Asthma susceptibility gene, medicine thereof and application of medicine

Abstract

The invention provides an asthma susceptibility gene, a medicine thereof and application of the medicine. The susceptibility gene is SGLT2. The medicine is an inhibitor canagliflozin of SGLT2. The potential causal association between the hypoglycemic drug canagliflozin and the asthma risk is found for the first time, the canagliflozin can significantly improve Th2 type airway inflammation, goblet cell metaplasia and pulmonary interstitial collagen deposition, the treatment effect is significant, and the anti-inflammatory protection effect is related to a PI3K / AKT pathway.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a susceptibility gene for asthma, its drug, and the application of the drug. Background Technology

[0002] Bronchial asthma (or simply asthma) is a complex chronic airway disease characterized by persistent airway inflammation. This inflammatory process involves various immune cells and inflammatory mediators, leading to airway hyperresponsiveness and airflow limitation. Persistent airway inflammation causes structural changes such as epithelial cell damage, smooth muscle cell proliferation, and collagen deposition, which can induce irreversible airway remodeling over a long period. Airway remodeling not only further leads to airflow limitation and decreased lung function in asthma patients but also increases the risk of acute exacerbations, hospitalization, and death. The synergistic effect of airway inflammation and airway remodeling in asthma not only exacerbates patients' clinical symptoms and disease burden but also affects long-term prognosis. Therefore, the main strategy for asthma treatment is to achieve good symptom control while minimizing the risk of asthma-related acute exacerbations and death, and slowing down or reversing airway remodeling as much as possible. Despite the availability of various treatment options, asthma control in children in my country remains unsatisfactory, with some children still experiencing poor asthma control even after standardized treatment. Therefore, developing new treatment strategies is of great significance for improving the effectiveness of asthma treatment and enhancing patients' quality of life.

[0003] Drug repurposing, due to its advantages such as short development cycle and low cost, has become an important direction for researchers to explore new treatments for various diseases. Hypoglycemic drugs are a class of drugs used to treat diabetes and related metabolic diseases, mainly including metformin, glucagon-like peptide-1 receptor agonists (GLP-1 RAs), sodium-glucose cotransporter-2 inhibitors (SGLT-2 inhibitors), thiazolidinediones (TZDs), sulfonylureas, and insulin and its analogues.

[0004] Canagliflozin (CANA) is an SGLT2 inhibitor that lowers blood glucose by inhibiting renal tubular reabsorption of glucose and promoting urinary glucose excretion. However, recent studies have revealed its pleiotropic effects, particularly its important roles in anti-inflammation, metabolic regulation, and immune modulation. Zhang et al. found that CANA can regulate inflammation and ferroptosis, thereby reducing lipotoxicity in cardiomyocytes. Niu et al. discovered that CANA can inhibit the activation of the NOD-like receptor pyrin domain-containing 3 (NLRP3) inflammasome and inflammation.

[0005] However, the traditional administration of canagliflozin cannot overcome the low concentration at the site of inflammation and the potential side effects caused by systemic exposure, which limits its application in the treatment of asthma. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a susceptibility gene for asthma, a drug thereof, and the application of the drug.

[0007] To address the aforementioned technical problems, the present invention provides a susceptibility gene for asthma, wherein the susceptibility gene is SGLT2.

[0008] Based on a general technical concept, the present invention provides a drug for treating asthma, wherein the drug is canagliflozin.

[0009] Based on a general technical concept, the present invention provides a drug for treating asthma, said drug being canagliflozin aerosol.

[0010] Furthermore, the preparation method of the aforementioned drug, canagliflozin aerosol, includes: S1. After ultra-fine pulverization of canagliflozin, it is added to the base solution for emulsification and mixing, and then ultrasonically dispersed to obtain the drug solution. S2. Cool the liquid medicine to 8°C, inject it into the aerosol can, introduce propellant, control the pressure at 1.0 MPa, and seal it by laser welding to obtain canagliflozin aerosol. The base solution consists of 50g ethanol, 30g deionized water, 2g Tween-80, and 0.5g citric acid.

[0011] Based on a general technical concept, the present invention provides the application of the aforementioned drug in the preparation of a medicament for treating asthma.

