Application of bifidobacterium animalis subsp. Lactis in preparation of preparation for preventing and / or treating allergic rhinitis

Abstract

The invention relates to application of bifidobacterium animalis subsp. Lactis in preparation of a preparation for preventing and / or treating allergic rhinitis. The bifidobacterium animalis subsp. Lactis comprises Bifidobacterium animalis subsp. Lactis BGI-N3, the Bifidobacterium animalis subsp. Lactis BGI-N3 is preserved in the China Center for Type Culture Collection on October 17, 2022, and the preservation number of the Bifidobacterium animalis subsp. Lactis BGI-N3 is CCTCC (China Center for Type Culture Collection) NO: M20221585. Based on an allergic rhinitis animal model, the action mechanism of the animal bifidobacterium subsp. Lactis in the inflammation process is deeply analyzed, it is found that the animal bifidobacterium subsp. Lactis can effectively prevent and / or treat allergic rhinitis, and a new strategy is provided for developing a scheme for treating allergic rhinitis.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to the application of Bifidobacterium animalis subsp. lactis in the preparation of a preparation for preventing and / or treating allergic rhinitis. Background Art

[0002] Allergic rhinitis, also known as allergic rhinitis (AR), is a chronic nasal mucosal inflammatory disease characterized by an immune response mediated mainly by immunoglobulin E (IgE) in susceptible individuals upon exposure to specific allergens, involving the participation of immune active cells and cytokines. The main clinical manifestations of this disease include episodic sneezing, runny nose, nasal congestion and other symptoms. Although allergic rhinitis usually does not cause death, it has a significant negative impact on the quality of life of patients. The frequent occurrence of rhinitis symptoms, such as nasal congestion, runny nose and sneezing, seriously affects the social interaction, work efficiency and learning ability of patients. In addition, if allergic rhinitis fails to be treated promptly and effectively, its long-term existence may lead to further immune system dysfunction, increasing the sensitivity of patients to external stimuli and forming a vicious cycle. Therefore, the early identification and treatment of allergic rhinitis are crucial for improving the quality of life of patients and preventing potential immune function disorders.

[0003] There are numerous treatment strategies for allergic rhinitis, including allergen avoidance, drug therapy, immunotherapy, and the emerging probiotic therapy. Allergen avoidance is the key to treatment, involving maintaining a clean indoor environment, using air purification equipment, and reducing exposure to common allergens such as dust mites and pollen. Drug therapy plays a central role in the treatment of allergic rhinitis, including oral and nasal antihistamines and glucocorticoids, which can effectively relieve symptoms such as nasal itching, sneezing, and runny nose. Leukotriene receptor antagonists, such as montelukast, have been shown to effectively improve the nocturnal symptoms of patients with allergic rhinitis. Mast cell stabilizers, such as sodium cromoglycate, reduce the release of inflammatory mediators by stabilizing the mast cell membrane. In addition, decongestants such as ephedrine can quickly relieve nasal congestion symptoms. Immunotherapy, also known as allergen-specific immunotherapy (ASIT), is a treatment method that trains the patient's immune system by gradually increasing the allergen dose, aiming to reduce sensitivity to allergens. This method has been proven to reduce the symptoms of allergic rhinitis and improve the quality of life of patients in the long term. In recent years, the emerging probiotic therapy, as a new treatment method, has also begun to play a role in the treatment of allergic rhinitis. Studies have shown that probiotics may help prevent and / or alleviate the symptoms of allergic rhinitis by regulating the host's immune response, balancing the gut microbiota, enhancing the gut barrier function, and having an anti-inflammatory effect. These findings provide a new perspective for the treatment of allergic rhinitis and open up new possibilities for future research and clinical practice.

[0004] Although the existing treatment methods for allergic rhinitis have their respective advantages, there are also certain limitations in practical applications. Although allergen avoidance is a key strategy for preventing allergic reactions, it is very challenging for patients to completely avoid commonly existing allergens such as pollen and dust mites in daily life. Although drug therapy can quickly relieve the symptoms of rhinitis, its accompanying side effects cannot be ignored. For example, antihistamines may cause adverse reactions such as drowsiness and headache, while long-term use of glucocorticoid drugs may lead to side effects such as thinning of the nasal mucosa, increased infection risk, and endocrine disorders in the body. As an allergy treatment method with relatively few side effects, immunotherapy still has many deficiencies such as a long treatment cycle, high cost, and poor patient compliance. In addition, non-drug treatment methods, such as probiotic intervention, are greatly affected by the biological characteristics of the probiotics themselves. Even different strains of the same genus or species of bacteria may show significant strain-specificity in terms of genetic metabolism characteristics and action mechanisms. In clinical interventions to improve allergic rhinitis, it is often difficult to ensure their actual application efficacy, which limits their wide application in clinical practice.

