Application of parabacteroides dielsii outer membrane vesicles in preparation of medicines for treating hepatic fibrosis

Abstract

The invention discloses an application of parabacteroides dielsii outer membrane vesicles in preparation of a medicine for treating hepatic fibrosis. The invention successfully prepares, extracts and systematically characterizes the outer membrane vesicles (OMV) derived from parabacteroides dielsii for the first time, and verifies the therapeutic potential of the OMV in a CCl4-induced liver injury and fibrosis model. Experimental results show that the OMV intervention significantly improves the pathological structure of liver tissue and significantly reduces collagen deposition, so that the OMV intervention is expected to become a novel treatment strategy for hepatic fibrosis.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to application of outer membrane vesicles of Parabacteroides dieldii in preparing a drug for treating liver fibrosis. Background Art

[0002] Hepatic fibrosis is a pathological process caused by chronic liver damage, characterized by excessive deposition of extracellular matrix and scar tissue in the liver. Without timely and effective intervention and treatment, hepatic fibrosis can progress to cirrhosis, severely impairing liver function. The development of hepatic fibrosis is associated with multiple factors, including viral hepatitis, alcoholic liver disease, non-alcoholic fatty liver disease, and autoimmune liver disease. Although the etiology of hepatic fibrosis is complex and remains to be fully elucidated, studies have shown that factors such as inflammation, oxidative stress, and activation of hepatic stellate cells (HSCs) play a key role in its development and progression. In recent years, therapeutic strategies for hepatic fibrosis have primarily focused on anti-inflammatory, antioxidant, and HSC activation inhibition. However, existing treatments, such as anti-inflammatory drugs, antioxidants, and immunomodulators, have limited efficacy and are associated with significant side effects and drug resistance. Therefore, the development of novel therapeutics for hepatic fibrosis is of great clinical significance.

[0003] Bacterial outer membrane vesicles (OMVs), also known as extracellular vesicles, are naturally secreted primarily by Gram-negative bacteria and possess a nanoscale lipid bilayer structure. Bacterial OMVs contain a variety of immunomodulatory components, such as nucleic acids, proteins, and lipids. Furthermore, bacterial OMVs possess numerous properties, including low toxicity, good biocompatibility, and high stability, making them potential alternatives to live bacterial therapies. However, little is known about whether OMVs derived from the human gut microbiome can inhibit liver inflammation and reduce liver fibrosis.

[0004] Parabacteroides distasonis is a Gram-negative anaerobic bacterium in the genus Bacteroides, widely present in the intestinal microbiome of humans and animals. As a potential next-generation probiotic, Parabacteroides distasonis modulates intestinal bile acid metabolism by upregulating bile salt hydrolase activity and inhibiting the farnesoid-X receptor, thereby downregulating the mitochondrial permeability transition (MPT)-caspase-11 necroptosis pathway and inhibiting hepatocyte pyroptosis. However, it is currently unclear whether outer membrane vesicles (OMVs) derived from Parabacteroides distasonis can improve the treatment of liver fibrosis. Further exploration of the potential therapeutic properties of Parabacteroides distasonis OMVs may provide new strategies for the treatment of liver fibrosis. Summary of the Invention

[0005] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the purpose of the present invention is to provide the use of outer membrane vesicles of Parabacteroides distichous in the preparation of a drug for treating liver fibrosis. The present invention successfully prepared, extracted and systematically characterized outer membrane vesicles (OMVs) derived from Parabacteroides distichous for the first time, and verified their therapeutic potential in a CCl4-induced liver injury and fibrosis model. Experimental results showed that OMV intervention significantly improved the pathological structure of liver tissue and significantly reduced collagen deposition, thus it is expected to become a new treatment strategy for liver fibrosis.

[0006] The first aspect of the present invention provides the use of outer membrane vesicles of Parabacteroides dieldii in the preparation of a medicament for treating liver fibrosis.

