Lactobacillus brevis IM01 and application thereof in preparation of drugs for treating allergic asthma

Abstract

The preservation number of the strain is CGMCC (China General Microbiological Culture Collection Center) NO.30979, the preservation date is June 17, 2024, the preservation classification is named as Lactobacillus brevis, and the preservation unit is the General Microbiological Culture Collection Center of the China Committee for Culture Collection of Microorganisms. The strain is harmless to animals and 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 an NF-kappa B channel and improving allergic airway inflammation. The invention also discloses application of the strain and the thallus component thereof in preparation of drugs for preventing and treating allergic asthma.

Description

Technical Field

[0001] The invention relates to a candidate strain of Lactobacillus curvatus and application of its bacterial components, belonging to the field of microorganisms. Background Art

[0002] Allergic asthma, also known as allergic asthma or atopic asthma, is an allergic disease caused by an abnormal immune response to allergens. Asthma affects as many as 300 million people worldwide. The U.S. National Health and Nutrition Examination Survey (NHANES) (2005-2006) indicated an asthma prevalence of 8.8%, of which 62.1% were allergic. It is estimated that there are approximately 30 million asthma patients in my country, and the number continues to rise annually.

[0003] Classic allergic asthma develops in two phases: the sensitization phase and the effector phase. Only after the activation of the effector phase, the bioactive substances released, trigger the pathological manifestations of an allergic reaction. Repeated allergen stimulation leads to recurrent and persistent airway inflammation, resulting in structural changes in the airways. Based on molecular mechanisms, asthma can be categorized as type 2 and non-type 2. Type 2 asthma, which accounts for 64%-73% of all asthma patients, is characterized by an increase in eosinophils and a significant increase in the secretion of T helper 2 (Th2) cell cytokines such as interleukin-4 (IL-4), IL-5, and IL-13. Th2 cytokines promote eosinophilia and activate B cells into plasma cells, releasing large amounts of specific immunoglobulin E (IgE). This IgE promotes mast cell degranulation, releasing multiple factors, including leukotrienes and histamine, triggering contraction and spasm of airway smooth muscle, leading to asthma symptoms. Current treatments primarily alleviate airway inflammation, reduce airway reactivity, inhibit histamine release, and reduce eosinophil infiltration, such as montelukast, loratadine, suplastat toluene, and sodium cromoglycate. These drugs primarily provide rapid symptom relief, but long-term treatment increases the risk of adverse reactions and drug resistance. Therefore, the development of safe and effective probiotic microecological preparations with anti-asthma properties is crucial for the search for new anti-asthma treatments.

[0004] NF-κB (Nuclear Factor kappa B) is a ubiquitous transcription factor that is phosphorylated by IκB kinase and activated upon dissociation of its inhibitor, κ-B subunit α (IκBα). It participates in regulating inflammation, innate and adaptive immune responses, development, cell survival, and proliferation. The NF-κB complex activates the transcription of genes involved in immune-inflammatory responses. Once activated, it initiates a cascade of proinflammatory cytokines, particularly TNF-α, IL-1β, and IL-6, which play a key role in the development of allergic airway inflammation. Studies have shown that NF-κB is persistently activated in allergic asthma, and that inhibition of NF-κB significantly alleviates ovalbumin (OVA)-induced asthma. NF-κB regulates CD4+ T cell differentiation and the transcription of the Th2 cytokines IL-4 and IL-13. NF-κB activation can occur through multiple pathways. TLRs (Toll-like receptors) activate the downstream IKK complex (IκB kinase) through MyD88 (Myeloid Differentiation Primary Response 88)-dependent or -independent pathways, leading to IκB phosphorylation and degradation, and the release of NF-κB (such as the p65 / p50 dimer), which enters the cell nucleus and initiates the transcription of inflammatory genes. IL-4 and IL-13 activate the JAK / STAT signaling pathway (anus kinase / signal transducer and activator of transcription) through their receptors (IL-4R and IL-13R), promoting IgE synthesis, activating NF-κB, and increasing inflammatory responses in airway epithelial cells, smooth muscle cells, and immune cells. IL-5 exacerbates chronic airway inflammation primarily by promoting the survival and recruitment of eosinophils, thereby activating NF-κB. NF-κB activation also promotes the recruitment and activation of various inflammatory cells. For macrophages, activation of the NF-κB signaling pathway enhances their inflammatory function, leading to the release of more inflammatory mediators, such as IL-6 and NO, which further exacerbate airway inflammation. For eosinophils, activation of the NF-κB signaling pathway can induce their migration to airway tissues and activation, leading to the release of various toxic proteins and inflammatory mediators, exacerbating airway inflammation and tissue damage. Therefore, the NF-κB signaling pathway is considered a potential target for asthma treatment, and inhibition of this pathway may be important for controlling asthma symptoms and improving patient outcomes.

