Application of parabacteroides gore outer membrane vesicles in preparation of drugs for neuroprotection and / or anxiety disorder improvement

Abstract

The invention discloses an application of parabacteroides gore outer membrane vesicles in preparation of drugs for neuroprotection and / or anxiety disorder improvement. Researches show that the parabacteroides gore outer membrane vesicles can effectively antagonize corticosterone-induced nerve cell injury, can relieve anxiety-like behaviors of chronic constraint stress mice, and have good neuroprotection and anxiety-resistant activity. The invention provides a new drug choice for clinically treating nerve injury and relieving anxiety disorder, and has a wide application prospect in the aspect of treating nervous system diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the use of *Pseudomonas gondii* outer membrane vesicles in the preparation of drugs for neuroprotection and / or improvement of anxiety disorders. Background Technology

[0002] With the acceleration of industrialization, intensified social competition, and the accumulation of diverse stressors such as information overload in the digital age, anxiety disorders have become one of the most prevalent mental illnesses globally. According to data from the Global Burden of Disease (GBD) study, the global incidence of anxiety disorders has been rising steadily over the past decade, and the affected population is showing a significant trend towards younger ages.

[0003] Clinical and basic research has confirmed that anxiety disorder is not simply a disorder of mood regulation, but a complex brain disease involving the disorder of the neuro-endocrine-immune network (NEI network). Long-term chronic anxiety can induce damage to the central nervous system through multiple pathological pathways: (1) Overactivation of the hypothalamic-pituitary-adrenal axis (HPA axis) leads to sustained high expression of glucocorticoids (GC), which can inhibit the regeneration of hippocampal neurons and promote neuronal apoptosis by regulating the glucocorticoid receptor (GR) signaling pathway, while damaging the synaptic transmission function of the amygdala mood regulation loop; (2) Enhanced neuroinflammatory response, excessive activation of microglia and release of pro-inflammatory factors such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), which damage the integrity of the blood-brain barrier and aggravate the disorder of the central nervous microenvironment; (3) Oxidative stress imbalance leads to the accumulation of large amounts of reactive oxygen species (ROS), which damages the mitochondrial function of neurons and reduces the expression level of synaptic plasticity-related proteins (such as BDNF). These pathological changes can further accelerate the progression of neurodegenerative diseases, forming a vicious cycle of chronic anxiety-HPA axis activation / neuritis / oxidative stress-neuronal damage-intensification of anxiety symptoms. This not only severely reduces patients' quality of life, cognitive function, and social adaptability, but also significantly increases the risk of complications such as depression and Alzheimer's disease, placing a heavy medical burden and economic pressure on families and society.

[0004] While current medications for treating anxiety disorders can alleviate symptoms to some extent, their limitations and inherent drawbacks have become key bottlenecks restricting treatment effectiveness. Mainstream drugs, such as selective serotonin reuptake inhibitors (SSRIs) and serotonin-norepinephrine reuptake inhibitors (SNRIs), generally suffer from slow onset of action, often requiring continuous use to achieve significant efficacy. During this period, patients' anxiety symptoms are difficult to control promptly, and some patients may even lose confidence in treatment due to the delayed effect. Furthermore, these drugs have a high incidence of side effects; common adverse reactions such as nausea, headache, insomnia, and sexual dysfunction not only reduce patient adherence but, in severe cases, necessitate adjustments to the medication regimen, affecting treatment continuity. In addition, while benzodiazepines have a faster onset of action, long-term use easily leads to tolerance and dependence, and withdrawal symptoms may occur upon discontinuation. They also pose certain cognitive impairments, limiting their long-term use. Therefore, there is an urgent need to develop novel therapeutic drugs with neuroprotective effects and anxiety-relieving properties.

[0005] Extracellular vesicles (EVs) are nanoscale particulate structures encased in a lipid bilayer, secreted by bacteria or eukaryotic cells. Currently, EVs produced in Gram-negative bacteria are called bacterial outer membrane vesicles (OMVs). OMVs are tiny spherical structures released from the bacterial outer membrane, ranging in diameter from 20 to 250 nm. They are primarily derived from the outer membrane and composed of various biomolecules such as lipids, proteins, phospholipids, DNA, RNA, and periplasmic proteins. Furthermore, OMVs carry various bacterial virulence components and immunostimulatory molecules, such as lipopolysaccharide (LPS) and virulence factors. These characteristics enable OMVs to play a crucial role in bacterial-host interactions, especially in the occurrence and development of infectious diseases. As research into OMVs deepens, their immense application potential in the biomedical field has been discovered. Bacterial vaccines based on OMVs have already been successfully developed, and in addition to vaccines, OMVs are increasingly being used as adjuvants, drug delivery carriers, cancer immunotherapy agents, and novel antibacterial agents.

