Lactobacillus paragasseri and application thereof in products for preventing and treating liver cancer

Abstract

The invention provides lactobacillus paragasseri and application thereof in a product for preventing and treating liver cancer, and belongs to the field of biological medicine. The invention provides lactobacillus paragasseri JMS024, the preservation number of the lactobacillus paragasseri JMS024 is CGMCC (China General Microbiological Culture Collection Center) No.33909, and the lactobacillus paragasseri JMS024 is preserved in the China General Microbiological Culture Collection Center on March 20, 2025. According to the invention, the extracellular vesicles are obtained by culturing the lactobacillus paragasseri JMS024 and separating, can significantly inhibit liver cancer cell proliferation, liver cancer cell migration and liver cancer cell autophagy, can significantly reduce the activity of liver cancer cells, and can be used for preparing products for treating liver cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to Lactobacillus paragaseri, and in particular to a strain of Lactobacillus paragaseri and its application in products for preventing and treating liver cancer. Background Art

[0002] Probiotics are a type of bacteria that are beneficial to the host. Currently, probiotics are mostly used as food supplements to maintain and improve the host's intestinal flora. A growing body of research supports the idea that probiotics can maintain a healthy intestinal microbiome and potentially improve gastrointestinal diseases, including gastrointestinal infections, inflammatory bowel disease, and even cancer. Probiotics may exert their effects in the body primarily by altering the host's microbiome, then by improving the barrier function of the intestinal mucosa, and finally by modulating the immune system.

[0003] Hepatocellular carcinoma (HCC), one of the most lethal malignancies worldwide, is closely associated with risk factors such as hepatitis B virus (HBV) and hepatitis C virus (HCV) infection, cirrhosis, and aflatoxin exposure. A growing body of research indicates that the gut-liver axis plays a crucial role in the development and progression of HCC. A normal intestinal barrier is composed of a healthy composition and distribution of the intestinal microbiota, the intestinal mucosal barrier, and the integrity of the immune system. Impaired intestinal barrier function leads to increased intestinal permeability, bacterial translocation, and LPS accumulation, all of which contribute to the development of HCC. Therefore, regulating the gut microbiota with probiotics may offer a novel approach for preventing and treating the development and progression of HCC. Modulating the gut microbiota with probiotics not only promotes gut microbial balance, improves intestinal inflammation and the mucosal barrier, but also significantly improves symptoms of cirrhosis and reduces the incidence of HCC. Modulating the gut microbiota with probiotics may represent a novel therapeutic approach for preventing the progression from hepatitis to cirrhosis and HCC. However, the mechanism of action of the intestinal flora in the development and progression of liver cancer is still unclear, so future research needs to explore the composition of the microbiome in healthy and diseased states and the factors that maintain its long-term stability. Currently, a growing number of studies have shown that vaginal intestinal flora and specific types of lactobacilli are associated with the development and progression of hepatitis and liver cancer through enterohepatic circulation. Probiotics such as lactobacilli and their metabolites can protect liver cells by changing the intestinal flora or directly acting on the liver.

[0004] Probiotic-derived extracellular vesicles (EVs) are double-layered nanoparticles with a diameter of approximately 20-400 nm, released by probiotics during their growth. They carry bioactive substances such as proteins, nucleic acids, lipids, and metabolites, becoming novel signaling molecules that mediate host-microbe interactions. Recent studies have shown that these vesicles can play an important role in anti-inflammatory, anti-tumor, and metabolic regulation through multi-system regulatory networks such as the gut-liver and gut-brain axes. Currently, research on the probiotic Lactobacillus paragaseri primarily focuses on gastrointestinal treatments, and there are no records of its use in the preparation of products for the treatment of liver cancer.

[0005] Prior art CN113604410B discloses that the Lactobacillus plantarum YT013 strain is cultured and fermented, and the resulting fermentation liquid is centrifuged and freeze-dried to prepare a Lactobacillus plantarum YT013 cell-free culture liquid; the cell-free culture liquid has a significant inhibitory effect on HepG2 liver cancer cells, and as the concentration of the Lactobacillus plantarum YT013 cell-free culture liquid increases, its inhibition rate on HepG2 liver cancer cells increases in a dose-dependent manner.

