Leuconostoc mesenteroides subspecies CGMCC No. 30904 and its application

Abstract

The present invention discloses a strain of Leuconostoc mesenteroides jonggajibkimchii subspecies, the deposit number of the strain is CGMCC No.30904, the deposit date is June 7, 2024, and the deposit classification is named Leuconostoc mesenteroides Leuconostoc mesenteroides The strain provided by the present invention is harmless to animals and has the significant efficacy of alleviating inflammatory infiltration of lung tissue caused by bronchial asthma, reducing the release of serum IgE and Th2 cytokines, inhibiting excessive macrophage replacement activation in the lungs, and improving allergic airway inflammation.

Description

Technical Field

[0001] The invention relates to a preserved strain of Leuconostoc mesenteroides subspecies jonggajibkimchii and application thereof, belonging to the field of microorganisms. Background Art

[0002] Bronchial asthma, also known as asthma, is a heterogeneous chronic inflammatory disease. Asthma is typically characterized by varying degrees of airflow limitation, accompanied by recurrent symptoms such as wheezing, shortness of breath, cough, and chest tightness. Prolonged disease progression can lead to airway remodeling. Asthma is one of the chronic diseases that pose a serious threat to public health worldwide. In recent years, epidemiological studies have confirmed that the number of people with asthma worldwide exceeds 358 million, with at least 250,000 deaths annually, and the prevalence has shown a significant upward trend since 1990. According to the 2022 Global Asthma Report, asthma ranks 24th among the leading causes of disability worldwide and 34th among the leading causes of disease burden. Globally, 96% of asthma-related deaths and 84% of related disabilities occur in low- and middle-income countries (developing countries), imposing a significant economic and medical burden on asthma patients and society.

[0003] Asthma heterogeneity is manifested in diverse etiologies, pathogenic mechanisms, clinical symptoms, and severity. According to the latest Global Initiative for Asthma (GINA) guidelines for asthma prevention and treatment, asthma can be divided into type 2 and non-type 2 types based on the presence of type 2 (T2) inflammation. This classification, representing distinct molecular mechanisms, is known as endotype. Type 2 asthma is the most common, accounting for 64%-73% of all asthma patients. It is primarily characterized by an increase in eosinophils and a significant increase in the T helper 2 (Th2) cell cytokines interleukin-4 (IL-4), IL-5, and IL-13. Th2 cytokines promote eosinophilia and activate B cells into plasma cells, leading to the release of large amounts of specific immunoglobulin E (IgE). This IgE promotes mast cell degranulation, releasing multiple factors, including leukotrienes and histamine, which trigger airway smooth muscle contraction and spasm, leading to asthma symptoms. This type of asthma generally responds well to glucocorticoids or newer biologics, but with some side effects. Non-type 2 asthma is more common in obesity-related asthma, neutrophilic asthma, and oligogranulocytic asthma. It is characterized by infiltration of Th1 and Th17 cells and neutrophils, as well as the production of NLR family, pyrin domain-containing protein 3 (NLRP3), as well as type I interferons, IL-1β, and IL-17. This type of asthma generally responds poorly to medications such as steroids. Therefore, the search for anti-asthma interventions without side effects is crucial.

[0004] The development of immune function and susceptibility to disease are strongly influenced by a variety of resident microorganisms or microbiota. Therefore, several studies have attempted to guide the development of immune function by administering specific microorganisms or probiotics, potentially preventing or treating allergic diseases. For example, Irina Spacova et al. found that the probiotic Lactobacillus rhamnosus can prevent the development of airway hyperresponsiveness, inhibit eosinophilia, and prevent the deterioration of airway function in allergic asthma. AnnaMarlene Schmid et al. reported that the probiotic Escherichia coli O83 can exert a protective effect against asthma by releasing outer membrane vesicles, inhibiting airway hyperresponsiveness, reducing airway eosinophils, Th2 cytokine production, and mucus secretion. However, research in this area is limited, and the number and variety of probiotic strains reported to have anti-asthma properties are limited, requiring further exploration.

