accounting for 80.67% of the total content in the category of organic acids and their derivatives. AEA was also detected in BL-S, accounting for 7.19% of that in CMVs ( Figure 14 Metabolite tracing analysis using KEGG, HMDB, and CHEBI databases showed that 698 metabolites were significantly upregulated and 185 metabolites were downregulated in BL-S compared with CMVs. KEGG enrichment pathway analysis of differential metabolites showed that ABC transporters, glycerophospholipid metabolism, amino acid biosynthesis, and purine metabolism were the main differential pathways ( Figure 15 The three differential metabolites worthy of attention are AEA, putrescine, and 2-hydroxyisocaproic acid (HICA). The contents of putrescine and AEA in CMVs are 15.68 times and 13.90 times higher than those in BL-S, respectively. The content of HICA, which has anti-pathogenic activity, in BL-S is significantly higher than that in CMVs. Figure 16 ).

[0078] (2) qPCR results showed that AEA significantly reduced the mRNA levels of inflammatory factors TNF-α, IL-6, and IL-1β in inflammatory iBMDM cells and HaCaT cells, while putrescine's anti-inflammatory activity was not as significant as that of AEA ( Figure 17 ).

[0079] (3) Flow cytometry results showed that putrescine had no significant effect on the ROS level of LPS-induced macrophages, while AEA significantly reduced the ROS level of LPS-induced macrophages ( Figure 18 ).

[0080] (4) The organic acid with the highest content in BL-S is 2-hydroxyisohexyl (HICA) which has anti-pathogenic activity ( Figure 18 ). The experiments confirmed that HICA and BL-S inhibited E. coli Effect of K-12 proliferation activity ( Figure 19-20 ).

[0081] Test Example 5 1. Investigate the effects of CMVs and BL-S prepared in Examples and Comparative Examples on the symptoms of IMQ-induced psoriasis in mice 1.1 Materials Imiquimod cream (Med shine); 4% paraformaldehyde tissue fixative (Biosharp); Anti-mouse-CD45-PerCP / Cyanine5.5 (Biolegend); Anti-mouse-CD11b-PE (Biolegend); Anti-mouse-F4 / 80-FITC (Biolegend); Anti-mouse-CD86-APC (Biolegend); Ki67 immunohistochemical antibody, F4 / 80 immunohistochemical antibody, CD206 immunohistochemical antibody; Hematoxylin (Servicebio); Yihong (Servicebio); Trizol lysis buffer (Vazyme, R401-01); LPS (MCE, HY-D1056); The reverse transcription kit and SYBR Green qPCR SuperMix were purchased from Beijing Quanshijin Company; All animal experiments were approved by the Experimental Animal Ethics Committee of Sun Yat-sen University and performed in accordance with the guidelines approved by the committee, with the ethics approval number: SYSU-IACUC-2024-001484.

[0082] 1.2 Construction of psoriasis mouse model and treatment plan (1) In this experiment, SPF-grade male BALB / c mice aged 6-8 weeks were acclimated in a pathogen-free environment for one week and given free access to food.

[0083] (2) After removing the hair on the back of the mice using a shaver and depilatory cream, the mice were randomly divided into five groups: Ctrl group, IMQ group, CMVs group, BL-S group, and Dex group. 24 hours after depilation, 62.5 mg of 5% imiquimod ointment was applied to the back skin of the mice in all groups except the Ctrl group once daily for 7 consecutive days to induce psoriasis symptoms.

[0084] (3) On days 4-7, mice in each group were gavaged with 100 μL PBS, CMVs (50 μg / mouse / day), or BL-S (50 μg / mouse / day), or dexamethasone cream was applied topically as a positive control group, once a day.

[0085] (4) The weight of the mice was measured and recorded daily, and the Psoriasis Area and Severity Index (PASI) was scored by observing the skin condition. The PASI assessment includes three clinical indicators: skin thickness, skin erythema, and skin scaling. The score range for each indicator is 0-4 points.

[0086] (5) On the 8th day, the mice were killed and the skin, heart, liver, spleen, lung, and kidney of the lesion site were collected for subsequent experiments.

[0087] 1.3. Tissue H&E staining, immunohistochemistry, and qPCR analysis: (1) H&E staining: The collected tissues were fixed in 4% paraformaldehyde solution, trimmed and rinsed, dehydrated, permeabilized, waxed, embedded, sliced, spread, fished out and baked to make paraffin-embedded sections. H&E staining was then completed after dewaxing, hydration, hematoxylin staining, dehydration, eosin staining, dehydration, permeabilization and sealing.

