Preparation method and application of clostridium butyricum-sourced extracellular vesicles

Abstract

The invention discloses a preparation method and application of clostridium butyricum-sourced extracellular vesicles, the extracellular vesicles (CBEVs) are separated and purified from clostridium butyricum, and through structural characterization, metabonomics and multi-function verification, the extracellular vesicles (CBEVs) have a good application prospect. Results show that the extracellular vesicle has multiple effects of resisting helicobacter pylori activity and gastric mucosa penetrability, inhibiting bacterial adhesion and biofilm formation, reducing gastric mucosa inflammatory factors and the like, and is non-toxic to gastric epithelial cells. Compared with the existing antibiotic and probiotic therapy, the vesicle has a remarkable antibacterial effect on sensitive helicobacter pylori and drug-resistant strains, solves the problems of high drug resistance, large side effect and the like of the existing antibiotic therapy for helicobacter pylori, avoids the problem of viable bacterium colonization failure of probiotics, and is higher in safety and suitable for long-term treatment and prevention. In addition, due to the natural source characteristic, excellent biocompatibility is achieved, and an innovative direction is provided for developing novel antibacterial drugs.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for preparing extracellular vesicles derived from Clostridium butyricum and its application. Background Technology

[0002] Helicobacter pylori is a Gram-negative pathogen closely associated with gastrointestinal diseases such as chronic gastritis, peptic ulcers, and gastric cancer. Currently, the main clinical approach is to eradicate Helicobacter pylori using combination antibiotic therapy. However, long-term use leads to increased bacterial resistance, intestinal flora imbalance, and frequent drug side effects, severely limiting the safety and effectiveness of treatment. Therefore, there is an urgent need to develop novel non-antibiotic treatment strategies.

[0003] Probiotics have been extensively studied due to their ability to regulate host immunity and inhibit pathogen colonization. While probiotics offer the advantage of low toxicity as a replacement therapy, live bacteria preparations suffer from bottlenecks such as poor stability and low gastrointestinal colonization rates, and their mechanism of action in inhibiting Helicobacter pylori is not fully understood. Furthermore, current probiotic products largely rely on the functions of the bacteria themselves, lacking in-depth development of the functions of their metabolites.

[0004] In recent years, extracellular vesicles (EVs) have attracted much attention. These are vesicle structures, approximately 30 nm to 5 µm in diameter, enclosed in a lipid bilayer and released by almost all cell types (including prokaryotes and eukaryotes). EVs can carry various bioactive molecules such as amino acids, lipids, and nucleic acids and mediate intercellular communication. Engineered EVs have been developed as drug delivery carriers, but their industrial preparation is costly and standardized. Research on EVs derived from natural microorganisms is still in its early stages, particularly in the development of dual functions such as targeted pathogen inhibition and host cell protection. Summary of the Invention

[0005] To address the shortcomings and deficiencies of the prior art, one of the objectives of this invention is to provide the application of Clostridium butyricum-derived extracellular vesicles in the preparation of anti-Helicobacter pylori drugs.

[0006] Clostridium butyricum ( Clostridium butyricum These are beneficial bacteria that regulate the balance of the human gut microbiota. They can repair the intestinal mucosal barrier and inhibit inflammatory responses by secreting short-chain fatty acids (such as butyric acid).

[0007] The Helicobacter pylori described in this invention includes clinically resistant anti-Helicobacter pylori and susceptible Helicobacter pylori, including but not limited to Hp SS1, ATCC 43504, Hp MLC, Hp M1, Hp L1, and Hp C1 strains.

[0008] The extracellular vesicles derived from Clostridium butyricum of this invention carry a variety of active metabolites, which can significantly inhibit the growth of Helicobacter pylori and reduce the mRNA transcription levels of Helicobacter pylori resistance genes (hefA) and virulence genes (cagA, ureB), thus exhibiting significant antibacterial effects against both sensitive and drug-resistant Helicobacter pylori strains.

[0009] The second objective of this invention is to provide the application of the combination of Clostridium butyricum-derived extracellular vesicles and clarithromycin in the preparation of anti-Helicobacter pylori drugs.

[0010] The extracellular vesicles derived from Clostridium butyricum of this invention have a scavenging effect on Helicobacter pylori biofilms. When used in combination with clarithromycin, the two have a synergistic effect, which can significantly enhance the anti-Helicobacter pylori effect, especially in efficiently clearing Helicobacter pylori biofilms and reducing the amount of biofilm formation.

[0011] A third objective of this invention is to provide the use of Clostridium butyricum-derived extracellular vesicles in the preparation of drugs for the prevention or treatment of gastrointestinal diseases caused by Helicobacter pylori infection.

[0012] The gastrointestinal diseases described in this invention include chronic gastritis, peptic ulcers, and gastric cancer caused by Helicobacter pylori infection.

[0013] The extracellular vesicles derived from Clostridium butyricum described in this invention have an average particle size of 130~200 nm and a surface charge of -41.2~-14.7 mV, exhibiting good stability in gastrointestinal fluids. In an artificial gastric mucosa model, the permeability is >55% after 180 min, demonstrating the ability to penetrate the gastric mucus layer. Furthermore, it can competitively inhibit Helicobacter pylori adhesion to GES-1 cells and remove already adhered bacteria, and can also target and track intracellular Helicobacter pylori.

