Ruminococcus lactaris, its plasma membrane vesicles and uses

Abstract

This invention relates to the field of biomedical technology, and particularly to *Lactobacillus rumenans*, its plasma membrane vesicles, and their applications. *Lactobacillus rumenans* and its derived plasma membrane vesicles can significantly reverse the elevation of serum liver function indicators and lipid metabolism parameters in mice with alcohol-related liver disease induced by liquid alcohol diets, and significantly alleviate liver histopathological damage and lipid droplet infiltration. Simultaneously, both can effectively inhibit local inflammatory responses in the liver and reduce the expression levels of inflammatory factors. These results indicate that *Lactobacillus rumenans* and its CMVs can significantly improve liver function impairment, histological lesions, and inflammatory status induced by ALD by regulating lipid metabolism and inflammatory signaling pathways, providing new experimental evidence and potential strategies for microbial intervention in ALD.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to rumen lactobacillus, its plasma membrane vesicles, and their applications. Background Technology

[0002] The liver is the core organ for metabolism and detoxification. Alcohol-related liver disease (ALD) accounts for a quarter of global liver disease deaths and is closely related to the occurrence and progression of liver fibrosis, cirrhosis, and liver cancer. ALD is a spectrum of diseases including alcoholic fatty liver, hepatitis, liver fibrosis, and cirrhosis, with a complex pathogenesis involving lipid metabolism, the gut-liver axis, and immune regulation. Ethanol is oxidized by CYP2E1 in the liver, producing reactive oxygen species (ROS) and damaging the intestinal barrier, allowing endotoxins to enter the liver via the portal vein, thus inducing ALD. With timely intervention, ALD can be alleviated, but currently, there are no specific drugs for treatment. This provides valuable research opportunities for the treatment of ALD.

[0003] The gut microbiota, often referred to as the "second brain" of the human body, has become a key factor in regulating the pathological process of alcohol-related liver disease. Several lactobacilli, including *Lactobacillus acidophilus*, *Lactobacillus rhamnosus*, and *Lactobacillus johnsonii*, have been shown to slow and prevent alcohol-related liver disease, but more potential gut lactobacilli require further investigation. Rumen lactobacilli (…) Ligilactobacillus ruminis, L.ruminis Lactobacillus ruminis is a Gram-positive bacterium belonging to the genus Lactobacillus, first isolated from the bovine rumen. Recent studies have found that it is also a component of the Firmicutes phylum of the human gut microbiota. Previous studies have reported that L. ruminis effectively inhibits rotavirus infection by activating the interferon signaling pathway and can also serve as a biomarker for ulcerative colitis. However, no studies have yet shown that L. ruminis plays a positive role in the treatment of alcoholic liver disease.

[0004] Cell membrane vesicles are lipid bilayer nanoparticles naturally extruded from the cell membrane by Gram-positive bacteria during their growth via budding. Cell membrane vesicles can encapsulate DNA, RNA, enzymes, toxins, and drug-resistant proteins. With their ideal properties such as nanoscale structure, low toxicity, good biocompatibility, high stability, and inherent immunogenicity, they have become a highly promising novel immunotherapeutic agent. In recent years, the application prospects of membrane vesicles in the prevention and treatment of liver diseases have gradually emerged. Existing studies have shown that membrane vesicles derived from the gut microbiota are key mediators of microbiota-host liver cell communication, suggesting the potential of cell membrane vesicles in the treatment of liver diseases. Currently, no studies have published the progress of research on the treatment of alcoholic liver disease using *Lactobacillus rumenans* or its cell membrane vesicles. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to solve at least one of the technical problems mentioned above.

[0006] The solution to the technical problem of this invention is: Firstly, this application provides rumen lactobacillus ( Ligilactobacillus ruminis The application of the *Lactobacillus rumeniformis* in the preparation of products for the treatment and / or prevention of alcohol-related liver diseases, wherein the *Lactobacillus rumeniformis* has the accession number ATCC No. 25644 and is deposited at the American Center for Type Culture Collection; the alcohol-related liver diseases include alcohol-induced liver injury, fatty liver disease, hepatic steatosis, alcoholic hepatitis, or alcoholic cirrhosis.

[0007] Secondly, this application provides rumen lactobacillus ( Ligilactobacillus ruminis The application of the rumen lactobacillus in the preparation of products that reduce the degree of liver tissue lesions, inflammatory cell infiltration or collagen fiber formation, wherein the rumen lactobacillus has the accession number ATCC No. 25644 and is deposited at the American Center for Type Culture Collection.