[0006] Wang Chang et al. used different concentrations of Lactobacillus plantarum CGMCC8198 supernatant (LpS) to treat HepG2 cells for different times, and used protein immunoblotting, oil red staining, and real-time quantitative fluorescence PCR to detect the effects of LpS on fatty degeneration of liver cancer cells and the key pathway of HMGCR / SMYD3 lipid metabolism; the MTT method, cell scratch test, and flow cytometry were used to detect the effects of LpS on the proliferation, migration, and apoptosis of HepG2 cells during lipid metabolism disorders; the results showed that LpS can inhibit the expression of genes such as HMGCR, SMYD3, and SREBP-2 in liver cancer cells with lipid metabolism disorders, and can also inhibit cell proliferation and migration in a dose-dependent manner and promote cell apoptosis (Wang Chang, Zhang Liyan, Zhang Tongcun, et al. Lactobacillus plantarum CGMCC8198 intervenes in lipid metabolism disorders to play a role in the prevention and treatment of liver cancer [J]. Bulletin of Microbiology, 2021.). Summary of the Invention

[0007] The present invention addresses the problem that no Lactobacillus paragasseri strain is currently available for use in the preparation of liver cancer treatment products. The present invention provides a strain of Lactobacillus paragasseri and its use in liver cancer treatment products. The present invention provides Lactobacillus paragasseri JMS024, deposited with CGMCC No. 33909. Extracellular vesicles of Lactobacillus paragasseri JMS024 can be isolated by liquid culture. The resulting extracellular vesicles significantly inhibit the proliferation and migration of liver cancer cells and can be used to prepare liver cancer treatment products.

[0008] To achieve the above object, the technical solution adopted by the present invention is as follows: In one aspect, the present invention provides a Lactobacillus paragaseri ( Lactobacillus paragasseri ) JMS024, deposited with CGMCC No. 33909, was deposited in the General Microbiology Center of China Culture Collection Administration on March 20, 2025.

[0009] In another aspect, the present invention provides a bacterial agent comprising the aforementioned Lactobacillus paragaseri or a preparation of the aforementioned Lactobacillus paragaseri.

[0010] Preferably, the preparation comprises the culture, culture extract, fermentation broth, fermentation broth precipitate, fermentation broth supernatant or fermentation broth extract of the Lactobacillus paragaseri.

[0011] Preferably, the bacterial agent is a solid preparation or a liquid preparation.

[0012] Preferably, the bacterial agent contains extracellular vesicles secreted by the Lactobacillus paragaseri.

[0013] On the other hand, the present invention provides an extracellular vesicle, which is prepared from the above-mentioned Lactobacillus paragastrii JMS024.

[0014] Preferably, the method for preparing the extracellular vesicles comprises: Lactobacillus paragastris JMS024 is cultured in a culture medium to obtain a culture supernatant, the supernatant is filtered to obtain a filtrate, and extracellular vesicles are separated from the filtrate.

[0015] On the other hand, the present invention provides the use of the aforementioned Lactobacillus paragaseri, the aforementioned bacterial agent or the aforementioned extracellular vesicles in the preparation of a product for treating cancer.

[0016] Preferably, the cancer comprises liver cancer.

[0017] Preferably, the product comprises a pharmaceutical product, and the pharmaceutical product further comprises a medically acceptable carrier.

[0018] Preferably, the product contains extracellular vesicles secreted by Lactobacillus paragastrii JMS024.

[0019] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention provides a Lactobacillus paragasseri JMS024, with a deposit number of CGMCC No. 33909, which was deposited in the General Microbiology Center of the China Culture Collection Administration on March 20, 2025.

[0020] 2. The present invention cultivates Lactobacillus paragastrium JMS024, and the isolated extracellular vesicles significantly inhibit the proliferation of liver cancer cells, inhibit the migration of liver cancer cells, inhibit the autophagy of liver cancer cells, and significantly reduce the vitality of liver cancer cells, and can be used to prepare products for the treatment of liver cancer.

[0021] Preservation Instructions Species name: Lactobacillus paragaseri, Latin name: Lactobacillus paragasseri , Strain number: JMS024, Depository: General Microbiology Center of China Culture Collection Administration of Microorganisms, Abbreviation of the depository institution: CGMCC, Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing Deposit date: March 20, 2025 The registration number of the CGMCC Collection Center is: CGMCC No.33909. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 These are the morphological identification results of Lactobacillus paragasseri JMS024, A is the Gram staining result of Lactobacillus paragasseri JMS024, and B is the colony of Lactobacillus paragasseri JMS024.

[0023] Figure 2 These are the morphological characteristics of extracellular vesicles under transmission electron microscopy.

[0024] Figure 3 This is a diagram showing the particle size analysis of extracellular vesicles of Lactobacillus paragaseri JMS024 characterized by nanoparticle tracer analysis.

[0025] Figure 4 This is a fluorescent image of extracellular vesicles of Lactobacillus paragaseri JMS024 entering liver cancer cells.