[0014] The extracellular vesicles derived from Clostridium butyricum described in this invention are non-toxic to GES-1 cells (survival rate >80%) in the concentration range of 1.56~1600 μg / mL, and can significantly reduce the levels of IL-6 and IL-8 secreted by GES-1 cells after Helicobacter pylori infection, thus exhibiting both safety and anti-inflammatory activity.

[0015] In some embodiments, the medicament of the present invention comprises extracellular vesicles derived from Clostridium butyricum and a pharmaceutically acceptable carrier.

[0016] In some embodiments, the pharmaceutically acceptable carrier includes, but is not limited to: solvents, diluents, disintegrants, precipitation inhibitors, surfactants, flow aids, binders, lubricants, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, flavorings, sweeteners, ion exchangers, release agents, coating agents, flavoring agents, and / or antioxidants.

[0017] The method for preparing extracellular vesicles derived from Clostridium butyricum according to the present invention includes the following steps: (1) Add Clostridium butyricum powder to the culture medium and suspend it in an anaerobic environment to obtain Clostridium butyricum culture medium; (2) Centrifuge the Clostridium butyricum culture medium, take the supernatant and concentrate it through a hollow fiber ultrafiltration membrane to obtain a concentrated solution; (3) Add a 33% sucrose solution to the bottom of the ultra-clean quick-seal centrifuge tube, then add concentrated liquid until the centrifuge tube is full, centrifuge at ultra-high speed, and collect the sucrose layer. (4) Add sterile PBS to the sucrose layer, remove the sucrose by ultrafiltration and centrifugation, and concentrate to obtain the extracellular vesicles of Clostridium butyricum.

[0018] This invention employs a combined process of "anaerobic culture-ultrafiltration-sucrose pad ultracentrifugation" to successfully obtain morphologically intact, uniformly sized, and negatively charged extracellular vesicles derived from Clostridium butyricum. Compared with traditional live bacterial preparations, this technology does not require complex bacterial culture and preservation conditions, has low vesicle preparation costs, and can be mass-produced, laying the foundation for industrial applications.

[0019] In some preferred embodiments, in step (1), the culture medium is MRS culture medium or MRS modified culture medium.

[0020] In some preferred embodiments, in step (1), the suspension culture conditions are: suspension culture at 37°C, 100~120 rpm, and anaerobic environment for 60~72 h.

[0021] In some preferred embodiments, in step (2), the centrifugation conditions are: centrifugation at 4°C and 8000~10000 rpm for 5~10 min.

[0022] In some preferred embodiments, in step (3), the conditions for ultracentrifugation are: ultracentrifugation at 4°C and 150,000 × g for 60 to 90 min.

[0023] Compared with the prior art, the present invention has the following advantages: This invention isolates and purifies extracellular vesicles (CBEVs) from Clostridium butyricum. Structural characterization, metabolomics, and multiple functional validations demonstrate that these CBEVs possess multiple effects, including anti-Helicobacter pylori activity, gastric mucosal penetration, inhibition of bacterial adhesion and biofilm formation, and reduction of gastric mucosal inflammatory factors, without toxicity to gastric epithelial cells. Compared to existing antibiotic and probiotic therapies, these vesicles exhibit significant antibacterial effects against both sensitive and drug-resistant Helicobacter pylori strains, solving the problems of strong drug resistance and significant side effects associated with existing antibiotic treatments for Helicobacter pylori. They also avoid the problem of probiotic colonization failure, resulting in higher safety and suitability for long-term treatment and prevention. Furthermore, their natural origin gives them excellent biocompatibility, providing an innovative direction for the development of novel antibacterial drugs. Attached Figure Description

[0024] Figure 1 This section covers the preparation and characterization of CBEVs (A. Schematic diagram of CBEV extraction and purification process; B. Transmission electron microscopy images of CBEVs, scale bar = 100 nm; C. Nanoparticle tracking analysis (NTA) results of CBEVs; D. Dynamic light scattering analysis (DLS) results of CBEVs; E. Zeta surface charge detection results of CBEVs; F. Non-targeted metabolomics analysis results of CBEVs; G. Disease relevance analysis of CBEVs). Figure 2 The results of the pharmaceutical property analysis of CBEVs are as follows: (A. Particle size changes of CBEVs in simulated gastric fluid; B. Particle size changes of CBEVs in simulated intestinal fluid; C. Schematic diagram of the gastric mucosa model; D. Permeability of CBEVs through the gastric mucosa model (n=3); E. Schematic diagram of the adhesion experiment; F. Laser confocal microscope images of the adhesion experiment, with red fluorescence indicating PKH26-labeled CBEVs and green fluorescence indicating FITC-labeled Hp MLCs, scale bar = 50 μm, and scale bar = 10 μm for the magnified view). Figure 3 This study examines the anti-Helicobacter pylori activity of CBEVs (A. CBEVs inhibition zone assay; B. Quantitative analysis of CBEVs inhibition zone assay (n=3); C. Effects of CBEVs on the mRNA transcription levels of resistance and toxic genes in Hp MLC (n=3); D. Removal of Hp MLC biofilm by CBEVs in combination with clarithromycin (CLR) (crystal violet assay); E. Quantitative analysis of the crystal violet assay (n=3). * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001). Figure 4The effects of CBEVs on GES-1 cells (A. CBEVs cytotoxicity to GES-1 cells as detected by CCK8 assay (n=3); B. Schematic diagram of the infected cell model; C. Effect of CBEVs on IL-8 levels in infected cells (n=3); D. Effect of CBEVs on IL-6 levels in infected cells (n=3); * indicates p<0.05, ** indicates p<0.01). Detailed Implementation