[0008] Thirdly, this application provides rumen lactobacillus ( Ligilactobacillus ruminis The application of this product in the preparation of products that improve liver lobule structure damage or liver lipid droplet deposition, wherein the rumen lactobacillus has the accession number ATCC No. 25644 and is deposited at the American Center for Type Culture Collection.

[0009] Fourthly, this application provides rumen lactobacillus ( Ligilactobacillus ruminis The application of this product in the preparation of products that reduce alanine aminotransferase (ALT) or aspartate aminotransferase (AST) levels, wherein the rumen lactobacillus has the accession number ATCC No. 25644 and is deposited at the American Center for Type Culture Collection.

[0010] Fifthly, this application provides rumen lactobacillus ( Ligilactobacillus ruminis The use of plasma membrane vesicles derived from rumen lactobacillus in the preparation of products for the treatment and / or prevention of alcohol-related liver diseases, wherein the rumen lactobacillus has the accession number ATCC No. 25644 and is deposited at the American Center for Type Culture Collection; wherein the alcohol-related liver diseases include alcohol-induced liver injury, fatty liver disease, hepatic steatosis, alcoholic hepatitis, or alcoholic cirrhosis.

[0011] Sixthly, this application provides rumen lactobacillus ( Ligilactobacillus ruminis The application of plasma membrane vesicles derived from this source in the preparation of products that reduce the degree of liver tissue lesions, inflammatory cell infiltration, or collagen fiber formation, wherein the rumen lactobacillus has the accession number ATCC No. 25644 and is deposited at the American Center for Type Culture Collection.

[0012] Seventhly, this application provides rumen lactobacillus ( Ligilactobacillus ruminisThe application of plasma membrane vesicles derived from this source in the preparation of products that improve liver lobule structure damage or liver lipid droplet deposition, wherein the rumen lactobacillus has the accession number ATCC No. 25644 and is deposited at the American Center for Type Culture Collection.

[0013] Eighthly, this application provides rumen lactobacillus ( Ligilactobacillus ruminis The application of plasma membrane vesicles derived from this source in the preparation of products that reduce alanine aminotransferase (ALT) or aspartate aminotransferase (AST) levels, wherein the rumen lactobacillus has the accession number ATCC No. 25644 and is deposited at the American Center for Type Culture Collection.

[0014] Furthermore, the number of rumen lactobacilli in the product as described in the first to fourth aspects is 10. 7 ~10 10 The preferred quantity is 10. 9 .

[0015] Furthermore, the number of rumen lactobacillus-derived plasma membrane vesicles in the product as described in aspects five to eight is 1-50 mg / kg. Preferably, the number is 5-10 mg / kg.

[0016] Specifically, the rumen lactobacillus or rumen lactobacillus-derived plasma membrane vesicles provided in this application, in the preparation of products for the treatment and / or prevention of alcohol-related liver disease, and in the preparation of products for the treatment and / or prevention of diseases caused by alcohol-related liver disease, may include a drug and may also include pharmaceutically acceptable excipients. The dosage form of the drug may be any one of injection, oral, tablet, capsule, emulsion, or granule.

[0017] Beneficial effects include rumen lactobacillus ( Ligilactobacillus ruminis Lactobacillus rumenella and its derived cell membrane vesicles (Lr CMVs) significantly reversed the elevation of serum liver function indicators (AST, ALT) and lipid metabolism parameters (total cholesterol, triglycerides) in mice with alcohol-associated liver disease (ALD) induced by liquid alcohol diets, and significantly reduced liver histopathological damage and lipid droplet infiltration. Simultaneously, both effectively inhibited local liver inflammation and reduced the expression levels of inflammatory factors. These results indicate that Lactobacillus rumenella and its CMVs can significantly improve ALD-induced liver function impairment, histological lesions, and inflammatory status by regulating lipid metabolism and inflammatory signaling pathways, providing new experimental evidence and potential strategies for microbial intervention in ALD. Attached Figure Description

[0018] Figure 1This is a comparison of liver function indicators AST and ALT, as well as liver TC and TG in the serum of mice from different groups of rumen lactobacillus in Example 1 of this application.

[0019] Figure 2 These are pathological images of mouse liver tissue stained with H&E and Oil Red O from different groups of rumen lactobacillus in Example 1 of this application.

[0020] Figure 3 This is a graph showing the RT-qPCR quantitative analysis of inflammatory factors and lipid metabolism-related genes in mouse liver tissue from different groups of rumen lactobacillus in Example 1 of this application.

[0021] Figure 4 This is a characterization image of rumen lactobacillus membrane vesicles (Lr CMVs) in Example 2 of this application.

[0022] Figure 5 This study compares the liver function indicators AST and ALT, as well as liver TC and TG, in the serum of mice from different groups of rumen lactobacillus membrane vesicles (Lr CMVs) in Example 3 of this application.

