Klebsiella pneumoniae HW2024 and use thereof in preparing a medicine for treating allergic rhinitis

Abstract

The application discloses a klebsiella oxytoca HW2024 and application of the klebsiella oxytoca HW2024 in preparation of a medicine for treating allergic rhinitis, and the preservation number of the klebsiella oxytoca HW2024 is CCTCC NO: M 20252524. The klebsiella oxytoca of the application is symbiotic bacteria, and is relatively safe and easy to obtain, so that the treatment cost of AR patients can be greatly reduced, and a new treatment option is provided for the treatment of AR. The medicine preparation for treating allergic rhinitis of the application has a simple and easy administration route and is easy to operate, and is convenient for patients to use.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a type of Klebsiella pneumoniae HW2024 and its application in the preparation of a medicine for treating allergic rhinitis. Background Technology

[0002] Allergic rhinitis (AR) is a common chronic inflammatory disease of the upper airway, with a prevalence as high as 20-30%. It not only seriously threatens patients' health and impairs their quality of life, but is also a significant risk factor for lower respiratory tract diseases and mental health disorders. However, the current clinical treatment outcomes for AR are far from satisfactory, and exploring its pathogenesis and conducting translational research will remain a hot topic and focus in otolaryngology-head and neck surgery for a considerable period of time.

[0003] (1) The IgE-mast cell-histamine signaling axis plays an important role in the pathogenesis of AR.

[0004] Previous studies have shown that the production and functional regulation of immunoglobulin E (IgE) is one of the core links in the pathogenesis of acute rheumatoid arthritis (AR): inhaled allergens induce the body to produce antigen-specific IgE, which binds to IgE receptors (FcεR1) on the surface of mast cells and basophils in the nasal mucosa, forming a sensitized state; when the body is exposed to the same allergen again, the allergen binds to IgE anchored on the surface of mast cells and basophils, thereby activating mast cells and basophils, inducing degranulation, and releasing inflammatory mediators such as histamine and type 2 cytokines. Histamine acts on sensory nerve endings and corresponding receptors in the vascular endothelium of the nasal mucosa, causing vasodilation and increased glandular secretion, leading to symptoms such as nasal itching, sneezing, and clear nasal discharge. It can also induce vascular endothelial cells and epithelial cells to express or secrete adhesion molecules, chemokines, and cytokines, recruiting and activating immune cells such as eosinophils, basophils, and type 2 helper T cells, resulting in chronic, persistent inflammation. Simultaneously, the recruited inflammatory cells further release inflammatory mediators, leading to nasal mucosal edema and nasal congestion. Therefore, the IgE-mast cell-histamine signaling axis plays a crucial role in the development of acute rhinitis (AR), and blocking histamine production or its downstream signaling pathways has potential therapeutic effects on AR. In fact, histamine receptor antagonists have become one of the commonly used drugs for treating AR in clinical practice; however, they easily cause adverse reactions such as nasal bleeding and headaches, and a considerable number of patients do not respond well to their treatment. Therefore, it is necessary to clarify the metabolic mechanism of histamine and, based on this, identify targeted therapeutic targets.

[0005] (2) Local histamine metabolism imbalance in the nasal mucosa is associated with the severity of AR disease.

[0006] Histamine is a nitrogen-containing compound primarily derived from mast cells, basophils, and platelets. It is produced from L-histidine by the catalysis of L-histidine decarboxylase (HDC). Histamine-degrading enzymes include histamine N-methyltransferase (HNMT) and diamine oxidase (DAO). HNMT is present in the cytoplasm and is widely expressed in various human cells, while DAO is a secreted protein mainly produced by the kidneys, intestines, and placenta. Therefore, HNMT and DAO are involved in regulating the degradation of intracellular and extracellular histamine, respectively. Previous studies have shown that both HNMT and DAO play a role in degrading histamine in the human nasal mucosa. Interestingly, the expression level of HDC in the nasal mucosa of AR patients was significantly increased compared to the control group, while the expression levels and histamine-degrading activities of the histamine-degrading enzymes HNMT and DAO were significantly decreased and correlated with disease severity. Therefore, the metabolic disorder of local histamine in the nasal mucosa is closely related to its pathogenesis, and promoting the degradation of local histamine in the nasal mucosa may be an effective treatment for AR.