[0026] Figure 5 This is a comparative experiment on the inhibition of liver cancer cell proliferation by extracellular vesicles of different Lactobacillus paragaseri; A is the experimental result for Hep3B cells, and B is the experimental result for HepG2 cells; ** indicates significant difference, P < 0.01.

[0027] Figure 6 These are the results of extracellular vesicles of Lactobacillus paragaseri JMS024 inhibiting the migration of liver cancer cells; A and B are the microscopic images and quantitative results of Hep3B cells, respectively, and C and D are the microscopic images and quantitative results of HepG2 cells, respectively.

[0028] Figure 7The results of extracellular vesicles of Lactobacillus paragaseri JMS024 inhibiting the expression of autophagy gene Beclin-1 in liver cancer cells; A is the experimental result for Hep3B cells, and B is the experimental result for HepG2 cells; ** indicates significant difference, P < 0.01.

[0029] Figure 8 This is the result of Lactobacillus paragaseri JMS024 extracellular vesicles inhibiting the expression of LC3BI and LC3BⅡ proteins in liver cancer cells.

[0030] Figure 9 Fluorescence image showing that extracellular vesicles of Lactobacillus paragaseri JMS024 inhibit the aggregation of GFP-LC3.

[0031] Figure 10 This is a microscopic image of extracellular vesicles of Lactobacillus paragaseri JMS024 inhibiting the formation of autophagosomes.

[0032] Figure 11 These are the results of extracellular vesicles of Lactobacillus paragastris JMS024 inhibiting the proliferation and metastasis of tumor cells; A is a comparison of liver tumor size before and after extracellular vesicle treatment, and B is a quantitative comparison of intrahepatic metastasis before and after extracellular vesicle treatment.

[0033] Figure 12 This is a protein analysis diagram of extracellular vesicles of Lactobacillus paragaseri JMS024 inhibiting autophagy through the AKT / mTOR signaling pathway.

[0034] Figure 13 Extracellular vesicles of Lactobacillus paragaseri JMS024 inhibit the expression of the autophagy gene Beclin-1 in tumor cells.

[0035] Figure 14 The extracellular vesicles of Lactobacillus paragaseri JMS024 inhibit tumor cell autophagy through the AKT / mTOR pathway; A is the protein analysis diagram, and B is the quantitative analysis diagram of the expression levels of p-AKT and p-mTOR. DETAILED DESCRIPTION

[0036] Unless otherwise specified, all raw materials and reagents used in the present invention were purchased from commercial suppliers, and experiments were performed according to the operating instructions. Unless otherwise specified, all instruments, equipment, devices, etc. used in the present invention were conventional instruments, equipment, devices, etc., and experiments were performed according to the operating instructions and supporting reagents.

[0037] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0038] Unless the context clearly dictates otherwise, as used herein, the expressions "a" and "an" include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0039] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0040] As used herein, the term "comprises" or "comprising" means "including but not limited to". The term is intended to be open-ended to specify the presence of any of the described features, elements, integers, steps or components, but does not exclude the presence or addition of one or more other features, elements, integers, steps, components or groups thereof. Therefore, the term "comprising" includes the more restrictive terms "consisting of" and "consisting essentially of". In one embodiment, the term "comprising" used throughout the application, particularly in the claims, may be replaced by the term "consisting of". The three-letter and one-letter codes for amino acids used herein are known to those skilled in the art or as described in J Biol. Chem, 243, p3558 (1968).

[0041] As used herein, the terms "optionally," "either," "any," or "any" mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur. For example, "optionally comprising an antibody heavy chain variable region" means that an antibody heavy chain variable region of a specified sequence may but need not be present.

[0042] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0043] As used herein, the terms "activity," "functional activity," or "biological activity," or the terms "biological property" or "biological characteristic" are used interchangeably herein and include, but are not limited to, epitope / antigen affinity and specificity, the ability to neutralize or antagonize antibody activity in vivo or in vitro, IC50, in vivo stability of the antibody, and the immunogenic properties of the antibody. Other identifiable biological properties or characteristics of antibodies known in the art include, for example, cross-reactivity (i.e., cross-reactivity with non-human homologs of the target peptide, or with other proteins or tissues), and the ability to maintain high protein expression levels in mammalian cells. The aforementioned properties or characteristics can be observed, measured, or assessed using techniques known in the art, including, but not limited to, ELISA, FACS, or BIACORE plasmon resonance analysis, in vitro or in vivo neutralization assays, receptor binding, cytokine or growth factor production and / or secretion, signal transduction, and immunohistochemistry of tissue sections from various sources (including humans, primates, or any other source).

