Application of Bacteroides monomorpha CGMCC No. 30981 in the preparation of asthma treatment drugs

Abstract

This invention discloses a strain of *Bacteroides monomorpha*, with accession number CGMCC No. 30981, accession date June 17, 2024, and accession classification name *Bacteroides monomorpha*. Bacteroides uniformis The depositary institution is the China General Microbiological Culture Collection Center. The strain provided by this invention has significant effects in reducing inflammatory infiltration of lung tissue caused by bronchial asthma, reducing the release of serum IgE and Th2 cytokines, inhibiting the activation of NLRP3 inflammasomes, and improving allergic airway inflammation.

Description

Technical Field

[0001] This invention relates to the application of Bacteroides monomorpha CGMCC No.30981 in the preparation of asthma treatment drugs, and belongs to the field of microbiology. Background Technology

[0002] Bronchial asthma, or simply asthma, is a heterogeneous chronic inflammatory disease. The typical characteristics of asthma are varying degrees of airflow limitation, accompanied by recurrent episodes of wheezing, shortness of breath, coughing, and chest tightness. As the disease progresses, it can lead to airway remodeling. Asthma is one of the most serious chronic diseases threatening public health worldwide.

[0003] The heterogeneity of asthma manifests in its diverse etiologies, pathogenic mechanisms, clinical symptoms, and severity. According to the latest Global Initiative for Asthma (GINA) guidelines, asthma can be classified into type 2 and non-type 2 based on the presence or absence of type 2 (T2) inflammation. This classification represents different molecular mechanisms and is called endotype. Type 2 asthma is the most common, accounting for 64%-73% of all asthma cases. It is primarily characterized by an increase in eosinophils and a significant increase in the levels of Th2 helper cell cytokines interleukin-4 (IL-4), IL-5, and IL-13. Th2 cytokines promote eosinophilia and lead to B cell activation into plasma cells, releasing large amounts of specific immunoglobulin E (IgE). IgE promotes mast cell degranulation, releasing leukotrienes, histamine, and other factors, causing airway smooth muscle contraction and spasm, resulting in asthma symptoms. This type of asthma usually responds well to glucocorticoids or novel biologics, but has certain side effects. Non-type 2 asthma is more common in obesity-related asthma, neutrophilic asthma, and oligogranulocytic asthma. It is characterized by Th1 and Th17 cell and neutrophil infiltration, and excessive production of NLR family Pyrin Domain Containing Protein 3 (NLRP3), as well as type I interferon, IL-1β, and IL-17. This type of asthma usually responds poorly to steroids and other medications. Therefore, finding anti-asthma interventions without side effects is essential.

[0004] Symbiotic flora are essential for maintaining the body's immune function. Studies have shown that resident symbiotic communities can influence and control basophil responses, thereby affecting the body's response to environmental allergens. Therefore, guiding the development of immune function and regulating the adaptive immune system through the administration of specific symbiotics is of great significance for the potential prevention or treatment of allergic diseases. For example, Vadim Pivniouk et al. found that intranasal administration of standardized human airway bacterial lysate OM-85 upregulated the proportion of activated regulatory T cells and attenuated dendritic cell responses, protecting allergen-sensitive animals from airway inflammation; Bilal Alashkar Alhamwe et al. reported that the symbiotic Acinetobacter lophiae can activate the epigenetics of T cells through the IL-6 / IL-10 axis, affecting the composition of the gut microbiota and exerting a protective effect against asthma. However, current research in this area is limited, and the number and types of probiotic or symbiotic strains with anti-asthmatic functions reported are limited, requiring further exploration.

[0005] Bacteroides monomorpha, also known as common Bacteroides, is a Gram-negative obligate anaerobic commensal bacterium involved in anti-inflammatory, metabolic, and immune regulation processes. Wenhui Pang et al. found that dysbiosis occurred in the gut microbiota of patients with allergic airway inflammation and mice, with a decrease in the biodiversity and number of Bacteroides. Oral administration of Bacteroides polymorpha could improve ovalbumin (OVA)-induced airway hyperresponsiveness in mice, reduce the secretion of Th2 cytokines in bronchoalveolar lavage fluid, and alleviate airway inflammation.