[0007] In some embodiments of the present invention, the drug is capable of reversing liver damage and oxidative stress induced by liver fibrosis.

[0008] In some embodiments of the present invention, the drug reduces or inhibits the increase in total bilirubin caused by liver damage.

[0009] In some embodiments of the present invention, the drug increases or inhibits the decrease in GSH caused by oxidative stress.

[0010] In some embodiments of the present invention, the average particle size of the outer membrane vesicles of Parabacteroides distichum is 55-80 nm.

[0011] In some embodiments of the present invention, the average particle size of the outer membrane vesicles of the Parabacteroides distichum is 56.44-79.18 nm.

[0012] In some embodiments of the present invention, the dosage forms of the drug include: powder, solution, capsule and granule.

[0013] In some embodiments of the present invention, the granules further include a pharmaceutically acceptable carrier.

[0014] In some embodiments of the present invention, the carrier comprises an enzyme-sensitive polymer and a redox-sensitive polymer.

[0015] In some embodiments of the present invention, the enzyme-sensitive polymer includes but is not limited to: amylose, chitosan, chondroitin sulfate, cyclodextrin, dextran, inulin, guar gum, pectin and locust bean gum, etc.

[0016] In some embodiments of the present invention, the redox-sensitive polymer includes but is not limited to: disulfide-bonded block copolymers (such as polyethylene glycol (PEG)-based materials, PEG-SS-PCL and PEO-PPO-SS-DTX), modified chitosan polymers (such as PEG-DMTK-SS-PLA and cationic peptide polymers) and disulfide-linked polyamides.

[0017] In some embodiments of the present invention, the outer membrane vesicles of Parabacteroides distichous are prepared using the following preparation method:

[0018] The outer membrane vesicles of Parabacteroides dieltii are obtained by primary filtration and then ultrafiltration.

[0019] In some embodiments of the present invention, the filtration pore size of the primary filtration is 0.2-0.25 μm.

[0020] In some embodiments of the present invention, the filtration pore size of the primary filtration is 0.22 μm.

[0021] In some embodiments of the present invention, the molecular weight cut-off of ultrafiltration is 95-110 KDa.

[0022] In some embodiments of the present invention, the molecular weight cut-off of ultrafiltration is 100 KDa.

[0023] In some embodiments of the present invention, the Parabacteroides dieltii cells are subjected to impurity removal treatment before primary filtration.

[0024] In some embodiments of the present invention, the impurity removal treatment includes centrifugation and filtration.

[0025] In some embodiments of the present invention, the centrifugation conditions are: centrifugation at 11000-12000 rpm for 1-3 times, each time for 20-30 minutes.

[0026] In some embodiments of the present invention, the centrifugation condition is: centrifugation at 12000 rpm for 2 times, each time for 30 minutes.

[0027] In some embodiments of the present invention, the filtration pore size of the suction filtration is 0.4-0.5 μm.

[0028] In some embodiments of the present invention, the filtration pore size of the suction filtration is 0.45 μm.

[0029] In some embodiments of the present invention, the preparation method further comprises: concentrating and purifying the outer membrane vesicles of Parabacteroides distichum after ultrafiltration.

[0030] In some embodiments of the present invention, the concentrating comprises: centrifugation.

[0031] In some embodiments of the present invention, the centrifugation condition is: centrifugation at 2500-3100 rpm for 25-40 min.

[0032] In some embodiments of the present invention, the centrifugation condition is: centrifugation at 3000 rpm for 30 min.

[0033] In some embodiments of the present invention, the purification comprises filtering and centrifuging the concentrated Parabacteroides distichous outer membrane vesicles.

[0034] In some embodiments of the present invention, the filtration pore size is 0.2-0.25 μm.

[0035] In some embodiments of the present invention, the filtration pore size of the filtration is 0.22 μm.

[0036] In some embodiments of the present invention, the centrifugation conditions are: greater than or equal to 120,000×g, and the centrifugation time is 1.5-2 h.