[0025] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. The described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Experimental reagents and materials: All Helicobacter pylori strains used in the examples were identified as Helicobacter pylori species using VITEK® MS fully automated rapid microbial mass spectrometry (V 3.2 version) (Table 1). The antibiotic resistance of all Helicobacter pylori strains used in the examples was determined by the standard Kirby-Bauer method (Table 1). The standard Helicobacter pylori strain ATCC 43504 and the Sydney strain of Helicobacter pylori (Hp SS1) were provided by Shenzhen Bolout Biological Products Co., Ltd., and strains 3-6 in Table 1 were isolated from gastric mucosal samples from clinical patients.

[0027] Table 1. Species identification and drug susceptibility test results of Helicobacter pylori strains

[0028] * The antibiotics tested included metronidazole, levofloxacin, clarithromycin, gentamicin, amoxicillin, rifampin, and furazolidone.

[0029] Helicobacter pylori frozen in glycerol was revived in solid culture medium (Shenzhen Bolout Biological Products Co., Ltd., catalog number: C04011), passaged twice, and then transferred to liquid culture medium for suspension culture at 120 rpm (culture environment: 37℃, 5% oxygen, 10% carbon dioxide, 85% nitrogen).

[0030] Helicobacter pylori liquid culture medium formula: 10% fetal bovine serum (Inner Mongolia Aopusai Biotechnology Co., Ltd., catalog number: BS-1101) + 1% Helicobacter pylori selective reagent (Shanghai Ruichu Biotechnology Co., Ltd., catalog number: T2105) + modified Brucella broth medium (Shanghai Ruichu Biotechnology Co., Ltd., catalog number: T1939).

[0031] Clostridium butyricum lyophilized powder was purchased from Chongqing Taiping Pharmaceutical Co., Ltd., and Clostridium butyricum MRS broth culture medium was purchased from Guangdong Huankai Microbial Technology Co., Ltd. (item number: 027312).

[0032] Hollow fiber ultrafiltration membrane (Wuhan Aidou Biotechnology Co., Ltd., product number: AHFUMPES100KD).

[0033] Example 1: Preparation of Clostridium butyricum-derived extracellular vesicles (CBEVs)

[0034] A schematic diagram of the preparation process of Clostridium butyricum-derived extracellular vesicles (CBEVs) is shown below. Figure 1 As shown in Figure A, the specific steps include the following: (1) Add Clostridium butyricum powder to sterilized fresh MRS medium, adjust the OD600 of the bacterial solution to 0.5, put the bacterial solution into an anaerobic culture bag, place it in a constant temperature shaker, and culture in a strict anaerobic environment at 37℃ and 120 rpm for 72 h. (2) After the culture was completed, the sample was centrifuged at 10,000 rpm for 5 min at 4℃, the bacterial precipitate was discarded, and 2,000 mL of the supernatant was loaded into a hollow fiber ultrafiltration membrane. The supernatant was concentrated using the tangential flow principle. The peristaltic pump was set to a speed of 200 rpm and the sample solution was concentrated to a volume of 100 mL. (3) Add 7 mL of 33% (g / 100 mL) sucrose solution to the bottom of the ultra-clean quick-seal centrifuge tube, then add concentrated liquid to fill the centrifuge tube, centrifuge at 150000×g for 90 min at 4℃, and collect the sucrose layer; (4) Add 4 times the volume of PBS filtered through a 0.22 μm microporous membrane to the sucrose layer, transfer the sample to a 10 kDa ultrafiltration centrifuge tube, and continuously ultrafilter and centrifuge at 3000 rpm at 4℃ to remove sucrose. Concentrate the sample volume to 2 mL to obtain the Clostridium butyricum-derived extracellular vesicles (CBEVs) sample.

[0035] Example 2: Characterization of Clostridium butyricum-derived extracellular vesicles (CBEVs)

[0036] (1) Transmission electron microscopy analysis: 20 μL of CBEV sample solution was dropped onto a 200-mesh carbon support copper grid and allowed to adsorb for 10 min. Excess droplets were removed from the side with filter paper and allowed to dry slightly. Then, 20 μL of uranium acetate staining solution was added and stained for 5 min. Excess staining solution was removed with filter paper. The sample was placed under an incandescent lamp to dry. The ultrastructure was observed and images were acquired under a transmission electron microscope (brand: JEOL, model: JEM-2100Plus). Under the transmission electron microscope, CBEVs were observed to be circular, membrane vesicle-like structures ( Figure 1 (B)