[0044] As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio, within the scope of sound medical judgment. As used herein, the term "pharmaceutically acceptable carrier, excipient, and / or diluent" refers to a carrier that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, and is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995). Pharmaceutically acceptable materials, compositions, or vehicles, such as liquid or solid fillers, diluents, excipients, solvents, media, encapsulating materials, manufacturing aids, or solvent encapsulating materials, are involved in maintaining the stability, solubility, or activity of the antibodies or antigen-binding fragments thereof of the present disclosure, and include, but are not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and their analogs. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, and sorbic acid. Stabilizers have the meanings generally understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in the drug, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolysate), etc.

[0045] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is further described in detail below with reference to the examples. If the specific conditions are not specified in the examples, the experiments shall be carried out under conventional conditions or the conditions recommended by the manufacturer. All reagents or instruments without the manufacturer specified are conventional products that can be purchased commercially. In order to better illustrate the present invention, many specific details are given in the specific embodiments below. The specific embodiments described here are only used to explain the present invention and are not intended to constitute any limitation to the present invention. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present invention. Such structures and technologies are described in many publications, such as "Molecular Cloning Laboratory Manual (Fourth Edition)" (Cold Spring Harbor Laboratory Science Press), Ausubel, FM et al., Current Protocols in Molecular Biology , Greene Publishing Assoc., and Wiley-Interscience.

[0046] SPSS 23.0 software was used for data analysis and statistical analysis, and one-way ANOVA test was used for significance analysis, with P < 0.05 indicating a significant difference.

[0047] Basic Example 1: Isolation of Lactobacillus paragaseri MRS medium (per liter) consists of the following: peptone 10.0 g; beef extract 10.0 g; yeast extract 5.0 g; diammonium hydrogen citrate 2.0 g; glucose 20.0 g; Tween 80 (1.0 mL); sodium acetate (CH3COONa·3H2O) 5.0 g; dipotassium hydrogen phosphate (K2HPO4·3H2O) 2.0 g; magnesium sulfate (MgSO4·7H2O) 0.58 g; and manganese sulfate (MnSO4·H2O) 0.25 g. Add distilled water to 1000 mL and adjust the pH to 6.2-6.6. Sterilize by autoclaving at 121°C for 20 min.

[0048] The separation steps are as follows: (1) Fecal samples from 70 healthy individuals with no signs of gastrointestinal inflammation or tumors were collected. The samples were numbered sequentially, inoculated into MRS liquid culture medium, and cultured anaerobically at 37°C for 24 h.

[0049] (2) Five single colonies were randomly selected from each sample and cultured in 2 mL of MRS liquid medium for 24 h, and the colonies were numbered in sequence.

[0050] (3) Bacteria were amplified for bacterial seed preservation and DNA preparation. Hundreds of isolated bacterial strains were identified; the supernatant was tested for its ability to inhibit liver cancer cells, and one strain, Lactobacillus paragaseri, was selected to have the strongest inhibitory effect on liver cancer cells and named JMS024.

[0051] Basic Example 2: Identification of Lactobacillus paragaseri The strain JMS024 obtained by screening in Basic Example 1 was subjected to the following identification: (1) Morphological identification: On the MRS lactobacillus culture medium plate, the isolated strain JMS024 showed a size of about 0.5-1.0 mm, with neat colony edges and protrusions, and Gram staining was positive. Under the microscope, the cell morphology was mostly short rods. Figure 1 A in the figure; Gram-positive, short rod-shaped, can be linked into chains, see Figure 1 B in.

[0052] (2) 16s rDNA sequence homology analysis The isolated strain JMS024 was cultured using conventional methods. Total DNA was extracted and used as a template for gene amplification. Universal primers 27F (SEQ ID NO: 1) and 1492R (SEQ ID NO: 2) were used to amplify the conserved region of the bacterial 16S rDNA gene. Information about the universal primers is provided in Table 1.

[0053] Table 1 Universal primers 27F and 1492R

[0054] The amplification system (25 μL) consisted of 1× PCR reaction buffer, 200 μmol / L dNTPs, 0.2 μmol / L each of the upstream and downstream primers, 1 U Taq DNA polymerase, and 1 μL template DNA. Reaction conditions included initial denaturation at 94°C for 5 min, followed by 25 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 40 s, and extension at 72°C for 30 s, followed by extension at 72°C for 10 min. PCR products were detected by 1% gel electrophoresis, and positive results were sequenced bidirectionally using universal primers 27F and 1492R. Sequence assembly and similarity analysis were performed using DNAStar software. Sequence alignment was performed online using the National Center for Biotechnology Information (NCBI) database (http: / / www.ncbi.nlm.nih.gov) and confirmed as Lactobacillus paragasseri. The 16S rDNA sequence of strain JMS024 is shown in SEQ ID NO. 3:

[0055] After the above identification, Lactobacillus paragasseri JMS024 was deposited in the General Microbiology Center of China Culture Collection Administration on March 20, 2025, with the deposit number CGMCC No.33909, and was classified as Lactobacillus paragasseri ( Lactobacillus paragasseri ).

