Lactobacillus paragasseri and application thereof in prevention and treatment of liver cancer

Abstract

This invention provides a strain of *Lactobacillus paragrigii* and its application in products for the prevention and treatment of liver cancer, belonging to the field of biomedicine. This invention provides a strain of *Lactobacillus paragrigii* (… Lactobacillus paragassers Lactobacillus parageri JMS024, with accession number CGMCC No. 33909, was deposited at the China General Microbiological Culture Collection Center on March 20, 2025. This invention describes the isolation of extracellular vesicles from Lactobacillus parageri JMS024 through culturing. These vesicles significantly inhibit the proliferation, migration, and autophagy of liver cancer cells, and significantly reduce their activity. They can be used to prepare products for the treatment of liver cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to Lactobacillus paragrigii, specifically to a strain of Lactobacillus paragrigii and its application in products for the prevention and treatment of liver cancer. Background Technology

[0002] Probiotics are a class of bacteria that are beneficial to the host. Currently, probiotics are mostly used as food supplements to maintain and improve the host's gut microbiota. A growing body of research supports the idea that probiotics, by maintaining a healthy gut microbiota, may improve gastrointestinal diseases, including gastrointestinal infections, inflammatory bowel disease, and even cancer. Probiotics exert their effects in the body, possibly first by altering the host's microbiota, second by improving the barrier function of the intestinal mucosa, and finally by modulating the immune system to exert their protective role.

[0003] Hepatocellular carcinoma (HCC), one of the deadliest malignant tumors worldwide, is closely associated with risk factors such as hepatitis B virus (HBV) and hepatitis C virus (HCV) infection, cirrhosis, and aflatoxin exposure. Increasing research shows that the gut-liver axis plays a crucial role in the development and progression of HCC. A normal gut barrier consists of the following components: a normally composed and distributed gut microbiota, the intestinal mucosal barrier, and the integrity of the immune system. Damage to the gut barrier increases intestinal permeability, leads to bacterial ectopic growth, and accumulates LPS, all of which contribute to the development of hepatocellular carcinoma. Therefore, probiotic regulation of the gut microbiota may be a novel approach to prevent and treat the development and progression of HCC. Altering the type and quantity of the gut microbiota not only promotes gut microbiota balance and improves intestinal inflammation and the mucosal barrier but also significantly improves symptoms of cirrhosis and reduces the incidence of HCC. Regulating the gut microbiota through probiotics may be a new therapeutic approach to prevent the progression from hepatitis to cirrhosis to HCC. However, the mechanism by which gut microbiota plays a role in the development and progression of liver cancer remains unclear. Therefore, future research needs to explore the composition of the microbiota in both healthy and disease states, as well as the factors that maintain its long-term stability. Currently, a growing body of research indicates that vaginal gut microbiota 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 hepatocytes by altering the gut microbiota or by acting directly on the liver.

[0004] Probiotic-derived extracellular vesicles (EVs) are bilayered membrane nanoparticles with a diameter of approximately 20-400 nm released by probiotics during their growth. These vesicles carry bioactive substances such as proteins, nucleic acids, lipids, and metabolites, serving as novel signaling molecules mediating 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-hepatic axis and gut-brain axis. Currently, research on the probiotic *Lactobacillus paragernii* mainly focuses on its application in treating the gastrointestinal tract, with no documented use of *Lactobacillus paragernii* in the preparation of products for treating liver cancer.

[0005] The prior art CN113604410B discloses that the fermentation broth obtained by culturing and fermenting Lactobacillus plantarum strain YT013 can be used to prepare cell-free culture medium of Lactobacillus plantarum YT013 after centrifugation and freeze-drying. The cell-free culture medium has a significant inhibitory effect on HepG2 liver cancer cells, and the inhibition rate of HepG2 liver cancer cells increases with the increase of the concentration of Lactobacillus plantarum YT013 cell-free culture medium, which is dose-dependent.