[0005] Lactic acid bacteria play an important role in the healthy development of life. They can maintain the homeostasis of intestinal microorganisms through various pathways and produce a variety of bioactive compounds such as peptides, bacteriocins, proteases, lipases and lactic acid during fermentation, which can improve the immunity and disease resistance of animals and other biological functions. Leuconostoc mesenteroides Leuconostoc mesenteroides (L. mesenteroides) is a facultative anaerobic Gram-positive bacterium and an important species of the genus Leuconostoc. It is listed on the "List of Bacteria Acceptable for Food Use" and possesses antimicrobial, antioxidant, and metabolic regulatory properties. Gao Kan et al. found that oral administration of Leuconostoc mesenteroides WHH1141 increased intestinal microbial diversity and short-chain fatty acid levels in mice, alleviating ovalbumin (OVA)-induced food allergy, suggesting that L. mesenteroides has immunomodulatory effects.

[0006] Macrophages can be divided into classically activated macrophages (M1) and alternatively activated macrophages (M2) based on their activation state. M1 macrophages release proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and IL-1β, exacerbating the inflammatory response. M2 macrophages, on the other hand, are divided into three subtypes: M2a, M2b, and M2c. They primarily produce anti-inflammatory cytokines such as IL-10 and possess potent phagocytic functions. The precise role of M2 macrophages in asthma is not fully understood, but they are known to promote Th2 immune responses, which are particularly important in allergic asthma. The number of M2 macrophages in asthmatic patients is correlated with the severity of asthma symptoms and promotes the Th2 inflammatory response in allergic asthma. In children with asthma, different macrophage subsets in the peripheral blood play distinct roles in asthma progression and prognosis. Studies have shown that the presence of M1 macrophages in the peripheral blood of children with asthma is positively correlated with asthma severity, while the presence of M2 macrophages is negatively correlated.

[0007] The purpose of the present invention is to provide a probiotic that can reduce lung inflammatory cell infiltration, lower lung Th2 cytokine and serum IgE levels, inhibit excessive macrophage replacement activation in the lungs, and alleviate allergic airway inflammation. Summary of the Invention

[0008] Based on the above invention purpose, the present invention first provides a strain of Leuconostoc mesenteroides jonggajibkimchii subspecies MY2024 (hereinafter referred to as MY2024), the deposit number of the strain is CGMCC No.30904, the deposit date is June 7, 2024, and the deposit classification is named Leuconostoc mesenteroides Leuconostoc mesenteroidesThe strain was isolated from black bean fermentation broth and is listed on the "List of Bacteria Applicable to Food" issued by the National Health Commission.

[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 a drug for treating or preventing allergic airway inflammation.

[0011] In a preferred embodiment, the allergic airway inflammation is asthma.

[0012] More preferably, the asthma is accompanied by an increase in serum IgE and / or Th2 cytokines in the airways.

[0013] More preferably, the asthma is accompanied by inflammatory cell infiltration, goblet cell hyperplasia, mucus production and collagen deposition in the airways.

[0014] More preferably, the asthma is accompanied by excessive enhancement of alternative activation of pulmonary macrophages.

[0015] Finally, the present invention provides a composition containing the aforementioned strain, which can be used in combination with other pharmaceutical ingredients to enhance or promote the therapeutic effect of asthma, including but not limited to probiotics, chemical molecules, protein molecules, and nucleic acid molecules that have therapeutic effects on asthma.

[0016] In a preferred embodiment, the composition further contains a pharmaceutically acceptable carrier and / or excipient.

[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 Leuconostoc mesenteroides with anti-asthma properties isolated and purified from black bean fermentation liquid. Experiments have shown that the isolated Leuconostoc mesenteroides subsp. jonggajibkimchii The MY2024 strain is harmless to animals and has been confirmed to have the function of improving asthma through animal experiments. Leuconostoc mesenteroides subsp. jonggajibkimchii The MY2024 strain can effectively reduce the serum IgE level of asthma model mice, reduce the production of Th2 cytokines in the lungs, alleviate lung inflammatory cell infiltration and airway mucus production, and reduce excessive macrophage replacement activation 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 The animal experimental protocols used in this invention;