[0037] (2) Nanoparticle Tracking Analysis (NTA): The standard solution of 100 nm polystyrene microspheres was diluted 250,000 times with ultrapure water, and 1 mL of the diluted standard solution was used for instrument automatic calibration. After the instrument automatic calibration was completed, the sample cell was cleaned with PBS buffer. The CBEV stock solution concentration was 300 μg / mL, which was diluted 3000 times with sterile PBS before being injected for detection. The nanoparticle tracking analyzer (brand: Fuliu Biotechnology, model: U 30E) performed automatic analysis and generated a test report. The NTA results showed that the particle concentration corresponding to CBEVs with a protein concentration of 300 μg / mL was 1.9 × 10⁻⁶. 11 Particles / mL, with over 90% of the particles distributed between 130 and 200 nm. Figure 1 (C)

[0038] (3) Particle size and zeta potential determination: Take CBEVs sample and dilute with ultrapure water to a concentration of 100 μg / mL. Pipette 1 mL and inject it into the cuvette of the Malvern laser nanoparticle size analyzer. Set the solution system to water and the detection temperature to 25℃. Then, take CBEVs sample and dilute with ultrapure water to a concentration of 100 μg / mL. Take about 0.8 mL and inject it into the zeta potential cuvette of the Malvern laser nanoparticle size analyzer, ensuring that the solution covers the electrode. Set the solution system to water and the detection temperature to 25℃. The average particle size of CBEVs is 136.8 ± 0.78 nm ( Figure 1 The surface charge of zeta (in D) is -29.71 mV. Figure 1 (E).

[0039] Example 3: Untargeted metabolomics analysis (substance identification) of CBEVs

[0040] Untargeted metabolomics analysis was performed using ultra-high pressure liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS).

[0041] (1) Sample pretreatment: Take 100 μL of sample, mix well, and add 300 μL of methanol-acetonitrile mixture (1:1). Vortex for 60 s. Centrifuge at 17000 g for 10 min at 4°C, and collect the supernatant into a new EP tube. After vacuum centrifugation at 35°C, add 100 μL of 50% methanol-water mixture, vortex for 60 s, and sonicate for 10 min. Centrifuge at 17000 g for 10 min at 20°C, and collect the supernatant into a sample vial for instrumentation.

[0042] (2) Instrument parameters: The experiment used a Waters H-Class ultra-high pressure liquid chromatography system equipped with an ACQUITY UPLC HSST3 column (1.8 μm, 2.1×100 mm). The mobile phases were an aqueous solution containing 0.1% formic acid (phase A) and an acetonitrile solution containing 0.1% formic acid (phase B). The gradient elution conditions were as follows: 95% A for 0-2.5 min, linearly reduced to 60% A for 2.5-14 min, reduced to 5% A for 14-24 min, maintained at 5% A for 24-27 min, and restored to the initial ratio for 27.1-30 min. The flow rate was 400 μL / min, the column temperature was 40°C, and the injection volume was 4 μL. Mass spectrometry analysis was performed simultaneously in positive and negative ion modes, using an electrospray ionization (ESI) source. The spray voltage was 5500 V in positive ion mode and -4500 V in negative ion mode. The sheath gas temperature was 550°C, the collision energy was 30 eV, the primary mass spectrometry scanning range was 50-1200 m / z, and 15 secondary fragment spectra were acquired per cycle.

[0043] (3) Data processing: After the data is converted to .abf format by AnalysisBaseFileConverter, peak extraction, comparison and database search are performed using MSDIAL 4.6 software (Metlin / MassBank / MoNA / HMDB v6.0). The parent ion mass deviation is set to ≤0.01 Da and the secondary fragment deviation is set to ≤0.05 Da. The compounds are identified by combining parameters such as retention time matching degree (RTmatched), isotope distribution (MS1 isotopes) and secondary spectrum similarity (Dot product>60%).

[0044] The results are shown in Table 2. A total of 124 high-confidence compounds were identified. These compounds are mainly alkaloids and amino acid compounds. Figure 1 The identified compounds were imported into FunMeta v1 (http: / / funmeta.oebiotech.cn / ) for metabolite-disease network analysis. The results showed that these compounds were highly correlated with amino acid metabolism disorders, fatty acid oxidation disorders, and digestive system diseases. Figure 1 (G).

[0045] Table 2. Compounds identified from CBEVs by non-targeted metabolomics analysis

[0046] Example 4: Stability of CBEVs in gastric / intestinal fluids Weigh 2.0 g of sodium chloride (Macklin, catalog number: S805275) and 3.2 g of pepsin (Macklin, catalog number: P916045-100g), add 800 mL of ultrapure water, adjust the pH to <2 with hydrochloric acid (Guangzhou brand, catalog number: GSCB11-GR-0.5L), and add ultrapure water to a final volume of 1000 mL to obtain artificial gastric juice.

[0047] Weigh 6.8 g of potassium dihydrogen phosphate (Aladdin, catalog number: P401704) and dissolve it in 250 mL of ultrapure water. Add 77 mL of 0.2 M sodium hydroxide solution (Meryer, catalog number: M71987-500G) and 500 mL of ultrapure water. Then add 10 g of trypsin (Macklin, catalog number: P916050-25g). After dissolving, adjust the pH of the solution to 6.80 ± 0.1 with 0.2 M sodium hydroxide or 0.2 M hydrochloric acid. Add ultrapure water to a final volume of 1000 mL to obtain the artificial intestinal fluid.