[0023] Figure 6 The images show H&E staining and Oil Red O staining pathological images of mouse liver tissue from different groups of rumen lactobacillus plasma membrane vesicles (Lr CMVs) in Example 3 of this application.

[0024] Figure 7 This is a graph showing the RT-qPCR quantitative analysis of inflammatory factors and lipid metabolism-related genes in mouse liver tissue from different groups of rumen lactobacillus plasma membrane vesicles (Lr CMVs) in Example 3 of this application. Detailed Implementation

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments have been briefly explained above. Obviously, the described drawings are only a part of the embodiments of the present invention, and not all of them. Those skilled in the art can obtain other design schemes and drawings based on these drawings without creative effort.

[0026] The following will clearly and completely describe the concept, specific structure, and technical effects of the present invention in conjunction with embodiments and accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention. Furthermore, all connections / linkages mentioned herein do not simply refer to direct connection of components, but rather to the ability to form a better connection structure by adding or reducing connecting accessories according to specific implementation conditions. The various technical features in this invention can be combined interactively without contradicting each other.

[0027] The following specific examples provide further details.

[0028] Alcohol is metabolized in the liver to produce reactive oxygen species (ROS) and disrupt the intestinal barrier, allowing LPS to enter the liver and activate the TLR4 and TGF-β pathways, inducing inflammation and collagen secretion from hepatic stellate cells. Therefore, LPS is a direct inducer of alcohol-related liver disease (ALD). The metabolic pathway of alcohol in mice is similar to that in humans; therefore, alcohol-induced ALD in mice can mimic the pathogenesis of ALD in humans. Thus, mice can be fed a liquid alcohol diet to induce ALD, and the therapeutic effects of rumen lactobacillus and its cytoplasmic membrane vesicles on ALD can be evaluated.

[0029] The rumen lactobacillus used in the embodiments of the present invention ( Ligilactobacillus ruminis It was purchased and has the accession number ATCC No. 25644. It is deposited at the American Center for Type Culture Collection.

[0030] Example 1: Rumen lactobacillus has an ameliorative effect on alcohol-related liver disease in animals. Construction and treatment of the experimental model: Mice fed with liquid alcohol were regularly administered an effective amount of rumen lactobacillus solution or an equivalent amount of inactivated rumen lactobacillus solution via gavage. Forty SPF-grade male C57BL / 6 mice were used in the experiment. After one week of acclimatization, they were administered a quadruple antibiotic mixture (0.5 g / L vancomycin, 1 g / L ampicillin, 1 g / L metronidazole, and 1 g / L neomycin sulfate) daily via gavage for one week to establish an antibiotic-induced pseudo-germ-free mouse model. The animals were then randomly divided into four groups of 10 mice each, with the following grouping and treatment: ① Control group: fed the control liquid diet, corresponding to... Figures 1-3 NC group; ② Alcohol model group (NIAAA): fed liquid alcohol diet, corresponding to Figures 1-3 ③ ALD group; ③ Live bacteria intervention group (NIAAA+) L.ruminis ): In addition to feeding liquid alcohol feed, administer 1×10 liters via gavage daily. 9 CFU / live rumen lactobacillus, corresponding to Figures 1-3 ALD+ in L.ruminis ④ Inactivated bacteria intervention group (NIAAA+Heat Kill) L.ruminis ): In addition to feeding liquid alcohol feed, administer 1×10 liters via gavage daily. 9 CFU / animal inactivated rumen lactobacillus (inactivation conditions: 70℃ water bath for 30 min, pasteurization), corresponding Figures 1-3 ALD+Heat Killed- L.ruminis or ALD+Heat- L.ruminis Feeding regimen: Except for the control group, the other three groups were acclimatized by gradually increasing alcohol concentration (1% → 5%) from day 1 to day 5 of the experiment, followed by continuous feeding at a 5% alcohol concentration for 10 days. During the intervention period, the live bacteria group and the inactivated bacteria group were administered the corresponding bacterial solutions by gavage daily. At the end of the experiment, blood samples were collected after anesthesia with sodium pentobarbital, the animals were euthanized, and tissue specimens were collected for subsequent pharmacodynamic evaluation.

[0031] (1) Serum biochemical index determination: Whole blood samples were collected from mice and allowed to stand at room temperature for 30 min. Serum was then separated by centrifugation at 4°C and 3000 rpm for 15 min. Using commercially available diagnostic kits and following the manufacturer's instructions, the activity levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum of each group of mice were determined by enzymatic reaction-spectrophotometry.

[0032] Test results are as follows Figure 1 As shown, compared with the control group, the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the model group mice were significantly increased. After intervention with rumen lactobacillus, all indicators showed a significant decrease, while no significant reduction was observed in the inactivated rumen lactobacillus group.

