Application of vitamin B7 in preparation of medicine for treating allergic rhinitis

Abstract

The invention discloses an application of vitamin B7 in preparation of a medicine for treating allergic rhinitis. According to the application, virtual screening is performed through clinically available medicines, and the vitamin B7 is locked as a candidate medicine possibly acting on METTL3. Furthermore, in-vitro nasal mucosa epithelial cell experiments and in-vivo mouse model researches prove that the vitamin B7 can effectively reduce the TSLP level and improve AR-related inflammatory reactions, and shows a remarkable treatment effect.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of vitamin B7 in the preparation of a drug for treating allergic rhinitis. Background Technology

[0002] Allergic rhinitis (AR) is a type I hypersensitivity reaction mediated by IgE after the nasal mucosa comes into contact with allergens. In recent years, the incidence of AR has remained high, affecting approximately 10-40% of the global population. Current treatments for AR mainly include drug therapy and allergen immunotherapy (AIT). Standard drug treatment regimens primarily include the use of oral antihistamines, leukotriene receptor antagonists, nasal antihistamines, and corticosteroids, either alone or in combination. However, even with strict adherence to standardized treatment protocols, nearly 20% of patients do not experience symptom improvement, exhibiting refractory AR. Therefore, it cannot fundamentally prevent the disease's progression, severely impacting quality of life and imposing a heavy economic burden on patients and society. Thus, exploring new intervention methods remains a key focus and challenge in current AR research.

[0003] Allergic rhinitis (AR) is considered a classic type 2 cytokine-mediated disease. After antigen stimulation, the antigen is presented by antigen-presenting cells, prompting naive T lymphocytes to differentiate into type 2 cells, inducing B cells to differentiate into plasma cells, producing IgE, and binding to mast cells and basophils to induce degranulation, leading to the release of inflammatory mediators such as histamine and leukotrienes, resulting in clinical symptoms such as nasal itching, sneezing, and runny nose. Recent studies have shown that nasal mucosal epithelial cells, by expressing pattern recognition receptors and protease-activated receptors, recognize microorganisms, allergens, and pollutants in the environment, releasing various bioactive molecules (such as TSLP, IL-25, and IL-33) to regulate the function of immune cells such as innate lymphocytes and dendritic cells, thus playing a key role in the occurrence and development of AR. However, the specific regulatory mechanisms remain unclear, and whether epigenetics is involved is unknown. It is well known that the pathogenesis of AR is jointly regulated by environmental and genetic factors, and epigenetic modifications, as an important bridge between the environment and genes, are receiving increasing attention for their role in allergic diseases. In recent years, with the rise of RNA epigenetics, RNA methylation modification, a hot topic, has provided a new research direction for exploring the pathogenesis of allergic rhinitis (AR). Finding ways to block the inflammatory response is of great significance for the treatment of allergic rhinitis.

[0004] RNA methylation occurs at gene modification sites including N1-methyladenosine (m1A), N6-methyladenosine (m6A), and 5-methylcytidine (m5C), and is widely involved in various biological behaviors of RNA, including transcription, splicing, nuclear export, translation, and degradation. Among these, m6A methylation is the most prevalent and abundant internal modification in eukaryotic genes. Studies have shown that m6A methylation is mainly mediated by methyltransferases (Writers), demethylases (Erasers), and methylation-reading proteins (Readers), which work together to influence the post-transcriptional processing of genes in eukaryotic cells, affecting the transmission of genetic information. This process is dynamic and reversible. Because m6A methylation is dynamic and reversible, targeting the balance between methyltransferases and demethylases to alter the overall RNA methylation level in the body may potentially reverse the occurrence and development of diseases, showing considerable promise for clinical translation and application. Methyltransferase-like protein 3 (METTL3), as the most important methyltransferase in m6A methylation, can significantly upregulate RNA methylation levels.