[0037] In some embodiments of the present invention, the centrifugation conditions are: 150,000×g, and the centrifugation time is 2 h.

[0038] In some embodiments of the present invention, the drug further contains a pharmaceutically acceptable adjuvant.

[0039] In some embodiments of the present invention, the pharmaceutically acceptable excipients include but are not limited to diluents (such as starch, dextrin, sucrose, lactose, mannitol, etc.), absorbents (such as calcium sulfate, calcium hydrogen phosphate, etc.), wetting agents (such as ethanol), binders (such as hydroxypropyl methylcellulose, povidone, etc.), disintegrants (such as sodium hydroxymethyl starch, cross-linked polyvinylpyrrolidone, etc.), lubricants (such as talc, hydrogenated vegetable oil, polyethylene glycol, etc.), colorants (such as titanium dioxide, methylene blue, etc.), coating materials, solvents, pH regulators, antibacterial agents (such as sodium sulfite, sodium thiosulfate, etc.), isotonic regulators (such as glucose, sodium chloride, etc.), and chelating agents (such as disodium EDTA).

[0040] The beneficial effects of the present invention are:

[0041] This invention successfully prepared, extracted and systematically characterized outer membrane vesicles (OMVs) from Parabacteroides dieldrinii for the first time, and verified their therapeutic potential in CCl4-induced liver injury and fibrosis models. It showed good biological activity and therapeutic potential and is expected to become a new treatment strategy for liver fibrosis.

[0042] The OMV-based intervention in this study significantly improved liver histopathology and reduced collagen deposition. Furthermore, compared with the commonly used anti-fibrotic drug pirfenidone (PFD) and the Parabacteroides distichum bacteria themselves, OMV demonstrated superior efficacy in restoring liver function-related biochemical indicators (ALT, AST, TBIL) and alleviating oxidative stress damage (SOD, GSH).

[0043] In the present invention, based on lipidomics analysis, it was found that OMVs are rich in lipid components with potential biological activity, such as phosphatidylcholine (PC), which is speculated to play an important role in the anti-liver fibrosis process. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is the sequencing alignment result of Parabacteroides distichous.

[0045] Figure 2 Flow chart of the preparation of Parabacteroides distichum outer membrane vesicles (OMVs).

[0046] Figure 3 This is the morphological result of outer membrane vesicles (OMV) of Parabacteroides distichous under electron microscope.

[0047] Figure 4 This is the particle size distribution diagram of the outer membrane vesicles (OMV) of Parabacteroides distichum.

[0048] Figure 5 This is a schematic diagram of animal experiment grouping in an embodiment of the present invention.

[0049] Figure 6 This is the effect of administration of Parabacteroides dieltii outer membrane vesicles (OMV) on CCl4-induced mouse liver tissue (appearance morphology).

[0050] Figure 7 The effect of administration of Parabacteroides distichum outer membrane vesicles (OMV) on CCl4-induced mouse liver tissue (H&E staining).

[0051] Figure 8 The effect of administration of Parabacteroides distichum outer membrane vesicles (OMV) on CCl4-induced mouse liver tissue (Sirius red staining).

[0052] Figure 9 The effect of administration of Parabacteroides distichum outer membrane vesicles (OMV) on CCl4-induced mouse liver tissue (Masson staining).

[0053] Figure 10 To investigate the effect of Parabacteroides distichous outer membrane vesicles (OMV) administration on CCl4-induced serum ALT levels in mice.

[0054] Figure 11To investigate the effect of Parabacteroides dieltii outer membrane vesicles (OMV) administration on CCl4-induced serum AST levels in mice.

[0055] Figure 12 The effect of Parabacteroides distichum outer membrane vesicles (OMV) administration on CCl4-induced serum TBIL levels in mice.

[0056] Figure 13 The effect of Parabacteroides dieltii outer membrane vesicles (OMV) administration on CCl4-induced serum SOD content in mice.