[0007] (3) Identification of histamine-degrading bacteria in the nasal cavity

[0008] In recent years, in the field of food safety, the addition of microbial agents, such as DAO-producing bacteria, has been used to degrade histamine levels in dairy products and other foods, thereby reducing the risk of histamine poisoning and improving symptoms associated with histamine intolerance. Guarcello et al. found that lactobacilli can secrete DAO to degrade histamine in cheese. Pashangeh et al. identified 27 out of 243 Staphylococcus strains in goat milk that exhibited histamine-degrading ability, with Staphylococcus epidermidis being the strongest. In fact, the nasal cavity also harbors a large number of bacteria. Studies have shown that the nasal flora of patients with acute rheumatoid arthritis (AR) undergoes significant changes, mainly characterized by an increase in Firmicutes and a decrease in Proteobacteria, Bacteroidetes, and Actinobacteria, and these changes are associated with the progression of AR. Therefore, whether histamine-degrading bacteria exist in the nasal cavity, whether they are related to the pathogenesis of AR, and whether these bacteria have a therapeutic effect on AR remain unclear.

[0009] Currently, there are no reports of drugs that utilize bacterial metabolism of histamine in the nasal mucosa to treat AR. Summary of the Invention

[0010] The purpose of this invention is to overcome the shortcomings of the prior art and provide a Klebsiella pneumoniae HW2024 and its application in the preparation of a drug for treating allergic rhinitis (AR). This invention identifies a bacterium that degrades histamine in the nasal cavity, namely Klebsiella pneumoniae HW2024, and its use in the preparation of a drug for treating allergic rhinitis has good economic value.

[0011] To achieve the above objectives, the technical solution designed by the present invention is as follows:

[0012] This invention provides a gas-producing Klebsiella pneumoniae ( Klebsiella aerogenes HW2024, its accession number is CCTCC NO: M 20252524.

[0013] The above-mentioned Klebsiella pneumoniae ( Klebsiella aerogenes HW2024 is deposited at the China Center for Type Culture Collection (CCTCC), accession number: CCTCC NO: M 20252524, deposit date: November 11, 2025, address: Wuhan University, Wuhan, China.

[0014] The present invention also provides the application of the above-mentioned Klebsiella pneumoniae HW2024 in the preparation of a medicine for treating allergic rhinitis.

[0015] The present invention also provides a pharmaceutical preparation for treating allergic rhinitis, comprising an effective amount of Klebsiella pneumoniae and pharmaceutically acceptable excipients.

[0016] Furthermore, the excipients are physiological saline, glucose, vitamin C, and amino acids.

[0017] Furthermore, the pharmaceutical preparation is a nasal drop, a spray, or a rinse.

[0018] Furthermore, the spray formulation is an atomizing agent, a spray, or a suspension.

[0019] Furthermore, the pharmaceutical preparation contains 10 kJ of Klebsiella pneumoniae. 6 -10 8 CFU / mL.

[0020] The principle of this invention:

[0021] 1) This invention uses in vitro experiments to screen and compare the ability of bacteria on the surface of the inferior turbinate mucosa in AR patients and normal controls to degrade bacteria. It was found that compared to the normal control group, the ability of the nasal flora in AR patients to degrade histamine was significantly weakened. Furthermore, using a selective culture medium with histamine as the sole carbon and nitrogen source, we screened and successfully identified *Klebsiella pneumoniae*. *Klebsiella pneumoniae* is a Gram-positive anaerobic bacillus that commonly colonizes the gastrointestinal tract. Further high-throughput sequencing of large samples revealed that *Klebsiella pneumoniae* could be detected in nearly 20% of nasal specimens, suggesting that *Klebsiella pneumoniae* is also an important component of the nasal bacterial genome. Further in vivo animal experiments confirmed that *Klebsiella pneumoniae* can significantly improve AR symptoms in mice and inhibit eosinophilic inflammation of the nasal mucosa, providing significant guidance for further research on the treatment of AR with *Klebsiella pneumoniae*.

[0022] 2) Based on histopathology, high-throughput sequencing, cell biology, molecular biotechnology and in vivo animal experiments, this invention proposes that Klebsiella pneumoniae degrades histamine and has a potential therapeutic effect on AR, thus proving that Klebsiella pneumoniae can be used to prepare drugs for the treatment of AR.

[0023] The beneficial effects of this invention are:

[0024] 1) The Klebsiella pneumoniae of the present invention is a symbiotic bacterium, which is relatively safe and easy to obtain, and thus can greatly reduce the treatment cost for AR patients and provide a new treatment option for AR.