[0005] In summary, there is an urgent need for a more effective and safer treatment plan to improve the quality of life of patients with allergic rhinitis and reduce side effects during the treatment process. Summary of the Invention

[0006] In view of the deficiencies of the prior art and the actual needs, the present invention provides the use of Bifidobacterium animalis subsp. lactis in the preparation of a preparation for preventing and / or treating allergic rhinitis, deeply analyzes Bifidobacterium animalis subsp. lactis BGI-N3, and develops its application in the field related to allergic rhinitis.

[0007] To achieve this purpose, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides the use of Bifidobacterium animalis subsp. lactis in the preparation of a preparation for preventing and / or treating allergic rhinitis; the Bifidobacterium animalis subsp. lactis includes Bifidobacterium animalis subsp. lactis BGI-N3, which was deposited at the China Center for Type Culture Collection on October 17, 2022, with the deposit number CCTCC NO: M20221585.

[0009] In the present invention, based on an animal model of allergic rhinitis, the mechanism of action of Bifidobacterium animalis subsp. lactis BGI-N3 in the inflammatory process was deeply analyzed, and it was found that it can effectively prevent and / or treat allergic rhinitis, providing a new strategy for developing a treatment plan for allergic rhinitis.

[0010] It can be understood that the preparation can be a related preparation containing the cells of the Bifidobacterium animalis subsp. lactis, such as freeze-dried bacterial powder, etc.

[0011] In a second aspect, the present invention provides a pharmaceutical composition for preventing and / or treating allergic rhinitis, and the pharmaceutical composition includes the Bifidobacterium animalis subsp. lactis described in the first aspect.

[0012] Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable excipient.

[0013] Preferably, the excipient includes at least one of a carrier, a wetting agent, a solubilizer, an osmotic pressure regulator, a coating material, a coloring agent, a pH regulator, an antioxidant, or a buffer.

[0014] In a third aspect, the present invention provides the use of the Bifidobacterium animalis subsp. lactis described in the first aspect in the preparation of a preparation for repairing nasal mucosa damage.

[0015] In the present invention, it was found that Bifidobacterium animalis subsp. lactis BGI-N3 can effectively repair nasal mucosa tissue damage and improve the adverse pathological state of nasal mucosa tissue.

[0016] Fourth aspect, the present invention provides the use of the Bifidobacterium animalis subsp. lactis described in the first aspect in the preparation of a preparation for inhibiting intestinal pathogenic bacteria.

[0017] Preferably, the intestinal pathogenic bacteria include Escherichia coli and / or Staphylococcus aureus.

[0018] In the present invention, it is found that Bifidobacterium animalis subsp. lactis BGI-N3 can effectively inhibit intestinal pathogenic bacteria such as Escherichia coli and Staphylococcus aureus, and has potential probiotic effects of maintaining the immune health of the body by regulating the balance of the intestinal flora. It can be used to develop related therapeutic drugs and can also be used to develop in vitro bacteriostatic agents for non-therapeutic purposes.

[0019] Fifth aspect, the present invention provides the use of the Bifidobacterium animalis subsp. lactis described in the first aspect in the preparation of a preparation for regulating the levels of serum cytokines.

[0020] Preferably, the cytokines include at least one of interleukin-12 (IL-12), interleukin-13 (IL-13), interferon-γ (IFN-γ), or transforming growth factor-β (TGF-β).

[0021] In the present invention, it is found that Bifidobacterium animalis subsp. lactis BGI-N3 can effectively regulate the levels of serum cytokines, reduce the level of IL-13, and increase the levels of IL-12, IFN-γ, and TGF-β. It can be used to develop related therapeutic drugs and can also be used to develop cytokine level regulators for non-therapeutic purposes for basic mechanism research.

[0022] Sixth aspect, the present invention provides the use of the Bifidobacterium animalis subsp. lactis described in the first aspect in the preparation of a preparation for regulating the levels of serum allergy markers.

[0023] Preferably, the serum allergy markers include at least one of immunoglobulin A (IgA), immunoglobulin E (IgE), platelet-activating factor (PAF), or immunoglobulin G1 (IgG1).

[0024] In the present invention, it is found that Bifidobacterium animalis subsp. lactis BGI-N3 can effectively regulate the levels of serum allergy markers, reduce the levels of IgE, IgG1, and PAF, and increase the level of IgA. It can be used to develop related therapeutic drugs and can also be used to develop serum allergy marker level regulators for non-therapeutic purposes for basic mechanism research.

[0025] Seventh aspect, the present invention provides the use of the Bifidobacterium animalis subsp. lactis described in the first aspect in the preparation of a preparation for regulating the differentiation of CD4 + T lymphocytes.

[0026] In the present invention, it is found that Bifidobacterium animalis subsp. lactis BGI-N3 can effectively regulate the differentiation of CD4 + T lymphocytes, increase the ratio of the number of Th1 / Th2 cells, and decrease the ratio of the number of Th17 / Treg cells, and can be used for the development of related therapeutic drugs.

