Lactobacillus-sourced extracellular vesicle and application thereof in prevention and treatment of necrotizing enterocolitis

Abstract

The invention relates to the technical field of biological medicine, in particular to a lactobacillus-sourced extracellular vesicle and application of the lactobacillus-sourced extracellular vesicle in prevention and treatment of necrotizing enterocolitis, the extracellular vesicle is prepared from lactobacillus, the lactobacillus is Lactobacillus rhamnosus GG, the strain is purchased in the American Center for Type Culture Collection, and the preservation number is ATCC 53103. The preparation method of the extracellular vesicles comprises the following steps: carrying out ultracentrifugation on a lactobacillus culture solution, removing thalli, then collecting supernate, carrying out primary microfiltration, taking micro-filtrate, carrying out differential centrifugation, resuspending precipitates by using PBS, then carrying out secondary microfiltration, collecting the micro-filtrate, carrying out density gradient centrifugation, and collecting a 30-45% interface vesicle layer. The invention discloses a synergistic effect mode between intestinal symbiotic flora and intestinal epithelial cell metabolic molecules for the first time, and the discovery is expected to construct a brand-new mechanism normal form for regulating the host intestinal barrier function by flora.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to an extracellular vesicle derived from lactobacillus and an application thereof in preventing and treating necrotizing enterocolitis. Background Art

[0002] Necrotizing enterocolitis (NEC) is a common and fatal gastrointestinal disease in premature infants and low birth weight infants with immature intestinal barrier development. Epidemiological data show that the incidence of NEC in very low birth weight infants (<1500 g) / extremely low birth weight infants (<1000 g) is as high as 11% to 22%, with an overall mortality rate of 23.5%, and the mortality rate of those requiring surgical intervention climbs to 50.9%. The disease is characterized by the collapse of the intestinal barrier, progresses rapidly, and lacks effective early warning signs. Currently, the first-line clinical treatment options mainly rely on antibiotics and intestinal resection, but the use of antibiotics can easily aggravate intestinal flora disorders. Although surgical intervention can save the lives of critically ill children, children with larger intestinal resections are prone to secondary short bowel syndrome, leading to irreversible damage to intestinal function. With declining fertility rates among the population of childbearing age and an increase in the proportion of pregnancies at advanced ages, the incidence of premature births and the proportion of small-for-gestational-age premature infants in my country have increased annually, with the annual number of premature births ranking among the highest in the world. NEC remains a major cause of death in premature infants under 32 weeks of age and a key disease affecting the survival of my country's newborn population. Therefore, identifying effective targets for the prevention and treatment of NEC is crucial for reducing neonatal mortality, improving the quality of my country's newborn population, and reducing the overall medical burden.

[0003] The pathogenesis of NEC remains incompletely elucidated. The current mainstream view is that it is associated with factors such as immature intestinal barrier function, intestinal microbial imbalance, and excessive inflammatory responses. Recent studies have shown that the colonization pattern of the intestinal microbiome is closely related to the development of the neonatal intestinal barrier, and their effects are bidirectional. With the continuous deepening of research, the mechanism by which the microbiome influences the intestinal barrier has expanded from single molecular pathways to the dialogue between the microbiome and the host in a complex symbiotic environment. This suggests that the dialogue mechanism within the complex symbiotic environment of the intestinal microbiome and host can provide new perspectives for understanding the pathogenesis of NEC. NEC still lacks a definitive and effective treatment. Current clinical treatments mainly rely on comprehensive medical management (such as fasting, antibiotics, parenteral nutrition, and circulatory support) and surgical intervention (such as resection of necrotic intestinal segments with fistula formation). However, broad-spectrum antibiotics can easily exacerbate microbial imbalance, and long-term parenteral nutrition may lead to liver damage. Surgery is invasive and carries a high risk of postoperative complications (such as short bowel syndrome). In recent years, the application of fecal microbiota transplantation and postbiotics (microbial metabolites) has provided new directions for the treatment of NEC: the former reduces inflammation by re-establishing the balance of the intestinal microecology; the latter improves intestinal barrier function by regulating intestinal microbiota metabolites such as short-chain fatty acids and bile acids. However, the heterogeneity of donors in fecal microbiota transplantation leads to unstable clinical efficacy, and the single effect of postbiotics is difficult to simulate the complex interactions of the microbiota, and its specific targets and pathways are also unclear. In this context, MVEVs have become a potential strategy to break through the bottleneck of existing technologies due to their metabolite synergy and precise delivery characteristics. As a natural metabolic complex for bacteria-host interactions, MVEVs can not only integrate a variety of active molecules, but also regulate the intestinal microenvironment through targeted delivery mechanisms. In summary, MVEVs can both simulate the overall regulation of the microbiota (overcoming the singleness of postbiotics) and achieve precise targeting (avoiding the heterogeneity of fecal microbiota transplantation), and are expected to open up a more controllable intervention path for the treatment of NEC. Summary of the Invention

[0004] In order to overcome the shortcomings and deficiencies in the prior art, the present invention aims to provide an extracellular vesicle derived from lactobacilli and its application in preventing and treating necrotizing enterocolitis.