[0012] Based on a general technical concept, the present invention provides the use of the aforementioned drug in the preparation of a drug for reducing peri-airway inflammatory cell infiltration and Th2 inflammatory response.

[0013] Based on a general technical concept, the present invention provides the use of the aforementioned drug in the preparation of a drug for reducing the degree of goblet cell metaplasia in bronchial epithelium of lung tissue.

[0014] Based on a general technical concept, the present invention provides the use of the aforementioned drug in the preparation of a drug for reducing collagen deposition in the pulmonary interstitium.

[0015] Compared with the prior art, the advantages of the present invention are as follows: (1) This invention provides an asthma susceptibility gene: SGLT2. This invention is the first to use a two-sample MR analysis method to explore the potential causal association between hypoglycemic drugs and asthma risk. The results show that SGLT2 inhibitors are causally associated with a reduced asthma risk, and the results are robust.

[0016] (2) This invention provides a drug for treating asthma: canagliflozin aerosol. Canagliflozin aerosol has significant advantages over its oral formulation. From the perspective of its mode of action, canagliflozin, originally an oral medication, needs to circulate throughout the bloodstream to exert its effects, which may cause some systemic adverse reactions. However, by changing to an aerosol, the drug can act directly on the airways, precisely targeting the lesion site, greatly reducing the amount of drug entering the systemic bloodstream, thus effectively avoiding systemic adverse reactions and significantly improving safety. At the same time, this formulation characteristic also expands its applicable population. Oral canagliflozin is mainly suitable for adults, while the aerosol, due to its local action and high safety, is more suitable for children, providing a more appropriate option for the treatment of related diseases in children.

[0017] (3) This invention provides an application of canagliflozin aerosol in the treatment of asthma. Canagliflozin aerosol can significantly improve Th2 type airway inflammation, goblet cell metaplasia and pulmonary interstitial collagen deposition, and the therapeutic effect is significant. The anti-inflammatory protective effect is related to the PI3K / AKT pathway.

[0018] In addition to the objectives, features, and advantages described above, the present invention has other objectives, features, and advantages. The invention will now be described in further detail with reference to the accompanying drawings and tables. Attached Figure Description

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0020] Figure 1 This is a confocal microscope image of H&E staining of mouse lung tissue in Experiment 1 of this invention.

[0021] Figure 2The results of the inflammatory infiltration score of mouse lung tissue in Experiment 1 of this invention are shown.

[0022] Figure 3 The qPCR method in Experiment 1 of this invention was used to detect the mRNA expression levels of IL-4, IL-13, and TNF-α in lung tissue.

[0023] Figure 4 The experiment in this invention used the ELISA method to detect the levels of IL-4, IL-13, and TNF-α in lung tissue homogenate.

[0024] Figure 5 This invention relates to Experiment 1, which involves the determination of the proportion of eosinophils in peripheral blood through peripheral blood cell classification and counting.

[0025] Figure 6 In Experiment 2 of this invention, PAS staining was used to assess the degree of goblet cell metaplasia in the bronchial epithelium of mouse lung tissue.

[0026] Figure 7 This is a statistical analysis chart of the goblet cell metaplasia rate in Experiment 2 of this invention.

[0027] Figure 8 The results of RT-qPCR analysis of goblet cell metaplasia-related genes Agr2, Foxa3, and Foxm1 in Experiment 2 of this invention are shown.

[0028] Figure 9 This is the immunohistochemical detection result of MUC5AC protein expression in mouse lung tissue in Experiment 2 of this invention.

[0029] Figure 10 The image shows an electron micrograph (a) of the mouse lung tissue surrounding the airway, obtained from Masson staining in Experiment 3 of this invention. The scale bar represents 100 μm. The image also shows a statistical analysis of the collagen deposition area ratio.

[0030] Figure 11 The figures show the results of Western blotting (WB) measurements of p-PI3K, PI3K, p-AKT, AKT, and GAPDH in Experiment 4 of this invention; 'a' in the figure is the WB analysis graph. 'b' in the figure shows the quantitative analysis of p-PI3K expression; 'c' in the figure shows the quantitative analysis of p-AKT expression.

[0031] Figure 12 The concentrations of ALT, AST, CREA, and BUN in the serum of mice in Experiment 5 of this invention are given.