[0006] NOD-like receptor thermal protein domain associated protein 3 (NLRP3) is a cytoplasmic complex that plays a crucial role in inflammatory responses. Activation of the NLRP3 inflammasome leads to the maturation and secretion of pro-inflammatory cytokines IL-1β and IL-18, which play key roles in airway inflammation in asthma. In asthma patients, NLRP3 inflammasome activation is associated with exacerbation of airway inflammation. NLRP3 inflammasome activation can occur through multiple pathways, including activation of pattern recognition receptors (PRRs), such as pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). Activated NLRP3 inflammasomes promote the maturation of IL-1β and IL-18, cytokines that can induce Th2 cell differentiation, promote airway inflammation, and increase airway hyperreactivity (AHR). The role of the NLRP3 inflammasome in asthma extends beyond promoting the release of inflammatory factors; it also involves airway epithelial cell damage and airway remodeling. Airway epithelial damage is a key marker of asthma pathogenesis, and activation of the NLRP3 inflammasome can exacerbate this damage. Furthermore, NLRP3 inflammasome activation is associated with pyroptosis, a programmed cell death mechanism that leads to cell membrane perforation, releasing inflammatory factors extracellularly and further promoting the inflammatory response. The role of the NLRP3 inflammasome varies across different asthma subtypes. In type 2 asthma, NLRP3 inflammasome activation is associated with eosinophil recruitment and activation, while in non-type 2 asthma, it is associated with neutrophil recruitment and activation. In summary, the NLRP3 inflammasome plays a multifaceted role in the pathogenesis of asthma, including promoting the release of inflammatory factors, exacerbating airway epithelial cell damage, and promoting pyroptosis. Therefore, the NLRP3 inflammasome is considered a potential therapeutic target for asthma, and its inhibitors may be significant for controlling asthma symptoms and improving patient prognosis.

[0007] The purpose of this invention is to provide the use of a strain or preparation of Bacteroides monomorpha with anti-asthmatic efficacy. The Bacteroides monomorpha IM01 of this invention has been verified to have anti-asthmatic efficacy, which can relieve asthma symptoms, reduce pulmonary inflammatory infiltration and airway mucus production, reduce pulmonary Th2 cytokine and serum IgE levels, and inhibit the activation of the pulmonary NLRP3 signaling pathway. Summary of the Invention

[0008] Based on the above-mentioned objectives, this invention first provides a strain of *Bacteroides monomorpha*, IM01 (hereinafter referred to as IM01), with accession number CGMCC No. 30981, accession date June 17, 2024, and accession classification name *Bacteroides monomorpha*. Bacteroides uniformis The depository is the China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Culture Collections. The strain was isolated from the feces of healthy individuals.

[0009] In a preferred embodiment, the 16S rRNA sequence of the strain is shown in SEQ ID NO.1.

[0010] Secondly, the present invention provides the application of the above-mentioned strains in the preparation of drugs for the treatment or prevention of allergic airway inflammation.

[0011] In one alternative implementation, the allergic airway inflammation is asthma.

[0012] In another alternative implementation, the asthma is accompanied by elevated serum IgE and / or airway Th2 cytokines.

[0013] In another alternative implementation, the asthma is accompanied by airway inflammatory cell infiltration, goblet cell proliferation, mucus production, and collagen deposition.

[0014] In another alternative implementation, the asthma is accompanied by activation of the NLRP3 inflammasome signaling pathway in the airway.

[0015] Third, the present invention provides a composition containing the above-mentioned strain, the composition containing a pharmaceutically acceptable carrier and / or excipient, the strain being used in combination with other ingredients to assist and / or enhance the pharmacodynamic effects of asthma.

[0016] In an alternative embodiment, the composition contains probiotics, plant extracts, chemical molecules, protein molecules, and nucleic acid molecules that have therapeutic effects on asthma.

[0017] In a more preferred embodiment, the composition is prepared as capsules, lyophilized powder, suspension, or tablets.

[0018] This invention relates to a strain of Bacteroides monomorpha that has anti-asthma properties, isolated and purified from the feces of healthy individuals. Experiments have demonstrated that the isolated strain... Bacteroides uniformis The IM01 strain has the function of improving asthma. Bacteroides uniformisThe IM01 strain can effectively reduce serum IgE levels in asthma model mice, reduce the production of Th2 cytokines in the lungs, alleviate inflammatory cell infiltration and airway mucus production in the lungs, and inhibit the activation of NLRP3 inflammasomes in the lungs, showing excellent application prospects in the preparation of drugs for the treatment and / or prevention of asthma. Attached Figure Description

[0019] Figure 1 This is the animal experimental protocol used in this invention;

[0020] Figure 2 The results showed that oral administration of IM01 significantly reduced the number of lung cells in the OVA mouse model. The horizontal axis represents different animal treatment groups, and the vertical axis represents the number of cells. * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001.