[0020] Figure 2 Oral administration of MY2024 significantly reduced the number of lung cells in OVA mouse models. The horizontal axis represents the 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] Figure 3-Figure 6 Oral administration of MY2024 significantly reduced the percentage of eosinophils and macrophages in the lungs of OVA mice, but did not affect the percentage of neutrophils and monocytes. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0022] Figure 3 Effects of oral administration of MY2024 on the percentage of eosinophils in mouse lung tissue;

[0023] Figure 4 Effects of oral administration of MY2024 on the percentage of macrophages in mouse lung tissue;

[0024] Figure 5 Effects of oral administration of MY2024 on the percentage of neutrophils in mouse lung tissue;

[0025] Figure 6 Effects of oral administration of MY2024 on the percentage of monocytes in mouse lung tissue;

[0026] Figure 7 Oral administration of MY2024 significantly reduced serum IgE levels in OVA mouse models. The horizontal axis represents different animal treatment groups, and the vertical axis represents serum IgE levels. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0027] Figures 8-10 Oral administration of MY2024 significantly reduced the levels of IL-4, IL-5, and IL-13 in the lung tissues of OVA mice. The horizontal axis represents the different animal treatment groups, and the vertical axis represents the corresponding cytokine content. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0028] Figure 8 Effects of oral administration of MY2024 on IL-4 levels in mouse lung tissue;

[0029] Figure 9 Effects of oral administration of MY2024 on IL-5 levels in mouse lung tissue;

[0030] Figure 10 Effects of oral administration of MY2024 on IL-13 levels in mouse lung tissue;

[0031] Figure 11 Shows the results of H&E staining comparison of lung tissue sections of mice in each group after oral administration of MY2024 (I);

[0032] Figure 12 Shows the comparison results of H&E staining of lung tissue sections of mice in each group after oral administration of MY2024 (B);

[0033] Figure 13 Shows the results of H&E staining comparison of lung tissue sections of mice in each group after oral administration of MY2024 (III);

[0034] Figure 14 Shows the results of PAS staining comparison of lung tissue sections of mice in each group after oral administration of MY2024 (I);

[0035] Figure 15 Shows the comparison results of PAS staining of lung tissue sections of mice in each group after oral administration of MY2024 (B);

[0036] Figure 16 Shows the results of PAS staining comparison of lung tissue sections of mice in each group after oral administration of MY2024 (I);

[0037] Figure 17 Shows the comparison results of MASSON staining of lung tissue sections of mice in each group after oral administration of MY2024 (I);

[0038] Figure 18 Shows the comparison results of MASSON staining of lung tissue sections of mice in each group after oral administration of MY2024 (B);

[0039] Figure 19 Shows the comparison results of MASSON staining of lung tissue sections of mice in each group after oral administration of MY2024 (III);

[0040] Figure 20-23 Oral administration of MY2024 significantly reduced the transcriptional levels of alternative activation marker genes in lung tissue macrophages in the OVA mouse model;

[0041] Figure 20 Effects of oral administration of MY2024 on the relative gene expression of CD206 in mouse lung tissue;

[0042] Figure 21 Effects of oral administration of MY2024 on the relative gene expression of Arg1 in mouse lung tissue;

[0043] Figure 22 Effects of oral administration of MY2024 on the relative gene expression of Fizz1 in mouse lung tissue;

[0044] Figure 23Effects of oral administration of MY2024 on the relative gene expression of Ym1 in mouse lung tissue;

[0045] Figure 24 It showed that oral administration of MY2024 could significantly reduce the protein level of Arg1, a marker gene for alternative activation of macrophages in the lung tissue of OVA mouse models. DETAILED DESCRIPTION

[0046] 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 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.

