Application of bacteroides simplex CGMCC (China General Microbiological Culture Collection Center) No.30981 in preparation of medicine for treating asthma

Abstract

The preservation number of the bacterial strain is CGMCC (China General Microbiological Culture Collection Center) No.30981, the preservation date is June 17, 2024, the preservation classification is named as the Bacteroides simplex, and the preservation unit is the General Microbiological Culture Collection Center of the China Committee for Culture Collection of Microorganisms. The strain provided by the invention has the remarkable effects of relieving lung tissue inflammatory infiltration caused by bronchial asthma, reducing release of serum IgE and Th2 type cell factors, inhibiting activation of NLRP3 inflammasome and improving allergic airway inflammation.

Description

Technical Field

[0001] The invention relates to application of Bacteroides monomorpha CGMCC No.30981 in preparing asthma therapeutic drugs, and belongs to the field of microorganisms. Background Art

[0002] Bronchial asthma, also known as asthma, is a heterogeneous chronic inflammatory disease. The typical characteristics of asthma are varying degrees of airflow limitation, accompanied by recurrent wheezing, shortness of breath, coughing, and chest tightness. As the disease progresses, it can lead to airway remodeling. Asthma is one of the chronic diseases that seriously threaten public health worldwide.

[0003] The heterogeneity of asthma is manifested in different causes, pathogenic mechanisms, clinical symptoms and severity. According to the latest Global Initiative for Asthma (GINA) asthma prevention and treatment guidelines, asthma can be divided into type 2 and non-type 2 types according to the presence or absence of type 2 (T2) inflammation. This classification method represents different molecular mechanisms and is called endotype. Type 2 asthma is the most common, accounting for 64%-73% of all asthma patients, and is mainly manifested by an increase in eosinophils and a significant increase in the T helper 2 cell (Th2) cytokines interleukin-4 (IL-4), IL-5 and IL-13. Th2 cytokines promote eosinophilia and lead to the activation of B cells into plasma cells, releasing a large amount of specific immunoglobulin E (IgE), which promotes mast cell degranulation, releases multiple factors such as leukotrienes and histamine, and causes airway smooth muscle contraction and spasm, resulting in asthma symptoms. This type of asthma usually responds well to glucocorticoids or new biological agents, but there are some side effects. Non-type 2 asthma is more common in obesity-related asthma, neutrophilic asthma, and oligogranulocytic asthma, which is characterized by the infiltration of Th1 and Th17 cells and neutrophils, the large production of NLR Family, Pyrin Domain Containing Protein 3 (NLRP3), and type I interferon, IL-1β, and IL-17. This type of asthma usually responds poorly to drugs such as steroids. Therefore, it is very necessary to find anti-asthma interventions without side effects.

[0004] Symbiotic flora is 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 allergens in the environment. Therefore, it is of great significance to guide the development of immune function and regulate the adaptive immune system by administering specific symbiotic bacteria, thereby potentially preventing or treating allergic diseases. For example, Vadim Pivniouk et al. found that nasal administration of standardized human airway bacterial lysate OM-85 could upregulate the proportion of activated regulatory T cells and weaken dendritic cell responses, protecting allergen-sensitive animals from airway inflammation; Bilal Alashkar Alhamwe et al. reported that the commensal bacterium Acinetobacter lofivifolia can activate the epigenetics of T cells through the IL-6 / IL-10 axis, affect the composition of the intestinal microbiota, and play a protective role in asthma. However, there are not many studies in this field at present, and the number and types of probiotics or symbiotic strains reported to have anti-asthma functions are limited, and further exploration is still needed.