[0006] Wang Chang et al. treated HepG2 cells with different concentrations of Lactobacillus plantarum CGMCC8198 lysate (LpS) for different durations. They used Western blotting, Oil Red staining, and real-time quantitative PCR to detect the effects of LpS on steatosis and the HMGCR / SMYD3 lipid metabolism pathway in liver cancer cells. The effects of LpS on the proliferation, migration, and apoptosis of HepG2 cells during lipid metabolism disorders were detected using the MTT assay, cell scratch assay, and flow cytometry. The results showed that LpS could inhibit the expression of genes such as HMGCR, SMYD3, and SREBP-2 in liver cancer cells with lipid metabolism disorders, and also dose-dependently inhibit cell proliferation and migration while promoting 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] This invention addresses the problem that existing technologies lack *Lactobacillus paragernii* strains suitable for preparing products for treating liver cancer, by providing a strain of *Lactobacillus paragernii* and its application in liver cancer prevention and treatment products. This invention provides *Lactobacillus paragernii* JMS024, with accession number CGMCC No. 33909. Extracellular vesicles of *Lactobacillus paragernii* JMS024 can be isolated through liquid culture. These extracellular vesicles significantly inhibit the proliferation and migration of liver cancer cells and can be used to prepare products for treating liver cancer.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] On the one hand, the present invention provides a *Lactobacillus paragrigii* (… Lactobacillus paragasseri JMS024, with accession number CGMCC No.33909, was deposited at the China General Microbiological Culture Collection Center on March 20, 2025.

[0010] On the other hand, the present invention provides a microbial agent comprising the above-mentioned Lactobacillus paragrigius or a preparation thereof.

[0011] Preferably, the preparation comprises a culture of Lactobacillus paragelius, a culture extract, a fermentation broth, a fermentation broth precipitate, a fermentation broth supernatant, or a fermentation broth extract.

[0012] Preferably, the microbial agent is a solid or liquid formulation.

[0013] Preferably, the bacterial agent contains extracellular vesicles secreted by the Lactobacillus paragernii.

[0014] On the other hand, the present invention provides an extracellular vesicle prepared from the aforementioned Lactobacillus paragernii JMS024.

[0015] Preferably, the method for preparing the extracellular vesicles includes:

[0016] Lactobacillus paragernii JMS024 was cultured in a culture medium, and the culture supernatant was obtained. The supernatant was filtered to obtain the filtrate, and extracellular vesicles were isolated from the filtrate.

[0017] On the other hand, the present invention provides the use of the above-mentioned Lactobacillus paragernii, the above-mentioned bacterial agent, or the above-mentioned extracellular vesicles in the preparation of cancer treatment products.

[0018] Preferably, the cancer includes liver cancer.

[0019] Preferably, the product includes a pharmaceutical product, and the pharmaceutical product further includes a medically acceptable carrier.

[0020] Preferably, the product contains extracellular vesicles secreted by Lactobacillus paragernii JMS024.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. This invention provides a Lactobacillus paragasseri JMS024, with accession number CGMCC No.33909, which was deposited at the China General Microbiological Culture Collection Center on March 20, 2025.

[0023] 2. The extracellular vesicles isolated by culturing Lactobacillus parageri JMS024 in this invention significantly inhibit the proliferation, migration, and autophagy of liver cancer cells, and significantly reduce the activity of liver cancer cells. These vesicles can be used to prepare products for the treatment of liver cancer.

[0024] Preservation Instructions

[0025] Strain name: Lactobacillus paragernii

[0026] Latin name: Lactobacillus paragasseri ,

[0027] Strain number: JMS024

[0028] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0029] The abbreviation for the depository institution is CGMCC.

[0030] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0031] Deposit date: March 20, 2025

[0032] Registered with the China National Collection Center (CGMCC) No. 33909. Attached Figure Description

[0033] Figure 1 The images show the morphological identification results of Lactobacillus paragrigii JMS024. A shows the Gram staining results of Lactobacillus paragrigii JMS024, and B shows the colonies of Lactobacillus paragrigii JMS024.

[0034] Figure 2 The morphological characteristics of extracellular vesicles under transmission electron microscopy.