[0047] Example 1. Isolation, preservation and identification of Leuconostoc mesenteroides MY2024

[0048] 1.1 Isolation of Leuconostoc mesenteroides MY2024

[0049] (1) Take 100 μL of black bean fermentation liquid sample and add it to an EP tube pre-filled with 900 μL of sterile PBS. Then perform gradient dilution of the sample in sequence until the concentration of the sample extract is diluted to 10 -6 times;

[0050] (2) Take 100 μL of samples with different dilutions and spread them on MRS solid culture medium and place it in an incubator;

[0051] (3) Culture at 37°C, 0.5% CO2 for 48 h;

[0052] (4) Remove the culture dish and use a sterile inoculation loop to pick colonies with different morphological characteristics. Transfer them to new MRS medium for purification. Culture them anaerobically at 37°C for 48 h. Transfer them three times continuously. Culture the purified strains in liquid MRS at pH 3.5. Screen strains with excellent acid-resistant growth for use in experiments or frozen storage.

[0053] 1.2 Culture preservation

[0054] This laboratory uses MRS medium containing 25% glycerol as the culture medium for freezing and preserving bacterial strains. The method is as follows:

[0055] (1) Sterilize a 2 mL sterilization tube at 121°C for 15 min before use.

[0056] (2) After the bacteria have been transferred to MRS solid medium three times, add 1.5 mL of sterile preservative solution to the culture dish;

[0057] (3) Use an L-rod to scrape the culture dish to ensure that the colonies are fully integrated into the bacterial preservation solution;

[0058] (4) Transfer the bacterial solution into a bacterial preservation tube, mix well, and store at -80℃.

[0059] 1.3 Observation of colony appearance and bacterial morphology

[0060] Leuconostoc mesenteroides subspecies jonggajibkimchii is a facultative anaerobe that grows well under anaerobic conditions. Its colonies are round, milky white, smooth, with neat edges and opaque appearance. Microscopic observation reveals Gram-positive cocci.

[0061] 1.4 Extraction of total bacterial DNA

[0062] A single colony was inoculated onto MRS solid medium and cultured anaerobically at 37°C overnight. DNA was extracted according to the instructions of the bacterial genomic DNA extraction kit (Nanjing Novozymes FastPure® DNA Extraction Kit).

[0063] 1.5 Accurately identify strains by comparing ANI and DDH with model strains

[0064] The genome of this strain was extracted and draft sequenced. Comparison with the type strain was then performed using genomic similarity analysis (digital DNA-DNA hybridization (dddh; dddh <70%) and average nucleotide identity (Ani; Ani <95%)) to further accurately identify the strain. Both methods, considered gold standards for prokaryotic species identification, are performed online (dddh, http: / / ggdc.dsmz.de; Ani, http: / / enve-omics.ce.gatech.edu / ani / ).

[0065] 1.5.1 Genome extraction and draft submission

[0066] This study used a DNA extraction kit produced by Nanjing Novozyme Biotechnology Co., Ltd. for gene extraction. The specific steps are as follows:

[0067] (1) Since the bacteria are Gram-positive, lysozyme solution needs to be added to the bacterial pellet and treated at 37°C for 2 hours to ensure that the cell wall is fully destroyed.

[0068] (2) Add 20 μl of proteinase K solution to the above mixture 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 precipitated in the solution.

[0069] (3) Add 150 μl of anhydrous ethanol and shake to mix. Flocculent precipitates will form 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.

[0070] (4) Place the adsorption column in 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.

[0071] (5) Add 600 μl of WB buffer, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, place it in a new collection tube, and centrifuge at 12,000 rpm for 2 minutes. Perform this step twice to ensure that the DNA is clean.

[0072] (6) Place the adsorption column in the collection tube, open the lid and leave it at room temperature for 3 to 5 minutes to dry, so that the residual rinse solution (mainly ethanol) in the adsorption column can evaporate completely.

[0073] (7) Transfer the adsorption column to a new EP tube, add 200 μl of Elution Buffer (preheated to 55°C) to the adsorption column membrane, leave at room temperature for 2-5 minutes, and centrifuge at 12,000 rpm for 1 minute to obtain pure bacterial genomic DNA. Transport the DNA to Beijing Tianyi Huiyuan Biotechnology Co., Ltd. via cold chain to complete the draft sequencing of the genome.

[0074] 1.5.2 Genomic Correlation Analysis

[0075] (1) Basic characteristics of the genome

[0076] The draft results showed that the genome size of the MY2024 strain was 198,873 bp and the GC content was 42.36%.