[0005] The object of the present invention is achieved by the following technical solution: an extracellular vesicle derived from lactobacillus, wherein the extracellular vesicle is prepared using lactobacillus, and the lactobacillus is Lactobacillus rhamnosus GG, which is purchased from the American Type Culture Collection with a preservation number of ATCC 53103.

[0006] Preferably, the method for preparing the extracellular vesicles is: subjecting the lactobacillus culture fluid to ultracentrifugation to remove the bacteria, then collecting the supernatant for a first microfiltration, taking the microfiltrate for differential centrifugation, resuspending the precipitate with PBS, then subjecting it to a second microfiltration, collecting the microfiltrate for density gradient centrifugation, and collecting a 30%-45% interface vesicle layer.

[0007] Preferably, the temperature of the ultracentrifugation is 3-5°C, the speed is 2500-3500g, and the time is 15-25min; the first microfiltration and the second microfiltration use a PVDF filter membrane with a pore size of 0.15-0.30μm; the parameters of the differential centrifugation are: first centrifugation at a centrifugal speed of 8000-12000g for 20-40min to remove large particle debris, and then centrifugation at a centrifugal speed of 80000-120000g for 100-140min, and the centrifugation temperature is 3-5°C; the density gradient centrifugation uses sucrose solutions of different concentrations to prepare a concentration gradient solution with a mass concentration of 10%-70%, and centrifugation at a centrifugal speed of 120000-180000g for 100-140min.

[0008] Another object of the present invention is achieved through the following technical solution: use of extracellular vesicles derived from lactobacilli in the preparation of a drug for preventing and treating necrotizing enterocolitis.

[0009] Preferably, the extracellular vesicles significantly improve the proliferation activity and migration ability of intestinal epithelial cells and promote intestinal damage repair.

[0010] Preferably, the extracellular vesicles contain CDCA, and the extracellular vesicle-mediated CDCA improves the proliferation and migration ability of intestinal epithelial cells through the activation of the GPR35 / ATPIP / TOMM20 signaling pathway.

[0011] Another object of the present invention is achieved through the following technical solution: a drug for preventing and treating necrotizing enterocolitis, comprising the extracellular vesicles described above and a pharmaceutically acceptable carrier.

[0012] Preferably, the extracellular vesicles significantly improve the proliferation activity and migration ability of intestinal epithelial cells and promote intestinal damage repair.

[0013] Preferably, the extracellular vesicles contain CDCA, and the extracellular vesicles mediate CDCA to improve the proliferation and migration ability of intestinal epithelial cells through the activation of the GPR35 / ATPIP / TOMM20 signaling pathway.

[0014] Another object of the present invention is achieved through the following technical solution: use of CDCA in the preparation of a drug for preventing and treating necrotizing enterocolitis.

[0015] The beneficial effects of the present invention are as follows: the present invention reveals for the first time the synergistic interaction pattern between the metabolic molecules of prokaryotic cells (intestinal symbiotic flora) and eukaryotic cells (intestinal epithelial cells). This discovery is expected to establish a new mechanistic paradigm for the regulation of host intestinal barrier function by the flora, opening up a new perspective for a deeper understanding of the complex interaction between intestinal flora and host cells. At the clinical translation level, the present invention further explores the development of a precise therapeutic strategy targeting intestinal barrier repair by engineering MVEVs to load them with specific metabolic molecules, providing a new approach and potential intervention targets for the prevention and treatment of NEC. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 , α-diversity, composition and differential expression of intestinal flora at the phylum and genus levels in NEC mice; AB are bar graphs of the relative abundance differences of intestinal flora at the phylum level between NEC and control groups; CD are bar graphs of the relative abundance differences of intestinal flora at the genus level between NEC and control groups; *P<0.05, ##P<0.001.

[0017] Figure 2 , Lactobacillus MVEVs significantly improved the proliferation activity and migration ability of intestinal epithelial cells and alleviated intestinal damage; A was observed under confocal microscopy that Lactobacillus MVEVs could smoothly enter IEC-6 cells; B was the CCK-8 experiment to detect the effect of Lactobacillus MVEVs on LPS-induced IEC-6 cell proliferation inhibition; C was the scratch experiment to evaluate the effect of Lactobacillus MVEVs on LPS-induced IEC-6 cell migration inhibition; D was Western blot detection of the expression of intestinal tight junction proteins after Lactobacillus MVEVs intervention; E was H&E staining of small intestinal tissue after Lactobacillus MVEVs intervention.

[0018] Figure 3 , the discovery of the isoenzyme system of Lactobacillus and CDCA; A is a phylogenetic tree showing the differentiation and kinship of different species in the evolutionary process, suggesting that there is an isoenzyme system similar to that of human mitochondria in Lactobacillus; B is a heat map showing that the metabolomics method was used to find that there were significant differences in the bile acid metabolite CDCA in the peripheral blood of children with NEC and the control group (red arrow).