[0033] (2) Measurement of liver biochemical indicators: Accurately weigh liver tissue samples from each group of mice and add pre-cooled phosphate-buffered saline (PBS, pH 7.4) at a ratio of 1:9 (w / v) to prepare a 10% tissue homogenate. Centrifuge the homogenate at 4°C and 12,000 rpm for 15 min, and collect the supernatant. Using a commercially available assay kit, and following the manufacturer's instructions, determine the levels of total cholesterol (TC) and triglycerides (TG) in the liver tissue.

[0034] Test results are as follows Figure 1 As shown, the trends of TC and TG changes in liver tissue were consistent with those in serum, with the levels in the model group being significantly higher than those in the control group. Intervention with *Lactobacillus rumenans* effectively reduced these levels. These results suggest that *Lactobacillus rumenans* has an ameliorative effect on liver damage in mice with alcoholic liver disease.

[0035] It is evident that rumen lactobacillus has an ameliorative effect on alcohol-related liver disease in mice.

[0036] (3) Hematoxylin-eosin staining (H&E staining): Mouse liver tissue was taken from the same location, fixed with paraformaldehyde for 24 hours, dehydrated, cleared, impregnated with paraffin, and embedded to prepare paraffin sections. The paraffin sections were baked in a 65℃ oven for 4 hours to melt the paraffin, and then successively immersed in xylene and ethanol solutions of different concentrations to dewax and rehydrate. They were then stained with hematoxylin and eosin, and finally mounted with neutral resin. The sections were stored under dry conditions at room temperature, and images were acquired by randomly selecting fields of view at room temperature.

[0037] Test results are as follows Figure 2 As shown, H&E staining results of mouse liver tissue showed that the model group mice had inflammatory cell infiltration, hepatocyte ballooning degeneration and destruction of liver lobule structure. After intervention with rumen lactobacillus, the above pathological changes were significantly reduced, the number of inflammatory cells decreased, and the hepatocyte structure was basically restored. However, there were no significant changes after intervention with inactivated rumen lactobacillus.

[0038] (4) Oil Red O staining: Mouse liver tissue was taken from the same location, embedded in OCT, and frozen sectioned (8 μm thick). The sections were thoroughly washed with distilled water, mixed with 6 mL of saturated Oil Red solution and 4 mL of distilled water, allowed to stand for 5–10 min, filtered, and stained with the resulting working solution in the dark for 10–15 min. Then, the sections were differentiated under a microscope with 60% ethanol until the stroma was clear. The sections were rinsed with distilled water 3 times for 5 min each time, and then stained with Marry's hematoxylin solution for 5–10 min. The sections were rinsed with distilled water 3 times for 5 min each time. Finally, the sections were mounted with glycerol gelatin, and images were acquired by randomly selecting fields of view.

[0039] Test results are as follows Figure 2 As shown, Oil Red O staining revealed significant lipid droplet accumulation in hepatocytes in the model group. After intervention, the lipid droplet deposition range decreased, while inactivated rumen lactobacillus showed no significant change after intervention, indicating that rumen lactobacillus can alleviate alcohol-induced liver tissue pathological damage.

[0040] It is evident that rumen lactobacillus can improve liver pathological damage in mice with alcohol-related liver disease.

[0041] (5) Detection of inflammation-related indicators: The mRNA transcription levels of inflammation-related factors in liver tissue were detected using reverse transcription real-time quantitative PCR (RT-qPCR). Specifically, total RNA was extracted from liver tissue using the TRIzol method, and after DNase I digestion to remove genomic DNA contamination, cDNA was synthesized using a reverse transcription kit. Subsequently, using Gapdh as an internal reference gene, the target gene sequences of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) were amplified using specific primers, and the relative expression levels of each gene were calculated.

[0042] Test results are as follows Figure 3 As shown, the mRNA expression of inflammatory factors (TNF-α, IL-1β, IL-6) in mouse liver was detected, and the expression levels in the model group were significantly upregulated compared with those in the control group. After treatment with rumen lactobacillus, the expression of the above factors was significantly inhibited. However, the inactivated rumen lactobacillus group could not reduce the expression level of inflammatory factors in the liver tissue of mice with alcohol-related liver disease, suggesting that rumen lactobacillus can alleviate alcohol-induced liver inflammation in mice.