[0056] Example 1: Isolation and identification of extracellular vesicles of Lactobacillus paragaseri JMS024 Culture method: Lactobacillus paragasseri JMS024 was inoculated into 150 mL of MRS medium and cultured anaerobically at 37°C. When the OD600 of the bacterial solution reached 0.8, the bacterial solution was inoculated into fresh 150 mL of MRS medium at a ratio of 2% v / v and cultured anaerobically at 37°C for 24 hours. The obtained culture medium was used for subsequent extracellular vesicle extraction.

[0057] The culture medium obtained above was ultracentrifuged to obtain the supernatant, which was filtered through a 0.22 μm filter membrane to remove the bacteria. The supernatant was then centrifuged at 100,000 rpm and 4°C for 60 min by ultracentrifugation. The supernatant was discarded, and 2 mL of PBS was added to rinse the precipitate from the ultracentrifuge tube. The volume was then made up to 10 mL with PBS, and the tube was centrifuged at 100,000 rpm and 4°C for 60 min. The PBS was discarded to obtain the precipitate, which was the extracellular vesicles (LcEVs) of Lactobacillus paragastris JMS024. The obtained LcEVs were resuspended in 200 μl of PBS buffer solution, aliquoted, and stored in a -80°C refrigerator.

[0058] The extracted extracellular vesicles were resuspended in 10-20 μl PBS, fixed with 2.5% glutaraldehyde, and placed on a 300-mesh copper grid. The copper grid was stained with 2% uranyl acetate and the morphological characteristics of the extracellular vesicles were observed under a transmission electron microscope. Figure 2 .

[0059] The isolated extracellular vesicles of Lactobacillus paragasseri JMS024 were detected on the ZetaView nanoparticle tracking analyzer. The Brownian motion of individual EVs was tracked using ZetaView 8.04.02 software, and the hydrodynamic diameter and concentration of extracellular vesicles of Lactobacillus paragasseri JMS024 were calculated using the Stockes-Einstein equation. The diameter of extracellular vesicle particles of Lactobacillus paragasseri JMS024 is mainly concentrated in the range of approximately 40-200 nm. Figure 3 .

[0060] Example 2: Extracellular vesicles of Lactobacillus paragaseri JMS024 can enter liver cancer cells In this example, two representative liver cancer cell lines used in liver cancer research were used: Hep3B cells purchased from ATCC, catalog number HB-8064; and HepG2 cells purchased from ATCC, catalog number HB-8065. Extracellular vesicles were prepared using the method of Example 1.

[0061] The general experimental process for experiments on two different cells is as follows: (1) Cell plating: Stable cells were added to DMEM culture medium to prepare a cell suspension with a volume of 500 μL per well containing 1.5×10 5 The cells were plated in confocal microplates and cultured overnight.

[0062] (2) Extracellular vesicle fluorescence staining and observation: 1) Using the ExoGlow™-Membrane EV Labeling Kit (System Biosciences) 2) Add 10 μL of vesicles to 300 μL of cell culture medium.

[0063] 3) Add 5 μL of ExoGlow Membrane (purchased from SystemBiosciences, USA, Catalog No. EXOGR800A-1) to the mixture and incubate at 37°C in the dark for 1 hour. Remove free probe from the labeled LpEVs-Membrane suspension using a GE PD Spintrap G-25 buffer exchange column.

[0064] a. Vortex the liquid in the PD Spintrap G-25.

[0065] b. Loosen the screw cap and unscrew the bottom closure.

[0066] c. Place the column into the collection tube provided with the package and centrifuge at 800 rpm for 1 minute to remove the original storage solution in the exchange column.

[0067] d. Add 0.4 mL of PBS to each tube and centrifuge at 800 rpm for 75 seconds.

[0068] e. Replace with a new collection tube.

[0069] f. Repeat steps d and e 4 times.

[0070] g. Replace with a new collection tube.

[0071] h. Slowly add the sample (140-180 μL) into the middle of the packed bed.

[0072] i. Centrifuge at 800 rpm for 2 minutes and recover the liquid at the bottom of the collection tube.