[0027] Compared with the prior art, the present invention has at least the following beneficial effects:

[0028] (1) In the present invention, it is discovered by excavation that Bifidobacterium animalis subsp. lactis BGI-N3 has good ability to inhibit intestinal pathogenic bacteria, and can restore and maintain the normal function of the immune system by balancing the intestinal flora;

[0029] (2) In the present invention, it is discovered by excavation that Bifidobacterium animalis subsp. lactis BGI-N3 can regulate the levels of serum allergy markers and cytokines, regulate the differentiation of CD4 + T lymphocytes, reduce the nasal mucosa damage caused by allergy, and effectively improve the symptoms of allergic rhinitis;

[0030] (3) Bifidobacterium animalis subsp. lactis BGI-N3 involved in the present invention provides a new direction for the treatment of allergic rhinitis, and has no toxic and side effects compared with other treatment methods, demonstrating the broad application prospects of BGI-N3 strain in the fields of food, health products and drugs, and indicating its huge potential in the industry. Description of the Drawings

[0031] Figure 1 It is a detection result diagram of the anti-inflammatory effect of Bifidobacterium animalis subsp. lactis BGI-N3 in Example 3, including the contents of TNF-α, IL-1β, IL-6 and IL-8 and the number of immune cells (* indicates p < 0.05 compared with the Control group; ** indicates p < 0.01 compared with the Control group; *** indicates p < 0.001 compared with the Control group; # indicates p < 0.05 compared with the LPS group; ## indicates p < 0.01 compared with the LPS group; indicates p < 0.001 compared with the LPS group; tested by ONE-WAY ANOVA);

[0032] Figure 2 It is a diagram of the allergic rhinitis symptom scores of rats in each group in Example 7, including typical rhinitis symptoms such as sneezing, nose rubbing, and runny nose (* indicates p < 0.05 compared with the model control group; ** indicates p < 0.01 compared with the model control group; *** indicates p < 0.001 compared with the model control group; tested by ONE-WAY ANOVA);

[0033] Figure 3Graphs of the levels of serum IgE, IgA, IgG1, and PAF in rats of each group in Example 7 (* indicates p < 0.05 relative to the model control group; ** indicates p < 0.01 relative to the model control group; *** indicates p < 0.001 relative to the model control group; tested by ONE-WAY ANOVA);

[0034] Figure 4 Graphs of the levels of TGF-β, IL-12, IL-13, and IFN-γ in serum of rats of each group in Example 7 (* indicates p < 0.05 relative to the model control group; ** indicates p < 0.01 relative to the model control group; *** indicates p < 0.001 relative to the model control group; tested by ONE-WAY ANOVA);

[0035] Figure 5 Graph of the CD4 + T lymphocyte count results in the spleens of rats of each group in Example 7, including the ratio of the number of Th1 / Th2 cells and the ratio of the number of Th17 / Treg cells (* indicates p < 0.05 relative to the model control group; ** indicates p < 0.01 relative to the model control group; *** indicates p < 0.001 relative to the model control group; tested by ONE-WAY ANOVA);

[0036] Figure 6 Graph of the histopathological analysis results of hematoxylin-eosin (HE) staining of nasal mucosa tissues of rats of each group in Example 7;

[0037] Figure 7 Graph of the histopathological analysis results of periodic acid-Schiff (PAS) staining of nasal mucosa tissues of rats of each group in Example 7. Detailed implementation mode

[0038] The technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and through specific implementation modes. However, the following examples are merely simple examples of the present invention and do not represent or limit the scope of the protection of the rights of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0039] For those not specifying specific techniques or conditions in the examples, they shall be in accordance with the techniques or conditions described in the literature in this field or in accordance with the product specifications. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through regular channels.

[0040] In a specific embodiment of the present invention, Bifidobacterium animalis subsp. lactis BGI-N3 was deposited at the China Center for Type Culture Collection (CCTCC) on October 17, 2022. The address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with a postal code of 430072, and the deposit number is CCTCC NO: M20221585.

[0041] Bifidobacterium animalis subsp. lactis involved in the present invention has a long application history at home and abroad. It has been included in the "List of Strains That Can Be Used in Food" issued by the National Health Commission. This strain has been widely recognized as having high safety, and no adverse reactions have been observed after long-term ingestion. Therefore, it is considered a probiotic suitable for long-term intake.

[0042] Example 1

[0043] In this example, an active probiotic powder of Bifidobacterium animalis subsp. lactis BGI-N3 was prepared.

[0044] Preparing Bifidobacterium animalis subsp. lactis BGI-N3 into an active probiotic powder is a key step for its application. The preparation of the active probiotic powder of Bifidobacterium animalis subsp. lactis BGI-N3 includes two parts: high-density fermentation and freeze-drying.