[0043] (6) Detection of lipid metabolism-related indicators: The mRNA transcription levels of lipid metabolism-related genes in liver tissue were detected using reverse transcription real-time quantitative PCR (RT-qPCR). Specifically, total RNA was extracted from liver tissue using the TRIzol method, and after DNase I digestion to remove genomic DNA contamination, cDNA was synthesized using a reverse transcription kit. Subsequently, using Gapdh as an internal control gene, the target gene sequences of sterol regulatory element binding protein 1 (Srebp1) and differentiation cluster 36 (Cd36) were amplified using specific primers, and the relative expression levels of each gene were calculated.

[0044] Test results are as follows Figure 3 As shown, the mRNA expression levels of lipid metabolism-related genes Srebp1 and Cd36 in the liver of mice in the alcoholic liver disease model group were significantly higher than those in the control group. After intervention with *Lactobacillus rumenae*, the expression of both genes was significantly downregulated. However, the inactivated *Lactobacillus rumenae* group could not reduce the expression levels of lipid metabolism-related genes in the liver tissue of mice with alcoholic liver disease, indicating that *Lactobacillus rumenae* may improve alcohol-induced liver lipid accumulation by regulating the expression of lipid metabolism-related genes.

[0045] The test results show that: Regarding liver function indicators, compared with the model group fed only liquid alcohol diet, rumen lactobacillus significantly reduced the levels of ALT, AST in mouse serum and TC and TG in liver. However, the inactivated rumen lactobacillus group did not reduce the levels of ALT, AST in mouse serum and TC and TG in liver, demonstrating that live rumen lactobacillus can effectively improve liver function, while inactivated rumen lactobacillus cannot improve liver function.

[0046] Pathological analysis of liver sections and staining revealed inflammatory cell infiltration, ballooning degeneration, and lobular destruction in the hepatocytes of mice in the model group. Administration of live rumen lactobacillus resulted in milder liver lesions, reduced inflammatory cell infiltration, and a return to normal hepatocyte structure. Administration of inactivated rumen lactobacillus showed no significant changes. Oil Red O staining revealed significant lipid droplet infiltration in the model group. Administration of rumen lactobacillus resulted in milder lipid droplet infiltration in the liver tissue of mice with alcohol-related liver disease compared to the model group. Administration of inactivated rumen lactobacillus showed no significant changes. These findings pathologically demonstrate the therapeutic effect of live rumen lactobacillus on alcohol-related liver disease.

[0047] Example 2 Characterization of plasma membrane vesicles derived from rumen lactobacilli Experimental Methods: Extraction of Rumen Lactobacillus Cell Membrane Vesicles (Lr CMVs): Rumen Lactobacillus was inoculated into GAM liquid medium and cultured in an anaerobic incubator at 37°C for 48 hours. The bacterial culture was collected, centrifuged at 15000g for 30 min, and the supernatant was filtered through a 0.45 μm filter. The supernatant was then enriched by a tangential flow system for 30 min. The concentrated sample was centrifuged at 150000g for 90 min to obtain Rumen Lactobacillus Cell Membrane Vesicles.

[0048] (1) NTA detection of particle size and density of rumen lactobacillus membrane vesicles (Lr CMVs): Take 100µL of rumen lactobacillus plasma membrane vesicle (Lr CMVs) suspension into a sterile centrifuge tube, add 900µL of sterile PBS, vortex at low speed for 20s, filter through a 0.45µm filter membrane, and then use an NS300 nanoparticle size analyzer (Malvern, Worcestershire, UK) to determine the particle size and density.

[0049] (2) Scanning electron microscopy to observe the morphology of rumen lactobacillus plasma membrane vesicles (Lr CMVs). Take 10 µL of rumen lactobacillus plasma membrane vesicle suspension, add 90 µL of sterile PBS solution, vortex at low speed for 10 s to prepare a diluted vesicle suspension; pipette 10 µL of the diluted suspension and drop it onto a 5 mm silicon wafer, let it stand at room temperature for 2–3 min. After the silicon wafer dries, place it in 1 mL of 4% glutaraldehyde solution and let it stand overnight at 4°C; then wash it 3 times with sterile PBS (5 min / wash). The silicon wafer is then dehydrated in 30%, 50%, 70%, 80%, and 90% ethanol solutions for 10 min each, followed by dehydration twice with anhydrous ethanol (10 min / wash), let it stand for 2 min, and then dry it; add isoamyl acetate for 10 min to replace the vesicles, and then dry it in a critical point dryer for 10 min, followed by gold sputtering using a vacuum coating machine. The prepared silicon wafers are observed and photographed using a field emission scanning electron microscope (S-4800, Hitachi).

[0050] (3) Measurement of Zeta potential of rumen lactobacillus plasma membrane vesicles (Lr CMVs): The zeta potential of rumen lactobacillus plasma membrane vesicles was determined using a dynamic light scattering system (DLS).