[0005] Bacteroides thetaiotaomicron, also known as common Bacteroides, is a Gram-negative obligate anaerobic symbiotic bacterium that participates in the body's anti-inflammatory, metabolic, and immune regulation processes. Wenhui Pang et al. found that allergic airway inflammation patients and mice had dysbacteriosis in the intestine, and the biodiversity and number of Bacteroidetes decreased. Oral administration of Bacteroides thetaiotaomicron can improve the symptoms of airway hyperresponsiveness induced by ovalbumin (OVA) in mice, reduce the secretion of Th2 cytokines in bronchoalveolar lavage fluid, and alleviate airway inflammation.

[0006] NOD-like receptor thermal proteindomain associated protein 3 (NLRP3) is a cytoplasmic complex that plays an important role in inflammatory responses. Activation of the NLRP3 inflammasome can lead to the maturation and secretion of proinflammatory cytokines IL-1β and IL-18, which play a key role in airway inflammation in asthma. In patients with asthma, activation of the NLRP3 inflammasome is associated with exacerbated airway inflammation. Activation of the NLRP3 inflammasome 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, which can induce Th2 cell differentiation, promote airway inflammation, and increase airway hyperreactivity (AHR). The role of NLRP3 inflammasome in asthma is not limited to promoting the release of inflammatory factors, but also involves airway epithelial cell damage and airway remodeling. Airway epithelial damage is an important hallmark of asthma, and activation of NLRP3 inflammasome can aggravate this damage. In addition, activation of NLRP3 inflammasome is also associated with pyroptosis, a form of programmed cell death that can lead to cell membrane perforation, allowing inflammatory factors to be released outside the cell, further promoting the inflammatory response. The role of NLRP3 inflammasome is also different in different subtypes of asthma. In type 2 asthma, activation of NLRP3 inflammasome is associated with the recruitment and activation of eosinophils, while in non-type 2 asthma, NLRP3 inflammasome is associated with the recruitment and activation of neutrophils. In general, NLRP3 inflammasome plays a multifaceted role in the pathogenesis of asthma, including promoting the release of inflammatory factors, aggravating airway epithelial cell damage, and promoting pyroptosis. Therefore, NLRP3 inflammasome is considered a potential target for asthma treatment, and its inhibitors may be of great significance for controlling asthma symptoms and improving patient prognosis.

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

[0008] Based on the above invention purpose, the present invention first provides a Bacteroides monomorpha strain IM01 (hereinafter referred to as IM01), the strain has a preservation number of CGMCC No.30981, a preservation date of June 17, 2024, and a preservation classification named Bacteroides monomorpha Bacteroides uniformis The depository is the General Microbiology Center of China Microbiological Culture Collection Administration. The strain is isolated from healthy human feces.

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

[0010] Secondly, the present invention provides the use of the above strain in preparing drugs for treating or preventing allergic airway inflammation.

[0011] In an alternative embodiment, the allergic airway disease is asthma.

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

[0013] In another alternative embodiment, the asthma is accompanied by inflammatory cell infiltration, goblet cell hyperplasia, mucus production, and collagen deposition in the airways.

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

[0015] Thirdly, the present invention provides a composition containing the above-mentioned strain, wherein the composition contains a pharmaceutically acceptable carrier and / or excipient, and the strain can be used in combination with other ingredients to assist and / or enhance the pharmacodynamic effect 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 powders, suspensions or tablets.