[0057] Figure 14 To investigate the effect of Parabacteroides distichous outer membrane vesicles (OMV) administration on CCl4-induced serum GSH content in mice.

[0058] Figure 15 This is a lipid composition analysis diagram of the outer membrane vesicles (OMV) of Parabacteroides distichum. DETAILED DESCRIPTION

[0059] The present invention is further described in detail below using specific examples. Unless otherwise specified, the raw materials, reagents, and apparatus used in the examples and comparative examples are available from conventional commercial sources or by conventional methods. Unless otherwise specified, all experimental or testing methods are conventional in the art.

[0060] Experimental Materials

[0061] In the following examples, Parabacteroides distichous is the commercial standard strain Parabacteroides distichous ATCC 8503 (purchased from ATCC).

[0062] Example 1

[0063] In this embodiment, a method for preparing outer membrane vesicles of Parabacteroides distichous is provided, and the specific steps include:

[0064] (1) Cultivation and identification of Parabacteroides distichous:

[0065] All experimental operations were carried out in a clean bench.

[0066] Use cotton wool soaked in 75% ethanol to disinfect the outer wall of the cryopreservation tube containing the ATCC 8503 strain of Parabacteroides dieldii. After the ethanol evaporates, remove the sealing film. After briefly burning and sterilizing the mouth of the cryopreservation tube with an alcohol lamp, open the cryopreservation tube, add 500 μL of sterile water to the freeze-dried bacterial powder, and dissolve and mix thoroughly. Use a sterile inoculation loop to dip the bacterial solution and streak it on the surface of a blood agar plate (containing 5% sheep blood). Place the inoculated plate in an anaerobic incubator at 37°C and culture for 48 hours. After the culture is completed, pick a single colony from the plate, transfer it to a fresh blood agar plate, and continue to culture under anaerobic conditions at 37°C for 48 hours.

[0067] To confirm the correctness of the cultured strain, genomic DNA was extracted from the cultured Parabacteroides distichous ATCC 8503 using Chelex100 reagent (purchased from Sigma-Aldrich). The OD260 / OD280 ratio was then measured using a Nanodrop 2000 instrument. A ratio of 1.7-1.9 was considered acceptable. PCR amplification was then performed using the qualified DNA as a template. The PCR amplification system is shown in the table below.

[0068] Table 1 PCR amplification system

[0069] Components content Taq PCR Master Mix 12.5μL DNA template 0.5μL Upstream primer 1 μL Downstream primer 1 μL Sterile water 10 μL total 25 μL

[0070] PCR amplification conditions were as follows: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1.5 min, for 30 cycles; and extension at 72°C for 10 min.

[0071] The upstream primer is: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 1); the downstream primer is: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 2).

[0072] The PCR amplification products were sent to Sangon Biotech (Shanghai) Co., Ltd. for Singer unidirectional sequencing, and then the sequencing results were compared using blast on the NCBI website to confirm that the bacterial species was correct.

[0073] The Singer unidirectional sequencing amplification primer is: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 3).

[0074] The blast comparison results are as follows Figure 1 shown.

[0075] It can be found that after sequencing comparison, the strain cultured in this example is indeed Parabacteroides distichous ATCC8503.

[0076] (2) Preparation of outer membrane vesicles of Parabacteroides distichum:

[0077] The process of preparing outer membrane vesicles of Parabacteroides distichum is as follows Figure 2 shown.

[0078] The specific steps are:

[0079] After resuscitation, confirmed Parabacteroides distichous ATCC 8503 was inoculated into BHI broth and cultured at 37°C under anaerobic conditions for 24 hours. After the strain was fully activated, it was inoculated into BHI broth at a 2% (v / v) inoculum and cultured at 37°C under anaerobic conditions for another 48 hours.