[0035] Figure 3 The particle size distribution of extracellular vesicles in Lactobacillus paragernii JMS024 is shown in the nanoparticle tracer analysis.

[0036] Figure 4 Fluorescent image of extracellular vesicles of Lactobacillus paragelius JMS024 entering liver cancer cells.

[0037] Figure 5 Comparative experiments on the inhibition of liver cancer cell proliferation by extracellular vesicles of different Lactobacillus paragerens; A represents the experimental results for Hep3B cells, and B represents the experimental results for HepG2 cells; ** indicates significant difference, P<0.01.

[0038] Figure 6The image shows the results of Lactobacillus paragelius JMS024 extracellular vesicles inhibiting the migration of liver cancer cells; where A and B are microscopic images and quantitative results for Hep3B cells, respectively, and C and D are microscopic images and quantitative results for HepG2 cells, respectively.

[0039] Figure 7 Figure 1 shows the results of Lactobacillus paragerens JMS024 extracellular vesicles inhibiting the expression of the autophagy gene Beclin-1 in liver cancer cells; A represents the experimental results for Hep3B cells, and B represents the experimental results for HepG2 cells; ** indicates a significant difference, P<0.01.

[0040] Figure 8 Figure showing the results of Lactobacillus paragelius JMS024 extracellular vesicles inhibiting the expression of LC3BI and LC3BI proteins in liver cancer cells.

[0041] Figure 9 Fluorescent image showing the inhibition of GFP-LC3 aggregation by extracellular vesicles of Lactobacillus paragelius JMS024.

[0042] Figure 10 Microscopic images showing how extracellular vesicles of Lactobacillus paragelius JMS024 inhibit the formation of autophagosomes.

[0043] Figure 11 The results show that extracellular vesicles of Lactobacillus parageri JMS024 inhibit 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.

[0044] Figure 12 This is a protein analysis diagram showing how extracellular vesicles of Lactobacillus paragelius JMS024 inhibit autophagy via the AKT / mTOR signaling pathway.

[0045] Figure 13 The extracellular vesicles of Lactobacillus paragelius JMS024 inhibit the expression of the autophagy gene Beclin-1 in tumor cells.

[0046] Figure 14 Extracellular vesicles of Lactobacillus paragelius JMS024 inhibit tumor cell autophagy through the AKT / mTOR pathway; A is a protein analysis diagram, and B is a quantitative analysis diagram of the expression levels of p-AKT and p-mTOR. Detailed Implementation

[0047] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0048] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0049] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0050] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0051] As used herein, the terms “comprises” or “comprising” mean “including, but not limited to”. This term is intended to be open-ended to specify the presence of any of the stated 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 “substantially consisting of”. In one embodiment, the term “comprising” used throughout the application, particularly in the claims, may be replaced by the term “consisting of”. The amino acid three-letter and single-letter codes used herein are as known to those skilled in the art, or as described in J Biol. Chem, 243, p3558 (1968).

[0052] As used herein, the terms “optional,” “any,” “arbitrary,” or “any one” mean that the event or circumstance described below may, but does not have to, occur, including the circumstances in which the event or circumstance may or may not occur. For example, “optionally containing one antibody heavy chain variable region” means that the antibody heavy chain variable region of a particular sequence may, but does not have to, be present.

[0053] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0054] The terms “activity,” “functional activity,” or “biological activity,” or “biological property,” or “biological characteristic,” as used herein, are used interchangeably 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 levels of protein expression in mammalian cells. The aforementioned properties or characteristics may be observed, measured, or evaluated using techniques known in the art, including, but not limited to, ELISA, FACS, or BIACORE plasma resonance analysis, unrestricted in vitro or in vivo neutralization assays, receptor binding, production and / or secretion of cytokines or growth factors, signal transduction, and immunohistochemistry of tissue sections from various sources, including human, primate, or any other source.