[0005] Given the increasing incidence of acute rheumatoid arthritis (AR) and the unmet treatment needs, coupled with the lack of reported applications of vitamin B7 in treating AR, there is an urgent need to develop a safer and more effective novel drug with broad-spectrum anti-inflammatory properties to treat AR inflammatory diseases. Summary of the Invention

[0006] This invention addresses the challenges in treating respiratory inflammatory diseases by providing an application of vitamin B7 in the preparation of drugs for treating allergic rhinitis. The invention discloses the mechanism of action of vitamin B7 in allergic rhinitis (AR) and, for the first time, reveals that vitamin B7 significantly inhibits the expression of TSLP in human and mouse nasal mucosal epithelial cells, and reduces inflammatory factors associated with allergic rhinitis in mice. Il-4, Il-13 The levels of IgE in mice and the serum levels of IgE provide a new perspective and target for the treatment of AR.

[0007] To achieve the above objectives, the technical solution designed by the present invention is as follows: This invention provides the application of vitamin B7 in the preparation of a drug for treating allergic rhinitis.

[0008] The present invention also provides a medicament for treating allergic rhinitis, the medicament comprising an effective amount of vitamin B7.

[0009] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0010] Furthermore, the effective concentration of vitamin B7 in the drug is 1~10μM.

[0011] Furthermore, the effective concentration of vitamin B7 in the drug is 4 μM.

[0012] Furthermore, the excipients are any one of physiological saline, glucose, vitamin C, and amino acids.

[0013] Furthermore, the pharmaceutical preparation is a spray, nasal drop, or rinse.

[0014] Furthermore, the spray formulation is any one of an atomizing agent, a spray, and a suspension.

[0015] Research basis of this invention Previous studies have found that the expression level of METTL3 in nasal mucosal epithelial cells of patients with acute rheumatoid arthritis (AR) is significantly upregulated, and METTL3 can regulate the expression of TSLP, a key pathogenic factor in AR. Therefore, intervening in the function of METTL3 may become a new target for the treatment of AR. This invention uses virtual screening of METTL3 protein, and the results show that vitamin B7, a drug already used in clinical practice, can bind to the active site of the METTL3 protease, blocking a series of reactions caused by the overexpression of the METTL3 enzyme. In an AR mouse model, the nasal mucosal inflammation level was significantly reduced in mice in the vitamin B7 nasal drop and drinking water supplementation groups. Il-4, Il-13, Tslp Expression decreased, and serum IgE levels in mice were also significantly downregulated. Furthermore, no significant side effects were observed with local or systemic administration in the mouse AR model group. Vitamin B7, as a water-soluble vitamin, has been used clinically to treat vitamin B7 deficiency with clear therapeutic effects and no toxic side effects, making it more readily accepted by patients than other medications.

[0016] The beneficial effects of this invention are: This invention, through systematic research, discovered that the key enzyme METTL3, an m6A methyltransferase, is abnormally highly expressed in allergic rhinitis (AR) and can regulate the level of TSLP, a key protein in AR pathogenesis, thereby participating in the development and progression of AR. Based on this, this invention, for the first time, proposes a virtual screening method using clinically available drugs to identify vitamin B7 as a potential candidate drug that may act on METTL3. Further in vitro nasal mucosal epithelial cell experiments and in vivo mouse model studies confirmed that vitamin B7 can effectively reduce TSLP levels and improve AR-related inflammatory responses, demonstrating a significant therapeutic effect. Details are as follows: 1. Clear mechanism of action: It reveals the important role of the METTL3-TSLP pathway in the pathogenesis of AR, providing a new theoretical basis for molecular targeted therapy of AR.

[0017] 2. Safe drug source: Virtual screening revealed that vitamin B7 has the potential to inhibit METTL3 function. As a commonly used vitamin in clinical practice, vitamin B7 has high safety and good prospects for clinical translation.

[0018] 3. Comprehensive verification methods: Combining histopathology, high-throughput sequencing, molecular and cell biology, in vitro cell culture and in vivo animal models, a complete chain of evidence has been formed from mechanism exploration to treatment verification, which is highly scientific and reliable.

[0019] 4. Outstanding application value: This study proposes a new use of vitamin B7 in the treatment of allergic rhinitis (AR) for the first time, expands the clinical indications of vitamin B7, and provides a safe, effective and easy-to-promote new strategy for the prevention and treatment of allergic rhinitis.