[0019] Figure 4 , Lactobacillus MVEVs mediate CDCA to improve the proliferation activity of intestinal epithelial cells; A is the transmission electron microscopy observation of lactobacilli releasing MVEVs in a natural state, indicated by the red arrow; B is the metabolomics test that found that the CDCA in the purified Lactobacillus MVEVs was significantly higher than that in the control group; C is the CCK-8 test of cell viability and proliferation activity of each group; **P<0.01, ***P<0.001.

[0020] Figure 5 , Lactobacillus MVEVs improve the proliferation and migration ability of intestinal epithelial cells through the GPR35 / ATPIP / TOMM20 pathway; A is the functional enrichment analysis and signal pathway annotation of differentially expressed genes; BD are qPCR detection of the mRNA expression levels of proteins related to the GPR35 / ATPIP / TOMM20 signaling pathway; E is qPCR detection of the mRNA expression levels of mitochondrial fission-related proteins Drp1, Fis1 and Mff; ***P<0.001. DETAILED DESCRIPTION

[0021] In order to facilitate the understanding of those skilled in the art, the following embodiments and accompanying drawings are provided. Figure 1-5 The present invention is further described, and the contents mentioned in the embodiment are not intended to limit the present invention.

[0022] Example 1

[0023] The invention discloses an extracellular vesicle derived from lactobacillus. The extracellular vesicle is prepared by using lactobacillus. The lactobacillus is Lactobacillus rhamnosus GG, which is purchased from the American Type Culture Collection and has a collection number of ATCC 53103.

[0024] The preparation method of the extracellular vesicles is as follows: subjecting the lactobacillus culture fluid to ultracentrifugation to remove the bacteria, then collecting the supernatant for a first microfiltration, taking the microfiltrate for differential centrifugation, resuspending the precipitate with PBS, then subjecting the microfiltration to a second microfiltration, collecting the microfiltrate for density gradient centrifugation, and collecting a 30%-45% interface vesicle layer.

[0025] The ultracentrifugation temperature is 4°C, the speed is 3000g, and the time is 20 minutes; the first and second microfiltrations use a PVDF filter membrane with a pore size of 0.22 μm; the parameters of the differential centrifugation are: first centrifugation at a centrifugal speed of 10,000g for 30 minutes to remove large particle debris, and then centrifugation at a centrifugal speed of 100,000g for 120 minutes, and the centrifugation temperature is 4°C; the density gradient centrifugation uses sucrose solutions of different concentrations to prepare concentration gradient solutions with mass concentrations of 30%, 45% and 60%, respectively, and centrifugation at a centrifugal speed of 150,000g for 120 minutes.

[0026] Application of extracellular vesicles derived from lactobacilli in the preparation of a medicine for preventing and treating necrotizing enterocolitis.

[0027] The extracellular vesicles significantly improve the proliferation activity and migration ability of intestinal epithelial cells and promote the repair of intestinal damage.

[0028] The extracellular vesicles contain CDCA, and CDCA mediated by the extracellular vesicles improves the proliferation and migration ability of intestinal epithelial cells through the activation of the GPR35 / ATPIP / TOMM20 signaling pathway.

[0029] A medicine for preventing and treating necrotizing enterocolitis, comprising the extracellular vesicles described above and a pharmaceutically acceptable carrier.

[0030] The extracellular vesicles significantly improve the proliferation activity and migration ability of intestinal epithelial cells and promote the repair of intestinal damage.

[0031] The extracellular vesicles contain CDCA, and the extracellular vesicles mediate CDCA to improve the proliferation and migration ability of intestinal epithelial cells through the activation of the GPR35 / ATPIP / TOMM20 signaling pathway.

[0032] Application of CDCA in the preparation of medicines for preventing and treating necrotizing enterocolitis.

[0033] Example 2

[0034] The difference between this embodiment and the above-mentioned embodiment 1 is that: the temperature of the ultracentrifugation is 3°C, the speed is 2500g, and the time is 25 min; the first and second microfiltrations use a PVDF filter membrane with a pore size of 0.15 μm; the parameters of the differential centrifugation are: first centrifugation at a centrifugal speed of 8000g for 40 minutes to remove large particle debris, and then centrifugation at a centrifugal speed of 80,000g for 140 minutes, and the centrifugation temperature is 3°C; the density gradient centrifugation uses sucrose solutions of different concentrations to prepare concentration gradient solutions with mass concentrations of 20%, 40% and 60%, respectively, and centrifugation at a centrifugal speed of 120,000g for 140 minutes.

[0035] Example 3

[0036] The difference between this embodiment and the above-mentioned embodiment 1 is that: the temperature of the ultracentrifugation is 5°C, the speed is 3500g, and the time is 15 min; the first and second microfiltrations use a PVDF filter membrane with a pore size of 0.30 μm; the parameters of the differential centrifugation are: first centrifugation at a centrifugal speed of 12000g for 20 minutes to remove large particle debris, and then centrifugation at a centrifugal speed of 120,000g for 100 minutes, and the centrifugation temperature is 5°C; the density gradient centrifugation uses sucrose solutions of different concentrations to prepare concentration gradient solutions with mass concentrations of 20%, 45% and 70%, respectively, and centrifugation at a centrifugal speed of 180,000g for 100 minutes.