[0073] (3) The labeled vesicles were diluted with PBS to a final concentration of 100 μg / mL per well, and cultured for 24 hours. The results were observed using a confocal fluorescence microscope. Figure 4 As shown, labeled extracellular vesicles of Lactobacillus paragastrii JMS024 were able to enter liver cancer cells.

[0074] Example 3: Comparative study on the inhibition of liver cancer cell proliferation by extracellular vesicles of different Lactobacillus paragaseri To better demonstrate the ability of extracellular vesicles of Lactobacillus paragastrii JMS024 to inhibit liver cancer cells, this example used other Lactobacillus paragastrii strains (JMS005, JMS013, JMS020, and JMS033) isolated in Basic Example 1 for comparison. Extracellular vesicles of each strain were prepared using the method of Example 1.

[0075] The specific steps are as follows: (1) Completely digest the Hep3B and HepG2 cells that have grown in the culture flask, add DMEM culture medium, repeatedly blow the digested cells to detach the cells from the cell wall and make a cell suspension, and centrifuge at 800 rpm for 5 minutes.

[0076] (2) Discard the supernatant, add an appropriate amount of culture medium to resuspend, draw 20 μl of cells into the counting area, and count them on a cell counter. Adjust the cell density to 1×10 4 pcs / ml.

[0077] (3) Add 100 μl of the cell suspension adjusted to a certain density to each well of the 96-well plate, so that the cells are evenly distributed in the well plate. Place the 96-well plate in a cell culture incubator at 37°C containing 5% CO2 and culture.

[0078] (4) After the cells adhered, the cell culture medium in the 96-well plate was aspirated and discarded. Fresh culture medium containing 100 μg / ml extracellular vesicles was added to the experimental wells. The blank control group (blank) was treated with fresh culture medium (without extracellular vesicles). The 96-well plate was placed in a cell culture incubator for 48 h.

[0079] (5) After the extracellular vesicle reaction is complete, add 10 μl of WST-1 solution (purchased from APExBIO, USA, No. B8301) to each well and incubate in a cell culture incubator for 1-2 hours. Measure the absorbance at 450 nm using a microplate reader.

[0080] The results are as follows Figure 5As shown, extracellular vesicles of Lactobacillus paragasseri JMS024 (100 μg / mL) significantly inhibited cell proliferation and significantly reduced the viability of Hep3B and HepG2 liver cancer cells after 48 hours of treatment. Other Lactobacillus paragasseri strains at the same concentration (100 μg / mL) had no significant inhibitory effect on these two liver cancer cells, and their efficacy was significantly lower than that of Lactobacillus gasseri JMS024. See Table 2 for specific data.

[0081] Table 2 Comparative study on the inhibition of liver cancer cell proliferation by extracellular vesicles of different Lactobacillus paragaseri

[0082] Note: In the same column of data, different letters indicate significant differences between the data, P<0.5; the same letters indicate no significant differences between the data, P>0.05.

[0083] Example 4: Extracellular vesicles of Lactobacillus paragastrii JMS024 inhibit liver cancer cell migration Cell culture: After thawing cells, when the adherent cells are fully grown, slowly pour the cell culture medium along the side of the culture flask and add 5 mL of PBS to wash twice. Digest the cells with 1 mL of 0.25% trypsin containing EDTA for about 4 to 5 minutes. When most of the cells begin to slide off the cell culture flask like quicksand under an inverted microscope, add DMEM culture medium to stop digestion. Passage the cells in the original flask at a ratio of 1:3, take the cell suspension and place it in a new culture dish, add DMEM to make up to 5 mL, and continue culturing. After the cells have grown stably, use a 6-well plate to add Hep3B and HepG2 cells. After the cells have attached, use a 200 μl pipette tip to scratch perpendicular to the marked line on the bottom of the plate, keeping the pipette tip vertical and the force consistent. Gently rinse with sterile PBS three times to remove detached cell debris. Serum-free medium containing extracellular vesicles of Lactobacillus paragastris JMS024 (final concentration 100 μg / ml) (Gibco, USA, Cat. No. 11965092) was added to the blank control group, which was treated with fresh medium (without extracellular vesicles) and all other procedures were the same. Observe the results after 24 hours, as shown in the following example. Figure 6 As shown, extracellular vesicles of Lactobacillus paragaseri JMS024 can significantly inhibit the migration of liver cancer cells.