[0045] High-density fermentation: Take a cryopreservation tube of Bifidobacterium animalis subsp. lactis BGI-N3 strain and inoculate it into 300 mL of TPY liquid medium (product number 027350, purchased from Guangdong Huankai Microbial Technology Co., Ltd.) at an inoculation amount of 1%. Anaerobically culture at 37°C for 24 h, and then inoculate it into 5 L of TPY liquid medium at an inoculation amount of 5%. Anaerobically culture at 37°C. When the cell density in the fermentation broth reaches 2.0×10 9 CFU / mL, collect the fermentation broth for the subsequent steps.

[0046] Freeze-drying: Centrifuge the fermentation broth at 4000 rpm for 10 min at 4°C, collect the cell sludge, add 110 g of skim milk powder, 140 g of trehalose, and 20 g of glycerol as freeze-drying protectants. Add 800 mL of sterile water to dissolve and mix evenly. Place it at -60°C for pre-freezing for 3 h, and then place it in a freeze-dryer. Complete the freeze-drying according to the instructions of the freeze-dryer. Finally, obtain the active probiotic powder of Bifidobacterium animalis subsp. lactis BGI-N3, and its active probiotic content is 100 billion CFU / g.

[0047] Example 2

[0048] This example detects the inhibitory ability of Bifidobacterium animalis subsp. lactis BGI-N3 against intestinal pathogenic bacteria.

[0049] The intestinal flora is closely related to the human immune system, and the balance of the intestinal flora is crucial for maintaining the normal function of the immune system. By measuring the inhibitory ability of Bifidobacterium animalis subsp. lactis BGI-N3 against common intestinal pathogenic bacteria in vitro, it helps to understand its regulatory effect on the balance of the intestinal flora.

[0050] Two common intestinal pathogenic bacteria, including Escherichia coli and Staphylococcus aureus, were activated using Brain Heart Infusion Broth Medium (BHI, product number HB8297-1, purchased from Qingdao Haibo Biotechnology Co., Ltd.). After activation, the cell concentration OD was adjusted using sterile Phosphate Buffered Saline (PBS, product number HB8507-1, purchased from Qingdao Haibo Biotechnology Co., Ltd.) 600nm = 0.7 for standby. The lyophilized active bacteria powder of Bifidobacterium animalis subsp. lactis BGI-N3 in Example 1 was activated using TPY Liquid Medium (product number 027350, Guangdong Huankai Microbial Science and Technology Co., Ltd.). The bacterial liquid was centrifuged at 7000 rpm for 5 min to obtain the probiotic fermentation supernatant, which was filtered through a 0.22 μm filter membrane for standby.

[0051] The inhibitory ability of Bifidobacterium animalis subsp. lactis BGI-N3 against common intestinal pathogenic bacteria was determined by the co-culture inhibition method. In the experimental group, 50 μL of the pathogen solution with adjusted concentration, 50 μL of the probiotic fermentation supernatant, and 50 μL of double-concentration BHI broth medium were added to a 96-well plate; in the control group, sterile normal saline was used instead of the probiotic fermentation supernatant. After adding the samples, the 96-well plate was placed in an incubator at 37 °C for 24 h, and the growth of different pathogenic bacteria was evaluated by OD 600nm absorbance. The experiment was repeated 3 times, and the results were expressed as mean ± standard deviation. The pathogen inhibition rate (%) = [1 - (OD 600试验组 / OD 600对照组 )] × 100%

[0052] The experimental results are shown in Table 1. The growth inhibition rate of Bifidobacterium animalis subsp. lactis BGI-N3 against Escherichia coli was 80.8 ± 6.4%; the growth inhibition rate against Staphylococcus aureus was 82.7 ± 5.2%. The above results indicate that Bifidobacterium animalis subsp. lactis BGI-N3 has a high inhibitory ability against the above common intestinal pathogenic bacteria and has the potential probiotic effect of maintaining the immune health of the body by regulating the balance of the intestinal flora.

[0053] Table 1 Inhibitory ability of Bifidobacterium animalis subsp. lactis BGI-N3 against intestinal pathogenic bacteria

[0054]

[0055]

[0056] Example 3

[0057] This example tests the inflammation inhibitory ability of Bifidobacterium animalis subsp. lactis BGI-N3.

[0058] By constructing an in vitro inflammatory macrophage model, the probiotic potential of Bifidobacterium animalis subsp. lactis BGI-N3 in immunomodulation was evaluated.

[0059] The Bifidobacterium animalis subsp. lactis BGI-N3 strain was inoculated into 50 mL of TPY liquid medium at an inoculum size of 2%, anaerobically cultured at 37 °C until the logarithmic growth phase, centrifuged at 4000 rpm for 10 min at 4 °C, the cells were collected, washed twice with sterile PBS, and finally resuspended in the cell basal medium, and the probiotic concentration was adjusted to 1.0×10 9 CFU / mL for subsequent experiments.