[0032] Figure 13 The images show H&E staining of the heart, liver, spleen, lungs, and kidneys of mice in each group of Experiment 5 of this invention. Detailed Implementation

[0033] The present invention will be further described below with reference to specific preferred embodiments, but this does not limit the scope of protection of the present invention. The materials, reagents, and instruments used in the following embodiments can all be purchased commercially. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods in the art.

[0034] Example 1 SGLT2 is a susceptibility gene for asthma.

[0035] Experiment 1: Causal relationship between SGLT2 gene and reduced asthma risk.

[0036] We employed a two-sample MR analysis using publicly available Genome-Wide Association Study (GWAS) datasets. The potential impact of glucose-lowering drugs on asthma risk was assessed by calculating the odds ratio (OR) and its 95% confidence interval (CI). MR utilizes single nucleotide polymorphism (SNP) variations as indices (IVs) to mimic drug intervention effects, thus avoiding confounding bias in causal inference. Genetic variations at gene loci encoding drug target proteins may affect their expression or function; these variations can be used to indirectly assess the potential impact of drug regulation of target proteins on clinical outcomes. Table 1 shows the instrumental variable screening and SNP results for glucose-lowering drugs.

[0037] Table 1: Instrumental variable screening and results of hypoglycemic drugs (SNPs).

[0038]

[0039] SNPs were selected as IVs based on strict screening criteria. Six SNPs were selected for sulfonylureas, two for GLP-1 receptor agonists, 31 for TZDs, and 19 for SGLT2 inhibitors. All SNPs had an F-value greater than 10, ensuring the effectiveness of the IVs.

[0040] To verify the effectiveness of the selected IVs, T2DM and glycated hemoglobin levels were analyzed as outcome indicators, and the results are shown in Table 2.

[0041] Table 2: MR results of hypoglycemic drugs targeting glycated hemoglobin and type 2 diabetes.

[0042]

[0043] The results in Table 2 show that ABCC8 was significantly associated with the outcome in the glycated hemoglobin analysis, but not statistically significant in the T2DM risk analysis. This may be related to its primary regulation of insulin secretion and its relatively short-lived effect, while the risk of T2DM involves more complex pathological mechanisms. PPARG did not show a significant association in the glycated hemoglobin analysis, but it did show a significant association in the T2DM risk analysis. This may be because it mainly works by improving insulin sensitivity rather than directly lowering blood glucose levels. Furthermore, GLP1R and SLC5A2 showed statistically significant associations in both analyses. These results indicate that the IVs selected in this invention have good validity.

[0044] Furthermore, four major asthma datasets were analyzed to assess the potential causal effects of glucose-lowering drug-related target genes on asthma risk. The results are presented in Table 3.

[0045] Table 3: Results of the analysis of the potential causal effects of hypoglycemic drug-related target genes on asthma.

[0046]

[0047] In separate MR analyses, the ukb-a-66 dataset (OR=0.780; 95% CI 0.595–0.951; P<0.001), the ebi-a-GCST90014325 dataset (OR=0.542; 95% CI 0.430–0.683; P<0.001), the ebi-a-GCST90038616 dataset (OR=0.689; 95% CI 0.563–0.789; P=0.001), and the ebi-aGCST90018795 dataset (OR=0.524; 95% CI 0.380–0.722; P<0.001) all showed a causal association between SLC5A2 and a reduced risk of asthma.

[0048] In the previous section, MR analysis revealed a causal association between SGLT2 inhibitors and the reduction of asthma risk. SGLT2 inhibitors mainly include empagliflozin, canagliflozin, and dapagliflozin.

[0049] Example 2 A drug for treating asthma: canagliflozin aerosol, the preparation method of which includes the following steps: (1) Take 10 g of canagliflozin (solid active ingredient) and pulverize it to a particle size of 8 μm under nitrogen protection.

[0050] (2) Mix 50 g ethanol, 30 g deionized water, 2 g Tween-80 and 0.5 g citric acid, and stir at 35℃ and 600 r / min for 18 min to obtain the base solution.

[0051] (3) Add the pulverized canagliflozin to the base solution, emulsify and mix at 30℃ and 2500 r / min for 30 min, and simultaneously turn on the ultrasonic dispersion at 25 kHz and 400 W to obtain the drug solution.