[0084] Example 5: Extracellular vesicles of Lactobacillus paragastrii JMS024 inhibit autophagy in liver cancer cells (1) Extracellular vesicles of Lactobacillus paragaseri JMS024 inhibit the expression of the autophagy marker gene Beclin-1 After culturing the cells according to the method of Example 4, the cells were cultured in starvation medium (EBSS medium, Gibco, USA, Catalog No. 14155063). The extracellular vesicles prepared in Example 1 (final concentration 100 μg / ml) were added and cultured for further 24 hours. Beclin-1 expression was detected by PCR. First, total cellular RNA was extracted and then reverse transcribed into cDNA for Real-Time PCR. The primers are shown in Table 3: Table 3 Description of primers for detecting Beclin-1 expression

[0085] The results are as follows Figure 7 As shown, extracellular vesicles of Lactobacillus paragaseri JMS024 inhibited the expression of the autophagy marker gene Beclin-1.

[0086] (2) Effect of extracellular vesicles of Lactobacillus paragaseri JMS024 on the expression of LC3B After culturing the cells according to the method in Example 4, the medium was switched to starvation medium (EBSS) and the extracellular vesicles prepared in Example 1 (final concentration 100 μg / ml) were added for further culture. After 24 hours, the expression level of LC3B-II was determined by western blot. Total protein was extracted using RIPA lysis buffer (Shanghai Beyotime Biotechnology Co., Ltd.), and total protein concentration was determined using a BCA protein assay kit (Shanghai Beyotime Biotechnology Co., Ltd.). After separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the proteins were transferred to a nitrocellulose membrane. The membrane was blocked with 5% skim milk powder for 2 hours at room temperature, followed by incubation with the primary antibody (LC3B, Abcam, ab192890) at 4°C overnight. After washing, the membrane was incubated with a horseradish peroxidase-conjugated secondary antibody (1:2000, Shanghai Beyotime Biotechnology Co., Ltd.) at room temperature for 1 hour. Electrochemiluminescence (ECL) kit (Biosharp, China) was used for development, and protein bands were analyzed using ImageJ software. The same method was used to detect the expression level of LC3B-I protein. Figure 8 As shown in the results, autophagy was significantly activated in both Hep3B and HepG2 cells, as evidenced by a significant increase in the expression of LC3B-I and LC3B-II; however, in the vesicle group, the expression of LC3B-I and LC3B-II was inhibited, indicating that extracellular vesicles of Lactobacillus paragastrium JMS024 inhibited the autophagy of liver cancer cells.

[0087] (3) Observation of GFP-LC3B expression distribution using laser confocal microscopy After culturing the cells according to the method of Example 4, the cells were cultured in starvation medium (EBSS medium). After transfection of GFP-LC3B into the cell line, the extracellular vesicles prepared in Example 1 (final concentration 100 μg / ml) were added and cultured for 24 hours. The expression distribution of GFP-LC3B was observed using a laser confocal microscope. When autophagy forms, the GFP-LC3 fusion protein translocates to the autophagosome membrane, forming multiple bright green fluorescent spots under a fluorescence microscope. One spot is equivalent to one autophagosome, and the level of autophagic activity can be evaluated by counting. The results are as follows. Figure 9 As shown, extracellular vesicles of Lactobacillus paragaseri JMS024 can inhibit the aggregation of GFP-LC3, that is, inhibit the formation of autophagosomes and inhibit the activity of autophagy.

[0088] (4) Electron microscopy observation of autophagosome formation After culturing the cells according to the method of Example 4, the cells were cultured in starvation medium (EBSS medium), and the extracellular vesicles prepared in Example 1 (final concentration 100 μg / ml) were added and cultured for a long time. After 24 hours, the formation of autophagosomes was detected by electron microscopy. The results are as follows: Figure 10 As shown in the figure, the number of autophagosomes formed in the extracellular vesicle treatment group was significantly less than that in the blank control group, indicating that the extracellular vesicles of Lactobacillus paragastris JMS024 can inhibit the autophagy of liver cancer cells.

[0089] Example 6: In vivo observation of the effect of extracellular vesicles on hepatocellular carcinoma Preparation of Hep3B cell suspension: Select Hep3B cells in good cell condition and in the logarithmic growth phase. When the cell density reaches 80%-90%, discard the original culture medium in the cell culture flask and rinse twice with PBS. After trypsinizing the cells, centrifuge at 800 rpm for 5 minutes, discard the supernatant, and resuspend the resulting pellet in PBS to adjust the cell density to 1×10 8 / ml. 1×10 6 Hep3B cells (blank control group), and 1×10 6 A combination of Hep3B cells and 100 μg / ml extracellular vesicles (EVs group) was injected into the left liver lobe of nude mice at a shallow angle. A distinct, translucent vesicle formed on the liver surface. The liver was harvested and observed 2 weeks later. After tumor formation, the liver was harvested and the tumor was observed and analyzed for intrahepatic metastasis. The results are shown in Figure 2. Figure 11 As shown in the data, the liver tumors in the extracellular vesicle group were smaller and had fewer intrahepatic metastases, while the liver tumors in the Hep3B group were larger and more prone to intrahepatic metastasis, indicating that the extracellular vesicles of Lactobacillus paragastris JMS024 inhibited the proliferation and metastasis of tumor cells.

