Application of microorganism-derived outer membrane vesicles in medicines for treating obesity and related metabolic diseases

Abstract

The invention relates to application of microorganism-derived outer membrane vesicles in medicines for treating obesity and related metabolic diseases, and belongs to the technical field of exosomes. In order to solve the technical problems that probiotics beneficial to intestinal tracts are weak in colonization ability in the intestinal tracts, are easily influenced by gastrointestinal tract environments and are unstable in individual response, the sphingosine-containing microbe-sourced outer membrane vesicle is provided, and the sphingosine content in the outer membrane vesicle is 50-65 ng / mg.

Description

Technical Field

[0001] This invention belongs to the field of exosome technology, specifically relating to the application of microbial-derived outer membrane vesicles in drugs for obesity and related metabolic diseases. Background Technology

[0002] For understanding the technical content of this invention:

[0004] The gut microbiota, acting as a "second genome," participates in obesity by regulating energy absorption, short-chain fatty acid (SCFA) synthesis, and inflammatory signaling pathways. Studies have shown that obese individuals exhibit an imbalance in the Firmicutes / Bacteroidetes ratio in their gut, with decreased abundance of butyric acid-producing bacteria and increased lipopolysaccharide (LPS)-producing bacteria, leading to metabolic endotoxemia and insulin resistance. While probiotics and prebiotics can alleviate obesity by regulating the gut microbiota, their effects are strain-specific and their mechanisms of action are unclear, necessitating the discovery of more targeted regulatory factors.

[0005] Specific strains such as *Parabacteroides goldsteinii* (Pg) and *Parabacteroides distasonis* (Pd), as key members of the gut symbiont, have shown a negative correlation between their abundance and obesity, suggesting they may exert anti-obesity effects by enhancing intestinal barrier function and inhibiting the expression of pro-inflammatory factors. Their secreted outer membrane vesicles (OMVs), acting as natural nanocarriers, can deliver bioactive molecules such as proteins and nucleic acids, mediating gut-host communication. OMVs possess low immunogenicity, high stability, and the ability to cross biological barriers, exhibiting unique advantages in regulating host immune metabolism (such as TLR signaling pathway activation and macrophage polarization), providing novel drug delivery systems and targets for obesity treatment.

[0006] Currently, there are no publicly available reports on OMVs derived from Parabacteroides distasonis and Parabacteroides goldsteinii in obesity and related metabolic diseases. Furthermore, the active components of OMVs remain poorly identified and their mechanisms of action are unclear. Therefore, exploring the functional properties of OMVs derived from Parabacteroides distasonis and Parabacteroides goldsteinii, identifying their key metabolites, and investigating their mechanisms of action will provide new treatment strategies for obesity and related metabolic syndromes, offer a theoretical basis for developing targeted delivery systems or microbiome-based combined therapies based on engineered OMVs, and drive a paradigm shift in obesity intervention strategies from "extensive microbiome regulation" to "precision nanotherapy."

[0007] Relevant patent documents retrieved: The document, published in China (CN114401727A) on April 6, 2022, discloses a method for treating metabolic diseases or conditions in mammalian subjects, comprising administering a composition to the gastrointestinal system of the subject, wherein the composition comprises inactivated *Parabacterium kiwifruit* (…). Parabacteroides goldsteinii ), growth medium of *Pseudomonas kiuri*, or vesicles derived from *Pseudomonas kiuri*.

[0008] The document, published in China (CN117159592A) on December 5, 2023, discloses the application of extracellular vesicles derived from *Pseudomonas dilatatus* in alleviating diseases caused by a high-fat diet. These vesicles can be used to alleviate conditions such as hyperlipidemia, hypertension, fatty liver, obesity, and diabetes caused by a high-fat diet.

[0009] Relevant non-patent literature retrieved: Journal or book title: *Cell Reports*, document title: Parabacteroides distasonis "Alleviates Obesity and Metabolic Dysfunctions via Production of Succinate and Secondary Bile Acids", Vol. 26, published on January 2, 2019, discloses that *Pseudomonas dignitaria* exerts a multi-target overall regulatory effect by producing succinate and secondary bile acids, which activate different signaling pathways. This significantly improves obesity, insulin resistance, lipid metabolism disorders, and non-alcoholic fatty liver disease symptoms in high-fat diet-induced obese (DIO) mice and ob / ob obese model mice. Summary of the Invention

[0010] The purpose of this invention is to provide: The application of microbial-derived outer membrane vesicles in drugs for obesity and related metabolic diseases, and related technologies, to address technical problems such as the weak colonization ability of beneficial probiotics in the gut, their susceptibility to the influence of the gastrointestinal environment, and unstable individual responses, or combinations thereof.

[0011] This patent provides an application of outer membrane vesicles derived from *Parabacteroides goldsteinii* (Pg-OMVs) and outer membrane vesicles derived from *Parabacteroides distasonis* (Pd-OMVs) in high-fat diet-induced obesity, combining the effects of inhibiting lipid accumulation, anti-inflammation, and antioxidant activity. Simultaneously, this patent also reveals the mechanism of action of sphinganine (d17:0), a key metabolite enriched in Pg-OMVs and Pd-OMVs, providing a theoretical basis and technical pathway for developing a standardized and mass-producible OMV functional component composition.