[0006] In recent years, the gut-brain axis mechanism has become a research hotspot in the field of neurological diseases. Extracellular vesicles, due to their ability to mediate intercellular signal transduction and easily cross biological barriers, have shown potential application value in the treatment of gut-brain axis-related diseases. However, research on extracellular vesicles derived from gut symbiotic bacteria is still relatively scarce, their biological activity has not been systematically developed, and their application in disease treatment has not been fully explored. Summary of the Invention

[0007] To overcome the aforementioned defects and deficiencies in the prior art, this invention provides the application of *Pseudomonas gondii* outer membrane vesicles in the preparation of drugs for neuroprotection and / or improvement of anxiety disorders.

[0008] The first objective of this invention is to provide the use of the outer membrane vesicles of *Pseudomonas gondii* as an active ingredient in the preparation of neuroprotective drugs.

[0009] A second objective of this invention is to provide the use of the outer membrane vesicles of *Pseudomonas gondii* as an active ingredient in the preparation of medicaments for improving anxiety disorders.

[0010] A third objective of this invention is to provide a neuroprotective drug.

[0011] A fourth objective of this invention is to provide a medicine for improving anxiety disorders.

[0012] This invention claims protection for the following: Application of outer membrane vesicles of *Pseudomonas gondii* as an active ingredient in the preparation of neuroprotective drugs.

[0013] Preferably, the *Pseudomonas gossypii* is *Pseudomonas gossypii* ATCC BAA-1180.

[0014] Application of outer membrane vesicles of *Pseudomonas gondii* as an active ingredient in the preparation of drugs to improve anxiety disorders.

[0015] Preferably, the *Pseudomonas gossypii* is *Pseudomonas gossypii* ATCC BAA-1180.

[0016] As one possible approach, the drug may also include pharmaceutically acceptable excipients.

[0017] As one possible approach, the dosage form of the drug includes, but is not limited to, tablets, capsules, granules, pills, powders, and mixtures.

[0018] A neuroprotective drug with outer membrane vesicles of *Pseudomonas gondii* as its active ingredient.

[0019] A drug to improve anxiety disorder, using the outer membrane vesicles of *Pseudomonas gondii* as the active ingredient.

[0020] Preferably, the *Pseudomonas gossypii* is *Pseudomonas gossypii* ATCC BAA-1180.

[0021] As one possible approach, the drug may also include pharmaceutically acceptable excipients.

[0022] Compared with the prior art, the present invention has the following beneficial effects: This invention discloses the application of *Pseudomonas gondii* outer membrane vesicles in the preparation of drugs for neuroprotection and / or improvement of anxiety disorders. The study shows that *Pseudomonas gondii* outer membrane vesicles can effectively antagonize corticosterone-induced neuronal damage and alleviate anxiety-like behavior in chronically restrained stress mice, exhibiting good neuroprotective and anti-anxiety activity. This invention provides a new drug option for the clinical treatment of nerve damage and anxiety disorders, and has broad application prospects in the treatment of neurological diseases.

[0023] Meanwhile, the *Pseudomonas gondii* of this invention is a human intestinal commensal bacterium, which has fewer adverse reactions and higher patient tolerance compared to chemical drugs. Attached Figure Description

[0024] Figure 1 Characterization results of outer membrane vesicles of *Pseudomonas gondii*; A: Transmission electron microscopy scan results; B: Particle size distribution of outer membrane vesicles of *Pseudomonas gondii*.

[0025] Figure 2 To investigate the protective effect of outer membrane vesicle treatment of *Pseudomonas gossyflores* against corticosterone-induced HT22 cell damage.

[0026] Figure 3 The effect of *Pseudomonas gossypii* outer membrane vesicle treatment on the improvement of anxiety in mice induced by chronic restraint stress; A: activity time in the central region; B: total activity distance. Detailed Implementation

[0027] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0028] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0029] The *Pseudomonas gondii* used in the examples is *Pseudomonas gondii* (… Parabacteroides goldsteinii )ATCCBAA-1180.