[0020] In summary, this invention is not only innovative in its mechanism research, but also has significant promotional value in practical applications. Attached Figure Description

[0021] Figure 1 A schematic diagram showing the significant upregulation of m6A modification levels and METTL3 gene and protein levels in AR patient tissues; In the figure, A is a schematic diagram of the colorimetric detection of m6A modification levels in tissues of normal individuals and AR patients; B is a schematic diagram of the m6A modification level in normal human and AR patient tissues detected by immunofluorescence assay; C represents the detection of nasal mucosal epithelial cells by RT-PCR. METTL3 A schematic diagram of gene expression levels; D is a schematic diagram showing the detection of METTL3 protein expression levels in the two groups using immunohistochemical staining.

[0022] Figure 2 A schematic diagram showing the significant upregulation of METTL3 gene and protein levels in epithelial cells of an AR mouse model; In the figure, A represents RT-PCR detection. Mettl3 A schematic diagram of gene expression levels in mouse nasal mucosa. B is a schematic diagram illustrating the detection of Mettl3 protein expression levels in mouse nasal mucosa using immunohistochemical staining.

[0023] Figure 3 A schematic diagram showing how downregulating TSLP expression in nasal mucosal epithelial cells can intervene in METTL3 expression. In the figure, A represents cells treated with siRNA. METTL3 A schematic diagram of gene expression levels; B represents cells treated with siRNA. TSLP A schematic diagram of gene expression levels; C is a schematic diagram showing the expression level of TSLP protein after siRNA treatment of cells.

[0024] Figure 4 This diagram illustrates how the levels of the TSLP gene and protein are significantly upregulated in nasal mucosal epithelial cells of AR patients and mouse models, and how these levels are positively correlated with METTL3. In the diagram, A is... TSLP A schematic diagram of gene expression levels in epithelial cells of normal individuals and AR patients; B is a schematic diagram illustrating the immunohistochemical detection of TSLP protein expression levels in epithelial cells of normal individuals and AR patients. C is TSLP and METTL3 A schematic diagram of gene-level correlations in nasal mucosal epithelial cells of AR patients; D is Tslp A schematic diagram of gene expression levels in the nasal mucosa of AR mice; E is a schematic diagram of the immunohistochemical detection of Tslp protein expression level in AR mouse nasal mucosal epithelial cells. F is Tslp and Mettl3 A schematic diagram of gene-level correlations in nasal mucosal epithelial cells of AR patients.

[0025] Figure 5 A schematic diagram of the docking of METTL3 with vitamin B7 molecules and a schematic diagram of vitamin B7 downregulating the level of m6A modification in human nasal mucosal epithelial cells. In the figure, A is a schematic planar diagram of the docking between the METTL3 protein and the vitamin B7 molecule; B is a 3D diagram showing the docking of the METTL3 protein with a vitamin B7 molecule. C is a schematic diagram showing the inhibitory effect of different concentrations of vitamin B7 on m6A modification in human nasal mucosal epithelial cells as detected by Dot-blot method; Figure 6 A schematic diagram illustrating how vitamin B7 reduces TSLP expression levels in human nasal mucosal epithelial cells. In the figure, A represents the RT-PCR detection of cells after vitamin B7 treatment. TSLP A schematic diagram of gene expression levels; B represents cells treated with vitamin B7, as detected by Western blot. TSLP A schematic diagram of protein expression levels; Figure 7 Schematic diagram of two administration methods for vitamin B7 to AR mice; Figure 8 This diagram illustrates how both administration methods of vitamin B7 can reduce inflammation levels in AR mice.

[0026] In the figure, A is a schematic diagram of the expression level of serum IgE in different treatment groups detected by ELISA; B represents the RT-PCR detection of nasal mucosa in mice from different treatment groups. Tslp A schematic diagram of gene expression levels; C represents the RT-PCR detection of different treatment groups of mice in the nasal mucosa. Il4 A schematic diagram of gene expression levels; D represents the RT-PCR detection of different treatment groups of mice in the nasal mucosa. Il13 A schematic diagram of gene expression levels; In the diagram, Vitanmin B7: Vitamin B7; m6A: N6-methyladenosine; METTL3: Methyltransferase-like protein 3; Control: Normal control group; AR: Allergic rhinitis; HDM: House dust mite; PBS: Phosphate-buffered saline solution; TSLP: Thymic stromal lymphopoietin; IgE: Immunoglobulin E; Il-4: Interleukin-4; Il-5: Interleukin-5; Il-13: Interleukin-13.