[0012] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0013] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0014] The definition of the standard chemical term can be found in the reference book "New Technologies and Applications of Probiotics", Chemical Industry Press, 2022 new edition, edited by Liu Zhenmin.

[0015] Unless otherwise stated, conventional methods within the scope of this art are employed. Unless specifically defined, the use of various commercially available products used herein employs standard techniques. For example, they may be implemented using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein are generally implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0016] To achieve the above objectives, the present invention provides the following technical solution: On the one hand, the present invention provides an outer membrane vesicle containing sphingosine.

[0017] Preferably, the sphingosine content in the outer membrane vesicles is 50-65 ng / mg.

[0018] The core functional component of outer membrane vesicles is outer membrane protein (OMP). The protein content is highly positively correlated with the total yield of outer membrane vesicles and is the most commonly used indicator for evaluating the total content of outer membrane vesicles in the laboratory. This invention uses protein concentration to quantitatively evaluate the concentration of outer membrane vesicles. As an example, this invention uses the BCA method to determine the concentration of outer membrane vesicles, such as using a BCA detection kit.

[0019] In this invention, the sphingosine content in the outer membrane vesicles is detected by chromatography.

[0020] Preferably, the sphingosine in the outer membrane vesicles is extracted with an organic solvent and then its content is determined using a liquid chromatography-mass spectrometry (LC-MS) system.

[0021] Preferably, the outer membrane vesicles are derived from *Parabacteroides*.

[0022] Preferably, the parabacterium includes Parabacteroides goldsteinii JCM13446 or Parabacteroides distasonis ATCC 8503.

[0023] On the other hand, the present invention provides a method for preparing the above-mentioned outer membrane vesicles.

[0024] Preferably, the preparation method includes: culturing *Pseudomonas jini* or *Pseudomonas diffusa*, collecting the culture supernatant, and separating the outer membrane vesicles.

[0025] Preferably, the separation method includes ultracentrifugation, ultrafiltration, tangential flow filtration (TFF), size exclusion chromatography (SEC), or affinity chromatography.

[0026] Preferably, the separation method is ultracentrifugation.

[0027] Preferably, the preparation method includes the following steps: S1. Bacteria are cultured in a culture medium containing vitamin K1 and heme chloride. After culture, the first supernatant is obtained by centrifugation. S2. Take the first supernatant and centrifuge it at ultraspeed to obtain the second supernatant; S3. The second supernatant is filtered and sterilized through a filter membrane.

[0028] Preferably, the culture medium in step S1 includes GAM culture medium; the ultracentrifugation in step D2 includes centrifugation at 40,000g for 1 hour, followed by centrifugation at 150,000g for 1.5 hours; and the filter membrane in step S3 includes a 0.22μm filter membrane.

[0029] On the other hand, the present invention provides a composition comprising the above-described outer membrane vesicles and a pharmaceutically acceptable carrier.

[0030] On the other hand, the present invention provides the use of the above-described outer membrane vesicles or the above-described composition in the preparation of medicaments for the prevention or treatment of metabolic diseases.

[0031] Preferably, the metabolic diseases include obesity, diabetes, hyperuricemia, hypertension, hyperlipidemia, hyperglycemia, metabolic syndrome, non-alcoholic fatty liver disease, insulin resistance, atherosclerosis, or inflammatory skin diseases.

[0032] Preferably, the drug includes drugs that inhibit lipid accumulation, anti-inflammatory drugs, antioxidant drugs, drugs that improve insulin sensitivity, or drugs that enhance mitochondrial function.

[0033] Accordingly, the application also includes use in combination with metabolic drugs or anti-inflammatory drugs.

[0034] Preferably, the metabolic drug includes metformin or a GLP-1 receptor agonist, and the anti-inflammatory drug includes an IL-17 inhibitor.

[0035] On the other hand, the present invention also provides a method for preventing or treating obesity and related metabolic diseases, namely, administering a therapeutically effective amount of the above-described outer membrane vesicles or the above-described composition to a subject in need.

[0036] The present invention has at least the following beneficial effects: 1. Discovery and application of novel functional OMV This study reveals for the first time the synergistic effects of outer membrane vesicles (OMVs) derived from *P. giardia* (Pg) and *P. di* (Pd) in the treatment of high-fat diet-induced obesity and related metabolic / inflammatory diseases, exhibiting multi-target effects including inhibiting lipid accumulation, anti-inflammation, anti-oxidation, and improving mitochondrial function.

[0037] 2. Screening and functional verification of sphingosine (SAd(17:0)), a key functional lipid in vesicles. For the first time, we discovered that sphingosine SA (d17:0), which is highly abundant in Pg-OMVs and Pd-OMVs, is a key active ingredient, and elucidated its mechanism of action by regulating macrophage polarization, mitochondrial homeostasis and ROS metabolism.

[0038] 3. Standardization and stability of OMV preparation technology A large-scale preparation process for Pg / Pd-OMVs was established. Through a specific culture medium formulation (with added vitamin K1 and heme chloride), two-step ultracentrifugation purification (40000g→150000g) and 0.22μm filtration sterilization technology, OMVs with uniform particle size (100-200 nm), intact structure and high purity were obtained to meet the needs of industrial development.