[0051] Centrifugation combined with a fully automated exosome purification system was used to extract and purify plasma membrane vesicles derived from rumen lactobacilli on a large scale. The test results of Example 2 are as follows: Figure 4 A, Figure 4 B and Figure 4 As shown in Figure C, the morphology of LrCMVs was observed by transmission electron microscopy, showing that Lr CMVs have spherical shapes and a bilayer lipid membrane; NTA showed that the average particle size of Lr CMVs was 184.6 ± 1.4 nm; the average zeta potential of Lr CMVs was −30.22 ± 4.66 mV.

[0052] Example 3: Membrane vesicles derived from rumen lactobacilli have an ameliorative effect on alcohol-related liver disease in mice. Ruminobacterium was cultured, and its cytoplasmic membrane vesicles were extracted. The density, particle size, and morphology of the cytoplasmic membrane vesicles were characterized, and the Zeta potential of the cytoplasmic membrane vesicles was measured. Mice fed with liquid alcohol diets were periodically administered low-dose or high-dose ruminobacterium-derived cytoplasmic membrane vesicles by gavage.

[0053] Experimental Methods: Construction of the experimental model, grouping and treatment: Forty SPF-grade male C57BL / 6 mice were used in the experiment. After one week of acclimatization, the animals were randomly divided into four groups of 10 mice each. The grouping and treatment were as follows: ① Control group: fed with control liquid diet, corresponding to... Figures 5-7 NC group; ② Alcohol model group (NIAAA): fed liquid alcohol diet, corresponding to Figures 5-7③ Low-dose membrane vesicle intervention group (NIAAA+Lr CMVs-L): In addition to being fed liquid alcohol feed, 5 mg / kg of rumen lactobacillus membrane vesicles were administered daily by gavage. Figures 5-7 The ALD+Lr CMVs-L group; ④ The high-dose membrane vesicle intervention group (NIAAA+Lr CMVs-H): In addition to being fed liquid alcohol feed, 10 mg / kg of rumen lactobacillus membrane vesicles were administered daily by gavage. Figures 5-7 The ALD+Lr CMVs-H group. Feeding regimen: Except for the control group, the other three groups were acclimatized by gradually increasing alcohol concentration (1%→5%) from day 1 to day 5 of the experiment, followed by continuous feeding at a 5% alcohol concentration for 10 days. During the intervention period, the membrane vesicle intervention group received the corresponding dose by gavage daily. At the end of the experiment, blood samples were collected after anesthesia with sodium pentobarbital, the animals were euthanized, and tissue specimens were collected for subsequent pharmacodynamic evaluation.

[0054] (1) Serum biochemical index determination Whole blood samples were collected from mice and allowed to stand at room temperature for 30 min. Serum was then separated by centrifugation at 4°C and 3000 rpm for 15 min. Using commercially available diagnostic kits and following the manufacturer's instructions, the activity levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum of each group of mice were determined by enzymatic reaction-spectrophotometry.

[0055] Test results are as follows Figure 5 As shown, compared with the control group, the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the model group mice were significantly increased. After Lr CMVs intervention, all indicators showed a significant decrease.

[0056] (2) Measurement of liver biochemical indicators Accurately weigh liver tissue samples from each group of mice and add pre-cooled phosphate-buffered saline (PBS, pH 7.4) at a ratio of 1:9 (w / v) to prepare a 10% tissue homogenate. Centrifuge the homogenate at 4°C and 12,000 rpm for 15 min, and collect the supernatant. Using a commercially available assay kit, and following the manufacturer's instructions, determine the levels of total cholesterol (TC) and triglycerides (TG) in the liver tissue.

[0057] Test results are as follows Figure 5As shown, the trends of TC and TG changes in liver tissue were consistent with those in serum, with significantly higher levels in the model group than in the control group, while significantly lower levels were observed in the intervention group. These results suggest that rumen lactobacillus plasma membrane vesicles have a certain alleviating effect on alcohol-induced liver injury in mice.

[0058] It is evident that rumen lactobacillus membrane vesicles (Lr CMVs) have an ameliorative effect on alcohol-related liver disease in mice.

[0059] (3) Hematoxylin-eosin staining (H&E staining) Mouse liver tissue was taken from the same location, fixed with paraformaldehyde for 24 hours, dehydrated, cleared, impregnated with paraffin, and embedded to prepare paraffin sections. The paraffin sections were baked in a 65℃ oven for 4 hours to melt the paraffin, and then successively immersed in xylene and ethanol solutions of different concentrations to dewax and rehydrate. They were then stained with hematoxylin and eosin, and finally mounted with neutral resin. The sections were stored under dry conditions at room temperature, and images were acquired by randomly selecting fields of view at room temperature.