[0018] The present invention is a strain of Bacteroides monomorpha with anti-asthma properties isolated and purified from healthy human feces. Experiments have shown that the isolated Bacteroides uniformis The IM01 strain has the function of improving asthma. Bacteroides uniformisThe IM01 strain can effectively reduce the serum IgE level of asthma model mice, reduce the production of Th2 cytokines in the lungs, alleviate the infiltration of inflammatory cells in the lungs and the production of airway mucus, and inhibit the activation of NLRP3 inflammasomes in the lungs, showing excellent application prospects in the preparation of asthma treatment and / or prevention drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Animal experimental protocols used in this invention; Figure 2 It shows that oral administration of IM01 can significantly reduce the number of lung cells in the OVA mouse model. The horizontal axis is the different animal treatment groups, the vertical axis is the number of cells, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001; Figure 3-Figure 6 It showed that oral administration of IM01 could significantly reduce 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. Figure 3 Effect of oral administration of IM01 on the percentage of eosinophils to total leukocytes in the lung tissue of mice; Figure 4 Effect of oral administration of IM01 on the percentage of basophils to total leukocytes in the lung tissue of mice; Figure 5 Effect of oral administration of IM01 on the percentage of neutrophils to total leukocytes in the lung tissue of mice; Figure 6 Effect of oral administration of IM01 on the percentage of monocytes to total leukocytes in the lung tissue of mice; Figure 7 It shows that oral administration of IM01 can significantly reduce the serum IgE level of OVA mouse model. The horizontal axis represents different animal treatment groups, and the vertical axis represents serum IgE content. * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001. Figure 8-12 It shows that oral administration of IM01 can significantly reduce the levels of IL-4, IL-5 and IL-13 in the lungs of OVA mouse models, where A is the release of IL-4; B is the release of IL-5; C is the release of IL-13, the horizontal axis is the different animal treatment groups, the vertical axis is the corresponding cytokine content, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001; Figure 8 Effects of oral administration of IM01 on IL-4 levels in mouse lung tissue; Fig. 9 Effects of oral administration of IM01 on IL-5 levels in lung tissue of mice; Fig.10 Effects of oral administration of IM01 on IL-13 levels in lung tissue of mice; Fig.11 Effects of oral administration of IM01 on IL-10 levels in lung tissue of mice; Fig.12 Effects of oral administration of IM01 on IL-TGF-β levels in mouse lung tissue; Fig.13 Shows the results of H&E staining comparison of lung tissue sections of mice in each group after oral administration of IM01 (I); Fig.14 Shows the results of H&E staining comparison of lung tissue sections of mice in each group after oral administration of IM01 (B); Fig.15 Shows the results of H&E staining comparison of lung tissue sections of mice in each group after oral administration of IM01 (III); Fig.16 Shows the comparison results of PAS staining of lung tissue sections of mice in each group after oral administration of IM01 (I); Fig.17 Shows the comparison results of PAS staining of lung tissue sections of mice in each group after oral administration of IM01 (B); Fig.18 Shows the results of PAS staining comparison of lung tissue sections of mice in each group after oral administration of IM01 (III); Fig.19 Shows the comparison results of MASSON staining of lung tissue sections of mice in each group after oral administration of IM01 (I); Fig. 20 Shows the comparison results of MASSON staining of lung tissue sections of mice in each group after oral administration of IM01 (B); Fig.21 Shows the comparison results of MASSON staining of lung tissue sections of mice in each group after oral administration of IM01 (III); Fig. 22 Shows the effect of oral administration of IM01 on the absolute expression of NLRP3, Caspase1 p20, and Phospho-NF-κB p65 proteins in the lungs of OVA mice model, where A is the absolute expression of NLRP3, Caspase1 p20, and Phospho-NF-κB p65 proteins in the NLRP3 signaling pathway of mouse lung tissue; Fig.23The effect of oral administration of IM01 on the relative expression of NLRP3, Caspase1 p20 and Phospho-NF-κBp65 proteins in the lungs of OVA mouse models compared with the internal reference gene GAPDH. The horizontal axis represents different genes, and the vertical axis represents the relative expression of proteins compared with GAPDH. * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001. DETAILED DESCRIPTION

[0020] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. 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.

[0021] Example 1. Isolation, preservation and identification of Bacteroides monomorpha IM01 strain 1.1 Isolation of Bacteroides monocytogenes IM01 strain (1) Take an appropriate amount of healthy human fecal sample and add it to an EP tube pre-filled with 900 μL sterile PBS. Dilute the sample in a gradient manner until the concentration of the specimen extract is diluted to 10 -6 times; (2) Take 100 μL of samples with different dilutions and spread them on BHI solid culture medium and place them in an incubator; (3) Culture at 37°C, 0.5% CO2 for 48 h; (4) Remove the culture dish and use a sterile inoculation loop to pick colonies with different morphological characteristics. Transfer them to new BHI solid medium for purification. Incubate anaerobically at 37°C for 48 h. Transfer the culture medium three times in a row. Culture the purified strains in liquid BHI at pH 3.5. Screen the strains with excellent growth for use in experiments or frozen storage.