[0021] Figures 3-6 The results showed that oral administration of IM01 significantly reduced the percentage of eosinophils in the lungs of OVA mouse models, but did not affect the percentage of basophils, neutrophils, and monocytes. The horizontal axis represents different groups, and the vertical axis represents the percentage of cells. * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001.

[0022] Figure 3 Effect of oral administration of IM01 on the percentage of eosinophils in total white blood cells in mouse lung tissue;

[0023] Figure 4 Effect of oral administration of IM01 on the percentage of basophils in total white blood cells in mouse lung tissue;

[0024] Figure 5 Effect of oral administration of IM01 on the percentage of neutrophils in total white blood cells in mouse lung tissue;

[0025] Figure 6 Effect of oral administration of IM01 on the percentage of monocytes in total white blood cells in mouse lung tissue;

[0026] Figure 7 The results showed that oral administration of IM01 significantly reduced serum IgE levels in the OVA mouse model. The x-axis represents different animal treatment groups, and the y-axis represents serum IgE content. * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001.

[0027] Figure 8-12Oral administration of IM01 significantly reduced the levels of IL-4, IL-5, and IL-13 in the lungs of OVA mouse models. A represents IL-4 release; B represents IL-5 release; and C represents IL-13 release. The x-axis represents different animal treatment groups, and the y-axis represents the corresponding cytokine levels. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0028] Figure 8 Effects of oral administration of IM01 on IL-4 levels in mouse lung tissue;

[0029] Figure 9 Effects of oral administration of IM01 on IL-5 levels in mouse lung tissue;

[0030] Figure 10 Effects of oral administration of IM01 on IL-13 levels in mouse lung tissue;

[0031] Figure 11 Effects of oral administration of IM01 on IL-10 levels in mouse lung tissue;

[0032] Figure 12 Effects of oral administration of IM01 on IL-TGF-β levels in mouse lung tissue;

[0033] Figure 13 The results of H&E staining of lung tissue sections from mice in different groups after oral administration of IM01 are shown in (I).

[0034] Figure 14 The results of H&E staining of lung tissue sections from mice in each group after oral administration of IM01 are shown in Part II.

[0035] Figure 15 The results of H&E staining of lung tissue sections from mice in each group after oral administration of IM01 are shown in Part III.

[0036] Figure 16 The results of PAS staining of lung tissue sections from mice in different groups after oral administration of IM01 are shown in (I).

[0037] Figure 17 The results of PAS staining of lung tissue sections from mice in different groups after oral administration of IM01 are shown in Part II.

[0038] Figure 18 The results of PAS staining of lung tissue sections from mice in each group after oral administration of IM01 are shown in Part III.

[0039] Figure 19 The results of MASSON staining of lung tissue sections from mice in each group after oral administration of IM01 are shown in (I).

[0040] Figure 20The results of MASSON staining of lung tissue sections from mice in each group after oral administration of IM01 are shown in Part II.

[0041] Figure 21 The results of MASSON staining of lung tissue sections from different groups of mice after oral administration of IM01 are shown in Part III.

[0042] Figure 22 This shows the effect of oral administration of IM01 on the absolute expression levels of NLRP3, Caspase1 p20, and Phospho-NF-κB p65 proteins in the lungs of an OVA mouse model, where A represents the absolute expression levels of NLRP3, Caspase1 p20, and Phospho-NF-κB p65 proteins in the NLRP3 signaling pathway of mouse lung tissue.

[0043] Figure 23 This diagram shows the effect of oral administration of IM01 on the relative expression levels of NLRP3, Caspase1 p20, and Phospho-NF-κBp65 proteins in the lungs of an OVA mouse model compared to the internal reference gene GAPDH. The horizontal axis represents different genes, and the vertical axis represents the relative protein expression levels compared to GAPDH. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001. Detailed Implementation

[0044] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result of the description. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0045] Example 1. Isolation, preservation and identification of Bacteroides monomorpha IM01 strain

[0046] 1.1 Isolation of Bacteroides monomorpha IM01 strain

[0047] (1) Take an appropriate amount of fecal sample from a healthy person and add it to an EP tube pre-filled with 900 μL of sterile PBS. Perform serial dilutions on the sample to dilute the extract to a concentration of 10. -6 times;

[0048] (2) Take 100 μL of samples with different dilutions and spread them on BHI solid culture medium and place them in an incubator;

[0049] (3) Incubate at 37 ℃ and 0.5% CO2 for 48 h;

[0050] (4) Take out the culture dish, pick up colonies with different morphological characteristics with a sterile inoculation loop, transfer them to a new BHI solid medium for purification, anaerobic culture at 37℃ for 48 h, transfer 3 times consecutively, culture the purified strain in liquid BHI at pH=3.5, and screen out strains with good growth for use in experiments or cryopreservation.