[0077] (2) dDDH and ANI

[0078] Selection of a type strain of Leuconostoc mesenteroides subsp. jonggajibkimchii Leuconostoc mesenteroides subsp. mesenteroides A comparison of dDDH and ANI between ATCC 8293 and MY2024 showed that the dDDH and ANI values ​​of the type strain ATCC 8293 and MY2024 were 86.20% and 98.87%, respectively, supporting the view that the two strains are the same bacterial species.

[0079] (3) 16S RNA sequence determination

[0080] A single colony of the bacteria was picked and added to 10 mL of MRS liquid medium, shaken at 37°C overnight, and transported to Beijing Tianyi Huiyuan Biotechnology Co., Ltd. for 16S RNA sequencing. Sequencing revealed that the 16S RNA sequence of the MY2024 strain is shown in SEQ ID NO. 1.

[0081] The deposit information of this strain is: the strain deposit number is CGMCC No.30904, the deposit date is June 7, 2024, and the deposit classification is named Enterococcus mesenteroides Leuconostoc mesenteroides The depository is the General Microbiology Center of China Culture Collection Administration, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, Tel: 8610-64807355.

[0082] Example 2 Evaluation of the Anti-asthma Function of Leuconostoc mesenteroides MY2024 Strain

[0083] 2.1 Sample: Activated culture of Leuconostoc mesenteroides MY2024 strain in logarithmic growth phase.

[0084] 2.2 Experimental animals and OVA animal model construction

[0085] Five-week-old female BALB / c mice, weighing 13–15 g, were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. (license number SCXK(Beijing)2064-0006). They were maintained at the Laboratory Animal Center of the Chinese Center for Disease Control and Prevention (license number SYXK(Beijing)2017-0021) in a barrier-type environment with a 12-h light / dark cycle, and mice had free access to water and food. All animal experiments were conducted 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).

[0086] According to the experimental requirements, mice were randomly divided into three groups: normal group (PBS group), model group (OVA group), and probiotic intervention group (MY2024), with 8 mice in each group. After one week of adaptation, mice in the PBS and OVA groups were gavaged with 200 μL PBS every day from 5 days before infection (day -5) to day 16 after infection (the day of infection was marked as day 0). Mice in the MY2024 group were gavaged with 200 μL MY2024 bacterial suspension (1×10 9CFU / mouse). On days 0 and 7, mice in the OVA and MY2024 groups were sensitized with intraperitoneal injections of 100 μL of ovalbumin suspension (prepared the same day, containing 100 μg of OVA), respectively. The PBS group was sensitized with an equal volume of PBS. From days 14 to 16, mice in the OVA and MY2024 groups were induced with asthma by inhalation of a 1% OVA solution for 20 minutes each day. The PBS group was sensitized with saline instead.

[0087] 2.3 Sample collection: Mice were sacrificed 24 h after the last nebulized challenge, and serum and lung tissue were collected.

[0088] (1) Blood sample collection

[0089] Collect blood from the eyeball. Trim the mouse's whiskers to avoid hemolysis; quickly remove the mouse's eyeball using curved forceps and collect whole blood in a 1.5 mL sterile EP tube. After collection, incubate in a 37°C water bath for 1 hour and centrifuge at 3500 rpm for 15 minutes. Aliquot the serum, label it, and store at -80°C. Be careful to avoid hemolysis during collection and avoid repeated freeze-thaw cycles.

[0090] (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 in size was taken from the middle lobe of the left lung and fixed with 4% paraformaldehyde for pathological sectioning. The right lung was taken and stored at -80°C. All mouse carcasses were sent to the Experimental Animal Center for recovery and unified treatment.

[0091] 2.4 Detection method:

[0092] 2.4.1 Serum IgE content detection: A commercial kit (IgE Mouse Uncoated ELISA Kit, Invitrogen, Cat#88-50460) was used for detection according to the instructions.

[0093] 2.4.2 Detection of Th2 cytokine levels (IL-4, IL-5, and IL-13) in lung homogenate supernatants: Commercially available kits were used according to the manufacturer's instructions. IL-4 (Mouse IL-4 Uncoated ELISA, Invitrogen, Cat#88-7044), IL-5 (Mouse IL-5 Uncoated ELISA, Invitrogen, Cat#88-7054), and IL-13 (Mouse IL-13 Uncoated ELISA, Invitrogen, Cat#88-7137) were assayed.