[0022] 1.2 Strain preservation This laboratory uses BHI medium containing 25% glycerol as the bacterial preservation medium for frozen preservation of bacterial strains. The method is as follows: (1) Sterilize a 2 mL sterilization tube at 121°C for 15 min before use; (2) After the bacteria have been transferred to BHI solid medium three times in succession, add 1.5 mL of sterile bacterial preservation solution to the culture dish; (3) Use an L-rod to scrape the culture dish to ensure that the colonies are fully integrated into the bacterial preservation solution; (4) Transfer the bacterial solution to a bacterial preservation tube, mix well, and store at -80℃.

[0023] 1.3 Observation of colony appearance and bacterial morphology Bacteroides monomorpha is a non-spore, non-flagellated, obligately anaerobic, gram-negative bacterium with varying lengths and grayish-white translucent colonies. It normally inhabits the intestines, mouth, upper respiratory tract, and reproductive tract of humans and animals.

[0024] 1.4 Extraction of total bacterial DNA A single colony was inoculated on 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 Novozyme FastPure® DNA Extraction Kit).

[0025] 1.5 Accurately identify strains by comparing ANI and DDH with model strains The genome of the strain was extracted and draft sequenced. The genome correlation analysis (digital DNA-DNA hybridization (dDDH; dDDH < 70%) and average nucleotide identity (Average Nucleotide Identity, ANI; ANI < 95%)) was performed by comparison with the model strain to further accurately identify the strain. Both methods are the gold standard for determining prokaryotic species and are completed online (dDDH, http: / / ggdc.dsmz.de; ANI, http: / / enve-omics.ce. gatech.edu / ani / ).

[0026] 1.5.1 Genome extraction and draft submission The present invention uses a DNA extraction kit produced by Nanjing Novozyme Biotechnology Co., Ltd. for gene extraction. The specific operation steps are as follows: (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 in a 55°C water bath for 10 min, invert and mix to ensure that there are no impurities in the solution.

[0027] (2) Add 150 μl of anhydrous ethanol and shake to mix. Flocculent precipitate will be generated during this process. Transfer all the liquid and floccules in the EP tube to the adsorption column FastPure gDNA Mini Columns II, centrifuge at 12000 rpm for 30 seconds, and discard the waste liquid.

[0028] (3) Place the adsorption column in the waste liquid collection tube, add 500 μl of buffer WA to the adsorption column, centrifuge at 12,000 rpm for 1 min, and discard the waste liquid.

[0029] (4) Add 600 μl of buffer WB, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, put it into a new collection tube, and centrifuge at 12,000 rpm for 2 minutes. Perform this step twice to ensure that the DNA is clean.

[0030] (5) Place the adsorption column in a collection tube, open the lid and leave it at room temperature for 3 to 5 minutes to dry. This is to allow the residual rinse solution (mainly ethanol) in the adsorption column to evaporate.

[0031] (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, place at room temperature for 2-5 minutes, and centrifuge at 12,000 rpm for 1 minute to obtain pure bacterial genomic DNA. Transport the cold chain to Beijing Tianyi Huiyuan Biotechnology Co., Ltd. to complete the draft sequencing of the genome.

[0032] 1.5.2 Genome correlation analysis (1) Basic characteristics of the genome The draft results showed that the genome size of strain IM01 was 3540728 bp and the GC content was 46.645%.