[0090] Example 7: Study on the mechanism of autophagy inhibition by extracellular vesicles of Lactobacillus paragaseri JMS024 After culturing the cells according to the method of Example 4, the cells were cultured in starvation medium (EBSS medium) and the extracellular vesicles prepared in Example 1 (final concentration 100 μg / ml) were added and cultured for a long time. Referring to the protein detection method of Example 5, the protein expression of p-AKT (Ser473) and p-mTOR (Ser2448) in the autophagy pathway was detected by western-blot after 24 hours. Activated AKT can further activate its downstream molecule mTOR through the TSC1 / 2 complex. mTOR exists in two different complex forms, namely mTORC1 and mTORC2. The former mainly inhibits autophagy. The results are shown in FIG. Figure 12 As shown in the figure, the expressions of p-AKT (Ser473) and p-mTOR (Ser2448) were upregulated, indicating that extracellular vesicles of Lactobacillus paragaseri JMS024 inhibited autophagy through the AKT / mTOR signaling pathway.

[0091] Liver cancer tissues were collected from the animal experiments and Beclin-1 mRNA expression levels in the two groups of tumor tissues were detected by RT-PCR according to the method of Example 5. The results are as follows: Figure 13 As shown in the figure, the Beclin-1 expression level in the vesicle-treated group was lower, indicating that extracellular vesicles of Lactobacillus paragastris JMS024 inhibited autophagy of tumor cells.

[0092] Animal experimental liver cancer tissues were taken and the expression levels of p-AKT and p-mTOR in the tumor tissues of the two groups of animals were detected by Western blot according to the method of Example 5. The results are as follows: Figure 14 As shown in the data, the expression levels of p-AKT and p-mTOR in the extracellular vesicle group were higher, and the difference was significant compared with the blank control group, which once again proved that extracellular vesicles of Lactobacillus paragastris JMS024 inhibited autophagy through the AKT / mTOR pathway.

[0093] Comparative Example 1: Comparison of different lactobacilli In addition, the present application was compared with Lactobacillus plantarum YT013 and Lactobacillus plantarum CGMCC8198 in the prior art, and the results are shown in Table 4.

[0094] Table 4 Comparison with lactic acid bacteria in the prior art

[0095] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A Lactobacillus paragaseri ( Lactobacillus paragasseri ) JMS024, characterized in that, The deposit number is CGMCC No.33909, and it was deposited in the General Microbiology Center of China Culture Collection Administration on March 20, 2025.

2. A bacterial agent, characterized in that The bacterial agent comprises the Lactobacillus paragaseri according to claim 1 or the preparation of the Lactobacillus paragaseri according to claim 1.

3. The microbial agent according to claim 2, characterized in that The preparation includes the culture, culture extract, fermentation broth, fermentation broth precipitate, fermentation broth supernatant or fermentation broth extract of the Lactobacillus paragaseri.

4. The bacterial agent according to claim 2 or 3, characterized in that The bacterial agent is a solid preparation or a liquid preparation.

5. The bacterial agent according to claim 2 or 3, characterized in that The bacterial agent contains extracellular vesicles secreted by the Lactobacillus paragaseri.

6. An extracellular vesicle, characterized in that The extracellular vesicles are prepared from the Lactobacillus paragaseri JMS024 according to claim 1.

7. The extracellular vesicle according to claim 6, characterized in that The preparation method of the extracellular vesicles comprises: Lactobacillus paragastris JMS024 is cultured in a culture medium to obtain a culture supernatant, the supernatant is filtered to obtain a filtrate, and extracellular vesicles are separated from the filtrate.

8. Use of the Lactobacillus paragaseri according to claim 1, the bacterial agent according to any one of claims 2 to 5, or the extracellular vesicles according to any one of claims 6 to 7 in preparing a product for treating cancer, characterized in that: The cancer includes liver cancer.

9. The use according to claim 8, characterized in that The product includes a drug, which also includes a medically acceptable carrier.

10. The use according to claim 9, characterized in that The product contains extracellular vesicles secreted by Lactobacillus paragaseri JMS024.

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