[0055] As used herein, the term "pharmaceutically acceptable" means 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, to the extent of reasonable medical judgment, and in proportion to a reasonable benefit / risk ratio. As used herein, the term "pharmaceutically acceptable carrier, excipient, and / or diluent" means 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 media, such as liquid or solid fillers, diluents, excipients, solvents, mediators, encapsulating materials, manufacturing aids, or solvent encapsulating materials, relate to maintaining the stability, solubility, or activity of the antibody or its antigen-binding fragment disclosed herein, and include, but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying 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, sorbic acid, etc. Agents for maintaining osmotic pressure include, but are not limited to, sugars, NaCl, and their analogues. Agents for delaying 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), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art as being able to stabilize the desired activity of an active ingredient in a drug, including but not limited to monosodium 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.

[0056] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed according to conventional conditions or the manufacturer's recommendations. All reagents or instruments without specified manufacturers are commercially available conventional products. To better illustrate this invention, numerous specific details are given in the following detailed embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concept of the invention. Such structures and techniques are found in many publications, such as *Molecular Cloning: A Laboratory Manual (4th Edition)* (Cold Spring Harbor Laboratory Science Press), Ausubel, FM et al. Current Protocols in Molecular Biology It is also described in Greene Publishing Association and Wiley-Interscience.

[0057] Data analysis and statistical analysis were performed using SPSS 23.0 software. One-way ANOVA was used for significance analysis, and P<0.05 was considered to be statistically significant.

[0058] Basic Example 1: Isolation of Lactobacillus parageri

[0059] The MRS medium (per liter) consists of the following components: 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; manganese sulfate (MnSO4·H2O) 0.25 g; distilled water to a final volume of 1000 mL; adjust pH to 6.2-6.6. Sterilization is performed using an autoclave at 121°C for 20 min.

[0060] The separation steps are as follows:

[0061] (1) Collect fecal samples from 70 healthy individuals who showed no signs of gastrointestinal inflammation or tumors during clinical testing. The samples were numbered sequentially, inoculated into MRS liquid culture medium, and anaerobically cultured at 37°C for 24 hours.

[0062] (2) Five single colonies were randomly selected from each sample and cultured in 2 mL of MRS liquid medium for 24 h for enrichment. The colonies were numbered sequentially.

[0063] (3) Amplification of bacteria for bacterial preservation and DNA preparation. Hundreds of isolated bacteria were identified; the ability of the supernatant to inhibit liver cancer cells was detected, and one strain of Lactobacillus parageri with the strongest inhibitory effect on liver cancer cells was screened and named JMS024.

[0064] Basic Example 2: Identification of Lactobacillus paragernii

[0065] The strain JMS024 obtained in Basic Example 1 was identified as follows:

[0066] (1) Morphological identification: On MRS Lactobacillus agar plates, the isolated strain JMS024 appeared to be approximately 0.5-1.0 mm in size, with neat colony edges and raised margins. It was Gram-positive, and under a microscope, the cells were mostly short rod-shaped. Figure 1 A in the sample is Gram-positive, short rod-shaped, and can form chains. Figure 1 B in the middle.

[0067] (2) Homology analysis of 16S rDNA sequence

[0068] The isolated strain JMS024 was cultured using conventional methods. Total DNA was extracted from the strain and used as a template for gene amplification. The conserved region of the bacterial 16S rDNA gene was amplified using universal primers 27F (SEQ ID NO:1) and 1492R (SEQ ID NO:2). Information on the universal primers is shown in Table 1.

[0069] Table 1 Universal primers 27F and 1492R

[0070]

[0071] The amplification system (25 μL) consisted of: 1×PCR reaction buffer, 200 μmol / L dNTPs, 0.2 μmol / L upstream and downstream primers each, 1 U Taq DNA polymerase, and 1 μL template DNA. Reaction conditions were: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 40 s, and 72℃ extension for 30 s, for a total of 25 cycles; followed by a 10 min extension at 72℃. PCR products were detected by 1% gel electrophoresis, and positive results were sequenced using universal primers 27F and 1492R. Sequence assembly and similarity analysis were performed using DNAStar software, and sequence alignment was performed online through the NCBI database (http: / / www.ncbi.nlm.nih.gov), confirming the strain as *Lactobacillus parageri*. The 16S rDNA sequence of strain JMS024 is shown in SEQ ID NO. 3.