[0048] CBEVs samples were taken and diluted with ultrapure water to a concentration of approximately 400 μg / mL. 250 μL of the sample solution was then added to 750 μL of artificial gastric fluid and artificial intestinal fluid, respectively. The control group was added to 750 μL of ultrapure water. Each group was incubated at 37℃ for 2, 3, and 4 h, respectively, and then loaded into a Malvern laser nanoparticle size analyzer to detect particle size distribution. Figure 2 As shown in Figures A and B, the particle size distribution of CBEVs did not change significantly during the incubation period, suggesting that CBEVs can withstand the corrosion of artificial gastric and intestinal fluids.

[0049] Example 5: Gastric mucosal permeability of CBEVs The FITC stock solution (Fluorescein Isothiocyanate) was purchased from Biosharp (catalog number BS096-50mg).

[0050] CBEVs were adjusted to a concentration of 1 mg / mL with ultrapure water, and 1 μL of FITC stock solution (5 mg / mL) was added to 1 mL of CBEVs. A FITC control group (1 μL of FITC stock solution added to 1 mL of ultrapure water) was used. The mixtures were incubated at 37°C for 1 h to obtain FITC-CBEVs and FITC aqueous solution samples. GES-1 cells were seeded at 10,000 cells / well in the upper layer of 12-well transwell culture chambers and cultured for 24 h until adherence. The culture medium was discarded, and residual medium was washed away with HBSS. 100 μL of a 3 mg / mL (0.3%) gastric mucin solution prepared with HBSS was added to the upper layer of the transwell chambers seeded with GES-1 cells to construct a gastric mucosal layer model (see...). Figure 2 (Diagram of C in the middle).

[0051] In the FITC-CBEVs group, 100 μL of FITC-CBEVs was added to the upper layer of the transwell chamber used to construct the gastric mucosal model. The FITC control group received an equal volume of FITC solution. The FITC-CBEVs model-free control group received an equal volume of FITC-CBEVs added to the upper layer of the blank transwell chamber. 1.5 mL of HBSS buffer was added to the lower layer of the transwell chamber in all groups. At 5, 10, 20, 30, 40, 60, 120, and 180 min, 100 μL of the lower layer liquid from each group was aspirated, and the fluorescence intensity (Ex=488 nm, Em=520 nm) of the FITC-CBEVs group (F1), FITC control group (F0), and FITC-CBEVs model-free control group (F2) was measured using a microplate reader. Transmittance = (F1-F0) / (F2-F0)*100% Experimental results showed that after 180 min of incubation, the permeability of CBEVs was >55%, suggesting that CBEVs have the potential to penetrate the gastric juice-mucosal layer.

[0052] Example 6: Effect of CBEVs on the adhesion ability of Helicobacter pylori GES-1 cells were seeded at 2000 cells / dish in laser confocal microscopy dishes and cultured for 24 h until cell adhesion. CBEVs were adjusted to a concentration of 1 mg / mL with sterile PBS, and the PKH26 fluorescent probe (APExBIO, catalog number: C8002-1154214-55-8) was added to a final concentration of 1 μM. The mixture was incubated at 37℃ for 1 h. CBEVs-PKH26 were precipitated according to the instructions of the exosome precipitation kit (Beyotime Biotechnology, catalog number: BB-3901), and its concentration was determined using the BCA protein quantification kit (Beyotime Biotechnology, catalog number: P0398S). After resuscitation and activation of Helicobacter pylori in solid culture medium, bacteria were scraped off with a cotton swab, and the bacterial suspension was adjusted with PBS to a McFarland standard turbidity of 0.5 (1.5 × 10⁻⁶). 8 Add FITC fluorescent dye to a final concentration of 100 µg / mL. Incubate at 37°C for 1 h. Centrifuge at 4500 rpm for 5 min, resuspend the bacterial pellet in sterile PBS, and measure the turbidity of the bacterial suspension.

[0053] For the competitive adhesion group, discard the old culture medium for GES-1 cells, add 50 µL of bacterial suspension and 50 µL of CBEVs to the GES-1 cells, and then add 100 µL of culture medium. The final concentration of Helicobacter pylori was MOI = 100:1, and the final concentration of CBEVs was 300 µg / mL. Incubate at 37°C for 1 h, discard the culture medium, gently wash the cells twice with pre-cooled PBS at 4°C, and add 200 µL of PBS to maintain cell morphology. Store the samples on ice until analysis.

[0054] For the de-adhesion group, discard the old culture medium for GES-1 cells and add 50 µL of bacterial suspension to the GES-1 cells to achieve a final Helicobacter pylori concentration of MOI = 100:1. Then add 100 µL of culture medium. Incubate at 37°C for 1 h. Next, add 50 µL of CBEVs to achieve a final CBEVs concentration of 300 µg / mL. Incubate at 37°C for 1 h, discard the culture medium, and gently wash the cells twice with pre-cooled PBS (4°C). Add 200 µL of PBS to maintain cell morphology. Store the samples on ice until analysis.