[0033] (2) dDDH and ANI Selection of a model strain of Bacteroides monocytogenes Bacteroides uniformis The dDDH and ANI values ​​of CL03T12C37 and IM01 were compared. The results showed that the dDDH and ANI values ​​of the model strain CL03T12C37 and IM01 were 81.00% and 97.67%, respectively, supporting that the two strains were the same bacterial species.

[0034] (3) 16S RNA sequence determination The single colony of the bacteria was picked up and added to 10 mL of BHI liquid culture medium, shaken at 37°C overnight, and transported to Beijing Tianyi Huiyuan Biotechnology Co., Ltd. for 16S RNA sequence determination. After sequence determination, the 16S RNA sequence of the IM01 strain is shown in SEQ ID NO.1.

[0035] The deposit information of this strain is as follows: the strain deposit number is CGMCC NO.30981, the deposit date is June 17, 2024, and the deposit classification is named Bacteroides uniformis IM01 strain, deposited by China Culture Collection Administration General Microbiology Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, Tel: 8610-64807355.

[0036] Example 2 Evaluation of the anti-asthma function of Bacillus monomorphus strain IM01 2.1 Sample: Activated culture of Bacteroides monotheca MY2024 strain in logarithmic growth phase.

[0037] 2.2 Experimental animals and OVA animal model construction 5-week-old female BALB / c mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., with a weight range of 13-15 g. The license number is SCXK (Beijing) 2064-0006. The breeding location is the Laboratory Animal Center of the Chinese Center for Disease Control and Prevention, license number SYXK (Beijing) 2017-0021, the breeding level is a barrier environment, 12-h light / dark cycle, and mice have free access to water and food. All animal experiments were carried out in accordance with the ethical guidelines for experimental animal welfare of the Laboratory Animal Center of the Chinese Center for Disease Control and Prevention (approval number: 2023-025).

[0038] According to the experimental requirements, the mice were randomly divided into three groups: normal group (PBS group), model group (OVA group) and probiotic intervention group (IM01 group), with 8 mice in each group. After one week of adaptation, the mice in the PBS group and the OVA group were gavaged with 200 μL PBS every day from 5 days before infection (day -5) to the 16th day after infection (the day of infection was recorded as day 0), and the mice in the IM01 group were gavaged with 200 μL IM01 bacterial suspension (1×10 9 CFU / mouse). On days 0 and 7, mice in the OVA group and IM01 group were sensitized by intraperitoneal injection of 100 μL ovalbumin suspension (prepared on the same day, containing 100ug OVA), and mice in the PBS group were injected with an equal volume of PBS. On days 14-16, mice in the OVA group and IM01 group were given nebulized inhalation of 1% OVA solution every day to stimulate asthma, each time lasting 20 minutes, while mice in the PBS group were given nebulized inhalation of normal saline instead.

[0039] 2.3 Sample collection: Mice were killed 24 hours after the last nebulization challenge, and serum and lung tissues were collected.

[0040] (1) Blood sample collection Blood was collected from the eyeball. The mouse whiskers were trimmed to avoid hemolysis; the eyeballs of the mice were quickly plucked with curved forceps, and the whole blood of the mice was collected in a 1.5 mL sterile EP tube. After the collection was completed, the blood was placed in a 37 ℃ water bath for 1 h, centrifuged at 3500 rpm for 15 min, and the serum was packaged and stored at -80 ℃ after being marked. Pay attention to avoid hemolysis during the collection process and avoid repeated freezing and thawing.

[0041] (2) Lung tissue: The chest was dissected to expose the heart and both lungs. The left lung was removed and the surrounding excess tissue was removed. Tissue of approximately 1 cm × 1 cm × 1 cm was obtained from the middle lobe of the left lung and fixed with 4% paraformaldehyde for pathological sectioning. The right lung was stored at -80°C. All mouse carcasses were sent to the Experimental Animal Center for recovery and unified treatment.