[0025] 2) The pharmaceutical preparation for treating allergic rhinitis of the present invention can be administered via nasal spray / nasal drops / rinse, which is simple, easy to operate, and convenient for patients to use. Attached Figure Description

[0026] Figure 1 A schematic diagram illustrating the histamine degradation capacity of the bacterial community on the surface of the inferior turbinate in normal controls and AR patients;

[0027] In the figure, a is the flowchart of sample collection and incubation with histamine;

[0028] b is a statistical graph of histamine degradation by nasal bacteria detected by LC-MS;

[0029] Figure 2 A schematic diagram for screening histamine-degrading bacteria—Klebsiella pneumoniae—from nasal bacteria;

[0030] In the figure, a is a flowchart for screening histamine-degrading bacteria in the nasal cavity;

[0031] b is a culture diagram of histamine-degrading bacteria in the nasal cavity;

[0032] c is a representative image of Klebsiella pneumoniae, identified by mass spectrometry as histamine-degrading bacteria in the nasal cavity.

[0033] d is a representative chromatogram of histamine degradation by Klebsiella pneumoniae detected by LC-MS;

[0034] e is a statistical graph showing the degradation of histamine by Klebsiella pneumoniae detected by LC-MS;

[0035] f is a statistical graph showing the histamine degradation detected by non-target metabolomics (left) and the resulting metabolites imidazole acetaldehyde (middle) and 1-methylimidazole acetaldehyde (right);

[0036] Figure 3 Diagram of the treatment protocol for Klebsiella pneumoniae and AR modeling in BALB / c mice;

[0037] Figure 4 The study showed that *Klebsiella pneumoniae* can alleviate eosinophilic inflammation in an AR animal model.

[0038] In the figure, a represents the expression of mouse serum-specific IgE;

[0039] b is a diagram showing the expression of histamine in mouse nasal wash fluid;

[0040] c shows the nasal symptom scoring chart for mice, including the scoring chart for the number of times the nose is scratched (left) and the scoring chart for the number of times the nose is sneezed (right).

[0041] d shows the expression of type 2 inflammatory factors (IL-4 (left), IL-5 (middle) and IL-13 (right)) in mouse nasal wash fluid;

[0042] e shows the expression of type 2 inflammatory factors (IL-4 (left), IL-5 (middle), and IL-13 (right)) in mouse nasal mucosa tissue;

[0043] f is a hematoxylin-hematoxylin staining image of eosinophil expression in mouse nasal mucosa tissue and a statistical graph of the number of eosinophils in the corresponding high-power field (400x magnification).

[0044] g shows the periodic acid Schiff reaction staining pattern of goblet cells in mouse nasal mucosa tissue and the statistical graph of the number of goblet cells in the corresponding high-power field (400x magnification);

[0045] Among them, saline is PBS solution, KA is a drug preparation for treating allergic rhinitis, and ovalbumin is PBS solution containing ovalbumin; Detailed Implementation

[0046] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0047] The following examples illustrate the grouping of study subjects and the collection and source of specimens.

[0048] 1. The research subjects are divided into:

[0049] ① The normal control group consisted of patients who underwent decompression surgery for simple nasal septum deviation, simple sinus cysts, nasal tumors, and traumatic optic nerve injury. Nasal swabs were collected from the surface of the inferior turbinate.

[0050] ②AR group: AR was diagnosed according to the ARIA guidelines (Brozek JL, et al. Journal of Allergy and Clinical Immunology 2017; 140: 950-8) and previous literature reports (Miao P, et al. Nature Microbiology 2023; 8:218-30). Nasal meatus swabs, nasal irrigation fluid and peripheral blood samples were collected from patients and healthy controls. Clinical data of patients such as age, sex and smoking history were collected.

[0051] This example excludes patients with nasal polyps who have had an acute upper respiratory tract infection or an acute asthma attack within 4 weeks, have received immunotherapy, have used systemic or local glucocorticoids or leukotriene receptor antagonists within one month prior to surgery, as well as patients with other nasal diseases such as posterior nasal polyps, fungal sinusitis, cystic fibrosis, primary ciliary dyskinesia, immunodeficiency, systemic vasculitis, and IgG4-related diseases.

[0052] Example 1: Comparison of the histamine degradation capacity of bacterial communities on the surface of the inferior turbinate between normal controls and AR patients.

[0053] Following the methods used in previous studies (Li D, et al. Cell Host Microbe. 2022;30:329-339; LiD, et al. Cell Metabolism. 2023; 35:685-694), the collected nasal meatus swabs were incubated in a culture medium with histamine as the sole carbon source, and the histamine content in the culture medium was detected by liquid chromatography-mass spectrometry.

[0054] like Figure 1 As shown, compared to the AR group, the nasal flora from the nasal cavity of the healthy control group showed stronger histamine degradation ability on the second and fourth days after incubation with histamine.