[0052] (4) Cool the drug solution to 8°C, inject it into a 100 ml aerosol can (filling volume 45 ml), introduce a propane-butane mixed propellant (mass ratio 1:1), control the pressure to 1.0 MPa, and seal it by laser welding.

[0053] Example 3 The application of canagliflozin aerosol of Example 2 in the preparation of a medicament for treating asthma.

[0054] (1) Establish an OVA-induced animal model of asthma.

[0055] 1.1 Experimental Groups: Thirty Balb / c mice (6 weeks old, male, weighing 16–20 g) were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd. All animal experiments were conducted and controlled according to the requirements of the Department of Animal Science, Central South University. During the rearing period, a regular day-night alternating lighting schedule was maintained, and ample food and water were provided. All experimental protocols were approved by the Ethics Committee of Xiangya Hospital, Central South University (CSU-2024-0052).

[0056] 1.2 Sensitization Procedure: OVA (100 μg / mouse) and aluminum hydroxide (2 mg / mouse) were dissolved in 200 μL of physiological saline and incubated in the dark (4℃, 30 min) to prepare the sensitization solution. Mice were sensitized by intraperitoneal injection (200 μL / mouse) on days 0, 7, and 14, respectively.

[0057] (2) Intervention and stimulation process: Administer the corresponding drugs or saline according to the following grouping scheme.

[0058] Ctrl group: Mice were sensitized with PBS and injected intraperitoneally with physiological saline. After 30 min, they were nebulized with 5% OVA solution (the control group used physiological saline) for 20 min.

[0059] OVA group: Mice were sensitized with OVA and injected intraperitoneally with physiological saline. 30 min later, they were nebulized with 5% OVA solution (the control group used physiological saline) for 20 min.

[0060] CANA group: Mice were sensitized with OVA; canagliflozin aerosol 50 mg / kg / day. After 30 min, they were nebulized with 5% OVA solution (the control group used physiological saline) for 20 min.

[0061] The body weight of mice in each group was observed and recorded daily during the experiment.

[0062] Experiment 1: Investigating how CANA reduces peri-airway inflammatory cell infiltration and Th2 inflammatory response in asthmatic mice.

[0063] 1.1 H&E Staining: Mouse lung tissue sections were heated in an oven for 30 min. Then, the sections were sequentially immersed in the following solutions: xylene (I, II, III) for 10 min each, anhydrous ethanol (I, II) for 10 min each, 95% ethanol (I, II) for 10 min each, 75% ethanol for 10 min, and finally rinsed with double-distilled water for 1 min. The sections were stained in hematoxylin solution for 2 min, followed by rapid differentiation with 1% hydrochloric acid ethanol (1 second), and then bluing in running water for 15 min. The sections were then immersed in eosin solution for 30 seconds, rinsed with running water, and then sequentially treated: immersed in anhydrous ethanol for 1 min, followed by immersion in xylene for 5 min. Finally, the sections were mounted with neutral resin.

[0064] Figure 1 The images show the H&E staining results of mouse lung tissue. As can be seen, the OVA group exhibited extensive inflammatory cell infiltration around the bronchial vascular units, with swollen and disorganized bronchial epithelial cells. Furthermore, the inflammatory infiltration score in the OVA group was significantly higher than that in the Ctrl group, indicating the successful establishment of the asthma mouse model. The CANA group improved the inflammatory cell infiltration in the lung tissue and restored the normal morphology and arrangement of the bronchial epithelium.

[0065] 1.2. Inflammatory infiltration score of mouse lung tissue.

[0066] A: The percentage of parabronchial / bronchial infiltration is divided into none (0), small amount (<25%, 1), moderate amount (25%-75%, 2) and large amount (>75%, 3).

[0067] B: The quality of parabronchial / bronchial infiltration is classified according to the degree of infiltration as none (0), mild (1, often interrupted inflammatory cell rings), moderate (2, complete rings or crescent rings) and severe (3, complete rings with ≥ layers).

[0068] C: Exudate in the bronchioles / bronchial lumen can be classified as none (0), small amount (1, lumen occlusion ≤25%) and large amount (2, lumen occlusion >25%).

[0069] D: Perivascular infiltration is classified as none (0), small amount (1), moderate amount (2) and large amount (3).