[0027] This indicates that P < 0.05. This indicates that P < 0.01. This indicates that P < 0.001. This means P < 0.0001. Detailed Implementation

[0028] 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.

[0029] The specific embodiments include the grouping of research subjects, specimen types, and equipment and reagents. 1. Study grouping and sample / clinical data collection 1) Study Groups: Referring to the "Guidelines for the Diagnosis and Treatment of Allergic Rhinitis (2015, Tianjin)" and the ARIA 2016 diagnostic criteria for AR (Brozek JL, et al. J Allergy Clin Immunol 2017 Oct;1404(4):950-958), the study subjects were divided into: ① Normal control group (Control): Patients with simple nasal septum deviation without AR or other respiratory diseases; ② Allergic Rhinitis Group (AR): Patients with deviated nasal septum and accompanying AR, specifically: a: The presence of two or more typical AR symptoms, such as nasal itching, nasal congestion, runny nose, and sneezing; b: Dermatophagoides pteronyssinus (Der p) positive skin prick test or serum Der p specific IgE ≥ 0.776 KU / L.

[0030] Study participants were those who had not used systemic or topical corticosteroids within one month prior to surgery, had not used topical or systemic anti-allergy medications within two weeks prior to surgery, and had not undergone immunotherapy. Patients with nasal diseases such as chronic sinusitis, fungal sinusitis, or cystic fibrosis, as well as those with systemic diseases such as immunodeficiency, tumors, neurological or psychiatric disorders, and pregnant or lactating women were excluded from this study. All enrolled patients signed informed consent forms.

[0031] 2) Sample Collection: Inferior turbinate tissue was collected from Control and AR patients, and nasal mucosal epithelial cells were scraped for later research use. The collected inferior turbinate tissue and nasal mucosal epithelial cells were uniformly numbered and managed. The tissue samples were divided into two parts: one part was placed in 4% paraformaldehyde for immunohistochemical studies; the other part was flash-frozen in liquid nitrogen and then transferred to a -80°C freezer for subsequent RT-PCR, WB, and other experiments. Epithelial cells were also divided into two parts: one part was used for ALI culture; the other part, along with the tissue, was first flash-frozen in liquid nitrogen and then transferred to a -80°C freezer for subsequent RT-PCR, WB, and other experiments.

[0032] Example 1: Detection of key proteins in nasal mucosal tissue and epithelial cells Using human tissues and epithelial cells, the m6A modification level and the expression of the key enzyme METTL3 in AR were initially detected by colorimetric methods, immunofluorescence, RT-PCR and immunohistochemistry.

[0033] like Figure 1 and Figure 4 As shown: m6A modification levels are increased in AR tissues ( Figure 1 A), and it mainly occurs in the nucleus of epithelial cells (A), and it mainly occurs in the nucleus of epithelial cells ( Figure 1 B). RNA methyltransferase METTL3 gene ( Figure 1 The expression level of METTL3 was significantly higher in AR group epithelial cells than in the normal control group. Immunohistochemistry revealed that METTL3 was expressed in both epithelial cells and stromal inflammatory cells, but the expression was more pronounced in AR epithelial cells. Figure 1 D). TSLP gene ( Figure 4 A) and protein ( Figure 4The expression level of B) was also significantly increased in epithelial cells of AR patients compared to the normal control group. Furthermore, the expression level of the gene and METTL3 was positively correlated. Figure 4 C).

[0034] Example 2: Cell culture, stimulation, and transfection experiments Based on the research in Example 1 above, METTL3 was knocked down at the cellular level using siRNA transfection, and then the changes in TSLP gene and protein levels were detected by RT-PCR and WB methods, respectively.

[0035] The results showed that knocking down METTL3 led to TSLP ( Figure 3 The levels of genes and proteins B and C decreased significantly. Adding different concentrations of vitamin B7 to cultured human nasal mucosal epithelial cells altered the overall intracellular m6A level. Figure 5 C), the gene and protein levels of TSLP are reduced ( Figure 6 (A and B).