[0037] In order to test the beneficial effects of the present invention, the inventors conducted the following experiments on the COL-OHA@PRP hydrogel obtained in Example 1:

[0038] 1. Experimental methods:

[0039] 1. H&E staining of small intestinal tissue after Lactobacillus MVEVs intervention

[0040] 1.1. Animal model construction and processing:

[0041] A. 6-8 week old C57BL / 6 mice were randomly divided into three groups (n=5):

[0042] i. Control group: daily gavage with PBS (200 μL / mouse);

[0043] ii. Injury group: free drinking of 3% DSS solution (MP Biomedicals, 160110) for 7 days;

[0044] iii. Intervention group: DSS treatment + Lactobacillus MVEVs (100 μg / mouse / day, dissolved in PBS and gavage).

[0045] B. The animals were sacrificed by anesthesia 24 hours after the intervention, and the mid-jejunum tissue (3 cm from the pylorus) was obtained.

[0046] 1.2 Tissue fixation and slice preparation:

[0047] A. Tissue blocks were fixed in 4% paraformaldehyde (Sigma, P6148) at 4°C for 24 h, then rinsed with PBS three times (10 min each time).

[0048] B. Gradient ethanol dehydration: 70% ethanol (1 h) → 80% ethanol (1 h) → 95% ethanol (1 h) → 100% ethanol (2 times, 1 h each).

[0049] C. Xylene transparency: Xylene I / II (30 min each) → wax immersion (60°C paraffin, 3 times, 1 h each).

[0050] D. Embedded in paraffin, 4 μm serial sections were prepared using a microtome (Leica RM2235).

[0051] 1.3 H&E staining

[0052] A. Dewaxing and rehydration: xylene I / II (10 min each) → gradient ethanol (100% → 95% → 80% → 70%, 5 min each) → distilled water immersion.

[0053] B. Hematoxylin staining: Harris hematoxylin (Sigma, HHS128) staining for 5 min → washing with running water for 5 min → differentiation with 1% hydrochloric acid and ethanol for 3 s → blueing with 0.2% ammonia solution for 30 s.

[0054] C. Eosin staining: stain with 0.5% eosin Y (Sigma, HT110116) for 2 min → rinse with running water for 10 s.

[0055] D. Dehydration and transparency: gradient ethanol (95% → 100%, 30 seconds each) → xylene I / II (2 minutes each).

[0056] E. Seal the slides with neutral gum (Sigma, 100483).

[0057] 1.4 Image Analysis and Quantification

[0058] A. Five fields of view were randomly photographed for each sample using an optical microscope (Nikon Eclipse Ci, 200×).

[0059] B. Quantitative indicators:

[0060] i. Villus height / crypt depth ratio (V / C ratio): measured using NIS-Elements software;

[0061] ii. Inflammatory infiltration score: 0 (no infiltration) - 3 (severe infiltration).

[0062] c. Data are expressed as mean ± SEM, and comparisons among groups were performed using one-way ANOVA (GraphPad Prism 9.0).

[0063] Key controls: normal untreated group, MVEVs-treated group (100 μg / mouse / day);

[0064] Reagent description: DSS (MW 36-50 kDa, MP Biomedicals 160110).

[0065] 2. Transmission electron microscopy observation of natural release of MVEVs by Lactobacillus

[0066] 2.1 Bacterial culture and sample preparation:

[0067] A. Lactobacillus rhamnosus GG (ATCC 53103) was inoculated into MRS broth (BD, 288130) and cultured anaerobically at 37°C until OD600 = 0.6.

[0068] B. Negatively stained samples:

[0069] i. Take 1 mL of bacterial culture and centrifuge at 12000 g for 5 min (4°C) → discard the supernatant.

[0070] ii. Resuspend the pellet in 20 μL of 2% phosphotungstic acid (PTA, Sigma P6391, pH 7.0) → stain at room temperature for 1 min.

[0071] iii. Add dropwise to a 200-mesh carbon-supported copper grid (Electron Microscopy Sciences, CF200-Cu) → dry with filter paper.

[0072] C. Ultrathin section samples:

[0073] i. Fix the bacterial pellet with 2.5% glutaraldehyde (Sigma, G5882) at 4°C for 2 h → rinse three times with PBS.

[0074] ii. Fix with 1% osmium hydroxide (Sigma, 75632) at 4°C for 1 h, then dehydrate with acetone in a gradient manner (30%-100%).

[0075] iii. Epon 812 resin (Sigma, 45345) was used for infiltration and embedding → polymerized at 70°C for 48 h.

[0076] iv. Ultramicrotome (Leica UC7) was used to cut 70 nm sections.

[0077] v. Double staining with uranyl acetate (15 min) + lead citrate (5 min).