[0088] (2) Immunohistochemistry: Tissue sections were dewaxed, hydrated, antigen retrieval, endogenous peroxidase removal, blocked, incubated with primary antibodies (anti-Ki67, F4 / 80, CD206), incubated with secondary antibodies, stained with DAB, dehydrated, and mounted for immunohistochemistry.

[0089] (3) qPCR analysis: The steps are the same as (2) in 1.2 of Experimental Example 3, and the primers mouse-β-actin, mouse-TNF-α, and mouse-IL-1β are the same as (1) in 1.2 of Experimental Example 4.

[0090] 1.4 Flow cytometry analysis of the polarization phenotype of mouse splenic macrophages The spleen was repeatedly ground thoroughly on ice using a mesh sieve, then centrifuged at 1500 g for 5 minutes to collect the cell suspension. Red blood cell lysis buffer was then used for 5 minutes on ice, and the cell pellet was collected by centrifugation. To the cell pellet in the experimental tube, 100 μL of cell staining buffer, 1 μL of anti-mouse-CD45-PerCP / Cyanine 5.5, 1 μL of anti-mouse-CD11b-PE, 1 μL of anti-mouse-F4 / 80-FITC, and 1 μL of anti-mouse-CD86-APC were added. Unstained tubes, single-stained tubes, and FMO tubes were also set up. After mixing, the cells were stained at 4°C in the dark for 15 minutes. After staining, the cells were collected by centrifugation, re-separated with PBS, filtered, and analyzed by flow cytometry.

[0091] 1.5. Exploration Results (1) The CMVs prepared in Example 1 can alleviate the symptoms of IMQ-induced psoriasis in mice A psoriasis-like mouse model was established by applying imiquimod (IMQ) ointment. After 7 consecutive days of induction, the mice showed typical symptoms such as epidermal thickening, erythema, and scaling, confirming the successful establishment of the model. Subsequently, the psoriasis mice were treated with oral CMVs and BL-S, or topical dexamethasone acetate (Dex) ointment as a positive control ( Figure 21 The results showed that the skin pathological symptoms of mice in the CMVs treatment group were significantly improved, while the BL-S treatment group also showed moderate symptom relief, but the effect was not as good as that of the CMVs treatment group ( Figure 22 During the experiment, the mice's disease progression was scored and recorded daily based on the clinical Psoriasis Area and Severity Index (PASI). The scoring results showed that the PASI scores of mice induced by IMQ were significantly increased, while the scores of mice in the CMVs, BL-S and Dex treatment groups were reduced in symptoms such as epidermal thickness, erythema and desquamation. Among them, the scores of the CMVs treatment group and the positive control Dex group decreased most significantly ( Figure 23 In addition, the weight of mice in the IMQ model group was significantly reduced compared with the healthy control group, while there was no significant difference in weight changes among the treatment groups ( Figure 24 At the same time, we also observed a reduction in spleen weight in the CMVs, BL-S, and Dex treatment groups, especially in the CMVs and Dex groups, indicating that splenomegaly was effectively alleviated ( Figure 24 ), suggesting a reduction in systemic inflammatory response and effective control of immune activation.

[0092] (2) The CMVs prepared in Example 1 can inhibit skin inflammation in psoriasis mice H&E staining results clearly showed that all treatment groups effectively inhibited IMQ-induced epidermal hyperkeratosis, especially the CMVs and Dex groups, which were significantly better than the BL-S group in reducing epidermal thickness ( Figure 25 At the same time, the infiltration of immune cells in the dermis and the reduction of epidermal thickness showed a synchronous downward trend ( Figure 25 A and C in the figure indicate that excessive immune activation in the lesion area was effectively controlled after treatment. In addition, Ki67 immunohistochemical staining results further revealed that CMVs treatment significantly inhibited the excessive proliferation of epidermal cells induced by IMQ ( Figure 25 D in the figure). Detection of the transcriptional levels of inflammatory factors in skin lesions further showed that CMVs treatment significantly reduced the expression levels of TNF-α and IL-1β mRNA ( Figure 25E in the figure indicates that the skin inflammatory response was effectively alleviated. Taken together, these results fully demonstrate the significant efficacy of CMVs in inhibiting skin inflammation in psoriatic mice, with an effect comparable to that of the positive control drug Dex, while the efficacy of BL-S was relatively weak.