[0005] Related research indicates that probiotics can not only enhance intestinal homeostasis but also maintain immune homeostasis. For example, Lactobacillus rhamnosus LGG can reduce airway hyperresponsiveness and alleviate airway inflammation by reducing inflammatory cell recruitment and inflammatory cytokine secretion. Bifidobacterium attenuates airway inflammation primarily by restoring the Th1 / Th2 balance and promoting Treg cell activation. A growing body of research indicates that the intestinal microbiome is closely linked to allergic diseases. Probiotics and their metabolites not only have immunomodulatory effects but can also significantly improve asthma symptoms. Clinical and laboratory studies have demonstrated that certain probiotics can alter the intestinal microbiota and modulate host immune function, alleviating allergic airway inflammation and pathological damage. For example, Lactobacillus rhamnosus LGG can reduce inflammatory cell recruitment and inflammatory cytokine secretion, thereby reducing airway hyperresponsiveness and alleviating airway inflammation. Oral administration of Lactobacillus bulgaricus N45.10 can inhibit lung inflammation and airway remodeling in a mouse model of allergic asthma. Currently, the overall clinical evidence for the role of probiotics in preventing and treating allergic asthma is inconclusive. Probiotic functions vary among strains, and the anti-asthma effects and mechanisms vary among strains. More experiments are needed to discover and evaluate probiotics with anti-asthma function and explore their immunomodulatory mechanisms against allergic asthma, thereby providing new methods and strategies for the prevention and treatment of asthma. The present invention aims to provide a probiotic with anti-asthma function. Summary of the Invention

[0006] Based on the above invention objectives, the present invention first provides a strain of Lactobacillus curvatus IM01 (hereinafter referred to as IM01), the preservation number of the strain is CGMCC NO.30979, the preservation date is June 17, 2024, and the preservation classification is named Lactobacillus curvatus Latilactobacillus curvatus The strain was isolated from a healthy human fecal specimen and is listed on the "List of Bacteria Suitable for Food Use" issued by the National Health Commission.

[0007] In a preferred embodiment, the sequence of 16S rRNA of the strain is shown as SEQ ID NO.1.

[0008] Secondly, the present invention also provides the use of the above-mentioned strain and its bacterial components in the preparation of drugs for treating or preventing allergic airway inflammation.

[0009] In a preferred embodiment, the allergic airway disease is asthma.

[0010] In another preferred embodiment, the asthma is accompanied by elevated serum IgE and / or Th2 cytokines in the airways.

[0011] In another alternative embodiment, the asthma is accompanied by inflammatory cell infiltration and goblet cell hyperplasia in the airways.

[0012] In another alternative embodiment, the asthma is accompanied by activation of the NF-κB signaling pathway in the airways.

[0013] Third, the present invention provides a composition containing the above-mentioned strain, which contains a pharmaceutically acceptable carrier and / or excipient. The strain can be used in combination with other ingredients to assist and / or enhance the pharmacodynamic effect of asthma. In an alternative embodiment, the composition contains probiotics, plant extracts, chemical molecules, protein molecules, and nucleic acid molecules that have therapeutic effects on asthma.