[0039] 4. Technological advantages that break through traditional therapies Compared with live probiotics, OMVs avoid the difficulties of intestinal colonization, gene transfer at the level of gene transfer and immune rejection, and have more stable biological activity. Compared with single-target small molecule drugs, OMVs and SA achieve more comprehensive metabolic regulation through multi-target synergy (lipid metabolism-inflammation-oxidative stress), and are especially suitable for the treatment of complex inflammatory diseases such as obesity accompanied by psoriasis.

[0040] 5. Novel Delivery and Treatment Strategies The stability and in vivo efficacy of OMVs and SA delivered orally in the digestive tract environment were verified, providing a basis for the development of oral probiotic preparations. The first "OMVs@SA" functional composition was created, which enhances the therapeutic effect through the synergistic effect of the carrier (OMVs) and the active molecule (SA). Attached Figure Description

[0041] Figure 1 The effect of different concentrations of PGOMV on the proliferation activity of iBMDM cells.

[0042] Figure 2 The effect of different concentrations of PDOMV on the proliferation activity of iBMDM cells.

[0043] Figure 3 Comparison of triglyceride accumulation in different groups of iBMDM.

[0044] Figure 4 The relative expression levels of the inflammatory factor IL-1β in iBMDM of different groups.

[0045] Figure 5 The relative expression levels of the inflammatory factor TNF-α in iBMDM of different groups.

[0046] Figure 6 To compare reactive oxygen species in different groups of iBMDM cells for flow cytometry analysis.

[0047] Figure 7 To compare mitochondrial reactive oxygen species in different groups of iBMDM cells for flow cytometry analysis.

[0048] Figure 8Comparative experiments of PGSPTi-OMV and PGOMV; A is a comparison of SA content; B is the effect on lipid accumulation inhibition, where NC group is the negative control, PBS group is the control group with PBS added, PGOMV group is the experimental group with PGOMV added, PGSPTi-OMV group is the experimental group with PGSPTi-OMV added, and PGSPTi-OMV+SA group is the experimental group with PGSPTi-OMV added and 10 μM SA added; C is the effect on IL-6 expression inhibition, where NC group is the negative control, FFA group is the control group induced by FFA, PGOMV group is the experimental group induced by FFA and with PGOMV added, PGSPTi-OMV group is the experimental group induced by FFA and with PGSPTi-OMV added, and PGSPTi-OMV+SA group is the experimental group induced by FFA and with PGSPTi-OMV added and 10 μM SA added.

[0049] Figure 9 To compare the effects of different concentrations of SA on the proliferation activity of iBMDM cells.

[0050] Figure 10 Comparison of triglyceride accumulation in different groups of iBMDM.

[0051] Figure 11 The relative expression levels of the inflammatory factor IL-1β in iBMDM of different groups.

[0052] Figure 12 For flow cytometry analysis of iBMDM cell polarization in different groups.

[0053] Figure 13 To compare reactive oxygen species in different groups of iBMDM cells for flow cytometry analysis.

[0054] Figure 14 To analyze the mitochondrial membrane potential of different groups of iBMDM cells by flow cytometry.

[0055] Figure 15 To compare mitochondrial reactive oxygen species in different groups of iBMDM cells for flow cytometry analysis.

[0056] Figure 16 Comparison of intracellular membrane lipid peroxide (MDA) accumulation in iBMDM cells of different groups.

[0057] Figure 17 Comparison of glucose metabolism rates in iBMDM cells from different groups.

[0058] Figure 18 The changes in glucose content in cell culture media for different groups.

[0059] Figure 19Comparison of OCR of oxygen consumption rate of iBMDM cells in different groups.

[0060] Figure 20 The relative expression levels of the inflammatory factor IL-1β in iBMDM of different groups.

[0061] Figure 21 The relative expression levels of the inflammatory factor TNF-α in iBMDM of different groups.

[0062] Figure 22 To compare reactive oxygen species in different groups of iBMDM cells for flow cytometry analysis.

[0063] Figure 23 To compare mitochondrial reactive oxygen species in different groups of iBMDM cells for flow cytometry analysis.

[0064] Figure 24 This is the chemical structure and molecular identifier of Sphinganine (d17:0).

[0065] Figure 25 This is a full-wavelength UV-Vis scan of SA (optical path 10 mm).

[0066] Figure 26 The standard curve of absorbance of SA at 224 nm versus concentration (0-2.87 mg / mL) is given by the linear regression equation y = 253.84x - 2.29424 (R² = 1).

[0067] Figure 27 Encapsulation efficiency (EE%) of PGOMV for SA load.

[0068] Figure 28 To establish a high-fat diet (HFD) induced obese mouse model, mice were treated with SA (2.68 μg) and PGOMV@SA (50 μg PGOMV loaded with 2.68 μg SA) orally every other day for two months, with normal-diet mice serving as controls.

[0069] Figure 29 Comparison of total cholesterol (TC) levels in the serum of mice in different groups.

[0070] Figure 30 Comparison of triglyceride (TG) levels in the serum of mice in different groups.

[0071] Figure 31 The results are from the Oral Glucose Tolerance Test (OGTT). A shows the comparison over time, and B shows the AUC statistics.

[0072] Figure 32 The results are from the Insulin Tolerance Test (ITT). A shows the comparison over time, and B shows the AUC statistics.