[0055] Example 2: Isolation and Identification of Histamine-Degrading Bacteria in the Nasal Cavity

[0056] 1. Isolation and Identification of Bacteria

[0057] (1) Following the methods of previous studies (Li D, et al. Cell Host Microbe. 2022;30:329-339; Li D, et al. Cell Metabolism. 2023; 35:685-694), the nasal meatus swabs with histamine-degrading ability from Example 1 were incubated for 27 days in a selective medium with histamine as the sole carbon source, and the isolated bacteria were then cultured (see Figure 2 a)

[0058] like Figure 2 As shown in b: We detected grayish-white, round colonies on the plate.

[0059] (2) Following the methods of previous studies (Li D, et al. Cell Host Microbe. 2022;30:329-339; Li D, et al. Cell Metabolism. 2023; 35:685-694), the strains obtained above were subjected to mass spectrometry detection and the strains were identified.

[0060] like Figure 2 As shown in c: Mass spectrometry identification confirmed that the bacteria is Klebsiella pneumoniae.

[0061] The gas-producing Klebsiella pneumoniae was named: Klebsiella pneumoniae ( Klebsiella aerogenes HW2024 is deposited at the China Center for Type Culture Collection (CCTCC), accession number: CCTCC NO: M 20252524, deposit date: November 11, 2025, address: Wuhan University, Wuhan, China.

[0062] 2. Klebsiella pneumoniae ( Klebsiella aerogenes Correlation between HW2024 and nasal histamine degradation

[0063] (1) Referring to previous research methods (Li D, et al. Cell Host Microbe. 2022;30:329-339; Li D, et al. Cell Metabolism. 2023; 35:685-694), the isolated and purified strains were incubated in a medium with histamine as the sole carbon source, and the histamine content in the medium was detected by liquid chromatography-mass spectrometry.

[0064] like Figure 2 As shown in de: When Klebsiella pneumoniae HW2024 was incubated with histamine, it was found that Klebsiella pneumoniae could significantly reduce histamine levels in a time-dependent manner.

[0065] (2) Referring to the methods of previous studies (Li D, et al. Cell Host Microbe. 2022;30:329-339; Li D, et al. Cell Metabolism. 2023; 35:685-694; Li JX, et al. Journal of Allergy and Clinical Immunology. 2022; 150:727-735), non-target metabolomics was used to detect metabolites in the culture supernatant and compared with histamine metabolites in the KEGG pathway.

[0066] like Figure 2 As shown in f: When Klebsiella pneumoniae HW2024 was incubated with histamine, a non-target metabolism assay revealed a decrease in histamine and an increase in its metabolite imidazole-4-acetic acid in the culture supernatant.

[0067] Example 3: Pharmaceutical preparation for treating allergic rhinitis

[0068] Preparation of pharmaceutical formulations for treating allergic rhinitis:

[0069] 1) Culture of Klebsiella pneumoniae HW2024: Clinical isolates of Klebsiella pneumoniae HW2024 were inoculated from glycerol stock solution into 10 mL of Clostridium tumefaciens medium (tryptone: 10 g / L, yeast extract: 5 g / L, sodium chloride: 10 g / L) and cultured anaerobically at 37 °C. When the bacteria reached the logarithmic growth phase, they were collected by centrifugation and transferred to 50 mL of Clostridium tumefaciens medium and cultured anaerobically at 37 °C for 40 hours. After centrifugation at 4000 g for 10 minutes, Klebsiella pneumoniae HW2024 cells were collected separately.

[0070] 2) The above-mentioned Klebsiella pneumoniae HW2024 cells were added to physiological saline to obtain a drug preparation for treating allergic rhinitis, wherein the content of Klebsiella pneumoniae in the drug preparation was 10. 6 -10 8 CFU / mL.

[0071] Example 4: Animal model study of the above-mentioned drug formulation regulating eosinophilic inflammation of the nasal mucosa in patients with acute rheumatoid arthritis (AR).

[0072] 1) Establishment of the mouse AR model and treatment with histamine-degrading bacteria and histamine-degrading enzymes: Based on previous studies (Liu Y, et al. Journal of Allergy and Clinical Immunology 2013;131:387-97; Wang H, et al. American Journal of Respiratory and Critical Care Medicine 2010; 181:908-16), this embodiment used OVA to induce a mouse AR model, such as... Figure 3 The method shown is as follows:

[0073] Sensitization phase: On days 0, 7, and 14, subcutaneous injections of saline (i.e., PBS solution) or 50 μg of ovalbumin OVA (50 μg of ovalbumin OVA in PBS solution containing 2 mg of aluminum hydroxide, final volume 200 μL) were administered.