[0014] In a more preferred embodiment, the composition is prepared as capsules, lyophilized powders, suspensions or tablets.

[0015] The present invention is to separate and purify the Lactobacillus curvatus strain with anti-asthma properties from the feces of healthy people. Experiments have shown that the isolated Latilactobacillus curvatus IM01 is harmless to animals and has been confirmed to have the function of relieving allergic asthma through animal experiments. Latilactobacillus curvatus IM01 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 goblet cell metaplasia, and inhibit the activation of the lung NF-κB signaling pathway, showing excellent application prospects in the preparation of asthma treatment and / or prevention drugs.

[0016] The present invention discovered a strain of Lactobacillus curvatus IM01. Animal experiments have shown that it has anti-asthmatic effects, alleviating asthma symptoms, reducing lung inflammation and airway mucus production, lowering lung Th2 cytokine levels and serum IgE levels, and inhibiting activation of the lung NF-κB signaling pathway. This strain has significant preventive and therapeutic effects on allergic asthma and holds promise as a new approach for the prevention and clinical adjuvant treatment of allergic asthma. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The animal experimental protocols used in this invention; Figure 2 Oral administration of IM01 significantly reduced the number of white blood cells in the lungs of OVA mice. 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, *** indicates p < 0.001, and **** indicates p < 0.0001. Figure 3-Figure 6Oral administration of IM01 significantly reduced the percentages of eosinophils, neutrophils, and monocytes in the lungs of OVA mice, but did not affect the percentage of basophils. The horizontal axis represents different groups, and the vertical axis represents the percentage of cells. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001. Figure 3 Effect of oral administration of IM01 on the percentage of eosinophils in total leukocytes in mouse lung tissue; 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 Effects of oral administration of IM01 on the percentage of monocytes to total leukocytes in the lung tissue of mice; Figure 7 Oral administration of IM01 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. Figure 8-12 Oral administration of IM01 significantly reduced the levels of IL-4, IL-5, and IL-13 in the lungs of OVA mice, and increased the levels of IL-10 and TGF-β. 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. Figure 8 Effects of oral administration of IM01 on IL-4 levels in mouse lung tissue; Figure 9 Effects of oral administration of IM01 on IL-5 levels in mouse lung tissue; Figure 10 Effects of oral administration of IM01 on IL-13 levels in mouse lung tissue; Figure 11 Effects of oral administration of IM01 on IL-10 levels in mouse lung tissue; Figure 12 Effects of oral administration of IM01 on IL-TGF-β levels in mouse lung tissue; Figure 13 Shows the results of H&E staining comparison of lung tissue sections of mice in each group after oral administration of IM01 (I); Figure 14 Shows the comparison results of H&E staining of lung tissue sections of mice in each group after oral administration of IM01 (B); Figure 15 Shows the results of H&E staining comparison of lung tissue sections of mice in each group after oral administration of IM01 (III); Figure 16 Shows the results of PAS staining comparison of lung tissue sections of mice in each group after oral administration of IM01 (I); Figure 17 Shows the comparison results of PAS staining of lung tissue sections of mice in each group after oral administration of IM01 (B); Figure 18 Shows the results of PAS staining comparison of lung tissue sections of mice in each group after oral administration of IM01 (III); Figure 19-23 Shows the effect of oral administration of LC IM01 on the expression of NF-κB p65, Phospho-NF-κB p65, NF-κB IκBα, and Phospho-NF-κB IκBα proteins in the lungs of OVA mouse models; Figure 19 Western blot analysis showed that oral administration of LC IM01 affects the expression of NF-κB p65, Phospho-NF-κB p65, NF-κB IκBα, and Phospho-NF-κB IκBα proteins in the lungs of OVA mouse models; Figure 20 The results show the effect of oral administration of LC IM01 on the relative expression of NF-κB p65 protein in the lungs of OVA mice compared with the internal reference gene β-actin. The horizontal axis represents different genes, and the vertical axis represents the relative expression of proteins compared with actin. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001. Figure 21 The results show the effect of oral administration of LC IM01 on the relative expression of Phospho-NF-κB p65 protein in the lungs of OVA mice compared with the internal reference gene β-actin. The horizontal axis represents different genes, and the vertical axis represents the relative expression of proteins compared with actin. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001. Figure 22 The results show the effect of oral administration of LC IM01 on the relative expression of NF-κB IκBα protein in the lungs of OVA mice compared with the internal reference gene β-actin. The horizontal axis represents different genes, and the vertical axis represents the relative expression of proteins compared with actin. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001. Figure 23The figure shows the effect of oral administration of LC IM01 on the relative expression of Phospho-NF-κB IκBα protein in the lungs of OVA mice model compared with the internal reference gene β-actin. The horizontal axis represents different genes, and the vertical axis represents the relative expression of proteins compared with actin. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001. DETAILED DESCRIPTION