[0078] 2.2 Electron microscopy observation and data analysis:

[0079] A. Transmission electron microscopy (Hitachi HT7800, 80 kV) observation, accelerating voltage 80 kV.

[0080] B. Twenty bacteria were randomly selected and the diameter of MVEVs within 50 nm of their edges was measured (ImageJ v1.53).

[0081] C. Calculation of MVEVs release density: number of vesicles / μm 2 Bacterial surface area (formula: total number of vesicles / bacterial circumference × π × bacterial radius).

[0082] Negative control: Sterile MRS medium was used to prepare negative staining and ultrathin section samples in the same manner.

[0083] 3. Scratch assay to evaluate the effect of MVEVs on LPS-induced IEC-6 cell migration inhibition

[0084] 3.1 Cell culture and treatment:

[0085] A. IEC-6 cells (ATCC CRL-1592) were cultured in DMEM (Gibco, 11965) + 10% FBS (Gibco, 10099) + 1% insulin (Sigma, 10516).

[0086] B. Inoculate into 24-well plates (5×105 / well) and culture at 37°C until 90% confluence.

[0087] 3.2 Scratch creation and intervention:

[0088] A. Use a 200 μL sterile pipette tip (Axygen, T-200) to make vertical scratches (same operator, uniform force).

[0089] B. Wash three times with PBS and replace with the following treatment medium:

[0090] i. Control group: normal culture medium;

[0091] ii. Injury group: 1 μg / mL LPS (Sigma, L4391);

[0092] iii. Intervention group: LPS + 20 μg / mL Lactobacillus MVEVs.

[0093] C. The scratch area was photographed under an inverted microscope (Olympus CKX53, 100×) at 0 h, 12 h, and 24 h.

[0094] 3.3、Quantification of migration area:

[0095] A. ImageJ software delineates the scratch area and calculates the area

[0096] B. The experiment was repeated three times independently, and the data are expressed as mean ± SEM.

[0097] Strict control: MVEVs alone treatment group (20 μg / mL); LPS concentration verification: CCK-8 test confirmed that the 24h cell survival rate was >85%.

[0098] 4. Confocal microscopy observation of MVEVs entering IEC-6 cells

[0099] 4.1 Fluorescent labeling of MVEVs

[0100] A. Purified MVEVs (ultracentrifugation) were resuspended in PBS and the concentration was adjusted to 1 mg / mL.

[0101] B. Add PKH26 red membrane dye (Sigma, PKH26GL) to a final concentration of 2 μM → vortex and incubate for 5 min in the dark.

[0102] C. Add an equal volume of 1% BSA to terminate the reaction → ultracentrifuge at 100,000 g for 1 h (4°C) → resuspend in PBS.

[0103] 4.2 Cell treatment and endocytosis observation:

[0104] A. IEC-6 cells were seeded on confocal culture dishes (NEST, 801001) at a density of 2×10 4 / dish.

[0105] B. Experimental Grouping:

[0106] i. Experimental group: PKH26-MVEVs (20 μg / mL);

[0107] ii. Dye control group: free PKH26 (2 μM);

[0108] iii. Low-temperature blocking group: pre-cooled at 4°C for 30 min and then added with PKH26-MVEVs (incubated at 4°C).

[0109] c. Incubate at 37°C for 2 h → wash three times with PBS → fix with 4% PFA for 15 min.

[0110] 4.3 Confocal imaging and quantification

[0111] A. Permeabilization with 0.1% Triton X-100 for 10 min → DAPI nuclei staining (1 μg / mL, 10 min).

[0112] B. The slides were sealed with antifade reagent (Invitrogen, P36930).

[0113] C. Laser confocal microscope (Zeiss LSM 900) parameters:

[0114] PKH26: excitation wavelength 551 nm, emission collection 567 nm (red channel)

[0115] DAPI: excitation wavelength 358 nm, emission collection 461 nm (blue channel)

[0116] D, Z-stack scanning (layer thickness 0.5 μm), 3D reconstruction analysis of intracellular localization.

[0117] E. Randomly analyze 30 cells:

[0118] i. MVEVs-positive cell rate = (number of cells with red fluorescence area > 5% of cytoplasmic area / total number of cells) × 100%;

[0119] ii. Mean fluorescence intensity (MFI): ImageJ quantified the grayscale value of the red channel in the cytoplasmic area.

[0120] Key verification: The MFI of the low-temperature blocking group must be significantly lower than that of the experimental group (p<0.01) to confirm active endocytosis.

[0121] 5. qPCR experiment

[0122] Total RNA was extracted using TRIzol reagent (Vazyme R401-01), and 1 μg of RNA was used for cDNA synthesis using a reverse transcription kit (Vazyme R223).

[0123] 5.1. Genomic DNA Removal: Prepare the following mixture in an RNase-free centrifuge tube: 1 μg template RNA, 4 μL 4×g DNA wiper mix, add RNase-free ddH2O to 16 μL, mix gently by pipetting, and incubate at 42°C for 2 min.

