Application of target expression inhibitor in preparation of medicine for preventing and treating asthma

Abstract

The invention discloses application of a target expression inhibitor to preparation of a medicine for preventing and treating asthma. The invention discovers that the expression of OR5W2 in an asthma cell model is significantly up-regulated, the overexpression of OR5W2 can significantly promote the expression of IL-33 and CCL20 in the asthma cell model, and the knock-down of OR5W2 can significantly inhibit the expression of IL-33 and CCL20 in the asthma cell model. Technicians in the field know that IL-33 and CCL20 are key factors for causing a series of asthma symptoms, when a body inhales allergens, airway epithelial cells can secrete chemotactic factors CCL20 to attract mononuclear cells and immature dendritic cells into the lung, meanwhile, IL-33 is secreted to activate the lung dendritic cells and promote Th2-type immune response, and therefore, the lung is prevented from being infected by the lung. A series of asthma symptoms are further caused. Therefore, the OR5W2 expression inhibitor has the prospect of being developed into the medicine for preventing and treating asthma.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to the use of a target expression inhibitor in the preparation of drugs for the prevention and treatment of asthma. Background Technology

[0002] Asthma, also known as bronchial asthma, is a common chronic respiratory disease. This disease is characterized by chronic airway inflammation involving multiple cells and cellular components, clinically manifesting as recurrent episodes of wheezing and shortness of breath, possibly accompanied by chest tightness or cough. Asthmatic patients exhibit hyperresponsive airways and variable airflow limitation; with disease progression, airway remodeling may also occur, leading to structural changes in the airways. Epidemiological studies of asthma show a global increasing prevalence. Asthma is commonly seen in individuals with a family history of asthma, allergic diseases, respiratory disorders, and gastroesophageal reflux disease. Furthermore, obesity, smoking, and allergen exposure are also contributing factors.

[0003] Typical symptoms of asthma include recurrent episodes of wheezing, shortness of breath, chest tightness, or cough, often occurring or worsening at night or in the early morning. Most patients experience spontaneous remission or relief with bronchodilators. Treatment for asthma includes avoiding triggering factors, medication, and education and self-management. Medications typically include inhaled corticosteroids (ICS), long-acting beta-2 agonists (LABA), leukotriene modifiers, sustained-release theophylline, and macrolides.

[0004] OR5W2 is an olfactory receptor protein belonging to the G protein-coupled receptor (GPCR) family. Currently, there are no reports of OR5W2 being associated with the prevention or treatment of asthma. Summary of the Invention

[0005] The purpose of this invention is to provide an OR5W2 expression inhibitor for use in the preparation of drugs for the prevention and treatment of asthma.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution:

[0007] The application of an OR5W2 expression inhibitor in the preparation of drugs for the prevention and treatment of asthma; wherein, the OR5W2 expression inhibitor refers to a substance that inhibits the expression of OR5W2.

[0008] Preferably, the OR5W2 expression inhibitor is a nucleotide sequence that inhibits OR5W2 expression or a vector containing such a nucleotide sequence.

[0009] Preferably, the OR5W2 expression inhibitor is a small molecule compound, peptide, or polysaccharide that inhibits OR5W2 expression.

[0010] Preferably, the drug shown uses an OR5W2 expression inhibitor as the active ingredient and is formulated into a pharmaceutically acceptable dosage form using pharmaceutically acceptable excipients.

[0011] More preferably, the excipient is a liquid, solid, or semi-solid excipient.

[0012] More preferably, the dosage form includes tablets, capsules, injections, sprays, and aerosols.

[0013] The application of OR5W2 as a drug target in the screening and discovery of drugs for the prevention and treatment of asthma; wherein, OR5W2 refers to the OR5W2 gene or protein.

[0014] The application of OR5W2 as a drug target in screening for biomarkers for the diagnosis, treatment, or prognosis of asthma; wherein, OR5W2 refers to the OR5W2 gene or protein.

[0015] Beneficial effects:

[0016] This invention reveals that OR5W2 expression is significantly upregulated in an asthma cell model. Overexpression of OR5W2 significantly promotes the expression of IL-33 and CCL20 in the asthma cell model, while knockdown of OR5W2 significantly inhibits the expression of IL-33 and CCL20 in the asthma cell model. Those skilled in the art know that IL-33 and CCL20 are key factors triggering a series of asthma symptoms. When the body inhales allergens, airway epithelial cells secrete the chemokine CCL20 to attract monocytes and immature dendritic cells to the lungs, while simultaneously secreting IL-33 to activate lung dendritic cells and promote a Th2 immune response, thereby triggering a series of asthma symptoms. Therefore, OR5W2 expression inhibitors show promise for development into drugs for the prevention and treatment of asthma. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the composition of plasmid pcDNA3.1-OR5W2;

[0018] Figure 2 The mRNA expression levels of IL-33, CCL20, and OR5W2 in 16HBE cells of each group were determined.