[0018] 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.

[0019] Example 1. Isolation, identification and preservation of Lactobacillus curvatus IM01 strain 1.1 Isolation of Lactobacillus curvatus IM01 (1) Prepare MRS agar medium, sterilize it by high pressure at 121°C for 15 min, pour it into a culture dish, and use it for strain isolation after solidification; (2) Take an appropriate amount of fecal specimens collected from healthy individuals and place them into a 1.5 mL Eppendorf tube pre-filled with 900 μL of sterile PBS, and perform a 10-fold gradient dilution; (3) Take 100 μL of samples with different dilutions and spread them on MRS solid culture medium, and incubate them at 37°C 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. Culture anaerobically at 37°C for 48 h, transfer them three times continuously, and culture the purified strains in liquid MRS at pH = 3.5. Screen the strains with excellent growth for use in experiments or frozen storage.

[0020] 1.2 Culture preservation This laboratory uses MRS medium containing 30% glycerol as the culture medium for freezing and preserving bacterial strains. The method is as follows: (1) Prepare the bacterium preservation solution, sterilize it under high pressure, and dispense it into sterile 2 mL bacterium preservation tubes; (2) After the bacteria have been transferred to BHI solid medium three times, add 1.5 mL of sterile culture medium 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 into a bacterial preservation tube, mix well, and store at -80℃.

[0021] 1.3 Observation of colony appearance and bacterial morphology Lactobacillus curvatus is a non-spore-forming, facultative anaerobic bacterium that grows well in a 37°C incubator. Its colonies are round, milky white, smooth, with raised, neatly marginated, and opaque. Gram-positive, curved rods can be observed under a Gram staining microscope.

[0022] 1.4 Extraction of total bacterial DNA A single colony was inoculated on BHI 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).

[0023] 1.5 Accurate identification of strains by comparing ANI and DDH with the model strain 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 / ).

[0024] 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 pellet and shake to remove protein impurities; then add 200 μl of buffer BCL and shake to mix; then place in a 55°C water bath for 10 min, and mix by inversion to ensure that there are no impurities precipitated in the solution; (2) 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. (3) 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; (4) Add 600 μl of WB buffer, 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. This step is repeated twice to ensure that the DNA is clean. (5) Place the adsorption column in the collection tube, open the cover and leave it at room temperature for 3-5 minutes to dry, so that the residual rinse solution (mainly ethanol) in the adsorption column can evaporate completely; (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, centrifuge at 12,000 rpm for 1 minute to obtain pure bacterial genomic DNA, and transport it to Beijing Novogene Technology Co., Ltd. via cold chain to complete the draft sequencing of the genome.

[0025] 1.5.2 Genomic Correlation Analysis (1) Basic characteristics of the genome The draft results showed that the genome size of strain IM01 was 1,931,328 bp and the GC content was 42.66%.

[0026] (2) dDDH and ANI Selection of a model strain of Lactobacillus curvatus Latilactobacillus curvatus strain Comparison of dDDH and ANI between the NBRC 15884 and IM01 strains showed that the dDDH and ANI values ​​between the model strain NBRC 15884 and IM01 were 97% and 99.64%, respectively, supporting the identification of the two strains as the same bacterial species.