[0051] 1.2 Preservation of microbial strains

[0052] In our laboratory, BHI medium containing 25% glycerol was used as the preservation solution for cryopreservation of bacterial strains, as follows:

[0053] (1) The 2 mL sterilization tubes were sterilized at 121°C for 15 min before use;

[0054] (2) After the bacteria were transferred three times on BHI solid medium, 1.5 mL of sterile bacterial preservation solution was added to the culture dish;

[0055] (3) Use an L-shaped rod to scrape the culture dish to ensure that the colonies are fully integrated into the preservation solution;

[0056] (4) Transfer the bacterial solution to a preservation tube, mix well, and store at -80℃.

[0057] 1.3 Observation of colony appearance and cell morphology

[0058] Bacteroides monomorpha are non-spore-forming, non-motile, obligate anaerobic, Gram-negative bacteria of varying lengths, with grayish-white, semi-transparent colonies. Under normal circumstances, they reside in the intestines, oral cavity, upper respiratory tract, and reproductive tract of humans and animals.

[0059] 1.4 Extraction of total bacterial DNA

[0060] Single colonies were inoculated onto BHI solid medium and cultured anaerobically overnight at 37°C. DNA was extracted according to the instructions of the bacterial genomic DNA extraction kit (Nanjing Novizan FastPure® DNA Extraction Kit).

[0061] 1.5 Accurate identification of strains by comparing ANI and DDH with the model strain

[0062] The genome of this strain was extracted and sequenced using a draft sequence. Genomic correlation analysis (digital DNA-DNA hybridization (dDDH; dDDH < 70%) and average nucleotide identity (ANI; ANI < 95%)) was performed by comparing it with the type strain to further accurately identify the strain. Both methods are the gold standard for identifying prokaryotic species and were performed online (dDDH, http: / / ggdc.dsmz.de; ANI, http: / / enve-omics.ce.gatech.edu / ani / ).

[0063] 1.5.1 Genome Extraction and Draft Submission

[0064] This invention uses a DNA extraction kit produced by Nanjing Novizan Biotechnology Co., Ltd. for gene extraction. The specific operation steps are as follows:

[0065] (1) Add 20 μl of proteinase K solution to the bacterial precipitate and shake to remove protein impurities; then add 200 μl of buffer BCL and shake to mix; then incubate at 55°C for 10 min and invert to mix to ensure that there are no impurities precipitating in the solution.

[0066] (2) Add 150 μl of anhydrous ethanol and shake to mix. During this process, flocculent precipitate will be generated. Transfer all liquid and flocculent matter in the EP tube to the FastPure gDNA Mini Columns II adsorption column, centrifuge at 12000 rpm for 30 s, and discard the waste liquid.

[0067] (3) Place the adsorption column into the waste liquid collection tube, add 500 μl of buffer WA to the adsorption column, centrifuge at 12000 rpm for 1 min, and discard the waste liquid.

[0068] (4) Add 600 μl of WB buffer, centrifuge at 12000 rpm for 30 s, discard the waste liquid, put it into a new collection tube, centrifuge at 12000 rpm for 2 min. This step is repeated twice to ensure that the DNA is washed clean.

[0069] (5) Place the adsorption column in the collection tube, open the cap and let it air dry at room temperature for 3-5 minutes. The purpose is to allow the residual rinsing solution (mainly ethanol) in the adsorption column to evaporate completely.

[0070] (6) Transfer the adsorption column to a new EP tube, add 200 μl of Elution Buffer (preheated to 55°C) to the adsorption column membrane, incubate at room temperature for 2-5 min, centrifuge at 12000 rpm for 1 min to obtain purified bacterial genomic DNA. Transport via cold chain to Beijing Tianyi Huiyuan Biotechnology Co., Ltd. to complete the draft sequencing of the genome.

[0071] 1.5.2 Genomic Correlation Analysis

[0072] (1) Basic characteristics of the genome

[0073] The draft results show that the genome size of strain IM01 is 3540728 bp and the GC content is 46.645%.