[0060] The culture conditions of U937 human macrophages (product number AC055, purchased from American Tissue Culture Collection, ATCC) were RPMI-1640 medium (product number 33823, purchased from Invitrogen Gibco) containing 100 U / mL penicillin, 100 μg / mL streptomycin, and 10% FBS; when the cell confluence reached 90%, the cells were collected by centrifugation at 800 rpm, the old medium was discarded, the cells were washed twice with 2 mL PBS, the supernatant was discarded after centrifugation at 800 rpm for 5 min, the cells were resuspended in the complete medium, subcultured in flasks, and the medium was changed every other day.

[0061] The macrophage inflammation inhibition test was divided into 4 groups: (1) Control group; (2) LPS group; (3) BGI-N3 group; (4) BGI-N3 + LPS group. Each group was inoculated with 1×10 4 logarithmic growth phase U937 cells per well in a 96-well cell culture plate, and incubated with complete medium containing 500 ng / mL phorbol myristate acetate (PMA) for 24 h to induce the maturation of macrophage U937 in each group.

[0062] The intervention methods for different groups are as follows: (1) For the Control group, after inducing and maturing U937 cells and culturing them normally for 12 h, replace the fresh complete medium and continue culturing for 24 h; (2) For the LPS group, after inducing and maturing U937 cells and culturing them normally for 12 h, use the complete medium containing 1 μg / mL lipopolysaccharide (LPS) and continue culturing for 24 h. (3) For the BGI-N3 group: After infecting the induced and mature U937 cells with the medium containing BGI-N3 (the ratio of BGI-N3 to the multiplicity of infection (MOI) of immune cells is 100:1) for 12 h, replace the fresh complete medium and continue culturing for 24 h. (4) For the BGI-N3 + LPS group: After infecting the induced and mature U937 cells with the medium containing BGI-N3 (the ratio of BGI-N3 to the multiplicity of infection (MOI) of immune cells is 100:1) for 12 h, replace the complete medium containing 1 μg / mL LPS and continue culturing for 24 h. After completion, use an ELISA detection kit to detect the contents of TNF-α, IL-1β, IL-6, and IL-8 in the cell supernatant, and use CCK8 to determine the number of immune cells.

[0063] The experimental results are shown in Table 2 and Figure 1 , and the data are presented in the form of mean ± standard deviation. Compared with the Control group, * p < 0.05, ** p < 0.01, *** p < 0.001; compared with the LPS group, # p < 0.05, ## p < 0.01, ### p < 0.001. The results show that: compared with the Control group, the cell viability of U937 cells in the LPS group decreased significantly, and the levels of TNF-α, IL-1β, IL-6, and IL-8 in the cell supernatant increased significantly; there were no obvious changes in the levels of TNF-α, IL-1β, IL-6, and IL-8 in the cell supernatant of the BGI-N3 group. After pre-incubating with BGI-N3 for 12 h, compared with the LPS group, the levels of TNF-α, IL-1β, IL-6, and IL-8 in the cell supernatant of the BGI-N3 + LPS group decreased to varying degrees, and the cell viability increased significantly. It shows that BGI-N3 can effectively inhibit the inflammatory response induced by LPS, protect U937 cells from inflammatory damage, and exert the probiotic effect of immune regulation.

[0064] Table 2 Detection results of the anti-inflammatory effect of Bifidobacterium animalis subsp. lactis BGI-N3

[0065] Group TNF-α (pg / mL) IL-1β (pg / mL) IL-6 (pg / mL) IL-8 (pg / mL) Cell viability (%) Control 112.59±10.86 90.73±11.87 20.89±2.53 276.17±25.43 100±6.41 LPS <![CDATA[423.64±38.16 *** > <![CDATA[361.33±21.15 *** > <![CDATA[223.86±21.52 *** > <![CDATA[1258.97±75.59 *** > <![CDATA[68.26±8.28 *** > BGI-N3 116.23±11.02 88.67±3.95 19.43±1.98 266.67±15.63 96.37±4.92 BGI-N3 + LPS <![CDATA[258.31±25.16 ### > <![CDATA[204.28±33.47 ### > <![CDATA[122.1±14.91 ### > <![CDATA[726.59±29.76 ### > <![CDATA[90.92±3.83 ## >

[0066] Comparative Preparation Example

[0067] This comparative preparation example is for the preparation of an improved anti-allergy agent.

[0068] The preparation method of the agent for improving allergic reactions includes the following steps: accurately weigh a total of 10.4 mg of ground loratadine powder (product number ZH10970410, purchased from Bayer Healthcare Co., Ltd.), dissolve it in 50 mL of sterile physiological saline, and stir the solution until it is completely mixed to obtain an anti-allergy agent with a drug concentration of 0.208 mg / mL.

[0069] Example 4

[0070] This example is for the preparation of a low-dose anti-allergy probiotic agent.