[0019] Figure 3 The effect of OR5W2 overexpression on IL-33 and CCL20 expression levels in 16HBE cells;

[0020] Figure 4 To investigate the effect of OR5W2 knockdown on the expression levels of IL-33 and CCL20 in 16HBE cells. Detailed Implementation

[0021] The following describes the substantive content of the present invention in detail with reference to embodiments, but this does not limit the scope of protection of the present invention.

[0022] I. Experimental Materials

[0023] Gradient PCR instrument (MJ mini purchased from Eppendorf, Germany); Vortex-Genie 2 vortex shaker (Scientific Industries, USA); RNA extraction kit, ChamQ Universal SYBR qPCR Master Mix (Nanjing Novizan Biotechnology Co., Ltd.); House dust mite (HDM, House Dust Mite, Greer, USA); 3000 (Thermo Fisher Scientific); Blank plasmid pcDNA3.1, plasmid pcDNA3.1-OR5W2 (provided by Suzhou Genewiz Biotechnology Co., Ltd., composition as follows) Figure 1 Negative control small interfering RNA and OR5W2 small interfering RNA (provided by Suzhou Genewise Biotechnology Co., Ltd., sequences are shown in Table 1); human bronchial epithelioid cells 16HBE cells (Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.); DMEM medium (Jiangsu Kaiji Biotechnology Co., Ltd.).

[0024] Table 1

[0025]

[0026] II. Experimental Methods

[0027] 1. Cell modeling of asthma

[0028] Human bronchial epithelioid cells (16HBE) in good growth condition were harvested, and the cell density was adjusted to 1×10⁻⁶. 5 Cells were seeded at a density of 1 / mL into 12-well plates, with 1 mL of cell suspension added to each well. The plates were then incubated for 4 hours. Two groups were set up, with 3 wells in each group. The two groups were treated with 1 mL of DMEM medium (98% DMEM medium + 2% fetal bovine serum) containing 75 μg / mL HDM and 1 mL of DMEM medium (98% DMEM medium + 2% fetal bovine serum) without HDM, respectively, for 48 hours to establish cell models. Cells were then collected for subsequent experiments.

[0029] 2. OR5W2 overexpression experiment

[0030] Human bronchial epithelioid cells (16HBE) in good growth condition were harvested, and the cell density was adjusted to 1×10⁻⁶. 5Cells were seeded at a density of 1 / mL into 12-well plates, with 1 mL of cell suspension added to each well. The plates were then incubated for 4 hours. Three groups were established, with three wells in each group. The cells were cultured in DMEM complete medium containing 1 mL of 1 μg blank plasmid pcDNA3.1 and 1.5 μL Lipofectamine 3000; DMEM complete medium containing 1 μg blank plasmid pcDNA3.1, 1.5 μL Lipofectamine 3000, and 75 μg / mL HDM; and DMEM complete medium containing 1 μg pcDNA3.1-OR5W2, 1.5 μL Lipofectamine 3000, and 75 μg / mL HDM. After 48 hours of incubation, cells were collected for subsequent experiments.

[0031] 3. OR5W2 knockdown experiment

[0032] Human bronchial epithelioid cells (16HBE) in good growth condition were harvested, and the cell density was adjusted to 1×10⁻⁶. 5 Cells were seeded at a density of 1 / mL into 12-well plates, with 1 mL of cell suspension added to each well. The plates were then incubated for 4 hours. Three groups were established, with three wells in each group. The cells were cultured in DMEM complete medium containing 1 mL of 50 nmol negative control siRNA and 2.4 μL Lipofectamine 3000; DMEM complete medium containing 50 nmol negative control siRNA, 2.4 μL Lipofectamine 3000, and 75 μg / mL HDM; and DMEM complete medium containing 50 nmol siOR5W2, 2.4 μL Lipofectamine 3000, and 75 μg / mL HDM. After 48 hours of incubation, cells were collected for subsequent experiments.

[0033] 4. Determine the expression levels of OR5W2 and asthma-related genes.

[0034] The expression of the target gene in cells was detected using qRT-PCR, with 2 -ΔΔCt The values ​​represent the relative gene expression levels. Primer information is shown in Table 2, synthesized by Shanghai Sangon Biotech Co., Ltd.