[0074] (2) dDDH and ANI

[0075] Select a model strain of Bacteroides monomorpha. Bacteroides uniformis The dDDH and ANI values ​​of strain CL03T12C37 and strain IM01 were compared. The results showed that the dDDH and ANI values ​​of the type strain CL03T12C37 and strain IM01 were 81.00% and 97.67%, respectively, supporting that the two strains belong to the same bacterial species.

[0076] (3) 16S RNA sequencing

[0077] A single colony of this bacterium was picked and added to 10 mL of BHI liquid medium, incubated overnight at 37°C with shaking, and then transported via cold chain to Beijing Tianyi Huiyuan Biotechnology Co., Ltd. for 16S RNA sequencing. The 16S RNA sequence of strain IM01 is shown in SEQ ID NO.1.

[0078] The preservation information for this strain is as follows: strain accession number CGMCC NO.30981, preservation date June 17, 2024, and preservation classification name. Bacteroides uniformis Strain IM01 is deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China Academy of Microbiology, Postcode: 100101, Tel: 8610-64807355.

[0079] Example 2: Evaluation of the anti-asthma function of monomorphic bacillus strain IM01

[0080] 2.1 Sample: Logarithmic growth phase culture of Bacteroides monomorpha strain MY2024 after activation.

[0081] 2.2 Construction of experimental animals and OVA animal models

[0082] Five-week-old female BALB / c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., with a body weight range of 13 - 15 g. The license number was SCXK (Beijing) 2064 - 0006. The animals were housed at the Laboratory Animal Center of the Chinese Center for Disease Control and Prevention, with the license number SYXK (Beijing) 2017 - 0021. The housing level was a barrier environment with a 12-hour light / dark cycle, and the mice had free access to water and food. All animal experiments were conducted in accordance with the guiding principles of laboratory animal welfare ethics of the Laboratory Animal Center of the Chinese Center for Disease Control and Prevention (approval number: 2023 - 025).

[0083] According to the experimental requirements, the mice were randomly divided into three groups: a normal group (PBS group), a model group (OVA group), and a probiotic intervention group (IM01 group), with 8 mice in each group. After one week of adaptation, mice in the PBS group and the OVA group were gavaged with 200 μL of PBS per mouse daily from 5 days before infection (day -5) until 16 days after infection (the day of infection was recorded as day 0). Mice in the IM01 group were gavaged with 200 μL of the IM01 bacterial suspension (1×10 9 CFU / mouse) during the same period. On days 0 and 7, mice in the OVA group and the IM01 group were intraperitoneally injected with 100 μL of ovalbumin suspension (prepared on the same day, containing 100 μg OVA) for sensitization, and mice in the PBS group were intraperitoneally injected with an equal volume of PBS. From days 14 to 16, mice in the OVA group and the IM01 group were challenged with asthma by aerosol inhalation of 1% OVA solution for 20 minutes each time, and mice in the PBS group were aerosolized with normal saline as a substitute.

[0084] 2.3 Sample collection: The mice were sacrificed 24 hours after the last aerosol challenge, and mouse serum and lung tissues were collected.

[0085] (1) Blood sample collection

[0086] Blood was collected from the orbital cavity. The mouse whiskers were cut off to avoid hemolysis; the mouse eyeballs were quickly removed using curved forceps, and the whole blood of the mouse was collected with a 1.5 mL sterile EP tube. After collection, the blood was incubated in a 37 °C water bath for 1 h, centrifuged at 3500 rpm for 15 min, the serum was aliquoted, labeled, and stored at -80 °C. Note that hemolysis should be avoided during the collection process, and repeated freezing and thawing should be avoided.

[0087] (2) Lung tissue: The chest was dissected to expose the heart and both lungs, and the left lung was removed. Excess peripheral tissues were removed, and a tissue sample of approximately 1 cm × 1 cm × 1 cm was taken from the middle lobe of the left lung and fixed with 4% paraformaldehyde for pathological sectioning. The right lung was stored at -80 °C, and all mouse carcasses were sent to the laboratory animal center for unified recycling and disposal.

[0088] 2.4 Detection methods:

[0089] 2.4.1 Serum IgE level detection: The serum IgE level was detected using a commercially available kit (IgE Mouse Uncoated ELISA Kit, invitrogen, Cat#88-50460) according to the instructions.