[0060] Test results are as follows Figure 6 As shown, H&E staining results of liver tissue revealed that mice in the model group exhibited inflammatory cell infiltration, hepatocyte ballooning degeneration, and destruction of liver lobule structure. However, after intervention with rumen lactobacillus and its CMVs, the above pathological changes were significantly alleviated, the number of inflammatory cells decreased, and the hepatocyte structure was basically restored.

[0061] (4) Oil Red O staining Mouse liver tissue was taken from the same location, embedded in OCT, and frozen sectioned (8 μm thick). The sections were thoroughly washed with distilled water, mixed with 6 mL of saturated Oil Red solution and 4 mL of distilled water, allowed to stand for 5–10 min, filtered, and stained with the resulting working solution in the dark for 10–15 min. Then, the sections were differentiated under a microscope with 60% ethanol until the stroma was clear. The sections were rinsed with distilled water 3 times for 5 min each time, and then stained with Marry's hematoxylin solution for 5–10 min. The sections were rinsed with distilled water 3 times for 5 min each time. Finally, the sections were mounted with glycerol gelatin, and images were acquired by randomly selecting fields of view.

[0062] Test results are as follows Figure 6 As shown, Oil Red O staining revealed significant lipid droplet accumulation in hepatocytes of the model group. After intervention, the lipid droplet deposition range decreased, also showing a dose-dependent effect, indicating that rumen lactobacillus plasma membrane vesicles can alleviate alcohol-induced liver tissue pathological damage.

[0063] It is evident that rumen lactobacillus membrane vesicles (Lr CMVs) have an ameliorative effect on alcohol-related liver disease in mice.

[0064] (5) Detection of inflammation-related indicators The mRNA transcription levels of inflammation-related factors in liver tissue were detected using reverse transcription real-time quantitative PCR (RT-qPCR). Specifically, total RNA was extracted from liver tissue using the TRIzol method, and after DNase I digestion to remove genomic DNA contamination, cDNA was synthesized using a reverse transcription kit. Subsequently, using Gapdh as an internal reference gene, the target gene sequences of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) were amplified using specific primers, and the relative expression levels of each gene were calculated.

[0065] Test results are as follows Figure 7 As shown, the mRNA expression of liver inflammatory factors (TNF-α, IL-1β, IL-6) was detected, and the expression levels in the model group were significantly upregulated compared with those in the control group. After treatment with rumen lactobacillus plasma membrane vesicles, the expression of the above factors was significantly inhibited, suggesting that this intervention can alleviate alcohol-induced liver inflammation in mice.

[0066] It is evident that rumen lactobacillus membrane vesicles (Lr CMVs) can improve inflammation levels in mice with alcohol-related liver disease.

[0067] (6) Detection of lipid metabolism-related indicators The mRNA transcription levels of lipid metabolism-related genes in liver tissue were detected using reverse transcription real-time quantitative PCR (RT-qPCR). Specifically, total RNA was extracted from liver tissue using the TRIzol method, and after DNase I digestion to remove genomic DNA contamination, cDNA was synthesized using a reverse transcription kit. Subsequently, using Gapdh as an internal control gene, the target gene sequences of sterol regulatory element binding protein 1 (Srebp1) and differentiation cluster 36 (Cd36) were amplified using specific primers, and the relative expression levels of each gene were calculated.

[0068] Test results are as follows Figure 7 As shown, the mRNA expression levels of lipid metabolism-related genes Srebp1 and Cd36 in the liver of mice in the alcoholic liver disease model group were significantly higher than those in the control group. After intervention with rumen lactobacillus plasma membrane vesicles, the expression of both genes was significantly downregulated, suggesting that they may improve alcohol-induced liver lipid accumulation by regulating the expression of lipid metabolism-related genes.

[0069] It is evident that rumen lactobacillus membrane vesicles (Lr CMVs) can improve lipid metabolism in mice with alcohol-related liver disease.

[0070] Current results indicate that, regarding liver function indicators, compared with the model group fed only a liquid alcohol diet, gavage administration of high or low doses of *Lactobacillus rumenella* cell membrane vesicles to mice fed a liquid alcohol diet significantly reduced serum ALT and AST levels and liver TC and TG levels, demonstrating that *Lactobacillus rumenella* cell membrane vesicles can effectively improve liver function. Regarding liver pathological sections and staining analysis, H&E staining results showed that the model group mice exhibited inflammatory cell infiltration, ballooning degeneration, and lobular destruction in their hepatocytes. After gavage administration of high or low doses of *Lactobacillus rumenella* cell membrane vesicles, the degree of liver tissue lesions was milder, inflammatory cell infiltration was reduced, and hepatocyte structure tended to be normal. Oil Red O staining results showed that the model group mice exhibited significant lipid droplet infiltration. After gavage administration of high or low doses of *Lactobacillus rumenella* cell membrane vesicles, the degree of lipid droplet infiltration in the liver tissue of mice with alcohol-related liver disease was milder than that in the model group, pathologically demonstrating the therapeutic effect of *Lactobacillus rumenella* cell membrane vesicles on alcohol-related liver disease.