[0055] Figure 2 The diagram in Figure E illustrates the adhesion experiment. The results show that CBEVs can remove *Hp* MLCs adhering to GES-1 cells; when both are co-incubated with GES-1 cells, CBEVs competitively inhibit the adhesion of *Hp* MLCs to GES-1 cells. Figure 2 (Middle F). Figure 2 The magnified view of the middle section (F) suggests that CBEVs and Hp MLC exhibit intracellular co-localization, and their intracellular transport pathways may overlap. This indicates that CBEVs have the potential to target and track intracellular Helicobacter pylori.

[0056] Example 7: Anti-Helicobacter pylori activity of CBEVs (1) Inhibition ring test Helicobacter pylori (Hp) SS1 in the logarithmic growth phase was collected and the bacterial concentration was adjusted to 1 McFarland standard turbidity using liquid culture medium. 100 µL of the bacterial suspension was evenly spread onto a solid culture medium. The culture dish was inverted and allowed to air dry. A 0.75 mg / mL clarithromycin solution (CLR) and a 1 mg / mL CBEVs solution were prepared. 20 µL of each drug solution was added to a blank drug sensitivity test disc, allowing it to fully absorb. A blank control group was prepared with 20 µL of PBS. The discs were then placed in the center of the solid culture medium containing the Hp. pylori and incubated for 72 h. The diameter of the inhibition zone was then measured using calipers. Figure 3As shown in Figures A and B, the diameter of the inhibition zone of CBEVs was 43.37 ± 0.12 mm, and the diameter of the inhibition zone of CLR was 77.21 ± 6.75 mm. The results suggest that CBEVs have the activity of inhibiting the growth of Helicobacter pylori.

[0057] (2) Determination of the minimum inhibitory concentration (MIC) of CBEVs Collect Helicobacter pylori bacteria in the logarithmic growth phase, adjust the bacterial concentration to 0.5 McFarland turbidity with liquid culture medium, and inoculate 100 µL / well into 96-well plates (the final inoculation density of Helicobacter pylori is 5 × 10⁻⁶). 7 (CFU / mL).

[0058] CBEVs were diluted twofold with Helicobacter pylori liquid culture medium, with 100 µL of drug solution added to each well, resulting in final concentrations of 1280, 640, 320, 160, 80, 40, 20, and 10 μg / mL. The growth control group received 100 µL of culture medium and 100 µL of bacterial suspension; the blank control group received 200 µL of culture medium. Each concentration was tested in triplicate. Helicobacter pylori was co-incubated with the drug for 24 h, and the lowest drug concentration (MIC) corresponding to the well where the culture medium remained clear was observed visually. The OD value at 600 nm was measured using a microplate reader. A reliable result was achieved when: ① visible turbidity was observed in the growth control group; ② the OD600 change below the MIC was <0.05. The MIC values ​​of CBEVs for strains 1–6 in Appendix 1 were determined, and the results are shown in Table 3. The MIC range of CBEVs for the tested Helicobacter pylori strains was 80–320 μg / mL. This indicates that CBEVs have an inhibitory effect on both antibiotic-sensitive and antibiotic-resistant strains.

[0059] Table 3. MIC range of CBEVs against various Helicobacter pylori strains (n=3)

[0060] (3) Antibiotic combined drug sensitivity test After resuscitation in solid medium, *Helicobacter pylori* (Hp) MLCs were passaged twice to ensure full activation. Bacteria were scraped off with a cotton swab and resuspended in *Helicobacter pylori* liquid medium. The bacterial concentration was adjusted to 0.5 McFarland turbidity, and inoculated into 96-well plates at 100 µL / well (final inoculation density 5 × 10⁻⁶). 7 (CFU / mL).

[0061] Following the checkerboard dilution method, CBEVs and antibiotics were combined in pairs, with 50 μL of each drug administered in combination. Taking the combined drug susceptibility test of CBEVs and clarithromycin as an example, the specific procedure was as follows: CBEVs were diluted twofold with Helicobacter pylori liquid medium, and 50 µL of the drug solution was added vertically to each well of a 96-well plate (dilution gradient from row A to row F), resulting in final concentrations of 1280, 640, 320, 160, 80, 40, 20, and 10 μg / mL. Clarithromycin stock solution was diluted twofold with Helicobacter pylori liquid medium, and 50 µL of the drug solution was added horizontally to each well of a 96-well plate (dilution gradient from column 1 to column 6), resulting in final concentrations of 8, 4, 2, 1, 0.5, 0.25, 0.125, and 0.0625 μg / mL. The growth control group received 100 µL of medium and 100 µL of bacterial suspension; the blank control group received 200 µL of medium. Three replicates were set up for each concentration in each group. After co-incubating Helicobacter pylori with the drug for 24 h, the combined MIC of the two drugs was used to calculate the partial inhibitory concentration index (FICI) according to the following formula: FICI = MIC A药联用 / MIC A药单用 + MIC B药联用 / MIC B药单用 FICI determination: When FICI ≤ 0.5, the two drugs have a synergistic effect; when 0.5 ≤ FICI ≤ 1.0, the two drugs have an additive effect; when 1.0 ≤ FICI ≤ 2.0, the two drugs have no correlation; when FICI > 2.0, the two drugs have an antagonistic effect. The experimental results are shown in Table 4. CBEVs have a synergistic effect with clarithromycin and metronidazole.