[0090] 2.4.2 Detection of Th2 cytokine (IL-4, IL-5, and IL-13) levels in lung homogenate supernatant: Commercially available kits were used, and the detection was performed according to the instructions. The kits included IL-4 (Mouse IL-4 Uncoated ELISA, invitrogen, Cat#88-7044), IL-5 (Mouse IL-5 Uncoated ELISA, invitrogen, Cat#88-7054), IL-13 (Mouse IL-13 Uncoated ELISA, invitrogen, Cat#88-7137), IL-10 (Mouse IL-10 Uncoated ELISA Kit, invitrogen, Cat#88-7105), and TGF-β (Human / Mouse TGF beta-1 Uncoated ELISA Kit, Cat#88-8350).

[0091] 2.4.3 Flow cytometry analysis of cell proportion:

[0092] (1) Sacrifice the mice, open the thoracic cavity, expose the heart and lung tissue, remove the mouse lung tissue and place it in pre-cooled RPMI 1640 medium.

[0093] (2) After rinsing the lung tissue with PBS, place a 40 μm nylon mesh in a 6-well plate, take 2 mL of RPMI 1640, place it in the nylon mesh, and slowly grind the cells with a grinding stick to allow them to pass through the filter mesh into the 6-well plate. Collect the grinding solution into a 15 mL centrifuge tube and centrifuge at 1500 rpm for 5 min.

[0094] (3) Collect the supernatant and store it at -80℃ for Th2 cytokine ELISA detection. Resuspend the cells in 2 mL of digestion solution (containing 2 mg / mL collagenase I), place the centrifuge tube in a 37℃ shaker and shake at 200 rpm / min for 20 min, then add RPMI 1640 medium containing 10% FBS to stop digestion, and centrifuge at 1500 rpm for 5 min.

[0095] (4) Discard the supernatant, add 2 mL of red blood cell lysis buffer, quickly disperse the cells, let stand at room temperature for 5 min, then add 2 mL of 10% FBS RPMI 1640 to stop the reaction, and centrifuge at 1500 rpm for 5 min. Discard the supernatant, add 1 mL of PBS to resuspend the cells, take a portion and count the cells using a cell counter, then centrifuge at 1500 rpm for 5 min.

[0096] (5) Adjust the cell concentration to 1×10 6 Cells / 100 μL of cell suspension were added to block the Fc receptor with anti-CD16 / 32 and incubated on ice in the dark for 10 min. Then, anti-CD11b-APC / Cyanine7 (Biolegend, Cat#101226), anti-F4 / 80-PE / Cyanine7 (Biolegend, Cat#123114), anti-CD45-AF700 (Biolegend, Cat#103128), anti-SiglecF-BV421 (Biolegend, Cat#155509), anti-Ly6G-PE (Biolegend, Cat#127608), and anti-Ly6C-Percp / Cyanine5.5 (Biolegend, Cat#128028) were added and incubated on ice in the dark for 30 min.

[0097] (6) Add 1 mL PBS, centrifuge at 400 g for 5 min, and repeat twice.

[0098] (7) Add 150 μL of Fixation Buffer (Biolegend, Cat#420801), incubate at room temperature in the dark for 20 min, then add 1 mL of 1×Permeabilization Wash Buffer (Biolegend, Cat#421002), and centrifuge at 400 g for 5 min. Discard the supernatant, add 1 mL of PBS to resuspend, centrifuge again and discard the supernatant. Add 300 μL of PBS to resuspend, sieve, and transfer to a flow cytometer for analysis. Fluorescence intensity is displayed as a percentage, and the results are analyzed using FlowJo software version 10.8.1.

[0099] 2.4.4 Lung pathological examination: The tissue was blocked with 4% paraformaldehyde for 24 hours in advance; the preparation of pathological sections, H&E staining, PAS staining and MASSON staining, and section scanning were all completed by Wuhan Saiwei Biotechnology Co., Ltd.; CaseViewer (version 2.3.0) was used for slide reading.