[0072]

[0073] Following the above identification, *Lactobacillus paragernii* JMS024 was deposited on March 20, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33909, and classified as *Lactobacillus paragernii* (…). Lactobacillus paragasseri ).

[0074] Example 1: Isolation and identification of extracellular vesicles of Lactobacillus parageri JMS024

[0075] Culture method: Lactobacillus paragerens 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 h. The culture medium obtained can be used for subsequent extracellular vesicle extraction.

[0076] The culture medium obtained above was centrifuged at high speed to obtain the supernatant. The supernatant was filtered through a 0.22 μm filter membrane to remove bacterial cells. The culture medium was then centrifuged at 100,000 rpm at 4°C for 60 minutes. The supernatant was discarded, and 2 mL of PBS was added to wash the precipitate from the centrifuge tube. The volume was then brought up to 10 mL with PBS. The precipitate was centrifuged at 100,000 rpm at 4°C for 60 minutes. The PBS was discarded. The resulting precipitate was Lactobacillus paragelii JMS024 extracellular vesicles (LcEVs). The LcEVs were resuspended in 200 μl of PBS buffer, aliquoted, and stored at -80°C.

[0077] The extracted extracellular vesicles were resuspended in 10-20 μl of 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. (See [link to transmission electron microscopy description]). Figure 2 .

[0078] Extracellular vesicles of isolated *Lactobacillus paragernii* JMS024 were analyzed using 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 *Lactobacillus paragernii* JMS024 extracellular vesicles were calculated using the Stockes-Einstein equation. The diameter of the *Lactobacillus paragernii* JMS024 extracellular vesicle particles was mainly concentrated in the range of approximately 40-200 nm. (See...) Figure 3 .

[0079] Example 2: Extracellular vesicles of Lactobacillus paragerens JMS024 can enter liver cancer cells.

[0080] In this example, two representative liver cancer cell lines used in liver cancer research were employed: 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 described in Example 1.

[0081] The general experimental procedure for experiments on two different cell types is as follows:

[0082] (1) Cell plating:

[0083] Stable cells were added to DMEM culture medium to prepare a cell suspension, with each well containing 500 μL of 1.5 × 10⁻⁶ cells. 5 One cell was spread in a confocal dish and cultured overnight.

[0084] (2) Fluorescent staining and observation of extracellular vesicles:

[0085] 1) Use the ExoGlow™-Membrane EV Labeling Kit (System Biosciences)

[0086] 2) Add 10 μL of vesicles to 300 μL of cell culture medium.

[0087] 3) Add 5 μL of ExoGlow Membrane (purchased from SystemBiosciences, USA, catalog number EXOGR800A-1) to the mixture and incubate at 37°C in the dark for 1 hour. Remove the free probe from the labeled LpEVs-Membrane suspension using a GE PD Spintrap G-25 buffer exchange column.

[0088] a. Vortex the liquid in the PD Spintrap G-25.

[0089] b. Loosen the screw cap and unscrew the bottom closing device.

[0090] c. Place the column into the collection tube provided with the packaging, centrifuge at 800 rpm for 1 minute to remove the original stock solution from the exchange column.

[0091] d. Add 0.4 mL of PBS to each tube, centrifuge at 800 rpm for 75 seconds.

[0092] e. Replace with a new collection tube.

[0093] f. Repeat steps d and e four times.

[0094] g. Replace with a new collection tube.

[0095] h. Slowly add the sample (140-180 μL) to the middle of the packed bed.

[0096] i. Centrifuge at 800 rpm for 2 minutes, and collect the liquid at the bottom of the collection tube.

[0097] (3) Dilute the labeled vesicles with PBS to a final concentration of 100 μg / mL per well, and continue culturing for 24 hours. Observe the results using a confocal fluorescence microscope. The results are as follows: Figure 4 As shown, the labeled Lactobacillus paragernii JMS024 extracellular vesicles can enter liver cancer cells.