[0094] 2.4.3 Flow cytometry detection of cell ratio:

[0095] (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.

[0096] (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.

[0097] (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 the digestion and centrifuge at 1500 rpm for 5 min.

[0098] (4) Discard the supernatant, add 2 mL of red blood cell lysis buffer, quickly blow off the cells, let stand at room temperature for 5 minutes, then add 2 mL of 10% FBS RPMI 1640 to stop the reaction, and centrifuge at 1500 rpm for 5 minutes. Discard the supernatant, add 1 mL of PBS to resuspend the cells, take a portion of the cells and count them using a cell counter, then centrifuge at 1500 rpm for 5 minutes.

[0099] (5) Adjust the cell concentration to 1×10 6 Anti-CD16 / 32 was added to block Fc receptors in 100 μL of cell suspension and incubated on ice for 10 min in the dark. Subsequently, 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 for 30 min in the dark.

[0100] (6) Add 1 mL of PBS and centrifuge at 400 g for 5 min. Repeat twice.

[0101] (7) Add 150 μL of Fixation Buffer (Biolegend, Cat#420801) and 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 and resuspend in 1 mL of PBS. After centrifugation, discard the supernatant. Add 300 μL of PBS and resuspend. After sieving, transfer to a flow cytometer and prepare for loading. Fluorescence intensity is displayed as a percentage, and the results are analyzed using FlowJo software version 10.8.1.

[0102] 2.4.4 Lung Pathology: Tissues were pre-blocked with 4% paraformaldehyde for 24 h. Pathological section preparation, H&E staining, PAS staining, and Masson staining, as well as scanning, were performed by Wuhan Sevier Biotechnology Co., Ltd. Slides were read using CaseViewer (version 2.3.0).

[0103] 2.4.5 Tissue RNA Extraction and Real-Time Quantitative PCR Detection: Tissues were placed in grinding tubes containing 1 mL of Trizol and one grinding bead was added to each tube. The tubes were then placed in a grinding module that had been pre-cooled at -80°C for 2 h and ground. After grinding, 200 μL of chloroform was added to each tube and the mixture was shaken and placed on ice for 20 min. The tubes were centrifuged at 12,000 rpm at 4°C for 20 min, and the upper layer (aqueous phase) was removed. 500 μL of isopropanol was added and mixed by inversion. The tubes were incubated at room temperature for 10 min, and then centrifuged at 12,000 rpm at 4°C for 20 min. The supernatant was discarded. The resulting white precipitate was washed with 75% ethanol to obtain RNA, which was then dissolved in 100 μL of enzyme-free water and the RNA concentration was determined. The reverse transcription reaction solution system (40 μL per tube) was prepared according to the reagent manufacturer's instructions. 500 ng of RNA was added to each tube. Reverse transcription was performed on the machine at 37°C for 15 min and 85°C for 5 min to obtain cDNA. Quantitative PCR reaction solution (20 μL per tube, containing 2 μL of DNA template) was prepared according to the manufacturer's instructions. Quantitative PCR was performed using the following reaction conditions: initial denaturation at 95°C for 30 seconds, followed by 40 cycles of denaturation at 95°C for 3 seconds and annealing at 60°C for 30 seconds. Fluorescence signals were collected at the end of each cycle. The relative expression of each target gene was calculated using the 2-ΔΔCt method, using β-actin as an internal reference gene.