[0042] 2.4 Detection method: 2.4.1 Detection of serum IgE content: Use a commercial kit (IgE Mouse Uncoated ELISA Kit, invitrogen, Cat#88-50460) for detection according to the instructions.

[0043] 2.4.2 Detection of Th2 cytokines (IL-4, IL-5 and IL-13) in lung homogenate supernatant: Detection was performed using commercial kits according to the instructions, including 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 ELISAKit, invitrogen, Cat#88-7105), and TGF-β (Human / Mouse TGF beta-1 Uncoated ELISA Kit, Cat#88-8350).

[0044] 2.4.3 Flow cytometry detection of cell ratio: (1) Kill the mice, open the chest cavity, expose the heart and lung tissue, remove the mouse lung tissue, and place it in pre-cooled RPMI1640 culture medium.

[0045] (2) After rinsing the lung tissue with PBS, place a 40 μm nylon mesh on a 6-well plate. Pipette 2 mL of RPMI 1640 and place it on the nylon mesh. Use a grinding rod to slowly grind the cells so that they pass through the filter mesh and flow into the 6-well plate. Collect the grinding solution into a 15 mL centrifuge tube and centrifuge at 1500 rpm for 5 min.

[0046] (3) Collect the supernatant and store at -80℃ for Th2 cytokine ELISA detection. Resuspend the cells with 2 mL of digestion solution (containing 2 mg / mL collagenase I), place the centrifuge tube in a 37℃ shaker at 200 rpm / min for 20 min, then add RPMI 1640 medium containing 10% FBS to terminate digestion, and centrifuge at 1500 rpm for 5 min.

[0047] (4) Discard the supernatant, add 2 mL of red blood cell lysis buffer, blow off quickly, let stand at room temperature for 5 min, then add 2 mL of 10% FBS RPMI 1640 to stop the process, and centrifuge at 1500 rpm for 5 min. Discard the supernatant, add 1 mL of PBS to resuspend the cells, take a portion to count the cells using a cell counter, and then centrifuge at 1500 rpm for 5 min.

[0048] (5) Adjust the cell concentration to 1×10 6 cells / 100 μL cell suspension, anti-CD16 / 32 was added to block Fc receptors, 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.

[0049] (6) Add 1 mL of PBS and centrifuge at 400 g for 5 min. Repeat twice.

[0050] (7) Add 150 μL Fixation Buffer (Biolegend, Cat#420801) and incubate at room temperature in the dark for 20 min. Then add 1 mL 1× Permeabilization Wash Buffer (Biolegend, Cat#421002) and centrifuge at 400 g for 5 min. Discard the supernatant, add 1 mL PBS to resuspend, centrifuge and discard the supernatant. Add 300 μL PBS to resuspend, sieve and transfer to a flow tube for loading. Fluorescence intensity is displayed as a percentage, and the results are analyzed using FlowJo software version 10.8.1.

[0051] 2.4.4 Pulmonary pathology: The tissues were sealed with 4% paraformaldehyde for 24 h in advance. Pathological section preparation, H&E staining, PAS staining, MASSON staining, and section scanning were all completed by Wuhan Saiweier Biotechnology Co., Ltd. CaseViewer (version 2.3.0) was used for reading.