[0098] Example 3: Comparative experiment on the inhibition of liver cancer cell proliferation by extracellular vesicles of different Lactobacillus paragerens.

[0099] To better demonstrate the ability of Lactobacillus paragernii JMS024 extracellular vesicles to inhibit liver cancer cells, this example uses other Lactobacillus paragernii strains (JMS005, JMS013, JMS020, JMS033) isolated in Basic Example 1 for comparison. Extracellular vesicles of each strain were prepared using the method in Example 1.

[0100] The specific steps are as follows:

[0101] (1) Digest the Hep3B and HepG2 cells that have grown in the culture flask completely, add DMEM culture medium, repeatedly pipette the digested cells to detach them from the cell wall and make a cell suspension, and centrifuge at 800 r / min for 5 min.

[0102] (2) Discard the supernatant, resuspend the cells in an appropriate amount of culture medium, and transfer 20 μl of cells to the counting area. Count the cells using a cell counter. Adjust the cell density to 1 × 10⁻⁶. 4 per ml.

[0103] (3) Add 100 μl of cell suspension with adjusted density to each well into the 96-well plate to ensure that the cells are evenly distributed in the well plate. Place the 96-well plate in a cell culture incubator containing 5% CO2 at 37°C for culture.

[0104] (4) After the cells adhered, the cell culture medium in the 96-well plate was aspirated. Fresh culture medium containing 100 μg / ml extracellular vesicles was added to each well. The blank control group was given only fresh culture medium (without extracellular vesicles). The 96-well plate was then placed in the cell culture incubator and cultured for 48 h.

[0105] (5) After the extracellular vesicle activity 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 h. Measure the absorbance at 450 nm using a microplate reader.

[0106] The results are as follows Figure 5 As shown, extracellular vesicles of *Lactobacillus paragrigius* JMS024 (100 μg / mL) significantly inhibited cell proliferation and reduced the viability of Hep3B and HepG2 liver cancer cells after 48 hours of treatment. Other *Lactobacillus paragrigius* strains at the same concentration (100 μg / mL) did not show significant inhibitory effects on these two liver cancer cell types, and their effects were significantly lower than those of *Lactobacillus paragrigius* JMS024. Specific data are shown in Table 2.

[0107] Table 2. Comparative experiments on the inhibition of liver cancer cell proliferation by extracellular vesicles of different Lactobacillus paragerens.

[0108]

[0109] Note: In the same column of data, different letter labels indicate significant differences between the data (P < 0.5); the same letter labels indicate no significant differences between the data (P > 0.05).

[0110] Example 4: Extracellular vesicles of Lactobacillus parageri JMS024 inhibit the migration of liver cancer cells

[0111] Cell Culture: After cell resuscitation, once the adherent cells have fully confluenced, slowly pour the cell culture medium along the sidewall of the culture flask and wash twice with 5 mL of PBS. Digest the cells with 1 mL of 0.25% EDTA-containing trypsin for approximately 4-5 minutes. When most cells begin to slip out of the culture flask in a quicksand-like manner under an inverted microscope, add DMEM culture medium to stop digestion. Passage the cells from the original flask at a 1:3 ratio, transfer the cell suspension to a new culture dish, and add DMEM to bring the volume to 5 mL. Continue culturing. Once the cells have stabilized, add Hep3B and HepG2 cells to a 6-well plate. After the cells adhere, use a 200 μl pipette tip to make a mark perpendicular to the bottom marking line of the plate, keeping the tip vertical and applying consistent pressure. Gently rinse three times with sterile PBS to remove detached cell debris. Serum-free culture medium (Gibco, USA, catalog number 11965092) containing *Lactobacillus paragelius* JMS024 extracellular vesicles (final concentration 100 μg / ml) was added; the blank control group consisted of fresh culture medium (without extracellular vesicles), with all other procedures performed the same. Results were observed after 24 hours. Figure 6 As shown, extracellular vesicles of Lactobacillus paragerens JMS024 can significantly inhibit the migration of liver cancer cells.