[0104] Table 1. Primer sequences used in PCR

[0105]

[0106] 2.4.6 Tissue protein extraction and Western blot analysis: Place the tissue in a grinding tube containing 1 mL of PBS and add one grinding bead to each tube. Place the grinding tube in a grinding module that has been pre-chilled at -80°C for 2 hours and grind on a grinding machine. After grinding, aspirate 200 μL of the homogenate and add an equal amount of RIPA protein lysis buffer (containing protease inhibitors). Mix thoroughly with a micropipette. Place on ice for 30 minutes and centrifuge at 12,000 rpm at 4°C for 10 minutes. Transfer the supernatant to a new 1.5 mL EP tube, retaining 2 μL of each sample for protein quantification. Add 1 / 4 of the remaining sample volume to 5× protein loading buffer and heat in a 95°C metal bath for 10 minutes. Centrifuge at 12,000 rpm at 4°C for 5 minutes. Collect the prepared protein samples and freeze them at -80°C for subsequent studies. A commercial kit (Pierce) was used. TM BCA protein quantification was performed using BCA Protein Assay Kits (Cat#23227) according to the manufacturer's instructions. Using commercial precast gels (Lablead, Cat#P01015), 30 μg of sample protein was added to the sample wells after adding running buffer. Electrophoresis was performed at 160 V for approximately 40 minutes. Nitrocellulose membranes (NC membranes) were activated with 1× transfer buffer and proteins were transferred to the membranes using a wet transfer method at a constant current of 250 mA for 47 minutes. The membranes were then blocked with 5% skim milk in 1× TBST for 1 hour at 37°C and washed three times with 1× TBST for 5 minutes each to remove residual skim milk. The desired bands were cut based on the protein marker and incubated with the primary antibody overnight at 4°C. The next day, the membranes were washed three times with 1× TBST and incubated with the corresponding HRP-conjugated secondary antibody for 1 hour at 37°C. The membranes were then washed three times with 1× TBST. Protein expression was detected by electrochemiluminescence (ECL).

[0107] 2.5 Statistical analysis methods:

[0108] 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 with Turkey's post hoc test for multiple comparisons. Data that did not conform to a normal distribution were log-transformed or subjected to nonparametric analysis.

[0109] 2.6 Results

[0110] To investigate the efficacy of Leuconostoc mesenteroides MY2024 in relieving asthma, we used 5-week-old female BALB / c mice weighing 13-15 g. We induced asthma in mice by intraperitoneal injection of OVA followed by aerosol challenge. From 5 days before the first sensitization (day -5) to the last aerosol challenge (day 16), mice were gavaged daily with 200 μL of Leuconostoc mesenteroides MY2024 (1×10 9 CFU / mouse). The model group and blank control group received daily oral gavage of the same volume of PBS. Mice were sacrificed 24 hours after aerosol challenge. The anti-asthmatic effect of the MY2024 strain was evaluated by comparing the total lung cell count, percentage of differentiated cells, serum IgE, lung Th2 cytokines, lung inflammatory cell infiltration, mucus secretion, and collagen deposition between the MY2024 and OVA groups. The anti-asthmatic mechanism of the MY2024 strain was explored by comparing the mRNA and protein expression of gene markers for alternative macrophage activation in the lung tissue of the MY2024 and OVA groups.

[0111] (1) If Figure 1 As shown, this study was divided into three groups: PBS group (normal group), OVA group (OVA sensitization challenge) and MY2024 group (OVA sensitization challenge and oral administration of MY2024 intervention). The results showed:

[0112] Figure 2 The results showed that oral administration of Leuconostoc mesenteroides MY2024 strain could significantly reduce the total number of lung cells in asthmatic mice (PBS group 1.67±0.10×10 6 / mL, OVA group 3.98±0.25×10 6 / mL, MY2024 group 2.64±0.33×10 6 / mL).

[0113] Figure 3-Figure 6 The results showed that the proportion of eosinophils and macrophages decreased after oral administration of Leuconostoc mesenteroides MY2024 strain, while there was no significant difference in the proportion of neutrophils and monocytes. Figure 3 The percentage of eosinophils in each group was shown to be: 0.47±0.12 in the PBS group, 18.47±3.38 in the OVA group, and 6.36±2.13 in the MY2024 group; Figure 4 The percentage of macrophages in each group was shown as follows: 0.83 ± 0.09 in the PBS group, 11.97 ± 2.64 in the OVA group, and 4.86 ± 1.08 in the MY2024 group; Figure 5 The neutrophil percentage of each group was shown as follows: 5.17±1.16 in the PBS group, 4.98±1.04 in the OVA group, and 5.43±1.91 in the MY2024 group; Figure 6The percentage of monocytes in each group was shown as follows: 5.80±1.29 in the PBS group, 6.01±0.98 in the OVA group, and 5.67±1.80 in the MY2024 group).