[0062] Table 4. Combined antibacterial activity of CBEVs with antibiotics (n=3)

[0063] Example 8: Effects of CBEVs on the transcriptional levels of drug resistance-related genes and virulence-related genes in Helicobacter pylori Helicobacter pylori (Hp) MLCs were diluted with fresh liquid medium to McFarland standard turbidity 1 and inoculated into 24-well plates at a rate of 500 µL / well. The CBEVs group received 500 µL of 320 μg / mL CBEVs, while the growth control group received an equal volume of complete medium. After co-incubation for 24 h, total RNA was extracted from the bacteria using a rapid RNA extraction kit (Altair Biotechnology (Guangzhou) Co., Ltd., catalog number: 400-100). The RNA was then reverse transcribed into cDNA using the Hifair® AdvanceFast 1st Strand cDNA SynthesisSuperMix for qPCR kit (Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number: 11155ES60). The PCR system was prepared according to the instructions of the KT AntiQ qPCR SYBR Green Fast Mix kit (Anhui Ketu Biotechnology Co., Ltd., catalog number: KQ410). The cDNA loading volume was 1000 ng, with two replicates per sample. Primer sequences are shown in Table 5.

[0064] Table 5 Primer sequences for RT-qPCR

[0065] The extracted cDNA was subjected to real-time PCR experiments using an ABI QuantStudio 5 real-time PCR instrument. PCR program: Pre-denaturation: 50℃, 2 min → 95℃, 10 min; Amplification cycles: 95℃, 5 s → 55℃, 15 s → 72℃, 30 s, 40 cycles; Melting curve analysis: 95℃, 15 s → 60℃, 1 min → 95℃, 1 s.

[0066] Using the bacterial survival gene rpoD as an internal control, the mRNA levels of the efflux pump gene hefA, the urease gene ureB, and the virulence protein gene cagA in each group of samples were relatively quantified using the 2^(-ΔΔCt) method. Figure 3 As shown in Figure C, CBEVs inhibit the mRNA transcription levels of the hefA, ureB, and cagA genes in Helicobacter pylori.

[0067] Example 9: Inhibitory effect of CBEVs on Helicobacter pylori biofilm Take Helicobacter pylori (Hp) MLC in the logarithmic growth phase and adjust the bacterial suspension to McFarland standard turbidity of 0.7 using low-nutrient liquid medium (2% fetal bovine serum). Add 1 mL of bacterial suspension to each well of a 24-well plate and incubate for 5 days to form a visible biofilm.

[0068] Carefully discard the old culture medium using a pipette, taking care not to damage the biofilm. Add 1 mL of drug solution to the CBEVs group at final concentrations of 160 μg / mL and 320 μg / mL, respectively. Add drug solution to the CLR group at final concentrations of 16 μg / mL and 32 μg / mL, respectively. Add 1 mL of a mixture of CBEVs and CLR to the combined drug group, achieving final concentrations of 160 μg / mL and 16 μg / mL, respectively. Add an equal volume of complete culture medium to the normal control group.

[0069] After 48 h of incubation, the free bacterial culture was aspirated with a pipette, and the biofilm was gently washed once with 1 mL of sterile PBS. The culture plate was transferred to a 37°C incubator, inverted, and air-dried. 1 mL of methanol was added to each well, and the plate was fixed at room temperature for 15 min. The methanol was then aspirated, the plate was inverted, and air-dried. Subsequently, 200 μL of 0.1% crystal violet solution (ECOTOP, catalog number: ES-8116-100ml) was added to each well, and staining was performed for 15 min. Excess crystal violet staining solution was aspirated from the wells, and the plates were gently washed twice with sterile PBS, wiping away any excess crystal violet from the well walls with a cotton swab. The plates were inverted and air-dried. The biofilm morphology was photographed and recorded. Then, 1 mL of 33% glacial acetic acid was added to each well, and the plate was destained for 10 min. 200 μL of the solution was transferred to a 96-well plate, and the OD value was measured at 595 nm using a microplate reader. Figure 3 As shown in Figures D and E, CBEVs can inhibit the biofilm of Helicobacter pylori to a certain extent, and the combination of CBEVs and CLR can significantly remove the biofilm of Helicobacter pylori.

[0070] Example 10: Protective effect of CBEVs on GES-1 cells (1) Cytotoxicity test Human gastric epithelial cells GES-1 were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics and continuously cultured at 37°C in a 5% CO2 environment. GES-1 cells in the logarithmic growth phase were harvested and cultured at a rate of 2 × 10⁻⁶ cells / year. 4 Cells were seeded at a density of [number] cells / well in 96-well plates and cultured for 24 h until cell adhesion. After removing the old culture medium, 200 µL of CBEVs diluted with DMEM complete medium to final concentrations of 1.56, 6.25, 25, 100, 400, and 1600 µg / mL were added. The control group received an equal volume of complete medium. After 48 h of treatment, 20 µL of CCK8 working solution was added to each well according to the CCK8 kit instructions. The cells were incubated at 37°C for 60 min, and OD values ​​were measured using a multi-mode microplate reader. 450 nm. Cell viability = OD CBEVs组 / OD 对照组 *100%.