[0112] Example 5: Extracellular vesicles of Lactobacillus parageri JMS024 inhibit autophagy in liver cancer cells

[0113] (1) Extracellular vesicles of Lactobacillus parageri JMS024 inhibit the expression of the autophagy marker gene Beclin-1.

[0114] After culturing cells according to the method in Example 4, the cells were cultured in starvation medium (EBSS medium, Gibco, USA, catalog number 14155063), and extracellular vesicles prepared in Example 1 (final concentration 100 μg / ml) were added for further culturing. After 24 hours, Beclin-1 expression was detected by PCR. First, total RNA was extracted from the cells, then reverse transcribed into cDNA, and Real-Time PCR was performed. The primers are shown in Table 3.

[0115] Table 3 Primer descriptions for detecting Beclin-1 expression

[0116]

[0117] The results are as follows Figure 7 As shown, extracellular vesicles of Lactobacillus parageri JMS024 inhibited the expression of the autophagy marker gene Beclin-1.

[0118] (2) Effect of extracellular vesicles of Lactobacillus paragerens JMS024 on LC3B expression

[0119] After culturing cells according to the method in Example 4, the cells were cultured in starvation medium (EBSS medium), and 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 detected by Western blot. Total protein was extracted from the cells using RIPA lysis buffer (Beyond Biotech, Shanghai), and the total protein concentration was detected using a BCA protein assay kit (Beyond Biotech, Shanghai). After separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the protein was transferred to a nitrocellulose membrane. The membrane was blocked with 5% skim milk powder at room temperature for 2 hours, followed by incubation overnight at 4°C with primary antibody (LC3B, Abcam, ab192890). After washing the membrane, horseradish peroxidase-labeled secondary antibody (1:2000, Beyond Biotech, Shanghai) was added and incubated at room temperature for 1 hour. Electrochemiluminescence (ECL) imaging was performed using a kit (Biosharp Biotechnology, China), and protein bands were analyzed using ImageJ software. The expression level of LC3B-I protein was detected using the same method. Results are as follows: Figure 8 As shown, 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, and the extracellular vesicles of Lactobacillus paragernii JMS024 inhibited autophagy in liver cancer cells.

[0120] (3) Observation of GFP-LC3B expression distribution by laser confocal microscopy

[0121] After culturing cells according to the method in Example 4, the cells were cultured in starvation medium (EBSS medium). After transfecting the cell line with GFP-LC3B, extracellular vesicles prepared in Example 1 (final concentration 100 μg / ml) were added and the cells were cultured for another 24 hours. The expression distribution of GFP-LC3B was observed using a laser confocal microscope. During autophagy formation, the GFP-LC3 fusion protein translocates to the autophagosome membrane, forming multiple bright green fluorescent spots under a fluorescence microscope. Each spot corresponds to one autophagosome, and the level of autophagy activity can be evaluated by counting the spots. The results are as follows... Figure 9 As shown, extracellular vesicles of Lactobacillus parageri JMS024 can inhibit the aggregation of GFP-LC3, that is, inhibit the formation of autophagosomes and inhibit autophagy activity.

[0122] (4) Electron microscopy observation of autophagosome formation

[0123] After culturing cells according to the method in Example 4, the cells were cultured in starvation medium (EBSS medium), and extracellular vesicles prepared in Example 1 (final concentration 100 μg / ml) were added and cultured for another 24 hours. The formation of autophagosomes was detected by electron microscopy, and the results were as follows: Figure 10 As shown, 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 paragernii JMS024 can inhibit autophagy in liver cancer cells.