[0052] 2.4.5 Tissue protein extraction and Western blot detection: Place the tissue in a grinding tube containing 1 mL PBS and add one grinding bead to each tube. Then place the grinding tube in a grinding module precooled at -80℃ for 2 hours and grind on the machine; after grinding, take 200 μL of homogenate and add an equal amount of RIPA protein lysis buffer (containing protease inhibitors), and mix thoroughly with a micropipette. After standing on ice for 30 minutes, centrifuge at 12000rpm at 4℃ for 10 minutes. Transfer the supernatant to a new 1.5 mL EP tube, retain 2 μL of each sample for protein quantification, and add 1 / 4 of its volume of 5× protein loading buffer to the remaining sample, and heat it in a 95℃ metal bath for 10 minutes. Centrifuge at 12000rpm at 4℃ for 5 minutes, collect the prepared protein samples, and freeze them at -80℃ for subsequent research. Use a commercial kit (Pierce TM BCA Protein Assay Kits, Cat#23227) was used for BCA protein quantification according to the instructions. Commercial precast gel (Lablead, Cat#P01015) was used. After adding electrophoresis buffer, 30 μg of sample protein was added to the spotting well, and electrophoresis was performed at 160 V constant voltage for about 40 min. 1× transfer buffer was used to activate the nitrocellulose membrane (NC membrane), and the protein was transferred to the NC membrane by wet transfer method at a constant current of 250 mA for 47 min. Then, 5% skim milk in 1× TBST was used to block at 37°C for 1 h, and the membrane was washed three times with 1× TBST for 5 min each time to remove the residual skim milk. The required bands were cut according to the protein marker and incubated with primary antibodies at 4°C overnight. The membrane was washed three times with 1× TBST the next day, and the bands were incubated with the corresponding HRP-labeled secondary antibody at 37°C for 1 h. Then, the membrane was washed three times with 1× TBST, and the expression of the corresponding protein was detected by electrochemiluminescence (ECL) method.

[0053] 2.5 Statistical analysis methods: All data were statistically analyzed using GraphPad Prism 9.1.0. Data are expressed as mean ± standard deviation (SD). Differences between groups were analyzed using one-way analysis of variance and Turkey test as post hoc multiple comparisons. Data that did not conform to normal distribution were logarithmically transformed or subjected to nonparametric analysis.

[0054] 2.6 Results To observe the effect of Bacteroides monocytogenes IM01 strain in relieving asthma, we used 5-week-old female BALB / c mice weighing 13-15g and induced asthma in mice by intraperitoneal injection of OVA sensitization and aerosol challenge. Figure 1As shown, mice were intragastrically administered 200 μL of Bacteroides monothecae IM01 strain (1×10 9 CFU / mouse), and the model group and the blank control group were gavaged with the same volume of PBS every day. The mice were killed 24 hours after aerosol challenge, and the total number of lung cells, percentage of classified cells, serum IgE, lung Th2 cytokines, lung inflammatory cell infiltration, mucus secretion, and collagen deposition levels of mice in the IM01 group and OVA group were compared to evaluate the anti-asthmatic effect of the IM01 strain.

[0055] (1) This study was divided into three groups: PBS group (normal group), OVA group (OVA sensitization challenge) and IM01 group (OVA sensitization challenge and oral IM01 intervention). The results showed that oral administration of Bacteroides monocytogenes IM01 strain could significantly reduce the total number of lung cells in asthmatic mice ( Figure 2 , PBS group: 1.37±0.25×10 6 / mL, OVA group 4.65±0.72×10 6 / mL, IM01 group 3.23±0.60×10 6 / mL), mainly manifested by 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). In addition, this strain can also effectively reduce the serum IgE of 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).

[0056] IM01 strain can effectively reduce the levels of Th2 cytokines IL-4, IL-5, and IL-13 in the lungs, among which 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 ( Fig. 9 : PBS group 12.21±2.51 pg / mL, OVA group 59.27±17.61 pg / mL, IM01 group 41.72±11.84 pg / mL) and IL-13 ( Fig.10 : PBS group 22.14±4.62 pg / mL, OVA group 54.21±15.39 pg / mL, IM01 group 30.77±7.65 pg / mL). At the same time, the bacteria can upregulate the anti-inflammatory cytokine IL-10 ( Fig.11 : PBS group 671.09±62.00pg / mL, OVA group 835.65±49.90pg / mL, IM01 group 1057.54±181.19pg / mL) and TGF-β ( Fig.12 : 50.02±3.15 pg / mL in PBS group, 56.62±3.62 pg / mL in OVA group, and 71.47±6.91 pg / mL in IM01 group).