[0036] Further cell culture and transfection experiments were conducted to verify the results, as follows: a. Culture of human nasal mucosal epithelial cells (HNECs) at the air-liquid interface To better simulate in vivo conditions, HNECs were cultured in an air-liquid interphase (AIP) plane according to a published method (Liu JX, et al. J Allergy Clin Immunol 2020 01;1451(1):160-72). Specifically, normal HNECs with a cell purity exceeding 98% were scraped off. The cells were washed twice with DMEM (dulbecco's modified eaglemedium) containing 10% penicillin and streptomycin antibiotics. Then, the cells were transferred to a 6-well plate coated with type I rat tail collagen and 2 mL of BEBM basal medium containing growth factors were added. When the cells reached the confluence of the culture plate, they were passaged into transwell chambers (0.4 μm) coated with collagen and cultured for another 21 days until cell differentiation. When the cells reached the confluence of the upper chamber, the medium in the upper chamber was aspirated, and the lower chamber was replaced with 1:1 BEBM / DMEM medium. The cells were cultured for another 21 days until cell differentiation.

[0037] b. METTL3 small interfering RNA transfection experiment (siRNA) HNECs were used for transfection experiments with METTL3 small interfering RNA (siRNA). The procedure was followed according to the transfection instructions. Preliminary experiments were conducted to determine the optimal sequence and concentration for knocking down the METTL3 target gene. The optimal sequence is as follows: Sequence 1 is 5'-GCUGCACUUCAGACGAAUUTT-3', SEQ ID NO.1; Sequence 2 is 5'-GCUCAACAUACCCGUACUATT-3', SEQ ID NO.2.

[0038] The final concentration was 100 nM siNC or si METTL3 (Hippocampus Biotechnology Co., Ltd., Huzhou, Zhejiang, China). After transfecting cells with RNAiMAX transfection reagent (Invitrogen Thermo Fisher Scientific, USA) for 8 hours, the cells were replaced with DMEM F12 complete medium for recovery for 16 hours. RNA was then collected for RT-PCR experiments.

[0039] The results showed that after transfection of cells with siMETTL3 small interfering RNA, the gene level of METTL3 was significantly reduced, indicating that the transfection experiment was successful and the knockout efficiency reached 80%. Figure 3 A) Cells were collected, RNA was extracted, and RT-PCR experiments confirmed that low expression of METTL3 significantly downregulated TSLP mRNA levels. Figure 3 B), after collecting the protein, Western blotting was performed to detect the protein expression level of TSLP after METTL3 knockdown. The results showed that the protein level of TSLP was also significantly reduced after METTL3 knockdown. Figure 3 C).

[0040] c. Vitamin B7 stimulation of epithelial cells experiment Mature HNECs were cultured via ALI, and different concentrations of vitamin B7 were added to the lower culture medium. After 24 hours, the cells were collected, and RNA was extracted for subsequent Dot-blot experiments. The results showed that vitamin B7 reduced intracellular m6A modification levels ( Figure 5 C), RT-PCR experiments were performed, and the results showed that vitamin B7 reduced the RNA level of TSLP ( Figure 6 A), protein was extracted and subjected to Western blotting experiments. The results showed that vitamin B7 reduced the protein level of TSLP (A). Figure 6 B).

[0041] The primer sequences (human specimens) used in the above experiment are as follows: Primer pair METTL3: METTL3-F: 5'-CATTGCCCACTGATGCTGTG-3', SEQ ID NO.3, METTL3-R: 5'-AGGCTTTCTACCCCATCTTGA-3', SEQ ID NO.4; Primer pair TSLP: TSLP-F: 5'-ATGTTCGCCATGAAAACTAAGGC-3', SEQ ID NO.5, TSLP-R: 5'-AGGCTTTCTACCCCATCTTGA-3', SEQ ID NO.6; Primer pair β-Actin: β-Actin-F: 5'-CACCATTGGCAATGAGCGGTTC-3', SEQ ID NO.7, β-Actin-R: 5'-AGGTCTTTGCGGATGTCCACGT-3', SEQ ID NO. 8.