[0030] Example 1: Preparation and characterization of outer membrane vesicles of *Pseudomonas gondii* I. Experimental Methods 1. Preparation of outer membrane vesicles of *Pseudomonas gondii* Prepare the culture medium: 41.7 g / L modified GAM medium, 15 g / L agar powder, then autoclave at 121℃ for 15 min, then pour the plates to obtain culture plates.

[0031] *Pseudomonas gondii* was spread onto culture plates and anaerobically cultured at 37°C for 48 h. After culture, colonies were eluted with 30 mL of PBS buffer to obtain a bacterial suspension. The suspension was centrifuged at 20,000 g for 30 min at 4°C, and the supernatant was filtered through a 0.22 μm filter to obtain the filtrate. 30 mL of the filtrate was extracted using an EXODUS H-600 automated exosome extraction system. Finally, the chip was washed with 400 μL of PBS buffer, and the eluent was collected to obtain the purified *Pseudomonas gondii* outer membrane vesicle (PG-OMVs) solution.

[0032] 2. Characterization of outer membrane vesicles of *Pseudomonas gondii* (1) Observation by transmission electron microscopy ① Take 10 μL of PG-OMVs solution and drop it onto the prepared Parafilm sealing film (the back of the Parafilm sealing film is attached to the table). Place the copper mesh of the carrier film face down and let it naturally absorb the suspension droplets for 15 min. Then use filter paper strips to absorb the excess droplets and let the copper mesh dry slightly. ② Take 10 μL of 2% phosphotungstic acid solution (w / v) as the staining solution and drop it onto Parafilm sealing film. Place the copper mesh face down with the staining solution and invert it to stand for 5 min. Use filter paper strips to absorb excess droplets, dry the copper mesh under an incandescent lamp, and observe and photograph it under a transmission electron microscope.

[0033] (2) Particle size and concentration The particle size distribution and concentration of PG-OMVs were determined using a nanoflow cytometer (NanoFCM, Flow NanoAnalyzer U30E).

[0034] II. Experimental Results Transmission electron microscopy scanning results as follows Figure 1 As shown in A, the results indicate that PG-OMVs exhibit a typical saucer-like vesicle structure, consistent with the morphological characteristics of bacterial outer membrane vesicles.

[0035] Particle size and concentration analysis results showed that ( Figure 1 The particle concentration of PG-OMVs (B) was 7.6 × 10⁻⁶. 7 particles / mL (dilution factor: 100), equivalent to an original concentration of 7.6 × 10⁻⁶. 9 The median particle size of PG-OMVs was 145.5 nm, indicating that PG-OMVs with intact morphology and concentrated particle size distribution were successfully prepared.

[0036] Example 2: Protective effect of outer membrane vesicles of *Pseudomonas griseus* on nerve cells I. Experimental Methods Place the frozen HT22 cell tubes in a 37°C water bath and shake rapidly until they are the size of soybeans. Transfer them to 15 mL centrifuge tubes, centrifuge at 800 rpm for 4 min, collect the pellet, resuspend it in complete culture medium (DMEM + 10% FBS + 1% penicillin-streptomycin, v / v), add it to a culture dish, and incubate at 37°C and 5% CO2 for 36 h.

[0037] When the cell aggregation reaches 75%, discard the culture medium, wash twice with PBS buffer, add 1 mL of 0.25% trypsin solution (v / v) and digest at 37°C for 40 s, add complete culture medium to stop digestion, pipette and centrifuge at 800 rpm for 4 min, resuspend the precipitate and add fresh complete culture medium to continue culturing.

[0038] HT22 cell suspension was seeded at 100 μL per well in a 96-well plate. After complete cell adhesion, cells were divided into the following groups for treatment: Control group: Replace 100 μL of complete culture medium in each well (this group contains no cells and is not treated with any drugs). Control group (con): 100 μL of complete culture medium was replaced in each well to maintain normal HT22 cell culture without drug intervention; Corticosterone group (cort): 100 μL of complete culture medium was replaced in each well, and corticosterone was added to a final concentration of 150 μM after 2 h. Outer membrane vesicle group (pg omv): Discard the original culture medium and add 100 μL of PG-OMVs (prepared in Example 1) solution diluted 100 times with complete culture medium to each well; Corticosterone + outer membrane vesicle treatment group (cort + pg omv): The original culture medium was discarded, and 100 μL of PG-OMVs (prepared in Example 1) solution diluted 100 times with complete culture medium was added to each well. After 2 h, corticosterone was added to a final concentration of 150 μM.