[0057] (2) Pathological sections were taken from the left lung lobes of mice 24 hours after the last nebulization challenge to observe the pathological changes in the lung tissue of mice. The results of H&E staining showed that compared with the mice in the PBS group, the bronchial mucosa of the lung tissue of the mice in the OVA group had different degrees of inflammatory changes, mainly manifested as the destruction, shedding and loss of airway epithelial cells; thickening of the airway wall, infiltration of a large number of inflammatory cells around the wall, increased connective tissue, thickening of local alveolar septa, and hyperplasia of airway smooth muscle. The infiltration of inflammatory cells in the IM01 group of mice was significantly reduced, and the destruction of the airway epithelium was significantly alleviated compared with the OVA group ( Figure 13-Figure 15 ). PAS staining showed that compared with the PBS group, the goblet cell proliferation of the lung tissue of the mice in the OVA group increased significantly, and the mucus secretion increased, while the mucus secretion of the IM01 group decreased significantly compared with the OVA group ( Figure 16-Figure 18 MASSON staining showed that compared with the PBS group, the lung tissue of the OVA group mice had obvious collagen deposition, while the collagen deposition in the IM01 group was significantly improved ( Figure 19-21 IM01 strain can significantly reduce the infiltration of inflammatory cells around the airway wall, reduce mucus secretion and collagen deposition, and reduce airway wall damage.

[0058] (3) After 21 days of IM01 intervention, the expression levels of NLRP3, Caspase1 p20, and Phospho-NF-κB p65 proteins in the lung tissue of mice were significantly lower than those in the OVA group ( Figure 22-Figure 23: NLRP3: 1.05±0.12 in the PBS group, 1.43±0.14 in the OVA group, and 0.65±0.03 in the IM01 group; Caspase1 p20: 0.48±0.07 in the PBS group, 1.27±0.18 in the OVA group, and 0.50±0.19 in the IM01 group; Phospho-NF-κB p65: 0.85±0.16 in the PBS group, 1.48±0.08 in the OVA group, and 0.75±0.04 in the IM01 group), indicating that the activation of the NLRP3 inflammasome induced by OVA may be improved, indicating that the IM01 strain can effectively inhibit the activation pathway of the NLRP3 inflammasome.

[0059] In summary, compared with the OVA group, oral administration of the IM01 strain can significantly improve the asthma symptoms of mice, reduce lung inflammatory cell infiltration, mucus secretion, collagen deposition and pulmonary fibrosis, and reduce the activation of NLRP3 inflammasomes, indicating that the Bacteroides monocytogenes IM01 strain 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, the deposit number of which is CGMCC No.30981, the deposit date is June 17, 2024, and the deposit classification is named Bacteroides monomorpha Bacteroides uniformis The preservation unit is the General Microbiology Center of China Microbiological Culture Collection Administration.

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

1.

3. Use of the strain according to claim 1 or 2 in the preparation of a drug for preventing or treating allergic airway inflammation.

4. The use according to claim 3, characterized in that: The allergic airway inflammation disease is asthma.

5. The use according to claim 4, characterized in that: The asthma is accompanied by an increase in serum IgE and / or Th2 cytokines in the airways.

6. The use according to claim 4, characterized in that: Asthma is associated with inflammatory cell infiltration, goblet cell hyperplasia, mucus production, and collagen deposition within the airways.

7. The use according to claim 4, characterized in that: Asthma is accompanied by activation of the NLRP3 inflammasome signaling pathway in the airways.

8. A composition containing the strain according to claim 1 or 2, characterized in that: The composition contains a pharmaceutically acceptable carrier and / or excipient.

9. The composition according to claim 8, characterized in that The composition contains one or more of probiotics, plant extracts, chemical molecules, protein molecules, and nucleic acid molecules.

10. The composition according to claim 8, characterized in that The composition is prepared as capsules, lyophilized powders, suspensions or tablets.

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