[0124] Example 6: In vivo observation of the effects of extracellular vesicles on hepatocellular carcinoma

[0125] Preparation of Hep3B cell suspension: Select Hep3B cells in good 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 wash twice with PBS. Digest the cells with trypsin, centrifuge at 800 r / min for 5 min, discard the supernatant, resuspend the resulting pellet in PBS, and adjust the cell density to 1×10⁻⁶ cells / mL. 8 1×10⁻⁶ cells / ml. 6 Hep3B cells (blank control group) and 1×10 6 A combination of Hep3B cells and 100 μg / ml extracellular vesicles (extracellular vesicle group) was injected into the left hepatic lobe of nude mice at a small angle, forming a distinct, semi-transparent vesicle on the liver surface. The liver was harvested two weeks later for observation. After tumor formation, the liver was harvested for observation of the hepatic tumor and analysis of intrahepatic metastasis; the results are as follows. Figure 11 As shown, the extracellular vesicle group had smaller liver tumors and fewer intrahepatic metastases, while the Hep3B group had larger liver tumors and were more prone to intrahepatic metastases, indicating that the extracellular vesicles of Lactobacillus parageri JMS024 inhibited the proliferation and metastasis of tumor cells.

[0126] Example 7: Study on the mechanism by which extracellular vesicles of Lactobacillus parageri JMS024 inhibit autophagy

[0127] After culturing cells according to the method in Example 4, the cells were cultured in starvation medium (EBSS medium), and extracellular vesicles prepared in Example 1 (final concentration 100 μg / ml) were added for further culture. Following the protein detection method in 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, mTORC1 and mTORC2, with the former mainly inhibiting autophagy. The results are as follows... Figure 12 As shown, the upregulation of p-AKT (Ser473) and p-mTOR (Ser2448) indicates that extracellular vesicles of Lactobacillus paragernii JMS024 inhibit autophagy through the AKT / mTOR signaling pathway.

[0128] Animal liver cancer tissues were collected, and the expression level of Beclin-1 mRNA in the two groups of tumor tissues was detected by RT-PCR according to the method in Example 5. The results are as follows. Figure 13 As shown, the Beclin-1 expression level was lower in the vesicle treatment group, indicating that the extracellular vesicles of Lactobacillus parageri JMS024 inhibited autophagy in tumor cells.

[0129] Animal liver cancer tissues were collected for experimentation. Following the method described in Example 5, the expression levels of p-AKT and p-mTOR in the tumor tissues of the two groups of animals were detected by Western blot. The results are as follows: Figure 14 As shown, the extracellular vesicle group showed higher expression levels of p-AKT and p-mTOR, and the difference was significant compared with the blank control group, which further proves that the extracellular vesicles of Lactobacillus paragernii JMS024 inhibit autophagy through the AKT / mTOR pathway.

[0130] Comparative Example 1: Comparison of different lactobacilli

[0131] In addition, this application was compared with the prior art Lactobacillus plantarum YT013 and Lactobacillus plantarum CGMCC8198, and the results are shown in Table 4.

[0132] Table 4 Comparison with existing technologies for lactic acid bacteria

[0133]

[0134] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A type of Lactobacillus paragerens ( Lactobacillus paragasseri JMS024, characterized in that, The accession number is CGMCC No.33909, and it was deposited at the China General Microbiological Culture Collection Center on March 20, 2025.

2. A microbial agent, characterized in that, The bacterial agent comprises *Lactobacillus paragrigius* as described in claim 1 or extracellular vesicles of *Lactobacillus paragrigius* as described in claim 1.

3. An extracellular vesicle, characterized in that, The extracellular vesicles were prepared from Lactobacillus paragernii JMS024 as described in claim 1.

4. The extracellular vesicle according to claim 3, characterized in that, The method for preparing the extracellular vesicles includes: Lactobacillus paragernii JMS024 was cultured in a culture medium, and the culture supernatant was obtained. The supernatant was filtered to obtain the filtrate, and extracellular vesicles were isolated from the filtrate.

5. The use of *Lactobacillus paragelii* as described in claim 1, the bacterial agent as described in claim 2, or the extracellular vesicles as described in any one of claims 3-4 in the preparation of cancer treatment products, characterized in that... The cancer in question is liver cancer.

6. The application according to claim 5, characterized in that, The product includes pharmaceuticals, and the pharmaceuticals also include medically acceptable carriers.

7. The application according to claim 6, characterized in that, The product contains extracellular vesicles secreted by Lactobacillus paragelius JMS024.

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