[0071] In summary, *Lactobacillus rumenans* and its derived cell membrane vesicles significantly reversed the elevation of serum liver function indicators (AST, ALT) and lipid metabolism parameters (total cholesterol, triglycerides) in mice with alcohol-related liver disease induced by liquid alcohol diets, and significantly reduced liver histopathological damage and lipid droplet infiltration. Simultaneously, both effectively inhibited local liver inflammation and reduced the expression levels of inflammatory factors. These results indicate that *Lactobacillus rumenans* and its CMVs can significantly improve liver function impairment, histological lesions, and inflammatory status induced by ALD by regulating lipid metabolism and inflammatory signaling pathways, providing new experimental evidence and potential strategies for microbial intervention in ALD.

[0072] This invention provides a method using rumen lactobacillus ( Ligilactobacillus ruminis This paper describes a novel approach to the treatment or prevention of alcohol-related liver disease using cellular membrane vesicles, and proposes its potential applications in the treatment of alcohol-related liver disease. It demonstrates an ameliorative effect on alcohol-related liver disease.

[0073] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. Rumen lactobacillus ( Ligilactobacillus ruminis The application of the *Lactobacillus rumeniformis* in the preparation of products for the treatment and / or prevention of alcohol-related liver diseases, wherein the *Lactobacillus rumeniformis* has the accession number ATCC No. 25644 and is deposited at the American Center for Type Culture Collection; the alcohol-related liver diseases include alcohol-induced liver injury, fatty liver disease, hepatic steatosis, alcoholic hepatitis, or alcoholic cirrhosis.

2. Rumen lactobacillus ( Ligilactobacillus ruminis The application of the rumen lactobacillus in the preparation of products that reduce the degree of liver tissue lesions, inflammatory cell infiltration or collagen fiber formation, wherein the rumen lactobacillus has the accession number ATCC No. 25644 and is deposited at the American Center for Type Culture Collection.

3. Rumen lactobacillus ( Ligilactobacillus ruminis The application of this product in the preparation of products that improve liver lobule structure damage or liver lipid droplet deposition, wherein the rumen lactobacillus has the accession number ATCC No. 25644 and is deposited at the American Center for Type Culture Collection.

4. Rumen lactobacillus ( Ligilactobacillus ruminis The application of this product in the preparation of products that reduce alanine aminotransferase or aspartate aminotransferase levels, wherein the rumen lactobacillus has the accession number ATCC No. 25644 and is deposited at the American Center for Type Culture Collection.

5. Rumen lactobacillus ( Ligilactobacillus ruminis The use of plasma membrane vesicles derived from rumen lactobacillus in the preparation of products for the treatment and / or prevention of alcohol-related liver diseases, wherein the rumen lactobacillus has the accession number ATCC No. 25644 and is deposited at the American Center for Type Culture Collection; wherein the alcohol-related liver diseases include alcohol-induced liver injury, fatty liver disease, hepatic steatosis, alcoholic hepatitis, or alcoholic cirrhosis.

6. Rumen lactobacillus ( Ligilactobacillus ruminis The application of plasma membrane vesicles derived from this source in the preparation of products that reduce the degree of liver tissue lesions, inflammatory cell infiltration, or collagen fiber formation, wherein the rumen lactobacillus has the accession number ATCC No. 25644 and is deposited at the American Center for Type Culture Collection.

7. Rumen lactobacillus ( Ligilactobacillus ruminis The application of plasma membrane vesicles derived from this source in the preparation of products that improve liver lobule structure damage or liver lipid droplet deposition, wherein the rumen lactobacillus has the accession number ATCC No. 25644 and is deposited at the American Center for Type Culture Collection.

8. Rumen lactobacillus ( Ligilactobacillus ruminis The application of plasma membrane vesicles derived from this source in the preparation of products that reduce alanine aminotransferase (ALT) or aspartate aminotransferase (AST) levels, wherein the rumen lactobacillus has the accession number ATCC No. 25644 and is deposited at the American Center for Type Culture Collection.

9. The application as described in any one of claims 1-4, characterized in that, The product contains 10 rumen lactobacilli. 7 ~10 10 .

10. The application as described in any one of claims 5-8, characterized in that, The product contains 1-50 mg / kg of plasma membrane vesicles derived from rumen lactobacillus.

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