[0027] (3) 16S RNA sequence determination A single colony of the bacteria was picked and added to 10 mL of BHI liquid medium, shaken at 37°C overnight, and transported under cold chain to Beijing Tianyi Huiyuan Biotechnology Co., Ltd. for 16S RNA sequencing. Sequencing revealed that the 16S RNA sequence of the IM01 strain is shown in SEQ ID NO. 1.

[0028] The preservation information of this strain is: The preservation number of Lactobacillus curvatus IM01 strain is CGMCC NO.30979, the preservation date is June 17, 2024, and the preservation classification is named Lactobacillus curvatus Latilactobacillus curvatus 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.

[0029] Example 2. Evaluation of the anti-asthma function of Lactobacillus curvatus IM01 2.1 Sample: Activated culture of Lactobacillus curvatus IM01 strain in the logarithmic growth phase.

[0030] 2.2 Experimental animals and OVA animal model construction Four-week-old female SPF BALB / c mice, weighing 13–15 g, were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. (license number SYXK(Beijing)2021-0006). They were housed at the Laboratory Animal Center of the Chinese Center for Disease Control and Prevention (license number SYXK(Beijing)2022-0029), with five mice per cage, in a barrier environment with a 12-h light / dark cycle. Mice had free access to water and food. All animal experiments were conducted in accordance with the Ethical Guidelines for Laboratory Animal Welfare of the Laboratory Animal Center of the Chinese Center for Disease Control and Prevention (approval number: 2023-025).

[0031] The animal experiment scheme used in the present invention is as follows Figure 1 As shown. Mice were randomly divided into three groups: normal group (PBS group), model group (OVA group), and probiotic intervention group (OVA+LC IM01 group). After one week of adaptation, mice in the PBS and OVA groups were gavaged with 100 μL PBS every day for 5 days before sensitization (day -5) until the end of the experiment (the day of sensitization was recorded as day 0). Mice in the OVA+LC IM01 group were gavaged with 100 μL IM01 bacterial suspension (2×10 9 CFU / mouse). On days 0 and 7, mice in the OVA and OVA+LC IM01 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. On days 14-16, mice in the OVA and OVA+LC IM01 groups were sensitized with 1% OVA solution via nebulized inhalation daily for 30 minutes to induce asthma. The PBS group was sensitized with nebulized saline instead.

[0032] 2.3 Sample collection: Mice were sacrificed 24 h after the last nebulized challenge, and serum and lung tissue were collected.

[0033] (1) Blood sample collection 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.

[0034] (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.

[0035] 2.4 Detection method: 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.

[0036] 2.4.2 Detection of Th2 cytokines (IL-4, IL-5, and IL-13) in lung homogenate supernatants: Commercially available kits were used for detection according to the manufacturer's instructions. These assays 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).

[0037] 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.

[0038] (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.

[0039] (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.

[0040] (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.

[0041] (5) Adjust the cell concentration to 1×106 cells / 100 μL cell suspension, add anti-CD16 / 32 to block Fc receptors, and incubate on ice in the dark for 10 min. Then add 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), and incubate on ice in the dark for 30 min.

[0042] (6) Add 1 mL of PBS and centrifuge at 400 g for 5 min. Repeat twice.

[0043] (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.

[0044] 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).