[0124] 5.2. Prepare the reverse transcription reaction system: Add 4 μL of 5×HiScript II qRTSuperMix II directly to the reaction tube in step 1 and mix gently by pipetting.

[0125] 5.3. Perform reverse transcription reaction: 50℃ for 15 min, 85℃ for 5 sec. Dilute the cDNA 10-fold before performing subsequent qPCR reaction.

[0126] 5.4. RNA levels were detected using qPCR (Vazyme Q711).

[0127] A. Prepare the following mixture in a qPCR tube: 10 μL of 2× ChamQ Universal SYBR qPCR Master Mix, 0.4 μL of primer 1 (10 μM), 0.4 μL of primer 2 (10 μM), 1 μL of cDNA, and 8.2 μL of ddH2O.

[0128] B. Perform qPCR reaction according to the following conditions: pre-denaturation at 95°C for 30 seconds, cycling (95°C for 10 seconds, 60°C for 30 seconds, for a total of 40 cycles), and extension at 72°C for 5 minutes.

[0129] The RNA expression level was normalized to the expression level of GAPDH, and the tsRNA expression level was normalized to the expression level of U6. -ΔΔCt The relative RNA expression was analyzed by the method.

[0130] The primer sequences are shown in the following table:

[0131]

[0132]

[0133] 6. Western Blot Experiment

[0134] 6.1 Sample Preparation

[0135] A. Protein extraction:

[0136] Tissue sample: 50 mg of small intestinal tissue was collected and added to 500 μL of RIPA lysis buffer (Beyotime P0013B) (containing 1% protease inhibitors). The sample was homogenized on ice (15,000 rpm, 3 × 10 s). The sample was allowed to stand at 4°C for 30 min, then centrifuged at 12,000 g for 15 min at 4°C and the supernatant was collected.

[0137] B. Concentration determination: Determine the protein concentration using a BCA kit (Beyotime P0009) (standard curve range: 0-2000 μg / mL). Adjust the sample to a uniform concentration (2 μg / μL) and add 5× Loading Buffer and boil for 10 min.

[0138] 6.2 SDS-PAGE electrophoresis

[0139] A. Gel preparation:

[0140] Separation gel: 12% acrylamide (Bio-Rad, 4561043)

[0141] Stacking gel: 5% acrylamide

[0142] B. Sample loading and electrophoresis:

[0143] Load 20 μg protein into each well → electrophoresis at 80V constant voltage (concentrating gel) → constant voltage of 120V (separating gel) → stop when bromophenol blue reaches the bottom of the gel.

[0144] Molecular weight marker: Prestained protein marker (Thermo, 26616)

[0145] 6.3. Transfer

[0146] A. Wet transfer method:

[0147] PVDF membrane (Millipore, IPVH00010) was activated with methanol for 1 min and then immersed in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol) together with the gel.

[0148] The membrane was transferred at a constant current of 300 mA for 90 min (4°C).

[0149] B. Transfer verification: Ponceau red staining (Sigma, P7170) was used to observe the integrity of the bands.

[0150] 6.4. Blocking and Antibody Incubation

[0151] A. Blocking: Block with 5% skim milk (Bio-Rad, 1706404) at room temperature for 1 h with shaking.

[0152] B. Primary Antibody Incubation:

[0153] Target protein ZO-1, primary antibody (CST 13663T), dilution ratio 1:1000, 4°C overnight;

[0154] The target protein β-actin, primary antibody (CST#4970), dilution ratio 1:2000, room temperature for 2h (internal reference).

[0155] C. Secondary antibody incubation: HRP-labeled goat anti-rabbit IgG (CST#7074, 1:5000), shake at room temperature for 1 h.

[0156] 6.5 Chemiluminescence Detection

[0157] A. Development: ECL luminescent solution (Thermo, 32106) evenly covers the membrane → Chemiluminescence analyzer (Bio-Rad ChemiDocMP) collects the signal.

[0158] B. Exposure optimization: multi-gradient exposure (5s-5min), select images without saturated bands.

[0159] 2. Experimental Results

[0160] 1. Alpha diversity, composition, and differential expression of intestinal microbiota at the phylum and genus levels in NEC mice

[0161] In previous experimental studies, we observed that NEC model mice showed intestinal flora imbalance characteristics compared with the normal control group through 16S rRNA sequencing analysis, with a significant increase in the relative abundance of Proteobacteria (P < 0.01) and a significant decrease in the abundance of Firmicutes (P < 0.05) ( Figure 1 A-1B). Further analysis revealed that the abundance of intestinal pathogenic bacteria such as Citrobacter and Acinetobacter in the NEC group showed a significant enrichment trend (increased by 2.8 times and 3.5 times, respectively), while the abundance of the beneficial bacteria genus Lactobacillus decreased significantly (decreased by more than 90%) ( Figure 1 C-1D), and intestinal flora imbalance is closely related to intestinal barrier function damage.