[0035] Table 2 Primer sequences

[0036]

[0037] 5. Statistical Analysis

[0038] Statistical tests were performed on the data using GraphpadPrism 9.0, employing one-way ANOVA and Dunnett's multiple comparison test. p < 0.05 was considered statistically significant.

[0039] III. Experimental Results

[0040] 1. Results of cell modeling in the asthma model

[0041] Airway epithelial cells play a crucial role in the pathogenesis of asthma. When the body inhales allergens, airway epithelial cells secrete the chemokine CCL20, attracting monocytes and immature dendritic cells to the lungs. Simultaneously, they secrete IL-33 to activate pulmonary dendritic cells and promote a Th2 immune response, thereby triggering a series of asthma symptoms. Figure 2 It was found that when 75 μg / mL HDM was administered, the mRNA levels of IL-33 and CCL20 significantly increased (p < 0.05), indicating that 16HEB, under HDM stimulation, exhibited the airway epithelial cell response characteristic of asthma, thus successfully establishing the model. Simultaneously, from Figure 2 It was found that, compared with the blank control group, the expression level of OR5W2 was significantly upregulated after HDM administration (p<0.05).

[0042] 2. The effect of OR5W2 overexpression on asthma

[0043] IL-33 and CCL20 are elevated in human bronchial epithelioid cells of asthmatic patients. Figure 3 It was found that by administering an overexpression plasmid to induce OR5W2 overexpression in 16HBE cells, the mRNA levels of IL-33 and CCL20 in 16HBE cells were significantly increased after administration of HDM (p < 0.05), and OR5W2 overexpression intervention could again significantly increase the mRNA levels of IL-33 and CCL20 (p < 0.05).

[0044] 3. The effect of OR5W2 knockdown on asthma

[0045] IL-33 and CCL20 are elevated in human bronchial epithelioid cells of asthmatic patients. Figure 4 It was found that by administering siRNA to reduce OR5W2 expression in 16HBE cells, the mRNA levels of IL-33 and CCL20 in 16HBE cells were significantly increased after HDM administration (p < 0.05), while OR5W2 expression knockdown intervention significantly reduced the mRNA levels of IL-33 and CCL20 (p < 0.05).

[0046] In summary, OR5W2 expression was significantly upregulated in the asthma cell model. Overexpression of OR5W2 significantly promoted the expression of IL-33 and CCL20 in the asthma cell model, while knockdown of OR5W2 significantly inhibited the expression of IL-33 and CCL20 in the asthma cell model. Those skilled in the art know that IL-33 and CCL20 are key factors in triggering a series of asthma symptoms. When the body inhales allergens, airway epithelial cells secrete the chemokine CCL20 to attract monocytes and immature dendritic cells to the lungs, while simultaneously secreting IL-33 to activate lung dendritic cells and promote a Th2 immune response, thereby triggering a series of asthma symptoms. Therefore, OR5W2 expression inhibitors show promise for development into drugs for the prevention and treatment of asthma.

[0047] The purpose of the above embodiments is to specifically illustrate the substantive content of the present invention, but those skilled in the art should know that the scope of protection of the present invention should not be limited to the specific embodiments.

Claims

1. The application of an OR5W2 expression inhibitor in the preparation of drugs for the prevention and treatment of asthma; wherein, The OR5W2 expression inhibitor refers to a substance that inhibits the expression of OR5W2.

2. The application according to claim 1, characterized in that: The OR5W2 expression inhibitor is a nucleotide sequence that inhibits OR5W2 expression or a vector containing that nucleotide sequence.

3. The application according to claim 1, characterized in that: The OR5W2 expression inhibitor is a small molecule compound, peptide, or polysaccharide that inhibits OR5W2 expression.

4. The application according to claim 1, characterized in that: The drug shown uses an OR5W2 expression inhibitor as its active ingredient and is formulated into a pharmaceutically acceptable dosage form using pharmaceutically acceptable excipients.

5. The application according to claim 4, characterized in that: The excipients are liquid, solid, or semi-solid excipients.

6. The application according to claim 4, characterized in that: The dosage forms include tablets, capsules, injections, sprays, and aerosols.

7. The application of OR5W2 as a drug target in screening and discovering drugs for the prevention and treatment of asthma; among which, OR5W2 refers to the OR5W2 gene or protein.

8. The application of OR5W2 as a drug target in screening for biomarkers for the diagnosis, treatment, or prognosis of asthma; among which, OR5W2 refers to the OR5W2 gene or protein.

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