[0073] Figure 33 Changes in liver weight in mice in different groups.

[0074] Figure 34 The levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum of mice in different groups were compared. Group A corresponds to the comparison of AST levels, and Group B corresponds to the comparison of ALT levels.

[0075] Figure 35 Flow cytometry analysis of splenic macrophages; CD11b F4 / 80 M1 (CD86) represents the macrophage ratio and quantification.

[0076] Figure 36 Histological analysis images of different groups.

[0077] Note: In the above figures, ns represents P > 0.05. This means P < 0.05. This means P < 0.01. This represents P < 0.001. This represents P < 0.0001. Detailed Implementation 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.

[0078] 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 under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0079] Data analysis and statistical analysis were performed using professional data processing software. One-way ANOVA was used for significance analysis, and P<0.05 was considered to indicate a significant difference.

[0080] Example 1: Preparation and Application of Outer Membrane Vesicles (OMVs) The bacterium used in this invention is *Pseudomonas jini*. Parabacteroides goldsteinii(JCM13446) and Parabacteroides distasonis (ATCC 8503).

[0081] Parabacterium ginseng JCM 13446 ( Parabacteroides goldsteinii (Pg) was purchased from the Institute of Microbiology, Guangdong Academy of Sciences (Guangdong Provincial Center for Microbial Analysis and Testing), accession number GDMCC 1.2815.

[0082] Parabacterium dilatatum ATCC 8503 ( Parabacteroides distasonis (Pd) was purchased from the Institute of Microbiology, Guangdong Academy of Sciences (Guangdong Provincial Center for Microbial Analysis and Testing), accession number GDMCC 1.1564.

[0083] I. Experimental Methods 1. Strains culture methods (1) Culture medium preparation: GAM basal medium was autoclaved at 121°C for 15 minutes and cooled to approximately 50°C. Under aseptic conditions, 1 mL of sterile 0.1% (v / v) vitamin K1 solution and 1 mL of heme chloride stock solution (5 mg / mL, dissolved in 0.01 M NaOH) were added to every 1000 mL of medium and mixed thoroughly before use. This culture medium was used for all resuscitation, activation, and scale-up culture steps.

[0084] (2) Strain resuscitation and activation: Remove the frozen bacterial strain from the -80°C freezer and thaw briefly on ice. In a clean bench, use a sterile inoculation loop to scrape a small amount of the frozen bacterial solution and streak it onto a GAM solid plate. Immediately place the plate into an anaerobic culture bag containing an anaerobic gas-generating bag and an oxygen indicator, seal it, and place it in a 37°C constant temperature incubator for 48-72 hours until a single colony is formed. Pick a single colony with typical morphology and inoculate it into 5 mL of liquid GAM medium for preliminary activation culture.

[0085] (3) Expanding the culture and collecting the bacterial cells: The activated bacterial solution was transferred to new GAM liquid medium at an inoculation rate of 2%-5% (v / v). The inoculated culture flasks were placed in anaerobic culture bags and incubated statically at 37°C for about 24-48 hours. During this period, the optical density (OD600) of the culture at 600 nm was monitored periodically using a spectrophotometer. When the OD600 value reached 1.0 (in the late logarithmic growth stage), the culture was terminated.

[0086] (4) Cryopreservation of bacterial culture: For long-term preservation, the bacterial culture can be mixed with sterile glycerol containing 50% (v / v) at a ratio of 1:1, mixed well, and then dispensed into sterile cryovials. Store the cryovials in an ultra-low temperature freezer at -80°C.

[0087] 2. OMV Extraction Method (1) After inoculating the bacterial strain into the culture flask at a ratio of 5% (v / v), the culture flask was placed in a 2.5L anaerobic culture bag. An anaerobic gas-generating bag was added to the culture bag to consume the oxygen in the culture bag. An anaerobic indicator was added to detect the oxygen content in the bag. The flask was placed in a constant temperature incubator at 37℃ for static culture. After about 48 hours, obvious bacterial precipitate appeared at the bottom of the conical flask. After mixing, the precipitate was dispensed into 50ml centrifuge tubes and centrifuged at 10000rpm for 30min. The bacterial precipitate was discarded and the supernatant was collected. The supernatant was loaded into an ultracentrifuge tube and an ultracentrifuge tube sleeve, balanced, and then centrifuged at 40000g for 1h to remove macromolecular impurities. The supernatant was then placed in an ultracentrifuge and centrifuged at 150000g for 1.5h. The supernatant was discarded and the precipitate was collected. The precipitate was resuspended in PBS to obtain OMV. The precipitate was then filtered through a 0.22μm filter membrane in a clean bench and stored at -80℃ for later use. Among them, the OMV extracted from the supernatant of the culture medium of *Pleurobacterium giardi* JCM 13446 (Pg) was named PGOMV, and the OMV extracted from the supernatant of the culture medium of *Pleurobacterium digile* ATCC 8503 (Pd) was named PDOMV.

[0088] (2) Preparation of PGSPTi-OMV The preparation method of PGSPTi-OMV is as described in "2. OMV Extraction Method" above. Specifically, during inoculation with *Pseudomonas kiwifruit*, polysaccharin (final concentration 1 μM) was added to the culture medium, followed by anaerobic incubation for 48 hours. After bacterial culture, the supernatant was collected by centrifugation and filtered through a 0.22 μm filter membrane. The secreted outer membrane vesicles (OMVs) were harvested and purified by ultracentrifugation. Finally, the obtained vesicles were resuspended in sterile PBS and stored at –80°C for later use.