[0114] In addition, the strain can also effectively reduce the serum IgE of asthmatic mice ( Figure 7 : PBS group 4.11±0.39 μg / mL, OVA group 52.4±10.02 μg / mL, MY2024 group 26.85±3.82 μg / mL). This strain can also effectively reduce the Th2 cytokine IL-4 in the lungs ( Figure 8 : PBS group 49.72±5.68 pg / mL, OVA group 98.41±9.69 pg / mL, MY2024 group 66.75±6.51 pg / mL), IL-5 ( Figure 9 : PBS group 198.30±2.62 pg / mL, OVA group 426.96±80.03 pg / mL, MY2024 group 266.39±25.25 pg / mL) and IL-13 ( Figure 10 : 14.79±0.66 pg / mL in the PBS group, 34.22±5.85 pg / mL in the OVA group, and 19.80±1.77 pg / mL in the MY2024 group).

[0115] (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. H&E staining results showed that compared with the PBS group, the bronchial mucosa of the lung tissue of the OVA group mice had different degrees of inflammatory changes, mainly manifested as 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 inflammatory cell infiltration of the MY2024 group mice was significantly reduced, and the damage to the airway epithelium was significantly alleviated compared with the OVA group ( Figure 11-13 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 MY2024 group decreased significantly compared with the OVA group ( Figure 14-16 MASSON staining showed that compared with the PBS group, the lung tissue of mice in the OVA group had obvious collagen deposition, while the collagen deposition in the MY2024 group was significantly improved ( Figure 17-Figure 19 ).

[0116] (3) After 21 days of MY2024 intervention, the expression of macrophage alternative activation marker genes (including CD206, Arg1, Fizz1, and Ym1 ) expression was significantly lower than that in the OVA group ( Figure 20-23The quantitative results are shown in Table 2). The expression of Arg1, a marker of macrophage alternative activation, was significantly lower in the OVA group than in the control group, suggesting that OVA-induced macrophage alternative activation may be improved.

[0117] Table 2. Comparison of relative expression levels of macrophage alternative activation genes in animals of each experimental group

[0118]

[0119] Figure 24 Oral administration of MY2024 significantly reduced the level of Arg1 protein, a marker gene for alternative activation of macrophages in the lung tissue of OVA mice models ( Figure 24 PBS group 0.09±0.07, OVA group 1.03±0.20, MY2024 group 0.60±0.30).

[0120] In summary, compared with the OVA group, oral administration of the MY2024 strain can significantly improve the asthma symptoms of mice, reduce lung inflammatory cell infiltration, mucus secretion, collagen deposition and pulmonary fibrosis, and reduce excessive macrophage replacement activation in the lungs, indicating that the MY2024 strain of Leuconostoc mesenteroides has a good anti-asthmatic effect and is of great significance for the prevention and treatment of asthma.

Claims

1. A strain of Leuconostoc mesenteroides subspecies jonggajibkimchii, the deposit number of which is CGMCC No. 30904, the deposit date is June 7, 2024, and the deposit classification is named Leuconostoc mesenteroides Leuconostoc mesenteroides The preservation unit is the General Microbiology Center of China Culture Collection Administration of Microorganisms.

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

3. The use according to claim 2, characterized in that The allergic airway inflammation is asthma.

4. The use according to claim 3, characterized in that The asthma is accompanied by an increase in serum IgE and / or Th2 cytokines in the airways.

5. The use according to claim 3, characterized in that Asthma is accompanied by inflammatory cell infiltration, goblet cell hyperplasia, mucus production, and collagen deposition in the airways.

6. The use according to claim 3, characterized in that Asthma is accompanied by an exaggerated alternative activation of pulmonary macrophages.

7. A composition comprising the strain according to claim 1.

8. The composition according to claim 7, characterized in that The composition further contains a pharmaceutically acceptable carrier and / or excipient.

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

Images