[0080] After the culture is completed, the Parabacteroides dieldrin culture is transferred to a centrifuge tube and centrifuged twice at 4°C and 12000 rpm for 30 minutes each time. The supernatant obtained by centrifugation is filtered using a suction filtration system (0.45 μm pore size) to remove residual bacteria and cell debris. The filtrate is collected and further filtered using a 0.22 μm pore size filter membrane, and then transferred to a 100 KDa ultrafiltration tube and centrifuged at 4°C and 3000 rpm for 30 minutes to complete the concentration. The concentrate after centrifugation is filtered through a 0.22 μm pore size filter membrane and then transferred to an ultrahigh-speed centrifuge and centrifuged at 4°C and 150,000 × g for 2 hours. The resulting precipitate is resuspended in PBS and centrifuged at 4°C and 150,000 × g for 2 hours to obtain purified Parabacteroides dieldrin outer membrane vesicles.

[0081] (3) Characterization of the outer membrane vesicles of Parabacteroides distichum:

[0082] Take 10 μL of the outer membrane vesicles of Parabacteroides dieldii purified in the above steps, add them dropwise to a carbon-coated copper grid, and incubate for 10 minutes. Carefully remove the residual liquid with filter paper, and then add 10 μL of 2% uranyl acetate solution to the copper grid to stain the outer membrane vesicles of Parabacteroides dieldii (incubate for 2 minutes). After staining, the copper grid was washed with distilled water and then air-dried in the dark. Using a transmission electron microscope, the morphology of the outer membrane vesicles of Parabacteroides dieldii was observed and images were taken at an accelerating voltage of 120 kV. The size distribution and particle concentration of the outer membrane vesicles of Parabacteroides dieldii were further determined using a nano flow cytometer (NanoFCM, Flow NanoAnalyzer U30E). Using the BCA protein assay kit (Biyuntian), the protein concentration of the outer membrane vesicles of Parabacteroides dieldii was measured according to the instructions for subsequent experiments.

[0083] Characterization results such as Figure 3-4 shown.

[0084] By using transmission electron microscopy (TEM) to observe the morphological characteristics of the outer membrane vesicles of Parabacteroides dieldrinii, it can be found that the outer membrane vesicles of Parabacteroides dieldrinii have a uniform hemispherical shape with a concave side, a clear bilayer structure, a diameter of about 65nm, a clear vesicle structure, and are distributed individually or in clusters.

[0085] By using nanoflow cytometry to measure the size of the outer membrane vesicles of Parabacteroides dieldii, it was found that the average particle size of the outer membrane vesicles of Parabacteroides dieldii was 67.81±11.37 nm.

[0086] Example 2

[0087] In this example, the therapeutic effects of the outer membrane vesicles of Parabacteroides distichous prepared in the above examples were tested through animal experiments.

[0088] Specific experimental methods include:

[0089] (1) Animal model establishment and drug intervention grouping:

[0090] Forty male BALB / c mice were randomly divided into control group (NC), carbon tetrachloride group (CCl4), Parabacteroides dieldrin outer membrane vesicle group (OMV), Parabacteroides dieldrin group (PD) and positive drug pirfenidone group (PFD) after one week of adaptive feeding, with 8 mice in each group. Figure 5 shown.

[0091] Among them, mice in the CCl4, OMV, PD, and PFD groups were intraperitoneally injected with 10% CCl4 twice a week for 6 weeks to establish a mouse liver fibrosis model. The NC group was intraperitoneally injected with an equal dose of olive oil. During modeling, the NC and CCl4 groups were gavaged with PBS bacterial solution once every 2 days, while the PFD and PD groups were gavaged with PFD solution and PD bacterial solution once every 2 days, respectively. The PD group was gavaged with 0.2 mL of PD bacterial solution (concentration of 1×10 9 CFU / mL). In the PFD group, pirfenidone was administered at a dose of 300 mg / kg mouse body weight. The OMV group received an intravenous injection of OMV every three days at a dose of 5 mg / kg mouse body weight. All groups received OMV until the end of the experiment (6 weeks after model establishment).