[0042] Example 3 Virtual screening of small molecule compounds targeting Human MTA70 (METTL3) protein Due to its ability to crosslink with S-adenosylmethionine (SAM), METTL3 (N6-adenosine-methyltransferase catalytic subunit, also known as MTA70) was identified as the catalytic component responsible for generating m6A-modified methyltransferase complexes. Preliminary investigations revealed that Human MTA70 (N6-adenosine-methyltransferase catalytic subunit, Gene: METTL3, Uniprot ID: Q86U44) has a resolved three-dimensional structure (PDB ID: 6Y4G, resolution: 1.90 Å, A, fragment length: 1-580), making it suitable for virtual screening. Therefore, virtual screening was performed on the Human MTA70 protein using its inhibitor, the natural nucleoside sininefungin, and its ligand molecule SFG, to obtain small molecule compounds with strong binding affinity to the target protein. The software used for virtual screening was Schrödinger Maestro 12.8, and the 3D mapping software was PyMol, as detailed below: a. Protein preparation: The 3D structure of Human MTA70 (PDB ID: 6Y4G) was downloaded from the PDB website, and hydrogenation of the protein was performed using the Protein Preparation Wizard module. Energy optimization was then performed (OPLS2005 force field, RMSD 0.30 Å). A grid file was created for the processed protein using the Receptor Grid Generation module, centering the grid file on the site of its inhibitor, the natural nucleoside sininefungin (i.e., the ligand SFG), with a box size of 20 Å × 20 Å × 20 Å.

[0043] b. Compound preparation: The 2D formats of the HY-L001V MCE Bioactive Compound Library (containing 20.5K compounds) and the HY-L0078V MCE Epigenetics Library (containing 30.8K compounds) were processed by hydrogenation, energy optimization and other methods using the LigPrep Module of Schrödinger software to output 3D structures for virtual screening.

[0044] c. Molecular docking: The Virtual Screening Workflow module is used for virtual screening. The prepared compounds are imported and molecular docking is performed using the Glide module. That is, the acceptor and ligand molecules dock with each other through geometric matching and energy matching.

[0045] HY-L001V MCE Bioactive Compound Library: First, the high-throughput screening (HTVS) mode in the Glide module is used to screen the small molecule compounds prepared in the database. The top 15% of the small molecule compounds with the highest scores are selected for a second round of screening using the standard (SP) mode. Then, the top 15% of the small molecule compounds with the highest scores are selected for a third round of screening using the high-precision (XP) mode to obtain the ranking of the small molecule compounds.

[0046] HY-L0078V MCE Epigenetics Library: First, the high-throughput screening (HTVS) mode in the Glide module is used to screen the prepared small molecule compounds in the database. The top 15% of small molecule compounds by score are selected for a second round of screening using the standard (SP) mode. Then, the top 15% by score are selected for a third round of screening using the high-precision (XP) mode to obtain the ranking of small molecule compounds. The binding force between the target and the compound, the compound structure, etc. are manually verified, and the top 200 compounds in the HY-L001V MCE Bioactive Compound Library and the top 200 compounds in the HY-L0078V MCEEpigenetics Library are output as the final results.

[0047] Among the screened clinical drugs, vitamin B7 has attracted attention. Figure 5 (AB). As a water-soluble vitamin, vitamin B7 has been used clinically to treat vitamin B7 deficiency. It is safe, effective, non-toxic, and has few side effects. To further verify the effect of vitamin B7 on the function of METTL3 protein, this example first conducts an in vitro cell experiment, stimulating nasal mucosal epithelial cells of ALI-cultured AR patients with different concentrations of vitamin B7.

[0048] The results showed that the intracellular m6A modification level decreased with increasing vitamin B7 concentration. Figure 5 C). Meanwhile, in vitro cell culture experiments showed that the gene and protein levels of TSLP were regulated by vitamin B7 (C). Figure 6 AB).

[0049] Example 4 Vitamin B7 was purchased from MCE (China) in powder form. Depending on the actual situation, vitamin B7 was dissolved in PBS to obtain a stock solution with a concentration of 40 μM for storage.

[0050] Medications for treating allergic rhinitis (nasal drops) include vitamin B7 at an effective concentration of 4 μM.

[0051] Medications (oral solutions) for treating allergic rhinitis include vitamin B7 at an effective concentration of 2 μM.

[0052] Depending on the actual situation, the above-mentioned drugs for treating allergic rhinitis can be prepared by diluting a stock solution with a concentration of 40uM, or by directly preparing vitamin B7 (powder). At the same time, various excipients (excipients can be any one of physiological saline, glucose, vitamin C and amino acids) can be added to prepare the drug.