[0045] 2.4.5 Tissue Protein Extraction and Western Blot Analysis: Tissues were placed in grinding tubes containing 1 mL of PBS and one grinding bead was added to each tube. The tubes were then placed in a grinding block pre-chilled at -80°C for 2 h and ground. After grinding, 200 μL of the homogenate was added to an equal volume of RIPA protein lysis buffer (containing protease inhibitors) and mixed thoroughly using a micropipette. The tubes were placed on ice for 30 min and then centrifuged at 12,000 rpm for 10 min at 4°C. The supernatant was transferred to a new 1.5 mL EP tube, and 2 μL of each sample was reserved for protein quantification. The remaining sample was added to one-fourth of the volume of 5× protein loading buffer and heated in a 95°C metal bath for 10 min. The samples were then centrifuged at 12,000 rpm for 5 min at 4°C. The prepared protein samples were collected and frozen at -80°C for subsequent analysis. BCA protein quantification was performed using a commercially available kit (Pierce™ BCA Protein Assay Kits, Cat# 23227) according to the manufacturer's instructions. A commercial precast gel (Lablead, Cat# P01015) was used. After adding electrophoresis buffer, 30 μg of sample protein was added to the sample wells and electrophoresis was performed at 160 V for approximately 40 minutes. Nitrocellulose membrane (NC membrane) was activated with 1× transfer buffer and transferred to the membrane using a wet transfer method at a constant current of 250 mA for 47 minutes. The membrane was then blocked with 5% skim milk in 1× TBST at 37°C for 1 hour and washed three times with 1× TBST for 5 minutes each to remove residual skim milk. The desired bands were cut according to the protein marker and incubated with the primary antibody overnight at 4°C. The next day, the membrane was washed three times with 1× TBST and incubated with the corresponding HRP-conjugated secondary antibody at 37°C for 1 hour. The membrane was then washed three times with 1× TBST and the expression of the corresponding protein was detected by electrochemiluminescence (ECL).

[0046] 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 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.

[0047] 2.6 Results To observe the effect of Lactobacillus curvatus 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 followed by aerosol challenge. Figure 1As shown, mice were gavaged with 100 μL of Lactobacillus curvatus IM01 (1×10 9 CFU / mouse). The model group and blank control group were gavaged with an equal volume of PBS daily. Mice were sacrificed 24 hours after aerosol challenge. The anti-asthmatic effect of the IM01 strain was evaluated by comparing the total number of lung leukocytes, percentage of differentiated cells, serum IgE, lung Th2 cytokines, lung inflammatory cell infiltration, goblet cell metaplasia, and changes in inflammatory pathways between the OVA+LC IM01 group and the OVA group.

[0048] 2.6.1 IM01 can effectively reduce serum IgE and Th2 cytokines in the lungs of asthmatic mice. This study was divided into three groups: PBS group (normal group), OVA group (OVA sensitization challenge), and OVA+LC IM01 group (OVA sensitization challenge and oral administration of Lactobacillus curvatus IM01 intervention). The results showed that oral administration of Lactobacillus curvatus IM01 can significantly reduce the number of white blood cells in the lungs of asthmatic mice ( Figure 2 , PBS group 2737±39.66, OVA group 6230±125.9, OVA+LCIM01 group 5252±441.6), mainly manifested by a significant decrease in the proportion of eosinophils, basophils, neutrophils, and monocytes ( Figure 3 Eosinophils: 1.329±0.3387 in the PBS group, 3.683±0.7831 in the OVA group, and 0.9297±0.4451 in the OVA+LCIM01 group; Figure 4 For basophils: 0.04194±0.02039 in the PBS group, 2.216±0.2857 in the OVA group, and 1.504±0.1289 in the OVA+LC IM01 group; Figure 5 Neutrophils: 2.771±0.574 in the PBS group, 45.52±4.623 in the OVA group, and 22.02±5.093 in the OVA+LC IM01 group; Figure 6 For monocytes: PBS group 0.6056±0.1508, OVA group 0.5661±0.09544, OVA+LC IM01 group 0.1876±0.05502). In addition, this strain can also effectively reduce the serum IgE of asthmatic mice ( Figure 7 , 1.35±0.26 μg / mL in the PBS group, 52.95±13.92 μg / mL in the OVA group, and 29.04±5.70 μg / mL in the OVA+LC IM01 group).