[0093] (3) The CMVs prepared in Example 1 can regulate the balance of macrophages in the skin and spleen of psoriasis mice Immunohistochemical staining of skin lesions for F4 / 80 and CD206 revealed significant F4 / 80+ cell infiltration in the dermis of the IMQ-induced psoriasis mouse model. However, this infiltration was significantly alleviated after treatment with CMVs, BL-S, or Dex, with CMVs and Dex showing a more pronounced therapeutic effect. Figure 26 It is noteworthy that in the psoriatic inflammatory microenvironment, the number of CD206+ cells in the dermis is usually low, but after CMVs and Dex treatment, the number of these cells increased significantly ( Figure 26 qPCR test results further confirmed that CMVs treatment could reduce the ratio of CD86 / CD206 mRNA levels in the skin ( Figure 26 In addition, further flow cytometry analysis revealed that the infiltration ratio of CD45+ CD11b+ F4 / 80+ cells in the spleen of mice treated with CMVs, BL-S, and Dex decreased, and CMVs and Dex treatment also significantly reduced the proportion of CD45+ CD11b+ F4 / 80+ CD86+ cells ( Figure 27 These data collectively point to a conclusion: CMVs treatment can effectively reduce the infiltration of pro-inflammatory macrophages in the skin and spleen of mice, thereby effectively regulating local and systemic immune imbalance.

[0094] (4) Biosafety of CMVs and BL-S Prepared in Example 1 During the experiment of oral administration of CMVs and BL-S to mice, the body weight of the mice did not show significant decrease during the observation period ( Figure 24 Furthermore, H&E staining of the heart, liver, spleen, lungs, and kidneys of mice revealed no significant abnormal changes in the tissue structure and cell morphology of the five organs in any treatment group. This result fully demonstrates that CMVs and BL-S derived from Lactobacillus plantarum have good biocompatibility and do not exhibit significant toxic reactions in vivo ( Figure 28 ).

[0095] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing probiotic membrane vesicles and probiotic-derived components, characterized in that: The following steps are involved: (1) Mix the probiotics with the culture medium, centrifuge, and collect the supernatant and precipitate; (2) The supernatant of the bacterial solution is filtered to obtain the cell-free fermentation supernatant; (3) The supernatant of the bacterial solution was filtered and centrifuged to obtain cytoplasmic membrane vesicles; (4) After the bacterial pellet is resuspended, it is broken and centrifuged. The supernatant and the pellet are collected. The supernatant is filtered to obtain the lysis supernatant. The pellet is dissolved and filtered to obtain the lysis pellet.

2. The preparation method according to claim 1, characterized in that The probiotics described in step (1) are Lactobacillus plantarum ATCC BAA-793.

3. The preparation method according to claim 1, characterized in that The culture medium in step (1) is MRS broth culture medium.

4. The preparation method according to claim 1, characterized in that The centrifugation conditions in step (1) are 6000-12000 g for 10-30 min; the centrifugation conditions in step (3) are 100000-150000 g for 80-120 min.

5. The preparation method according to claim 1, characterized in that The filtration in step (2) is through a 0.22-0.25 μm microporous membrane; the filtration in step (3) is through a 0.45-0.48 μm microporous membrane.

6. The preparation method according to claim 1, characterized in that The rupture method in step (4) is high-pressure homogenization, ultrasonication or lysozyme method; the centrifugation condition is 8000-15000g for 15-45min; the supernatant is filtered through a 0.22-0.25μm microporous membrane; the precipitate is filtered through a 0.22-0.25μm microporous membrane.

7. The probiotic membrane vesicles and probiotic-derived components prepared by the preparation method according to any one of claims 1 to 6, characterized in that: The derived components are cytoplasmic membrane vesicles, lysis supernatant, lysis precipitation and cell-free fermentation supernatant.

8. Use of the probiotic membrane vesicles and probiotic-derived components prepared by the preparation method according to any one of claims 1 to 6 in the preparation of drugs for treating inflammatory diseases.

9. The use according to claim 8, characterized in that The inflammatory disease is an inflammatory skin disease.

10. The use according to claim 8, characterized in that The dosage form of the medicine is powder, granule, capsule, tablet, oral liquid or pill.

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