[0089] (3) The OMVs prepared by transmission electron microscopy, electrophoresis and NTA particle size analysis are concentrated between 100 and 200 nm. They have complete structure, high purity, and conform to the characteristics of typical bilayer membrane nanoparticles. They are of stable quality and suitable for subsequent biological research and industrial development.

[0090] 3. Determination of OMV concentration and SA content OMV concentration determination: After separating and purifying OMVs, the concentration of OMVs was determined by the BCA method, specifically using a BCA assay kit (purchased from Beyotime, catalog number P0009).

[0091] Determination of SA content: OMVs stored at -80°C were thawed on ice, and all subsequent operations were performed on ice. 100 µg of OMVs was mixed with 500 µL of 80% methanol-water solution containing an internal standard, vortexed for 30 seconds, followed by three freeze-thaw cycles (5 minutes in liquid nitrogen, 5 minutes on dry ice, and 5 minutes on ice). After centrifugation at 4°C and 12,000 rpm for 10 minutes, the supernatant was collected and freeze-dried until completely dry. The residue was resuspended in 50 µL of 70% methanol, vortexed for 3 minutes, and sonicated in an ice bath for 10 minutes. The solution was then centrifuged again at 4°C and 12,000 rpm for 15 minutes, and 40 µL of the supernatant was analyzed by LC-MS / MS.

[0092] Analysis was performed using a SCIEX Triple Quad™ 5500 LC-MS / MS system. Chromatographic separations were performed on an ACQUITYUPLC HSS T3 column (2.1 mm × 5 mm, 1.8 µm) at 30°C. The mobile phase consisted of 0.1% aqueous formic acid (A) and 0.1% formic acid acetonitrile solution (B), with a flow rate of 0.3 mL / min. The injection volume was 5 µL.

[0093] 4. CCK-8 cell proliferation detection iBMDMs (8,000 cells / well) were treated with 0.75 mM FFA (PA:OA=1:2) alone or with 0.75 mM FFA in combination with 10 or 20 μg / mL OMVs for 24 hours, then incubated with 10% CCK-8 reagent (APE×BIO K1018) for 30 minutes, and the absorbance at 450 nm was measured.

[0094] Proliferation rate = [(OD treatment group - OD blank group) / (OD control group - OD blank group)] × 100%.

[0095] 5. Cellular triglyceride detection Cells were lysed after treatment with 0.75 mM FFA alone or in combination with 10 or 20 μg / mL OMVs for 24 hours. Intracellular triglyceride levels were measured using the Amplex Red kit (S0219S), and the results were normalized to total protein content.

[0096] 6. Real-time quantitative PCR (qPCR) (1) Sample pyrolysis treatment Tissue samples: Take an appropriate amount of mouse tissue and place it in a grinding tube containing 1 mL Trizol reagent. Add ceramic beads and grind thoroughly using a tissue homogenizer. After grinding, transfer the lysis buffer to an RNase-free 1.5 mL EP tube using an RNase-free pipette tip and place on ice for later use.

[0097] Cell samples: For adherent cells, after washing with PBS, add 1 mL of Trizol reagent directly; for suspended cells, centrifuge to collect the precipitate, wash once with PBS, and then add 1 mL of Trizol. Repeat pipetting until the cells are fully lysed.

[0098] (2) RNA extraction Add 200 μL of chloroform to Trizol lysis buffer, vortex for 15 s, and incubate on ice for 5 min. Centrifuge at 12000 rpm for 20 min at 4 °C, and transfer the upper aqueous phase to a new RNase-free EP tube. Add an equal volume of isopropanol, mix well, and incubate on ice for 10 min. Centrifuge at 12000 rpm for 15 min at 4 °C, discard the supernatant, and obtain the RNA precipitate. Gently wash the precipitate twice with 0.75 mL of 75% ethanol (4 °C, 12000 rpm, 5 min / wash). Discard the ethanol and air dry at room temperature until the precipitate is clear. Dissolve the RNA in an appropriate amount of DEPC water and incubate on ice for 10 min. Determine the RNA concentration and purity using Nanodrop; the A260 / A280 ratio should be between 1.8 and 2.0.

[0099] (3) Reverse transcription to synthesize cDNA Prepare a 20 μL reaction mixture according to the reverse transcription kit instructions. After gentle mixing, run the following program in a PCR instrument: incubate at 42°C for 15 min; heat at 85°C for 5 s; store at 4°C.

[0100] (4) Real-time quantitative PCR (qPCR) Prepare a 20 μL reaction mixture: 1 μL cDNA template, 0.6 μL forward primer, 0.6 μL reverse primer, 7.8 μL LEPC water, and 10 μL SYBR Green dye. Mix well and aliquot into 8-tube strips, with 3 replicates per sample. Set the qPCR program according to the reagent instructions. Simultaneously detect the expression of an internal control gene (e.g., β-Actin) in all samples to standardize the expression level of the target gene.