[0071] The preparation method of a low-dose probiotic agent of Bifidobacterium animalis subsp. lactis BGI-N3 for improving allergic reactions includes the following steps: accurately weigh a total of 0.01 g of freeze-dried powder of Bifidobacterium animalis subsp. lactis BGI-N3 (containing 100 billion CFU / g of active probiotics), dissolve it in 99.99 mL of sterile physiological saline, and stir the solution until it is completely mixed to obtain an anti-allergy probiotic agent with a cell concentration of 1.0×10 7 CFU / mL.

[0072] Example 5

[0073] This example is for the preparation of a medium-dose anti-allergy probiotic agent.

[0074] The preparation method of a medium-dose probiotic agent of Bifidobacterium animalis subsp. lactis BGI-N3 for improving allergic reactions includes the following steps: accurately weigh a total of 0.10 g of freeze-dried powder of Bifidobacterium animalis subsp. lactis BGI-N3 (containing 100 billion CFU / g of active probiotics), dissolve it in 99.90 mL of sterile physiological saline, and stir the solution until it is completely mixed to obtain an anti-allergy probiotic agent with a cell concentration of 1.0×10 8 CFU / mL.

[0075] Example 6

[0076] This example is for the preparation of a high-dose anti-allergy probiotic agent.

[0077] The preparation method of a high-dose probiotic agent of Bifidobacterium animalis subsp. lactis BGI-N3 for improving allergic reactions includes the following steps: accurately weigh a total of 1.00 g of freeze-dried powder of Bifidobacterium animalis subsp. lactis BGI-N3 (containing 100 billion CFU / g of active probiotics), dissolve it in 99.00 mL of sterile physiological saline, and stir the solution until it is completely mixed to obtain an anti-allergy probiotic agent with a cell concentration of 1.0×10 9 CFU / mL.

[0078] Example 7

[0079] This example conducts an experiment on the efficacy of improving allergic rhinitis.

[0080] By constructing a rat model of allergic rhinitis, the efficacy of the agents or probiotic agents prepared in Comparative Preparation Example and Examples 4 - 6 that can improve allergies in allergic rhinitis was verified, as follows:

[0081] Thirty-six SPF-grade SD female rats, weighing 300 ± 20 g, were cultured in a group-raising manner, 5 rats per cage. The breeding temperature and humidity were 20 - 26°C and 40 - 70% respectively, with 12h:12h day-night intermittent lighting; the conditions of the breeding room were always kept stable to ensure the reliability of the test results. The experiment was divided into 6 groups, including a normal control group, a model control group, a positive control group (loratadine), and low-dose, medium-dose, and high-dose intervention groups of Bifidobacterium animalis subsp. lactis BGI-N3, with 6 rats in each group. The specific grouping is shown in Table 3.

[0082] Table 3 List of Groupings and Dose Designs

[0083]

[0084] The test stage was divided into three stages: a sensitization period (days 0 - 15), an intervention period (days 22 - 50), and a challenge period (days 42 - 50). An ovalbumin (OVA)-induced rat model of allergic rhinitis was used. The sensitization reagent contained 1 mg / mL OVA and 21 mg / mL Al(OH)3, and the challenge reagent contained 30 mg / mL OVA.

[0085] During the sensitization period, except for the normal control group which was injected with 1 mL of sterile saline, the remaining groups of rats were intraperitoneally injected with 1 mL of the sensitization reagent on days 0, 3, 6, 9, 12, and 15; during the intervention period, each dose intervention group of BGI-N3 was gavaged with the corresponding probiotic dose, with a daily gavage dose of 1 mL for 28 consecutive days. The normal control group and the model control group were given an equal amount of saline instead of the sample. The positive control group was given a loratadine solution instead of the sample; during the challenge period, except for the normal control group, the remaining groups of rats were instilled with 0.1 mL / rat / nare of the challenge reagent on days 42, 44, 46, 48, and 50. The normal control group was instilled with an equal amount of sterile saline at the same time. After the last test was completed, the symptoms of nose rubbing, sneezing, runny nose, etc. of each rat within 10 min were observed and recorded, and the scoring was carried out according to the "Animal Allergic Rhinitis Symptom Scoring Table (Table 4)".

[0086] Table 4 Animal Allergic Rhinitis Symptom Scoring Table

[0087] Allergic symptoms Normal (0 points) Mild (1 point) Moderate (2 points) Severe (3 points) Sneezing None ≤ 3 times 4 - 10 times ≥ 11 times Rubbing nose None Gently scratching nose and face Frequently scratching nose and face Acute friction Running nose None Flowing in front of nostrils Flowing past nostrils Flowing on face

[0088] After completing the allergic rhinitis symptom scoring, the rats were euthanized, and then serum samples, nasal mucosa tissue samples, and spleen samples were collected, and allergic-related biological indicators were measured.

[0089] Method for collecting nasal mucosa samples and detection content: Referring to the method described in the academic paper "Improvement of mouse nasal lavage and nasal mucosa acquisition method" published by Zhang Junyan et al. in the "Chinese Journal of Otorhinolaryngology-Skull Base Surgery", the nasal mucosa tissue was obtained, and the nasal mucosa tissue was fixed with paraformaldehyde and prepared for paraffin sections. Subsequently, hematoxylin-eosin (HE) staining and periodic acid Schiff (PAS) staining were performed to evaluate the pathological changes of the nasal mucosa.