[0070] E: Pulmonary parenchymal inflammation is scored as no (0), few (3, scattered infiltration) and many (5, confluent infiltration).

[0071] The final score formula is: Score (0-26 points) = A + 3(B + C) + D + E.

[0072] Figure 2 The results show the inflammation infiltration score of mouse lung tissue. As can be seen from the figure, the CANA group showed the most significant effect, with a significant reduction in inflammation score (P<0.0001).

[0073] 1.3 RT-qPCR analysis: Add 600 µL of Trizol to each well and extract RNA according to the instructions of the RNA extraction kit. After extraction, determine the concentration of RNA sample using an ultra-micro nucleic acid analyzer. Each sample was measured three times, and the average value was taken.

[0074] According to the reverse transcription kit instructions, prepare the reverse transcription reaction system on ice (the reverse transcription reaction solution system includes: 1 μg total RNA, 4 μL 5x TransScript Uni All-in-one SuperMix for qPCR, 1 μL gDNARemover, and RNase-free water to a total volume of 20 μL). Gently mix the reverse transcription reaction system, incubate at 50°C for 5 min, and heat at 85°C for 5 s to obtain cDNA.

[0075] The reaction mixture (10 μl of Hieff UNICON® qPCR SYBR, Green Master Mix, 1 μL of 10 μM Forward Primer, 1 μL of 10 μM Reverse Primer, 4 μL of cDNA, and DEPC water to a final volume of 20 μL) was thoroughly mixed and added to a 96-well plate (3 sub-wells for each indicator). The 96-well plate was placed in a PCR instrument, and the program was started. The plate was pre-denatured at 95°C for 30 s, pre-denatured at 95°C for 30 s, and annealed at 60°C for 10 s. This denaturation, annealing, and extension cycle was repeated 40 times. The primer sequences used are shown in Table 4.

[0076] Table 4: Primer information used in RT-qPCR.

[0077] Figure 3The mRNA expression levels of IL-4, IL-13, and TNF-α in lung tissue were detected by qPCR. The figure shows that the mRNA expression levels of Th2 cytokines IL-4 and IL-13, and the inflammatory factor TNF-α in the lung tissue of mice in the OVA group were significantly higher than those in the Ctrl group (P<0.0001, P<0.0001, P<0.0001), which is consistent with the high Th2 response state of the eosinophilic asthma model. After treatment with canagliflozin aerosol, the mRNA levels significantly decreased (P<0.0001, P<0.0001, P<0.0001).

[0078] 1.4 ELISA Analysis: Equilibrate the kit to room temperature for 20 minutes and prepare washing buffer. Fix the ELISA plate onto the plate holder, and set up standard wells (6 concentration gradients, 50 μL / well) and sample wells (10 μL sample + 40 μL diluent / well, blank wells with 50 μL diluent), all in duplicate. Add 100 μL of HRP detection antibody to each well and incubate at 37°C for 60 minutes. Discard the liquid and wash 5 times (300 μL / wash, pat dry after the last wash). In a dark room, add 50 μL each of TMB substrate A and B to each well, and develop at 37°C in the dark for 15 minutes. Immediately add 50 μL of stop solution and mix well. Measure the OD value using a microplate reader (450 nm / 630 nm) within 15 minutes, plot a standard curve, calculate the sample concentration, and take the mean of the replicates.

[0079] Figure 4 The levels of IL-4, IL-13, and TNF-α in lung tissue homogenates were detected by ELISA. The results showed the same trend as RT-qPCR (OVA vs Ctrl: P<0.0001, P<0.0001, P<0.0001; CANA vs OVA: P<0.001, P<0.05, P<0.0001).

[0080] 1.5. Routine peripheral blood test in mice: Mice were anesthetized with 0.3% sodium pentobarbital, and whiskers were removed from the corners of the mouth to avoid hemolysis during the procedure. One eye was quickly enucleated, and whole blood samples were directly dropped into 1.5 mL centrifuge tubes containing EDTA anticoagulant and stored at 4°C. Cell classification and counting were performed on the anticoagulant whole blood samples using an XN-1000-B1 fully automated hematology analyzer, followed by statistical analysis.