[0101] 7. Detection of cellular and mitochondrial reactive oxygen species (ROS) iBMDMs at 1×10 5Cells were evenly seeded in 12-well plates and cultured for 14 hours. Then, 0.75 mM FIFA and the drug (10 μg / ml PGOMV / PDOMV) were added, and the plates were cultured for another 24 hours. After removing the culture medium, the cells were washed once with PBS. Except for the unstained group, 200 μM 2'-7-dichlorofluorescein diacetate probe (DCFH-DA, Sigma-Aldrich, D 6883) or HKSOX-1m (5 / 6- mixture) (1:1000) (mitochondrial reactive oxygen species detection) was added to each well. The cells were incubated at 37°C in the dark for 20 minutes. After incubation, the culture medium was removed in the dark, the cells were washed with PBS, and the cells were digested with 0.25% trypsin to collect the cells. After centrifugation, the cells were resuspended in PBS and the FITC channel fluorescence was detected using a flow cytometer (CytoFLEX LX, Beckman Coulter). The obtained flow cytometry data were analyzed using FlowJo X software, and quantitative statistical analysis was performed.

[0102] 8. Flow cytometry detection of macrophage polarization Splenic cells or iBMDMs were stained with PerCP / Cy5.5-CD11b (BioLegend 101228), FITC-F4 / 80 (BioLegend 123108), PE-CD86 (BioLegend 105008), or FITC-CD80 (BioLegend 16-10A1) and analyzed using FlowJo X. 9. Mitochondrial membrane potential detection iBMDMs (1×10 5 Cells / wells were treated with 0.75 mM MFA alone or in combination with 10 μM SA, stained with JC-1 dye (Beyotime, C2006) according to the manufacturer's instructions, and analyzed by flow cytometry (CytoFLEX LX, Beckman Coulter). The ratio of red (aggregates) to green (monomers) fluorescence was calculated.

[0103] 10. Determination of malondialdehyde (MDA) content in cells Cells were treated with 0.75 mM FFA alone or in combination with 10 μM SA for 24 hours. After treatment, cells were lysed, and intracellular MDA levels were quantified using a malondialdehyde (MDA) assay kit (BC0020). Results were normalized to total protein content.

[0104] 11. Oxygen Consumption Rate (OCR) Measurement Add 0.5 mL of complete culture medium to each well of a 24-well S.NEST sensor plate. Cover the plate with a standard cell culture cap and pre-incubate in a cell culture incubator for 30 minutes. After pre-incubation, aspirate the culture medium. Then seed iBMDM cells at a density of 100,000 cells per well in 1 mL of phenol red-free high-glucose DMEM. Place the plate in an incubator (37°C, 5% CO2) to allow cells to adhere for 6 hours. Subsequently, treat cells alone with 0.75 mM FFA or co-treated with SA (10 μM), gently mixing. Seal the plate with a dedicated S.NEST 24-well cap and transfer to the S.NEST system (Cytena Bioprocess Solutions) for 48 hours of real-time monitoring.

[0105] 12. Measurement of glucose and lactic acid concentrations Glucose and lactate levels were monitored using the LiCellMo live-cell metabolic analysis system (PHC Corporation). The system was calibrated using a two-step procedure: First, calibration solution A (12.5 mM glucose, 6 mM lactate) was added to each well of a 24-well plate at 1 mL, and the plate was placed in the LiCellMo instrument for 24 hours. After the initial calibration period, solution A was removed and replaced with calibration solution B (25 mM glucose, 12 mM lactate), again at 1 mL per well, and another 24-hour calibration was performed.

[0106] iBMDM cells were then seeded at a density of 100,000 cells per well in 24-well plates, with a final volume of 1 mL per well. Cells were treated alone with 0.75 mM FFA or in combination with SA (final concentration: 15 μM). The plates were then transferred to a LiCellMo system for 24-hour real-time metabolic monitoring.

[0107] 13. Engineering-based PGOMV@SA build For drug loading, 0.2 mg SA was mixed with PGOMVs (protein concentration: 1 mg / mL) in an electroporation cuvette. Electroporation was performed using a MicroPulser electroporator (Bio-Rad, USA) under the following conditions: 300 V, 150 μF, and an electrode gap of 4 mm. After electroporation, the sample was incubated on ice for 30 min, followed by centrifugation at 4,000 rpm for 10 min each time using a 100 kDa ultrafiltration tube, and washed twice with ice-cold PBS buffer.

[0108] Quantify the amount of unloaded SA and calculate the drug encapsulation efficiency (EE%) using the following formula: EE% = (Drug load / Total drug load) × 100%.

[0109] 14. Establishment of an obese mouse model After one week of acclimatization to a standard diet, mice were randomly assigned to two groups: a normal diet control group (NCD, n = 5) and a high-fat diet group (HFD, n = 15). HFD (60% fat, 20% protein, 20% carbohydrate) was administered for 12 weeks to induce obesity. Subsequently, the NCD group continued as the control group (1) NCD (normal diet + PBS), while the HFD-fed mice were randomly assigned to three subgroups (n = 5 per group) for 8 weeks of gavage treatment: (2) HFD (high-fat diet + PBS), (3) HFD+SA (HFD+SA, 2.68 μg / 200 μL), and (4) HFD + PGOMV@SA (HFD + PGOMV@SA, 50 μg / 200 μL, containing 2.68 μg SA), administered every other day.