[0092] During the experiment, the mental state of the mice was observed and the weight changes were recorded every day. Each group of mice was fed with normal feed and had free access to food and water.

[0093] At the end of the sixth week, mice were fasted but not deprived of water. The following day, after anesthesia, blood was collected from the eye sockets to obtain serum samples, which were then stored at -80°C for subsequent biochemical analysis. Liver samples were obtained after autopsy and photographed. Subsequently, portions of the liver tissue were fixed in 4% paraformaldehyde for pathological staining.

[0094] The specific steps of pathological staining are as follows:

[0095] Liver tissue was immersed in 4% paraformaldehyde for 48 h. Before dehydration, it was embedded in paraffin and cut into 4 μm sections. Tissue sections were then dewaxed, hydrated, and stained using a hematoxylin-eosin (H&E) kit, a Sirius red kit, and a Masson trichrome kit, respectively. H&E-stained sections, Sirius red-stained sections, and Masson trichrome-stained sections were observed under a light microscope. H&E staining was used to observe tissue structure, Sirius red staining was used to show collagen fiber distribution, and Masson staining was used to assess the degree of fibrosis.

[0096] The specific steps of each staining method can be referred to the instruction manual, and the specific steps are as follows:

[0097] H&E staining:

[0098] Place the paraffin sections in xylene and dewax them thoroughly. Then immerse the dewaxed sections in gradient ethanol (anhydrous ethanol, 90% ethanol, 80% ethanol and 70% ethanol) in sequence to complete the hydration process, and finally transfer them to distilled water. Immerse the hydrated sections in hematoxylin stain for 8-15 minutes, remove excess stain with running water, and then place them in 1% hydrochloric acid alcohol differentiation solution for differentiation, and then wash them again with running water. Then immerse the sections in eosin stain for 2-5 minutes, rinse with running water, and then immerse them in 80% ethanol, 90% ethanol and anhydrous ethanol in sequence to complete the dehydration process. Immerse the dehydrated sections in xylene for transparent treatment, and then use neutral gum to seal the sections.

[0099] Sirius red staining:

[0100] Place the paraffin sections in xylene and dewax thoroughly. Then, immerse the dewaxed sections in a gradient of ethanol (absolute ethanol, 90% ethanol, 80% ethanol, and 70% ethanol) to complete the hydration process, and finally transfer to distilled water. Immerse the hydrated sections in Sirius stain for 1 hour, remove the excess stain with running water, and then soak in 0.5% acetic acid for 20 seconds. After rinsing with running water, immerse in anhydrous ethanol and xylene in sequence to complete the dehydration and transparent treatment, and then use neutral gum to complete the sealing of the sections.

[0101] Masson staining:

[0102] Place the paraffin sections in xylene and dewax them thoroughly. Then immerse the dewaxed sections in gradient ethanol (anhydrous ethanol, 90% ethanol, 80% ethanol and 70% ethanol) in sequence to complete the hydration process, and finally transfer them to distilled water. The hydrated sections are stained with hematoxylin, ponceau red and brilliant green in sequence. After removing excess dye with running water, immerse the above sections in 95% ethanol and anhydrous ethanol in sequence to complete the dehydration process. Immerse the dehydrated sections in xylene for transparent treatment, and then use neutral gum to seal the sections.

[0103] At the same time, commercially available kits were used to detect the following physiological and biochemical indicators of mouse serum: aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TBIL) and oxidative stress indicators (superoxide dismutase (SOD) and glutathione (GSH)).

[0104] The results are as follows Figure 6-14 shown.