[0081] Figure 5The proportion of eosinophils in peripheral blood was determined by peripheral blood cell differential counting. The results showed that the proportion of eosinophils in the OVA group was significantly higher than that in the Ctrl group. After CANA treatment, this proportion significantly decreased (P<0.0001, P<0.01), confirming the effectiveness of CANA in alleviating eosinophilic inflammation.

[0082] Experiment 2: To investigate the degree to which CANA reduces goblet cell metaplasia in the bronchial epithelium of asthmatic mice.

[0083] 2.1 PAS staining of mouse lung tissue: Following the HE staining method, dewaxing and hydration were performed. The tissue was treated with PAS oxidant for 5 min, rinsed with running water, and then soaked in double-distilled water for 5 min. The sections were then placed in Schiff staining solution and treated in the dark for 10 min, followed by rinsing with running water for 10 min. After soaking in hematoxylin staining solution for 2 min, differentiation was performed using 1% hydrochloric acid ethanol (1 second), followed by blueing in running water for 15 min. The sections were then treated with anhydrous ethanol for 1 min, xylene for 5 min, and mounted with neutral resin. The sections were observed under a microscope, images were acquired, and subsequent analysis was performed.

[0084] Figure 6 PAS staining was used to assess the degree of goblet cell metaplasia in the bronchial epithelium of mouse lung tissue, with a scale bar of 100 μm. PAS staining results showed that the OVA group mice had a large number of goblet cell metaplasia in the bronchial epithelium. This pathological change is one of the characteristic histological changes of asthma. In the CANA group, this goblet cell metaplasia was significantly reversed.

[0085] Statistical analysis of PAS staining was performed to quantify the goblet cell metaplasia rate (the ratio of PAS-stained area to the total area of ​​the entire field of view). Figure 7 The statistical analysis of goblet cell metaplasia rate shows that the goblet cell metaplasia rate in the OVA group was significantly higher than that in the control group, while in the CANA group, this rate decreased (P<0.0001, P<0.001).

[0086] 2.2 Real-time quantitative PCR was used to detect the mRNA expression levels of Arg2, Foxa3 and Foxm1 in lung tissue.

[0087] Figure 8 The results are RT-qPCR. The figure shows that the mRNA expression levels of goblet cell metaplasia-related genes Agr2, Foxa3, and Foxm1 were significantly upregulated in the OVA group and significantly decreased in the CANA group (P<0.0001; P<0.0001; P<0.0001; P<0.001, P<0.01, P<0.01).

[0088] 2.3 Immunohistochemical Detection of Lung Tissue Sections: Add antigen retrieval solution to lung tissue sections, place the sections in a microwave oven, microwave on medium heat for 8 min, turn off for 7 min, and microwave on low heat for 8 min; remove the staining chamber and cool to room temperature. Wash three times with PBS, 5 min each time. Incubate with 3% H2O2 at room temperature for 10 min (to inactivate endogenous peroxidase). Wash twice with PBS, 5 min each time. Add blocking solution to the tissue sections, ensuring even coverage of the entire tissue area. Place the sections in a humidified chamber and incubate at 37°C for 30 min. Add diluted primary antibody (Muc5ac 1:100) and incubate overnight at 4°C in a humidified chamber. Add HRP-labeled secondary antibody and incubate at room temperature for 1 hour. Wash three times with PBS, 5 min each time (to thoroughly remove unbound secondary antibody). Add DAB working solution (freshly prepared), control the staining time under a microscope, and rinse with running water after staining to stop the reaction. Stain the nuclei with hematoxylin for 1 min, allow differentiation solution to turn blue, dehydrate with graded alcohols, and mount with neutral resin. After completing all staining steps, tissue sections typically need to be dehydrated to remove excess water. Sections are sequentially immersed in 70%, 85%, and 95% ethanol, anhydrous ethanol I, and anhydrous ethanol II for 1 minute each. After dehydration, they are cleared twice with xylene for 1 minute each time to ensure the mounting medium is evenly distributed on the section. A drop of neutral resin is placed on the tissue section, and then gently covered with a coverslip, ensuring no air bubbles are formed and that the coverslip adheres tightly to the section. The mounting medium is allowed to air dry naturally for long-term storage and immediate observation.