[0110] 15. Metabolic index measurement After the experiment, blood samples were collected from mice, and serum was separated. The levels of total cholesterol (TC) (Nanjing Jiancheng, A111-1-1), triglycerides (TG) (Nanjing Jiancheng, A110-1-1), aspartate aminotransferase (AST) (Nanjing Jiancheng, C010-2-1), and alanine aminotransferase (ALT) (Nanjing Jiancheng, C009-2-1) were measured using commercial kits.

[0111] 16. Oral glucose tolerance test (OGTT) Mice were isolated in clean cages with ample water access, and fasted for 12-16 hours. After fasting, baseline blood glucose levels were measured and recorded. Mice were then administered 2 g / kg glucose via gavage, and subsequently returned to their cages, allowed to move under normal conditions. Blood glucose levels were measured at 0, 15, 30, 60, 90, and 120 minutes after gavage. At each predetermined time point, blood glucose levels were measured via tail vein sampling using a glucometer. Gentle pressure was applied to the sampling site after each sampling to achieve hemostasis. Blood glucose levels were recorded at each time point, and a blood glucose-time curve was plotted.

[0112] 17. Insulin Tolerance Test (ITT) Mice were placed individually in clean cages with ample water access and fasted for 4-6 hours. After fasting, baseline blood glucose levels were measured and recorded. Mice were then intraperitoneally injected with 0.75 U / kg insulin. Timing was initiated immediately after injection, and blood glucose levels were measured at 0, 15, 30, 60, 90, and 120 minutes using a glucometer drawn from the tail vein. After each blood draw, gentle pressure was applied to the puncture site to stop bleeding. Blood glucose levels were recorded at each time point, and a blood glucose-time curve was plotted.

[0113] 18. Histological analysis Tissue was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with H&E or Oil Red O. Sections were scanned using a KF-PRO-12-HI digital scanner.

[0114] II. Results and Analysis like Figure 1 As shown, treatment of macrophages with different concentrations of PGOMV resulted in no effect on cell proliferation within a concentration range of 50 μg / ml. Figure 2 As shown, different concentrations of PDOMV treated macrophages showed that concentrations up to 10 μg / ml did not affect cell proliferation, while concentrations exceeding 20 μg / ml inhibited cell growth.

[0115] like Figures 3-7 As shown, immortalized bone marrow-derived macrophages (iBMDMs) were treated with free fatty acid (FFA) to simulate lipotoxic stress. Both PGOMV and PDOMV significantly inhibited FFA-induced triglyceride (TG) lipid accumulation and the expression of pro-inflammatory cytokines IL-1β and TNF-α, while also significantly reducing intracellular reactive oxygen species (ROS) and mitochondrial ROS (mtROS) levels. These results indicate that PGOMV and PDOMV have a potent inhibitory effect on FFA-induced mitochondrial oxygen stress in macrophages, alleviating lipotoxic stress and blocking the vicious cycle of inflammation.

[0116] This invention uses the SPT inhibitor polysaccharin (1 μM, non-toxic to bacteria) to treat *Pleurobacterium kiwifruit* and extract OMVs (PGSPTi-OMV). Chromatographic analysis confirmed that polysaccharin reduced the SA level in PGSPTi-OMV, with the SA content in PGSPTi-OMV being 12.51 ± 0.42 ng / mg OMV, and the SA content in PGOMV being 58.725 ± 3.80 ng / mg (50-65 ng / mg) OMV. Figure 8 In the FFA-induced iBMDM model, only PGOMV (not PGSPTi-OMV) inhibited lipid accumulation (A). Figure 8 The expression of B) and the pro-inflammatory cytokine IL-6 ( Figure 8 (C). Crucially, exogenous SA (10 μM) supplementation restored the efficacy of PGSPTi-OMV to the PGOMV level (see C). Figure 8 (B and C), confirming that SA is a key active ingredient mediating metabolism and anti-inflammatory effects.

[0117] Cellular experiments showed that, within the concentration range of 15 μM, SA could promote the proliferation of iBMDMs cells. Figure 9), significantly inhibited FFA-induced intracellular TG accumulation ( Figure 10 ) and gene expression of the pro-inflammatory factor IL-1β ( Figure 11 Furthermore, SA can inhibit macrophage polarization towards the pro-inflammatory M1 phenotype. Figure 12 ), and reduce the accumulation of intracellular reactive oxygen species (ROS). Figure 13 This invention further explores the regulatory role of SA in mitochondrial homeostasis. The study found that SA significantly increases mitochondrial membrane potential (…). Figure 14 ), inhibiting mitochondrial ROS release ( Figure 15 ), and reduce the accumulation of lipid peroxidation product MDA ( Figure 16 Simultaneously, SA enhances glucose metabolism and increases cellular oxygen consumption rate (OCR). Figure 17-19 The results indicate that OMVs significantly improve mitochondrial respiratory efficiency. These findings suggest that OMVs provide experimental evidence for nanotargeted therapy of obesity-related metabolic inflammation through a multi-dimensional protective mechanism that mediates lipolysis, inhibits cellular inflammation, and promotes cellular antioxidant formation.