[0039] After culturing for another 24 h, cell viability was assessed using a CCK-8 assay kit. 10 μL of CCK-8 solution was added to each well, and the cells were incubated in the dark for 1.5 h. The absorbance at 450 nm was then measured using a microplate reader, and cell viability was calculated using the formula: Cell viability (%) = [(experimental wells - blank wells) / (control wells - blank wells)] × 100%.

[0040] II. Experimental Results The results are as follows Figure 2As shown, corticosterone treatment significantly reduced the survival rate of HT22 cells (P<0.05). Treatment with PG-OMVs alone had no significant effect on cell survival, but PG-OMVs treatment effectively reversed the corticosterone-induced decrease in cell survival (P<0.05). These results indicate that PG-OMVs have a significant protective effect against corticosterone-induced neuronal damage.

[0041] Example 3: The ameliorative effect of *Pseudomonas gondii* outer membrane vesicles on anxiety in mice. I. Experimental Methods 1. Laboratory animals and grouping Three-week-old male C57BL / 6J mice were acclimatized for one week and then randomly divided into three groups: CON, PBS, and PG, with nine mice in each group.

[0042] 2. Intervention Program Starting two days before the chronic restraint stress (CRS) model was established, all mice were administered the drug by gavage every two days until the CRS model was completed. The PG group was administered PG-OMVs (0.5 mg / kg, prepared in Example 1) by gavage, while the CON and PBS groups were administered an equal volume of PBS by gavage. The PBS and PG groups received restraint stimulation for 6 hours daily in the restraint tube for 14 days. During restraint, the mice only experienced limited movement; the restraint tube did not compress the body or tail. All mice were deprived of food and water during the restraint period, and were provided with normal water and food after each day's restraint.

[0043] Open field experiments were conducted after CRS modeling and drug administration.

[0044] 3. Open field experiment During the experiment, mice were removed from their cages and placed in the center of a 50 cm × 50 cm × 30 cm white open area, facing away from the experimenter. They were allowed to move freely for 5 minutes. VisuTrack animal behavior analysis software was used to monitor the mice's movement in real time. After each mouse's experiment, their feces and urine were cleaned up, and 75% alcohol (v / v) was sprayed into the open area to completely remove any odor left by the mice. The next round of the experiment was conducted after the alcohol had completely evaporated. The time spent in the central area and the total distance traveled within 5 minutes were recorded.

[0045] II. Experimental Results The central region activity time of mice in each group is as follows: Figure 3 As shown in A, compared with CON, the activity time of mice in the PBS group was significantly shorter in the central region (P<0.05), while the activity time of mice in the PG group was not significantly different from that in the CON group and was significantly higher than that in the PBS group (P<0.05).

[0046] There was no statistically significant difference in the total activity distance of the three groups of mice within 5 minutes. Figure 3 (B in the figure) indicates that the restraint stimulus did not affect the basic motor ability.

[0047] The results above indicate that restraint stimulation can induce anxiety-like behavior in mice, while PG-OMVs can effectively alleviate this behavior, suggesting that the outer membrane vesicles of *Pseudomonas gossypii* have anti-anxiety activity.

[0048] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Use of outer membrane vesicles of Parabacteroides goldsteinii as an active ingredient in the preparation of a medicament for neuroprotection.

2. Use according to claim 1, characterized in that, The Parabacteroides goldsteinii is Parabacteroides goldsteinii ATCC BAA-1180.

3. Use of outer membrane vesicles of Parabacteroides goldsteinii as an active ingredient in the preparation of a medicament for improving anxiety.

4. Use according to claim 3, characterized in that, The Parabacteroides goldsteinii is Parabacteroides goldsteinii ATCC BAA-1180.

5. Use according to any one of claims 1 to 4, characterized in that, The medicament further comprises a pharmaceutically acceptable adjuvant.

6. Use according to any one of claims 1 to 4, characterized in that, The dosage form of the medicament includes, but is not limited to, tablets, capsules, granules, pills, powders, and mixtures.

7. A medicament for protecting nerves, characterized by comprising a compound of the formula (I) as an active ingredient. The outer membrane vesicles of Parabacteroides goldsteinii are used as an active ingredient.

8. A medicament for ameliorating anxiety, characterized by comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof. The outer membrane vesicles of Parabacteroides goldsteinii are used as an active ingredient.

9. The medicament according to claim 7 or 8, characterized in that, The Parabacteroides goldsteinii is Parabacteroides goldsteinii ATCC BAA-1180.

10. The medicament according to claim 7 or 8, characterized in that, The medicament further comprises a pharmaceutically acceptable adjuvant.

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