[0071] like Figure 4 As shown in Figure A, within the experimental concentration range of 1.56–1600 µg / mL, CBEVs did not exhibit significant cytotoxicity against GES-1 cells, and the cell viability of each concentration group was greater than 80%.

[0072] (2) Anti-inflammatory activity GES-1 cells in logarithmic growth phase were harvested at a rate of 8 × 10⁻⁶. 5 Cells were seeded per well in 6-well plates and cultured for 24 h until cell adhesion was achieved. The old culture medium was then removed. After resuscitation and activation of *Helicobacter pylori* (Hp) MLC in solid medium, bacteria were scraped off with a cotton swab, resuspended in DMEM complete medium, and the McFarland standard concentration of the bacterial suspension was determined. The bacterial density was adjusted to a cell:bacteria ratio of 1:100. 2 mL of bacterial suspension was added to each well of the 6-well plate, and the bacteria and cells were co-cultured at 37 ℃ and 5% CO2 for 24 h to construct an infected cell model. Figure 4 (B) The normal control group was given an equal volume of DMEM complete culture medium.

[0073] Discard the old culture medium from the 6-well plate and add 2 mL of CBEVs diluted to a final concentration of 160 µg / mL using DMEM complete medium. Add 2 mL of DMEM complete medium to both the model group and the normal control group. After 24 h of treatment, collect the cell culture medium. Centrifuge at 500 rpm for 3 min, collect the supernatant, and use an ELISA kit to detect the expression levels of IL-6 and IL-8 in the cell supernatant. Figure 4 As shown in C and D, CBEVs significantly reduced the levels of IL-6 and IL-8 in infected cells, suggesting that CBEVs can reduce the secretion of inflammatory factors by gastric epithelial cells infected with Helicobacter pylori.

[0074] In summary, the results of multiple functional studies demonstrate that the CBEVs prepared in this invention exhibit excellent anti-Helicobacter pylori activity, effective against both sensitive and drug-resistant strains. They inhibit bacterial growth, reduce the transcription levels of drug-resistant and toxic genes, and can enhance antibacterial effects and scavenge biofilms when used in combination with clarithromycin. Furthermore, CBEVs are resistant to gastrointestinal corrosion, possess the ability to penetrate the gastric mucus layer, competitively inhibit Helicobacter pylori adhesion to gastric epithelial cells and target intracellular bacteria, and are non-toxic to gastric epithelial cells. They significantly reduce the levels of inflammatory factors in infected cells, exhibiting both safety and anti-inflammatory activity. Therefore, they can be used to prepare drugs against Helicobacter pylori and reduce gastric mucosal inflammation, showing broad application prospects in the prevention and treatment of Helicobacter pylori-related gastrointestinal diseases.

Claims

1. Application of Clostridium butyricum-derived extracellular vesicles in the preparation of anti-Helicobacter pylori drugs.

2. Application of the combination of extracellular vesicles derived from Clostridium butyricum and clarithromycin in the preparation of anti-Helicobacter pylori drugs.

3. The application according to claim 1 or 2, characterized in that, The Helicobacter pylori mentioned includes clinically resistant anti-Helicobacter pylori and susceptible Helicobacter pylori.

4. Application of extracellular vesicles derived from Clostridium butyricum in the preparation of drugs for the prevention or treatment of gastrointestinal diseases caused by Helicobacter pylori infection.

5. The application according to claim 4, characterized in that, The gastrointestinal diseases mentioned include chronic gastritis, peptic ulcers, and gastric cancer.

6. The application according to claim 1, 2, or 4, characterized in that, The extracellular vesicles derived from Clostridium butyricum have an average particle size of 130~200 nm, a surface charge of -41.2~-14.7 mV, and a permeability of >55% in an artificial gastric mucosa model after 180 min.

7. The application according to claim 1, 2, or 4, characterized in that, The method for preparing extracellular vesicles derived from Clostridium butyricum includes the following steps: (1) Add Clostridium butyricum powder to the culture medium and suspend it in an anaerobic environment to obtain Clostridium butyricum culture medium; (2) Centrifuge the Clostridium butyricum culture medium, take the supernatant and concentrate it through a hollow fiber ultrafiltration membrane to obtain a concentrated solution; (3) Add a 33% sucrose solution to the bottom of the ultra-clean quick-seal centrifuge tube, then add concentrated liquid until the centrifuge tube is full, centrifuge at high speed, and collect the sucrose layer. (4) Add sterile PBS to the sucrose layer, remove the sucrose by ultrafiltration and centrifugation, and concentrate to obtain the extracellular vesicles of Clostridium butyricum.

8. The application according to claim 7, characterized in that, In step (1), the suspension culture conditions are: suspension culture at 37℃, 100~120 rpm, and anaerobic environment for 60~72 h.

9. The application according to claim 7, characterized in that, In step (2), the centrifugation conditions are: centrifugation at 4℃ and 8000~10000 rpm for 5~10 min.

10. The application according to claim 7, characterized in that, In step (3), the conditions for ultracentrifugation are: ultracentrifugation at 4℃ and 150000×g for 60~90 min.

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