[0162] 2. Lactobacillus MVEVs significantly improved the proliferation activity and migration ability of intestinal epithelial cells and alleviated intestinal damage

[0163] According to literature reports, there are significant functional differences in MVEVs released by different bacterial communities. Does the Lactobacillus, which is significantly reduced in NEC, affect the intestinal barrier function through the MVEVs it secretes? To further explore this issue, we co-cultured Lactobacillus MVEVs with an intestinal epithelial cell injury model. The results showed that MVEVs released by Lactobacillus have the effect of promoting intestinal damage repair ( Figure 2 ).

[0164] 3. Discovery of Lactobacillus isoenzyme system and CDCA

[0165] To explore how MVEVs exert their biological functions in NEC, we conducted an extensive literature review and obtained the following important findings: ① The new concept of intestinal bacterial-derived host isozymes: In the long-term co-evolution of the intestinal flora with the host, there are some enzymes that have the same catalytic function as host enzymes and can regulate the physiological and pathophysiological functions of the host. The research team first proposed the new concept of "intestinal bacterial-derived host isozymes" and published the relevant research results in Science. They found that the dipeptidyl peptidase 4 isozyme derived from the intestinal flora can be secreted into the host body and degrade the host's glucagon-like peptide-1, thereby inducing impaired glucose tolerance. By synthesizing small molecule inhibitors with high activity and strong selectivity, it is possible to specifically inhibit the dipeptidyl peptidase 4 isozyme, thereby improving impaired glucose tolerance. ② Microbial gene clusters compensate for the lack of human uricase: Most mammals metabolize uric acid through uricase, but the human uricase gene was inactivated early in evolution, resulting in the unclear metabolic mechanism of uric acid in the intestine. Studies have found that intestinal bacteria degrade urea that the host cannot metabolize by expressing urea metabolism gene clusters, thereby alleviating hyperuricemia and complications. This suggests that microorganisms have acquired metabolic functions that are missing from the host through evolution and maintain a new mechanism for the homeostasis of host uric acid. Through bioinformatics analysis, we found that there is indeed an isoenzyme system similar to that of human mitochondria in Lactobacillus ( Figure 3 A). The above suggestions, from the perspective of intestinal microbiota-host isoenzymes, may provide new ideas for exploring how MVEVs derived from intestinal flora communicate with the host in NEC. This discovery has aroused our great interest.

[0166] 4. Lactobacillus MVEVs mediate CDCA to improve intestinal epithelial cell proliferation activity

[0167] The core mechanism of action of intestinal bacteria-host isoenzymes includes metabolic interactions and evolutionary complementation. We cultured Lactobacillus and observed using transmission electron microscopy that Lactobacillus can release MVEVs ( Figure 4 A). A model of intestinal epithelial cell injury was established and co-cultured with Lactobacillus MVEVs. The results showed that it could significantly restore the proliferation capacity of intestinal epithelial cells ( Figure 2 B), and alleviate the inhibitory effect of LPS on cell migration ability ( Figure 2 C). NEC animal model level detection found that Lactobacillus MVEVs intervention can effectively improve the expression of tight junction proteins in the mouse intestine ( Figure 2 D), promotes intestinal epithelial cell proliferation and intestinal villus repair, and reduces the degree of intestinal necrosis ( Figure 2E). Metabolomics methods were used to detect peripheral blood in NEC patients and controls, and it was found that the bile acid metabolite CDCA was significantly different between NEC and controls ( Figure 3 B). Subsequently, the culture supernatant of Lactobacillus was collected and the Lactobacillus MVEVs components were separated by ultracentrifugation. The CDCA content in the purified MVEVs was detected by metabolomics. The results showed that CDCA was enriched in the purified Lactobacillus MVEVs ( Figure 4 B). We selected the IEC-6 intestinal epithelial cell line as the experimental subject and constructed an NEC cell model by LPS modeling. We set up a "normal group," "LPS group," "LPS+Lactobacillus group," "LPS+MVEVs group," and "LPS+CDCA group." After 72 hours of culture, we used the CCK-8 kit to detect cell viability and proliferation activity in each group. The results confirmed that the CDCA in the MVEVs secreted by Lactobacillus had the main effect on improving the NEC cell model. Figure 4 C). These results indicate that Lactobacillus MVEVs host isozymes mediate cross-species transmission of CDCA and play an important role in the proliferation and differentiation of intestinal epithelial cells.