[0090] Method for collecting blood samples and detection content: After collecting the blood samples of the rats, they were left to clot at room temperature for 60 min, centrifuged at 3000 rpm for 10 min at 4 °C, and the upper-layer serum was aspirated. Using the corresponding Elisa detection kit, the levels of immunoglobulin A (IgA), immunoglobulin E (IgE), platelet-activating factor (PAF), immunoglobulin G1 (IgG1), and cytokines interleukin-12 (IL-12), interleukin-13 (IL-13), interferon-γ (IFN-γ), and transforming growth factor-β (TGF-β) were measured.

[0091] Method for collecting spleen samples and detection content: The spleen was quickly removed. Referring to the method described in the academic paper "Taurohyodeoxycholic acid alleviates trinitrobenzene sulfonic acid induced ulcerative colitis via regulating Th1 / Th2 and Th17 / Treg cells balance" published by Lv et al. in "Life Sciences", flow cytometry was used to count the CD4 + T lymphocytes in the rat spleen and calculate their proportions.

[0092] Method for processing experimental data: All data were expressed in the form of mean ± standard deviation, and one-way ANOVA test analysis was used to perform statistical analysis on the above experimental data. When p < 0.05, it indicated that there were significant differences between groups.

[0093] Results of the anti-allergic efficacy experiment:

[0094] (1) The probiotic agent of Bifidobacterium animalis subsp. lactis BGI-N3 effectively improved the symptoms of allergic rhinitis: The rhinitis symptom score is an important basis for evaluating the severity of the rhinitis symptoms in rats. This score consists of behaviors such as sneezing, nose rubbing, and runny nose. Figure 2The symptom scores of allergic rhinitis in rats of each group in Example 7. Compared with the normal control group of rats, the rhinitis symptom score of the model control group of rats increased significantly (4.67±0.82). After intervention with the probiotic agents described in Examples 4-6, the rhinitis symptom score of the rats decreased significantly. After intervention with the probiotic agent described in Example 5, the rhinitis symptom score decreased to 1.83±0.75 at the lowest. The above results indicate that the probiotic agent of Bifidobacterium animalis subsp. lactis BGI-N3 effectively improved allergic rhinitis symptoms such as sneezing, nose rubbing, and runny nose.

[0095] (2) The probiotic agent of Bifidobacterium animalis subsp. lactis BGI-N3 effectively improved the levels of serum allergy markers in rats with allergic rhinitis and regulated the levels of cytokines in the serum: Figure 3 and Figure 4 are the levels of immunoglobulin A (IgA), immunoglobulin E (IgE), platelet-activating factor (PAF), and immunoglobulin G1 (IgG1) in the serum of rats in each group in Example 7, as well as the levels of cytokines interleukin-12 (IL-12), interleukin-13 (IL-13), interferon-γ (IFN-γ), and transforming growth factor-β (TGF-β) in the serum. Compared with the normal control group of rats, the levels of serum PAF, IgE, IgG1, and the Th2-type cytokine IL-13 in the serum of the model control group of rats increased significantly, while the levels of serum IgA, TGF-β in the serum, and the Th1-type cytokines IL-12 and IFN-γ decreased significantly. The above results indicate that the Th2-type immune response activity in rats with allergic rhinitis is enhanced, while the Th1-type immune response activity is weakened, suggesting an allergic reaction. After intervention with the probiotic agents described in Examples 4-6, the serum allergy markers IgE and PAF in the above rats with allergic rhinitis, as well as the Th2-type cytokine IL-13 in the serum, decreased significantly, while the Th1-type cytokines IL-12 and IFN-γ increased significantly. The above indicators all approached the normal control group after intervention, suggesting the restoration of Th1 / Th2 balance and the alleviation of allergic reactions. This result indicates that probiotic agents of different doses of BGI-N3 effectively regulate the body's immunity, thereby achieving the purpose of improving rhinitis.

[0096] (3) The probiotic agent of Bifidobacterium animalis subsp. lactis BGI-N3 effectively regulated the differentiation of CD4 + T lymphocytes in the spleen of rats with allergic rhinitis: Figure 5 are the CD4 in the spleen of rats in each group in Example 7 +T lymphocyte count results, including the ratio of the number of Th1 / Th2 cells and the ratio of the number of Th17 / Treg cells. Compared with the rats in the normal control group, the ratio of the number of Th1 / Th2 cells in the model control group of rats was significantly decreased, while the ratio of the number of Th17 / Treg cells was significantly increased, indicating that in rats with allergic rhinitis, there was an imbalance in Th1 / Th2 and a bias towards Th2, and at the same time, there was an imbalance in Th17 / Treg and a bias towards Th17. At this time, the rats were in a state of allergy and exacerbated inflammatory response. After intervention with the probiotic agent described in Examples 4-6, the ratio of the number of Th1 / Th2 cells in the spleen of the rats was significantly increased, and the ratio of the number of Th17 / Treg cells was significantly decreased, and the ratio tended to be that of the normal control group. This result suggests that the imbalance of Th1 / Th2 and the imbalance of Th17 / Treg were significantly improved. This result indicates that the probiotic agent of Bifidobacterium animalis subsp. lactis BGI-N3 can improve the symptoms of allergic rhinitis by regulating the immune response, especially having an impact on the differentiation of CD4 + T lymphocytes.