[0100] 2.4.5 Tissue Protein Extraction and Western Blot Detection: Tissue samples were placed in grinding tubes containing 1 mL PBS, with one grinding bead added to each tube. The tubes were then placed in a grinding module pre-chilled at -80℃ for 2 hours for grinding. After grinding, 200 μL of homogenate was aspirated and an equal volume of RIPA protein lysis buffer (containing protease inhibitors) was added. The mixture was thoroughly mixed by micropipette. The mixture was incubated on ice for 30 min, then centrifuged at 12000 rpm for 10 min at 4℃. The supernatant was transferred to a new 1.5 mL EP tube. 2 μL of each sample was reserved for protein quantification. The remaining sample was added to 1 / 4 of its volume of 5× protein loading buffer and heated in a 95℃ metal bath for 10 min. The samples were centrifuged at 12000 rpm for 5 min at 4℃. The prepared protein samples were collected and stored at -80℃ for subsequent studies. A commercially available kit (Pierce) was used. TM BCA protein quantification was performed using the BCA Protein Assay Kit (Cat#23227) according to the manufacturer's instructions. A commercially available precast gel (Lablead, Cat#P01015) was used. After adding electrophoresis buffer, 30 μg of sample protein was added to each well, and electrophoresis was performed at a constant voltage of 160 V for approximately 40 min. The nitrocellulose membrane (NC membrane) was activated using 1× transfer buffer, and the protein was transferred to the NC membrane using a wet transfer method at a constant current of 250 mA for 47 min. The membrane was then blocked with 5% skim milk prepared with 1×TBST at 37°C for 1 h, followed by washing three times with 1×TBST for 5 min each time to remove residual skim milk. The desired bands were cut according to the protein markers and incubated with primary antibody overnight at 4°C. The next day, the membrane was washed three times with 1×TBST, and the bands were incubated with the corresponding HRP-labeled secondary antibody at 37°C for 1 h. The membrane was then washed three more times with 1×TBST, and the expression of the corresponding proteins was detected using electrochemiluminescence (ECL).

[0101] 2.5 Statistical Analysis Methods:

[0102] All statistical analyses were performed using GraphPad Prism 9.1.0. Data are expressed as mean ± standard deviation (SD). Differences between groups were analyzed using one-way ANOVA and the Turkey test for post-hoc multiple comparisons. Data that did not conform to a normal distribution underwent logarithmic transformation or nonparametric analysis.

[0103] 2.6 Results

[0104] To observe the effect of Bacteroides monomorphosum IM01 strain in alleviating asthma, we used 5-week-old female BALB / c mice weighing 13-15g. Asthma was induced in the mice via intraperitoneal injection of OVA followed by nebulized challenge. The mouse induction protocol is as follows: Figure 1As shown, mice were administered 200 μL of Bacteroides monomorphosum IM01 strain (1 × 10⁻⁵) daily by gavage from day -5 (day -5) until the last nebulization challenge (D16). 9 (CFU / mouse), while the model group and blank control group were administered the same volume of PBS by gavage daily. Mice were sacrificed 24 h after nebulization challenge. The anti-asthmatic effect of the IM01 strain was evaluated by comparing the total number of lung cells, the percentage of differentially classified cells, serum IgE, lung Th2 cytokines, lung inflammatory cell infiltration, mucus secretion, and collagen deposition levels between the IM01 group and the OVA group.

[0105] (1) This study was divided into three groups: the PBS group (normal group), the OVA group (OVA sensitization challenge), and the IM01 group (OVA sensitization challenge combined with oral IM01 intervention). The results showed that oral administration of Bacteroides monomorphosum IM01 strain significantly reduced the total number of lung cells in asthmatic mice ( Figure 2 The PBS group had a concentration of 1.37 ± 0.25 × 10⁻⁶. 6 / mL, OVA group 4.65±0.72×10 6 / mL, IM01 group 3.23±0.60×10 6 / mL, mainly manifested as a decrease in the proportion of eosinophils, while there was no significant difference in the proportion of basophils, neutrophils, and monocytes. Figure 3 Eosinophils: PBS group 0.19±0.01, OVA group 2.80±1.10, IM01 group 0.25±0.08; Figure 4 Basophils: PBS group 0.01±0.00, OVA group 0.38±0.06, IM01 group 0.40±0.17; Figure 5 Neutrophils: PBS group 0.53±0.19, OVA group 7.92±3.47, IM01 group 5.54±0.86; Figure 6 Monocytes: PBS group 0.03±0.01, OVA group 0.18±0.10, IM01 group 0.05±0.02). Additionally, this strain can effectively reduce serum IgE in asthmatic mice. Figure 7 (PBS group: 1.61±0.64 μg / mL; OVA group: 49.70±5.89 μg / mL; IM01 group: 37.57±6.23 μg / mL).