[0168] 5. Lactobacillus MVEVs improve the proliferation and migration of intestinal epithelial cells through the GPR35 / ATPIP / TOMM20 pathway

[0169] How does CDCA work? It was previously discovered that the isoenzyme system in lactobacilli is similar to that in human mitochondria ( Figure 3 A). Mitochondria, as the cell's energy factory and metabolic control center, play a key role in the development, regeneration, and functional maintenance of intestinal epithelial cells. The research team used high-throughput sequencing technology to perform transcriptomic analysis, functional enrichment analysis of differentially expressed genes, and signal pathway annotation. They found that Lactobacillus MVEVs-mediated CDCA promoted intestinal epithelial cell proliferation and migration, which was related to improved mitochondrial function ( Figure 5 A). We detected by qPCR that GPR35, ATPIP, and TOMM20 were significantly overexpressed in the Lactobacillus MVEVs intervention group ( Figure 5B-5D). Literature review: GPR35 is mainly expressed in intestinal epithelial cells, and its expression is regulated by the intestinal microbiota. It may act as a sensor of microbial metabolites and play a key role in body diseases by regulating intestinal microbial metabolism; ATPIP is a mitochondrial protein that participates in the process of protein targeting to mitochondria; TOMM20 is one of the main receptors of the mitochondrial outer membrane preprotein translocase TOM complex, and plays an important role in protein transport, cellular energy metabolism, and cell apoptosis. Further qPCR method was used to detect that the mRNA levels of mitochondrial fission-related proteins Drp1, Fis1 and Mff were significantly overexpressed in the Lactobacillus MVEVs intervention group ( Figure 5 E).

[0170] From the above results, it can be seen that the ability of Lactobacillus MVEVs to improve the proliferation and migration of intestinal epithelial cells is closely related to the activation of the GPR35 / ATPIP / TOMM20 signaling pathway and mitochondrial fission.

[0171] With the advancement of perinatal maternal and infant rescue technology and intervention concepts, the survival rate of small-for-gestational-age premature infants has continued to increase, and NEC has become one of the key diseases affecting their survival and health. However, the current prevention and treatment methods are extremely limited. The present invention reveals for the first time at the basic research level the synergistic effect pattern between the metabolic molecules of prokaryotic cells (intestinal symbiotic flora) and eukaryotic cells (intestinal epithelial cells). This discovery is expected to construct a new mechanism paradigm for the regulation of host intestinal barrier function by flora, opening up a new perspective for a deeper understanding of the complex interaction between intestinal flora and host cells. At the clinical translation level, the present invention further explores the development of a precise treatment strategy targeting intestinal barrier repair by engineering MVEVs to load them with specific metabolic molecules, providing a new approach and potential intervention targets for the prevention and treatment of NEC.

[0172] The above embodiments are preferred implementation schemes of the present invention. In addition, the present invention can also be implemented in other ways. Any obvious replacement without departing from the concept of the present invention is within the scope of protection of the present invention.

Claims

1. An extracellular vesicle derived from Lactobacillus, wherein the extracellular vesicle is prepared using Lactobacillus, characterized in that: The lactobacillus is Lactobacillus rhamnosus GG, which was purchased from the American Type Culture Collection with a collection number of ATCC 53103.

2. The extracellular vesicle derived from Lactobacillus according to claim 1, characterized in that: The preparation method of the extracellular vesicles is as follows: subjecting the lactobacillus culture fluid to ultracentrifugation to remove the bacteria, then collecting the supernatant for a first microfiltration, taking the microfiltrate for differential centrifugation, resuspending the precipitate with PBS, then subjecting the microfiltration to a second microfiltration, collecting the microfiltrate for density gradient centrifugation, and collecting a 30%-45% interface vesicle layer.

3. The extracellular vesicle derived from Lactobacillus according to claim 2, characterized in that: The ultracentrifugation temperature is 3-5°C, the speed is 2500-3500g, and the time is 15-25min; the first and second microfiltrations use a PVDF filter membrane with a pore size of 0.15-0.30μm; the parameters of the differential centrifugation are: first centrifugation at a centrifugal speed of 8000-12000g for 20-40min to remove large particle debris, and then centrifugation at a centrifugal speed of 80000-120000g for 100-140min, and the centrifugation temperature is 3-5°C; the density gradient centrifugation uses sucrose solutions of different concentrations to prepare a concentration gradient solution with a mass concentration of 10%-70%, and centrifugation at a centrifugal speed of 120000-180000g for 100-140min.

4. Use of the extracellular vesicles derived from lactobacilli according to any one of claims 1 to 3 in the preparation of a medicament for preventing and treating necrotizing enterocolitis.

5. The use according to claim 4, characterized in that: The extracellular vesicles significantly improve the proliferation activity and migration ability of intestinal epithelial cells and promote the repair of intestinal damage.

6. The use according to claim 4, characterized in that: The extracellular vesicles contain CDCA, and CDCA mediated by the extracellular vesicles improves the proliferation and migration ability of intestinal epithelial cells through the activation of the GPR35 / ATPIP / TOMM20 signaling pathway.

7. A drug for preventing and treating necrotizing enterocolitis, characterized in that: The drug comprises the extracellular vesicle according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier.

8. The drug according to claim 7, characterized in that: The extracellular vesicles significantly improve the proliferation activity and migration ability of intestinal epithelial cells and promote the repair of intestinal damage.

9. The drug according to claim 7, characterized in that: The extracellular vesicles contain CDCA, and the extracellular vesicles mediate CDCA to improve the proliferation and migration ability of intestinal epithelial cells through the activation of the GPR35 / ATPIP / TOMM20 signaling pathway.

10. Use of CDCA in the preparation of drugs for preventing and treating necrotizing enterocolitis.

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