[0097] (4) The probiotic agent of Bifidobacterium animalis subsp. lactis BGI-N3 effectively improved the pathological state of the nasal mucosa tissue of rats with allergic rhinitis: Figure 6 and Figure 7 are respectively the histopathological analysis result diagrams of hematoxylin-eosin (HE) staining and periodic acid-Schiff (PAS) staining of the nasal mucosa tissue of rats in each group in Example 7. The nasal mucosa structure of the rats in the normal control group was clear, the tissue was intact, arranged in an orderly manner, the epithelial cells were intact, no obvious inflammatory cell infiltration was seen, and no glandular hyperplasia was seen. However, in the model control group of rats, there were various adverse pathological changes such as loose arrangement and shedding of nasal mucosa epithelial cells, glandular hyperplasia, goblet cell hyperplasia, and epithelial cilia breakage. After intervention with the probiotic agent described in Examples 4-6, the arrangement of nasal mucosa epithelial cells in rats with allergic rhinitis was significantly improved, and the phenomena of glandular and goblet cell hyperplasia were significantly reduced, indicating that the nasal mucosa damage was improved. The above detection results show that the intervention of the probiotic agent of Bifidobacterium animalis subsp. lactis BGI-N3 effectively repaired the damage of the nasal mucosa tissue of rats with allergic rhinitis and improved the adverse pathological state of the nasal mucosa tissue.

[0098] In summary, the present invention deeply analyzed Bifidobacterium animalis subsp. lactis BGI-N3 and found that it has good ability to inhibit intestinal pathogenic bacteria, can restore and maintain the normal function of the immune system by balancing the intestinal flora, can regulate the levels of serum allergy markers and cytokines, and regulate the differentiation of CD4 + T lymphocytes, reduce the nasal mucosa damage caused by allergy, and effectively improve the symptoms of allergic rhinitis. These mechanisms of action indicate that the BGI-N3 probiotic agent can regulate the immune response, inhibit inflammation, and repair nasal mucosa damage, providing a new direction for the prevention / treatment of allergic rhinitis.

[0099] The applicant declares that the above description is only a specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by any person skilled in the art within the technical scope disclosed by the present invention fall within the protection scope and the disclosure scope of the present invention.

Claims

1. Use of Bifidobacterium animalis subsp. lactis in the preparation of a preparation for preventing and / or treating allergic rhinitis; The Bifidobacterium animalis subsp. lactis includes Bifidobacterium animalis subsp. lactis BGI-N3, which was deposited at the China Center for Type Culture Collection on October 17, 2022, with the deposit number CCTCC NO: M20221585.

2. A pharmaceutical composition for preventing and / or treating allergic rhinitis, characterized in that, The pharmaceutical composition includes the Bifidobacterium animalis subsp. lactis described in claim 1; Preferably, the pharmaceutical composition further includes pharmaceutically acceptable excipients; Preferably, the excipients include at least one of a carrier, a wetting agent, a solubilizer, an osmotic pressure regulator, a coating material, a colorant, a pH regulator, an antioxidant or a buffer.

3. Use of the Bifidobacterium animalis subsp. lactis described in claim 1 in the preparation of a preparation for repairing nasal mucosa injury.

4. Use of the Bifidobacterium animalis subsp. lactis described in claim 1 in the preparation of a preparation for inhibiting intestinal pathogenic bacteria.

5. The application according to claim 4, characterized in that The intestinal pathogenic bacteria include Escherichia coli and / or Staphylococcus aureus.

6. Use of the Bifidobacterium animalis subsp. lactis described in claim 1 in the preparation of a preparation for regulating the levels of serum cytokines.

7. The application according to claim 6, characterized in that, The cytokines include at least one of interleukin-12, interleukin-13, interferon-γ or transforming growth factor-β.

8. Use of the Bifidobacterium animalis subsp. lactis described in claim 1 in the preparation of a preparation for regulating the levels of serum allergy markers.

9. The application according to claim 8, characterized in that, The serum allergy markers include at least one of immunoglobulin A, immunoglobulin E, platelet-activating factor or immunoglobulin G1.

10. Use of the Bifidobacterium animalis subsp. lactis described in claim 1 in the preparation of a preparation for regulating the differentiation of CD4 + T lymphocytes.

Images