[0105] It was found that compared with the NC group, the livers of the mice in the CCl4 group showed typical fibrosis characteristics, including: rough surface, yellow color and hard texture (such as Figure 6 (As shown). H&E staining showed that CCl4 modeling resulted in disordered liver lobule architecture and significant inflammatory cell infiltration. Sirius red and Masson staining further demonstrated a significant increase in collagen fiber deposition. This confirms that CCl4 can cause liver fibrosis in mice. Following administration of OMVs, the positive drug PFD, and PD, the liver's appearance regained its rosy redness, its texture approached normal, inflammatory infiltration decreased, and collagen deposition decreased. These results confirm that OMVs can significantly improve CCl4-induced liver fibrosis, with therapeutic efficacy comparable to that of the clinical antifibrotic drug PFD.

[0106] To further evaluate the protective effect of Parabacteroides distichous outer membrane vesicles (OMVs) on liver function, the levels of liver function biomarkers ALT, AST, and TBIL in the serum of each group of mice were detected based on commercially available kits. The results showed that compared with the NC group, the serum ALT, AST, and TBIL levels in the CCl4 group were significantly increased, confirming that the liver fibrosis model was successfully established. Both OMV and PD intervention significantly reduced ALT and AST levels, restoring them to levels close to those in the NC group. It is worth noting that although OMV significantly alleviated the CCl4-induced increase in TBIL, PD itself did not produce this effect. After PD treatment, TBIL did not significantly decrease. It can be determined that OMV produces a new effect relative to PD itself, which can effectively reverse CCl4-induced liver damage.

[0107] Studies in the prior art have revealed that exposure to CCl4 can induce oxidative stress and the accumulation of lipid peroxides, which are key factors in the development of liver fibrosis. Therefore, in-depth assessment of the content of oxidative stress-related indicators in the liver can effectively understand the degree of liver damage and fibrosis. Figure 13-14 As shown in the results, compared with the NC group, the serum SOD and GSH levels of mice in the model CCl4 group were significantly reduced, confirming the elevated levels of oxidative stress induced by CCl4. OMV and PD treatment significantly increased SOD activity. However, it is worth noting that compared with the CCl4 group, OMV administration significantly upregulated GSH content, while PD administration failed to induce a statistically significant improvement in GSH. These results further confirm the above conclusion that OMV produces a novel effect relative to PD itself, which can effectively reverse CCl4-induced oxidative stress.

[0108] Example 3

[0109] In order to further explore why the OMV group and the PD group produced different therapeutic effects, the inventors conducted a component analysis of the outer membrane vesicles of Parabacteroides distichum in this example. The specific experimental method is as follows:

[0110] Take 80 μL of the outer membrane vesicles of Parabacteroides distichous obtained in the above example (if frozen, thaw in an ice bath), freeze-dry in a 96-well plate, add 10 μL of water to reconstitute, then add 150 μL of methanol solution containing 5 mM ammonium acetate, and vortex mix for 20 minutes. Then centrifuge the 96-well plate at 4000 × g for 20 minutes. Take 20 μL of the supernatant in a new 96-well plate, add 80 μL of methanol solution containing 5 mM ammonium acetate and mix evenly. Use ultra-performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-TQMS) to complete the detection of the targeted lipidome to obtain the components of the outer membrane vesicles of Parabacteroides distichous.

[0111] The UPLC-TQMS used an Acquity UPLC BEH C18 column (21×100 mm). Instrument parameters were: column temperature: 40°C, phase A: acetonitrile / water (6:4, dissolved in 5 mM ammonium formate + 0.1% formic acid), and phase B: isopropanol / acetonitrile (9:1, dissolved in 5 mM ammonium formate + 0.1% formic acid). Elution conditions were: 0-0.5 min (60% phase B), 0.5-11 min (60% to 100% phase B), 11-13 min (100% phase B), 13-13.5 min (100% to 60% phase B), and 13.5-15 min (60% phase B).

[0112] The results are as follows Figure 15 shown.