[0074] Stimulation phase: From day 21 to day 27, administer saline (i.e., PBS solution) or OVA solution (10 μL per nostril, once daily, 20 μL containing 0.8 mg OVA). From day 18 to day 20, administer the above-mentioned drug preparation (i.e., KA) or saline (i.e., PBS solution) (see...). Figure 3 ).

[0075] 2) Sample Collection: After deep anesthesia of mice, nasal irrigation fluid was collected and stored at -70℃. After euthanasia of some mice, the heads were separated, the scalp skin and soft tissue were removed, and the nasal cavity and sinuses were separated. After decalcification, fixation, and paraffin embedding, 4μm thick coronal serial sections were prepared. The nasal cavity and sinuses of some mice were dissected under a microscope, and the nasal and sinus mucosa was obtained and stored at -70℃. The following research and analysis are planned after obtaining the above specimens:

[0076] ① Histomorphological observation: HE and PAS staining methods were used to observe the infiltration of inflammatory cells and the number of goblet cells in the nasal mucosa;

[0077] ② Immunofluorescence assay was used to detect changes in the expression of eosinophils and neutrophils in the nasal cavity of mice;

[0078] ③RT-PCR was used to detect changes in the expression of type 2 inflammatory cytokines such as IL-4, IL-5 and IL-13 in the nasal mucosa of mice;

[0079] ④ ELISA was used to detect the expression levels of type 2 inflammatory cytokines such as IL-4, IL-5 and IL-13 in mouse nasal lavage fluid.

[0080] like Figure 4 As shown in a: Klebsiella pneumoniae does not affect the expression level of serum IgE, indicating that Klebsiella pneumoniae does not exert its effect by inhibiting systemic IgE production, suggesting that its therapeutic mechanism does not depend on systemic humoral immune regulation and may mainly act on local mucosal immunity.

[0081] like Figure 4 As shown in b~c: Klebsiella pneumoniae downregulated the expression level of histamine in nasal wash fluid; the reduction of histamine improved the relief of AR symptoms in mice, indicating that Klebsiella pneumoniae can inhibit the local production of histamine in the nasal mucosa.

[0082] like Figure 4 As shown in d~e: Klebsiella pneumoniae downregulated the expression levels of type 2 cytokines IL-4, IL-5 and IL-13 in nasal wash fluid; and Klebsiella pneumoniae downregulated the expression levels of type 2 cytokines IL-4, IL-5 and IL-13 in mouse nasal mucosa; Klebsiella pneumoniae may block allergic inflammation from the upstream by inhibiting Th2 immune response;

[0083] like Figure 4As shown in f: Klebsiella pneumoniae downregulated the infiltration of eosinophils in the nasal mucosa of mice, consistent with the downregulation of IL-5, indicating that inflammatory cells were suppressed and tissue inflammatory damage was reduced;

[0084] like Figure 4 As shown in g: Klebsiella pneumoniae downregulated the expression of goblet cells in the nasal mucosa of mice, further verifying the role of IL-13 and explaining the improvement of symptoms such as reduced nasal secretion from a histological perspective.

[0085] In summary, Klebsiella pneumoniae may reduce eosinophil infiltration, goblet cell proliferation, and histamine production by regulating local mucosal immune balance and inhibiting type 2 inflammatory response (Th2 cytokine release). This suggests that Klebsiella pneumoniae can significantly improve AR symptoms in mice and downregulate type 2 inflammatory factors in the nasal mucosa, thus demonstrating its therapeutic effect on AR.

[0086] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A type of gas-producing Klebsiella pneumoniae ( Klebsiella aerogenes HW2024, its accession number is CCTCC NO: M20252524.

2. The use of the Klebsiella pneumoniae HW2024 of claim 1 in the preparation of a medicament for treating allergic rhinitis.

3. A pharmaceutical preparation for treating allergic rhinitis, characterized in that: It consists of an effective amount of the gas-producing Klebsiella pneumoniae HW2024 as described in claim 1 and pharmaceutically acceptable excipients.

4. The pharmaceutical preparation according to claim 3, characterized in that: The excipients are physiological saline, glucose, vitamin C, and amino acids.

5. The pharmaceutical preparation according to claim 3 or 4, characterized in that: The pharmaceutical preparation is a nasal drop, spray, or rinse preparation.

6. The pharmaceutical formulation for treating AR according to claim 5, characterized in that: The spray formulation is an atomizing agent, spray, or suspension.

7. The pharmaceutical formulation for treating AR according to claim 3, characterized in that: The pharmaceutical preparation contains 10 kJ of Klebsiella pneumoniae HW2024. 6 -10 8 CFU / mL.

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