[0049] 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 26.60±8.84 pg / mL, OVA group 76.23±11.87 pg / mL, OVA+LC IM01 group 50.28±8.61 pg / mL), IL-5 ( Figure 9 , PBS group 2.30±0.18 pg / mL, OVA group 60.45±15.23 pg / mL, OVA+LC IM01 group 21.98±5.18 pg / mL) and IL-13 ( Figure 10 , PBS group 6.15±0.80 pg / mL, OVA group 67.70±52.35 pg / mL, OVA+LC IM01 group 25.34±7.10 pg / mL). At the same time, the bacteria can upregulate the anti-inflammatory cytokine IL-10 ( Figure 11 , PBS group 698.29±52.35 pg / mL, OVA group 717.37±50.92 pg / mL, OVA+LC IM01 group 860.43±142.71 pg / mL) and TGF-β ( Figure 12 , 61.80±6.88 pg / mL in the PBS group, 78.89±4.49 pg / mL in the OVA group, and 97.77±18.32 pg / mL in the OVA+LC IM01 group).

[0050] 2.6.2 IM01 can effectively reduce lung pathological damage in asthmatic mice. 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 the mice. H&E staining results showed that compared with the PBS group mice, the bronchial mucosa of the lung tissue of the OVA group mice had varying 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 infiltration of inflammatory cells in the OVA+LC IM01 group mice was significantly reduced, and the damage to the airway epithelium was significantly alleviated compared with the OVA group ( Figure 13-15 PAS staining showed that compared with the PBS group, the goblet cell metaplasia of the lung tissue airway wall of the OVA group mice was significantly increased, while the degree of goblet cell metaplasia in the OVA+LC IM01 group was significantly decreased compared with the OVA group ( Figure 16-18 ).

[0051] 2.6.3 Effect of IM01 on the pulmonary inflammatory pathway in asthmatic mice. After 21 days of IM01 intervention, the expression of NF-κB p65, Phospho-NF-κB p65, NF-κB IκBα, and Phospho-NF-κB IκBα proteins in the lung tissues of mice were significantly lower than those in the OVA group ( Figure 19-23 NF-κB p65: 1.87±0.52 in the PBS group, 0.98±0.16 in the OVA group, and 0.83±0.12 in the OVA+LC IM01 group; Phospho-NF-κB p65: 0.82±0.11 in the PBS group, 0.97±0.32 in the OVA group, and 0.34±0.16 in the OVA+LC IM01 group; NF-κB IκBα: 1.08±0.14 in the PBS group, 0.83±0.10 in the OVA group, and 1.11±0.09 in the OVA+LC IM01 group; Phospho-NF-κB IκBα: 0.27±0.14 in the PBS group, 0.85±0.14 in the OVA group, and 0.85±0.14 in the OVA+LC IM01 group 0.51±0.15), indicating that the IM01 strain can effectively inhibit the activation of the NF-κB inflammatory pathway induced by OVA, suggesting that inhibition of the NF-κB inflammatory pathway by IM01 is one of the possible mechanisms of the bacteria in the anti-asthma effect.

[0052] In summary, compared with the OVA group, oral administration of Lactobacillus curvatus IM01 strain can significantly improve the asthma symptoms of mice, reduce lung inflammatory cell infiltration and goblet cell metaplasia, and inhibit the activation of the NF-κB inflammatory pathway, indicating that Lactobacillus curvatus IM01 strain has good anti-inflammatory and anti-asthma effects, which is of great significance for the prevention and treatment of asthma.

Claims

1. A strain of Lactobacillus curvatus, the deposit number of which is CGMCC NO.30979, the deposit date is June 17, 2024, and the deposit classification is named Lactobacillus curvatus Latilactobacillus curvatus The preservation unit is the General Microbiology Center of China Culture Collection Administration of Microorganisms.

2. The Lactobacillus curvatus 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 or bacterial component 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 is asthma. The use according to claim 3, wherein the asthma is accompanied by an increase in serum IgE and / or Th2 cytokines in the airways.

6. 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.

7. The use according to claim 3, characterized in that Asthma is accompanied by activation of the NF-κB inflammatory signaling pathway in the airways.

8. A composition containing the strain according to claim 1 or 2, characterized in that The composition contains pharmaceutically acceptable carriers and / or excipients.

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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