[0113] It was found that the outer membrane vesicles (OMVs) of Parabacteroides distichous contain 13 lipid components, of which the five with the highest relative abundances are triglycerides (TAG, 35.48%), phosphatidylcholine (PC, 17.56%), sphingomyelin (SM, 10.39%), phosphatidylethanolamine (PE, 9.32%), and ceramide (Cer, 8.24%). Notably, several of these lipids have been shown to be closely involved in the regulation of liver fibrosis. Among them, ceramide, a key intermediate in sphingolipid metabolism, can activate hepatic stellate cells (HSCs) through signaling pathways such as TGF-β / Smad and JNK, promoting collagen deposition and thereby exacerbating the process of liver fibrosis. SM, a precursor of Cer, can be hydrolyzed by sphingomyelinase to produce Cer under stress conditions, indirectly participating in the profibrotic process. PC plays an important protective role in maintaining the stability of hepatocyte membrane structure and bile secretion, and PC deficiency has been reported to be closely associated with liver damage and fibrosis. In addition, an imbalance in the ratio of PC to PE may also lead to cell membrane instability and increased cell apoptosis, thereby promoting liver damage. In summary, the therapeutic effect of OMVs depends on the balance and interaction between multiple lipids.

[0114] More importantly, combined with the results of in vivo experiments, it was found that OMV treatment can significantly improve the pathological state of liver fibrosis induced by CCl4 in mice, including reducing serum ALT, AST, and TBIL levels, and increasing the content of antioxidant indicators SOD and GSH, revealing that OMV has a clear anti-fibrotic effect. In this regard, the main reason for this effect may be that the protective lipids in OMV, such as PC, synergize with other functional active ingredients to offset the effects of pro-fibrotic factors such as Cer, thereby achieving an overall anti-fibrotic effect. In addition, OMV may also intervene in the fibrosis process from multiple levels by regulating the lipid metabolism, immune inflammatory response or redox state of hepatocytes. Based on the particularity of the lipid composition in OMV, it exerts a better anti-fibrotic therapeutic effect that the PD group cannot achieve.

[0115] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Application of outer membrane vesicles of Parabacteroides distichum in the preparation of drugs for the treatment of liver fibrosis.

2. The use according to claim 1, characterized in that The drug can reverse liver damage and oxidative stress induced by liver fibrosis; Preferably, the drug reduces or inhibits the increase in total bilirubin caused by liver damage; Preferably, the drug increases or inhibits the decrease in GSH caused by oxidative stress.

3. The use according to claim 1, characterized in that The average particle size of the outer membrane vesicles of Parabacteroides dieldii is 55-80 nm, preferably 56.44-79.18 nm.

4. The use according to claim 1, characterized in that The dosage forms of the drug include: powder, solution, capsule and granule; Preferably, the granules further include a pharmaceutically acceptable carrier.

5. The use according to claim 1, characterized in that The outer membrane vesicles of Parabacteroides dieltii are prepared by the following preparation method: The Parabacteroides dieltii bacteria are subjected to primary filtration and then ultrafiltration to obtain the outer membrane vesicles of Parabacteroides dieltii; Among them, the filtration pore size of the primary filtration is 0.2-0.25μm; The molecular weight cut-off of ultrafiltration is 95-110KDa.

6. The use according to claim 5, characterized in that The Parabacteroides dieltii bacteria are subjected to impurity removal treatment before primary filtration; Preferably, the impurity removal treatment includes centrifugation and filtration.

7. The use according to claim 5, characterized in that The preparation method further comprises: concentrating and purifying the outer membrane vesicles of Parabacteroides dieldii after ultrafiltration.

8. The use according to claim 7, characterized in that The concentration comprises: centrifugation; Preferably, the centrifugation condition is: 2500-3100 rpm for 25-40 min.

9. The use according to claim 7, characterized in that The purification comprises: filtering and centrifuging the concentrated Parabacteroides dieldii outer membrane vesicles; Preferably, the filtration pore size of the filtration is 0.2-0.25 μm; Preferably, the centrifugation conditions are: greater than or equal to 120,000×g, and the centrifugation time is 1.5-2 h.

10. The use according to claim 1, characterized in that The medicine also contains pharmaceutically acceptable adjuvants.

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