[0106] The IM01 strain can effectively reduce the levels of Th2 cytokines IL-4, IL-5, and IL-13 in the lungs. Among them, IL-4 ( Figure 8: PBS group 25.87±4.33 pg / mL, OVA group 87.22±19.03 pg / mL, IM01 group 46.17±6.17 pg / mL), IL-5 ( Figure 9 The levels of PBS (12.21±2.51 pg / mL), OVA (59.27±17.61 pg / mL), and IM01 (41.72±11.84 pg / mL) and IL-13 ( Figure 10 The levels of IL-10 in the lungs of asthmatic mice were 22.14±4.62 pg / mL in the PBS group, 54.21±15.39 pg / mL in the OVA group, and 30.77±7.65 pg / mL in the IM01 group. Simultaneously, this bacterium was able to upregulate the anti-inflammatory cytokine IL-10 in the lungs of asthmatic mice. Figure 11 The levels of PBS (671.09±62.00 pg / mL), OVA (835.65±49.90 pg / mL), and IM01 (1057.54±181.19 pg / mL) and TGF-β ( Figure 12 The levels were as follows: PBS group 50.02±3.15 pg / mL, OVA group 56.62±3.62 pg / mL, IM01 group 71.47±6.91 pg / mL.

[0107] (2) Pathological sections were prepared from a portion of the left lung lobe of the mice 24 hours after the last nebulization challenge to observe the lesions in the lung tissue. H&E staining results showed that, compared with the PBS group, the OVA group mice exhibited varying degrees of inflammatory changes in the bronchial mucosa, mainly manifested as destruction, shedding, and loss of airway epithelial cells; thickening of the airway wall, extensive infiltration of inflammatory cells around the airway wall, increased connective tissue, thickening of local alveolar septa, and proliferation of airway smooth muscle. The IM01 group showed significantly reduced inflammatory cell infiltration and significantly less airway epithelial damage compared with the OVA group. Figures 13-15 PAS staining revealed that, compared with the PBS group, the OVA group showed significantly increased goblet cell proliferation in the airway wall of mice, and increased mucus secretion, while the IM01 group showed significantly decreased mucus secretion compared with the OVA group. Figures 16-18 MASSON staining revealed that, compared to the PBS group, the OVA group showed significant collagen deposition in the lung tissue of mice, while the IM01 group showed a significant improvement in collagen deposition. Figures 19-21 The IM01 strain can significantly reduce inflammatory cell infiltration around the airway wall, reduce mucus secretion and collagen deposition, and reduce airway wall damage.

[0108] (3) After IM01 intervention for 21 days, the expression levels of NLRP3, Caspase1 p20, and Phospho-NF-κB p65 proteins in mouse lung tissue were significantly lower than those in the OVA group. Figures 22-23The values ​​for NLRP3 (PBS group 1.05±0.12, OVA group 1.43±0.14, IM01 group 0.65±0.03), Caspase1 p20 (PBS group 0.48±0.07, OVA group 1.27±0.18, IM01 group 0.50±0.19), and Phospho-NF-κB p65 (PBS group 0.85±0.16, OVA group 1.48±0.08, IM01 group 0.75±0.04) suggest that OVA-induced activation of the NLRP3 inflammasome may be improved, indicating that strain IM01 can effectively inhibit the activation pathway of the NLRP3 inflammasome.

[0109] In conclusion, compared with the OVA group, oral administration of strain IM01 significantly improved asthma symptoms in mice, reduced inflammatory cell infiltration, mucus secretion, collagen deposition and pulmonary fibrosis in the lungs, and decreased activation of NLRP3 inflammasomes, indicating that Bacteroides monomorpha strain IM01 has a good anti-asthmatic effect and is of great significance for the prevention and treatment of asthma.

Claims

1. A strain of *Bacteroides monomorpha*, wherein the accession number is CGMCC No. 30981, the accession date is June 17, 2024, and the accession classification name is *Bacteroides monomorpha*. Bacteroides uniformis The depository is the China General Microbiological Culture Collection Center of the China Association for the Preservation and Management of Microbial Cultures.

2. The strain according to claim 1, characterized in that, The 16S rRNA sequence of the strain is shown in SEQ ID NO.

1.

3. The use of the strain according to claim 1 or 2 in the preparation of drugs for the prevention or treatment of asthma.

4. The application according to claim 3, characterized in that, The asthma was accompanied by elevated serum IgE and / or elevated Th2 cytokines in the airways.

5. The application according to claim 3, characterized in that, The asthma is accompanied by airway inflammatory cell infiltration, goblet cell proliferation, mucus production, and collagen deposition.

6. The application according to claim 3, characterized in that, The asthma is accompanied by activation of the NLRP3 inflammasome signaling pathway in the airway.

7. A composition containing the strain of claim 1 or 2, characterized in that, The composition contains a pharmaceutically acceptable carrier and / or excipient.

8. The composition according to claim 7, characterized in that, The composition is prepared as capsules, lyophilized powder, suspension or tablets.

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