[0053] Example 5: Animal experiments of the above-mentioned drug for treating allergic rhinitis a. Using wild-type C57BL / 6 mice, an AR mouse model was established, and the expression of Mettl3 and Tslp in AR mice was detected.

[0054] The results showed that the Mettl3 gene ( ) was present in the nasal mucosa of AR mice. Figure 2 A) and protein ( Figure 2 B) Expression levels were significantly increased in Tslp gene (B) in nasal mucosal epithelial cells of AR mouse models. Figure 4 D) and protein ( Figure 4 E) expression levels also increased significantly. Furthermore, Tslp gene levels were positively correlated with Mettl3 gene levels. Figure 4 F).

[0055] b. For the administration method of vitamin B7 intervention in AR mice, see [link to relevant documentation]. Figure 7 ; ① Sensitization phase: On days 0, 1 and 2, mice in the AR model group were given 1 μg of house dust mite extract (HDM, 1 μg / 20 μL PBS) via nasal drip.

[0056] ② Challenge phase: From day 7 to day 9, day 14 to day 16, and day 21 to day 23, 10 μg of HDM extract (10 μg / 20 μL PBS) was instilled into the nasal cavity daily. Control group mice were sensitized and challenged with PBS instead of HDM.

[0057] ③Grouping: Vitamin B7 nasal drops group: Mice were instilled with the above-mentioned nasal drops into their nasal cavities during this process, 10 μL in each nostril, for a total of 20 μL in both nostrils; Vitamin B7 drinking water group: Mice were given the above oral solution orally, about 6 ml per day.

[0058] ④ Mice were euthanized under anesthesia on day 24, and specimens were collected. Nasal mucosal epithelial cells were collected and stored at -80°C for subsequent RT-PCR and other experiments. Serum was used to detect IgE.

[0059] The results showed that in AR mice treated with oral liquid and nasal drops, the serum IgE level was significantly downregulated. Figure 8 A), and the expression levels of Tslp, Il-4, and Il-13 genes in the nasal mucosa were significantly suppressed ( Figure 8 (BD); This indicates that drugs containing vitamin B7 can effectively improve AR-related inflammatory responses, demonstrating a significant therapeutic effect.

[0060] In the above embodiments, the primer sequences (small mouse book) are as follows: primer pairs Il-4 Il4- F: 5'-GGTCTCAACCCCCAGCTAGT-3', SEQ ID NO.9, Il4- R: 5'-GCCGATGATCTCCTCAAGTGAT-3', SEQ ID NO.10; primer pairs Il-13 Il13- F: 5'-TGAGCAACATCACACAAGACC-3', SEQ ID NO.11, Il13- R: 5'-GGCCTTGCGGTTACAGAGG-3', SEQ ID NO.12; primer pairs Tslp Tslp- F: 5'-ACGGATGGGCTAACTTACAA-3', SEQ ID NO.13, Tslp-R: 5'-AGTCCTCGATTTGCTCGAACT-3', SEQ ID NO.14; primer pairs β-Actin β-actin- F: 5'-GTGACGTTGACATCCGTAAAGA-3', SEQ ID NO.15, β-actin- R: 5'-GCCGGACTCATCGTACTCC-3', SEQ ID NO. 16.

[0061] 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. The application of vitamin B7 in the preparation of a drug for treating allergic rhinitis.

2. The application according to claim 1, characterized in that: The drug contains an effective amount of vitamin B7.

3. The application according to claim 2, characterized in that: The drug also includes pharmaceutically acceptable excipients.

4. The application according to claim 2 or 3, characterized in that: The effective concentration of vitamin B7 in the drug is 1~10uM.

5. The application according to claim 4, characterized in that: The effective concentration of vitamin B7 in the drug is 4 μM.

6. The application according to claim 2 or 3, characterized in that: The excipients are any one of physiological saline, glucose, vitamin C, and amino acids.

7. The application according to claim 6, characterized in that: The pharmaceutical preparation is a spray, nasal drop, or rinse.

8. The application according to claim 7, characterized in that: The spray formulation is any one of atomizing agents, sprays, and suspensions.

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