[0118] Furthermore, this invention also validated SA function in an LPS-induced bacterial infection inflammation model. In the bacterial infection model, PGOMV effectively reversed the LPS-stimulated expression of pro-inflammatory factors IL-1β and TNF-α mRNA (…). Figure 20-21 Simultaneously, flow cytometry results showed that LPS stimulation significantly increased both reactive oxygen species (ROS) and mitochondrial ROS in iBMDM cells, and co-incubation with PGOMV significantly inhibited the release of intracellular ROS. Figure 22 ) and the release of reactive oxygen species from mitochondria ( Figure 23 This demonstrates that PGOMV may enhance cellular antioxidant capacity by inhibiting mitochondrial oxidative stress-induced dysfunction. In conclusion, PGOMV can effectively inhibit inflammatory responses related to bacterial infection and metabolic imbalance, and enhance cellular anti-inflammatory and antioxidant effects.

[0119] SA belongs to the dihydrosphingosine class of compounds, and its chemical structure and information are as follows: Figure 24 As shown, it has a 17-carbon skeleton. Based on the significant inhibitory effect of SA on lipid accumulation in in vitro cell models, this invention will further verify its therapeutic effect in mice. The maximum absorption peak of SA was determined at 224 nm using UV-Vis full-wavelength scanning, and an absorbance-concentration standard curve was established. Figure 25 The SA was encapsulated into the PGOMV using electroporation, resulting in a PGOMV@SA with an average encapsulation yield (EE%) of 26.8%. Figure 26-27 ).

[0120] In a 12-week obesity model induced by a high-fat diet (HFD, 60% fat) in C57BL / 6J mice, an 8-week intervention was performed to evaluate the therapeutic effects of free SA and PGOMV@SA. Results showed that PGOMV@SA significantly inhibited weight gain, with better efficacy than free SA (…). Figure 28 Regarding metabolic indicators, both PGOMV@SA and free SA significantly reduced serum TG levels, but only PGOMV@SA significantly reduced total TC levels. Figures 29-30 The oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) further confirmed that PGOMV@SA effectively restores glucose homeostasis and improves insulin sensitivity. Figures 31-32 ).

[0121] PGOMV@SA treatment significantly reduced liver weight ( Figure 33 The significant reduction in serum AST and ALT levels indicates an improvement in liver damage. Figure 34 ), significantly reduced the infiltration of pro-inflammatory M1 macrophages (CD11b+F4 / 80+CD86+) in the spleen ( Figure 35 Histological analysis showed that it effectively inhibited HFD-induced lipid droplet accumulation and inflammation levels in hepatocytes. Figure 36 ).

[0122] In summary, the vesicle structure and efficient delivery capability of PGOMV significantly enhance the bioavailability and targeting of SA, thereby amplifying its regulatory effects on metabolism and inflammation. These findings provide a promising application strategy for the treatment of metabolic diseases using engineered bacterial outer membrane vesicle-based delivery systems.

[0123] Pg-OMVs, Pd-OMVs, and PG-OMVs@SA can be delivered via gavage, and experiments have demonstrated their stability in the digestive tract environment, indicating good oral potential. Compared to traditional probiotics, Pg-OMVs and Pd-OMVs avoid problems such as difficulty in live bacteria colonization, gene transfer, and immune risks. Compared to small molecule drugs, their components are natural and have multiple targets, making them suitable for adjunctive treatment of obesity, metabolic syndrome, non-alcoholic fatty liver disease, atherosclerosis, and inflammatory skin diseases associated with obesity (such as psoriasis and atopic dermatitis). They also provide a research foundation and technological reserves for the development of novel functional microbial preparations.

[0124] 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. An outer membrane vesicle containing sphingosine, characterized in that, The sphingosine content in the outer membrane vesicles is 50-65 ng / mg.

2. The outer membrane vesicle according to claim 1, characterized in that, The outer membrane vesicles are derived from parabacterium.

3. The outer membrane vesicle according to claim 2, characterized in that, The parabacteroides include Parabacteroides goldsteinii JCM 13446 or Parabacteroides distasonis ATCC 8503.

4. A method for preparing the outer membrane vesicles according to claim 1, characterized in that, The preparation method includes: culturing *Pseudomonas jini* or *Pseudomonas diffusa*, collecting the culture supernatant, and separating the outer membrane vesicles.

5. A composition, characterized in that, It comprises the outer membrane vesicles as described in any one of claims 1-3 and a pharmaceutically acceptable carrier.

6. The use of the outer membrane vesicles according to any one of claims 1-3 or the composition according to claim 5 in the preparation of a medicament for the prevention or treatment of metabolic diseases.

7. The application according to claim 6, characterized in that, The metabolic diseases mentioned include obesity, diabetes, hyperuricemia, hypertension, hyperlipidemia, hyperglycemia, metabolic syndrome, non-alcoholic fatty liver disease, insulin resistance, atherosclerosis, or inflammatory skin diseases.

8. The application according to claim 6, characterized in that, The drugs include drugs that inhibit lipid accumulation, anti-inflammatory drugs, antioxidant drugs, drugs that improve insulin sensitivity, or drugs that enhance mitochondrial function.

9. The application according to claim 6, characterized in that, The application also includes use in combination with metabolic drugs or anti-inflammatory drugs.

10. The application according to claim 9, characterized in that, The metabolic drugs include metformin or GLP-1 receptor agonists, and the anti-inflammatory drugs include IL-17 inhibitors.

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