[0089] Figure 9 Immunohistochemical results of MUC5AC protein expression in mouse lung tissue. The results showed that MUC5AC protein expression was significantly increased in the OVA group and significantly decreased in the CANA group.

[0090] Experiment 3: To investigate the effect of CANA on reducing collagen deposition in the lung interstitium of asthmatic mice.

[0091] Masson staining: Following the HE staining method, dewaxing and hydration were performed. Hematoxylin was used to stain the nuclei for 2 min, followed by differentiation with 1% hydrochloric acid-ethanol for 1 second, and then blueing in running water for 15 min. The sections were then immersed in Ponceau S-Acid Fuchsin solution for 15 min, treated with 1% glacial acetic acid for 15 seconds, and rinsed with running water for 15 seconds. After immersing the sections in 1% phosphomolybdic-phosphotungstic acid solution for 15 min, they were immediately stained with aniline blue for 15 min, then treated with ultrapure water and 1% glacial acetic acid for 1 second, and finally mounted with neutral resin. The sections were observed under a microscope, images were acquired, and subsequent analysis was performed.

[0092] Figure 10 Masson staining was used to assess peri-airway collagen deposition in mouse lung tissue. Figure a shows an electron micrograph, with the scale bar representing 100 μm; figure b shows a statistical analysis of the collagen deposition area ratio. Masson staining results showed a significant increase in stained areas in the OVA group, indicating a marked increase in collagen fiber deposition in the lung tissue. In the CANA group, collagen fiber deposition was reduced. To further quantify the degree of collagen fiber deposition, a statistical analysis was performed on the ratio of collagen fiber area to the total field of view in the stained images. The results showed that the collagen fiber area ratio in the OVA group was significantly higher than that in the Ctrl group, while the collagen fiber area ratio in the CANA group was significantly lower than that in the OVA group (P<0.0001; P<0.01).

[0093] Experiment 4: Investigate how CANA inhibits the activation of the PI3K / AKT pathway in asthmatic mice.

[0094] Figure 11 The results show the levels of p-PI3K, PI3K, p-AKT, AKT, and GAPDH measured by Western blotting (WB); figure a is the WB analysis diagram. Figure b shows the quantitative analysis of p-PI3K expression; figure c shows the quantitative analysis of p-AKT expression. WB results indicated that the expression levels of p-PI3K and p-AKT were significantly increased in the lung tissue of mice in the OVA group, a phenomenon highly consistent with the key role of the PI3K / AKT pathway in asthma progression. CANA intervention significantly reversed these changes, as evidenced by a significant decrease in p-PI3K and p-AKT phosphorylation levels (a, b, c; P < 0.001; P < 0.001), suggesting that CANA may exert its effect by inhibiting PI3K / AKT pathway activation.

[0095] Experiment 5: In vivo safety assessment.

[0096] Figure 12 The concentrations of ALT, AST, CREA, and BUN in mouse serum are given.

[0097] Figure 13 H&E staining images of the heart, liver, spleen, lungs, and kidneys of mice in each group.

[0098] The results showed that the indicators in all treatment groups were within the normal range. In addition, the physiological structures of the heart, liver, spleen, lungs and kidneys of the mice in each group were intact, and no obvious pathological abnormalities were observed.

[0099] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention using the methods and techniques disclosed above, or modify them into equivalent embodiments with equivalent changes, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solutions of the present invention shall still fall within the protection scope of the technical solutions of the present invention.

Claims

1. A susceptibility gene for asthma, characterized in that, The susceptibility gene is SGLT2.

2. A drug for treating asthma, characterized in that, The drug in question is canagliflozin.

3. A drug for treating asthma, characterized in that, The drug in question is canagliflozin aerosol.

4. The drug according to claim 3, characterized in that, The preparation method of the canagliflozin aerosol includes: S1. After ultra-fine pulverization of canagliflozin, it is added to the base solution for emulsification and mixing, and then ultrasonically dispersed to obtain the drug solution. S2. Cool the liquid medicine to 8°C, inject it into the aerosol can, introduce a propellant, control the pressure at 1.0 MPa, and seal it by laser welding to obtain canagliflozin aerosol. The base solution comprises 50 g ethanol, 30 g deionized water, 2 g Tween-80, and 0.5 g citric acid.

5. The use of any one of claims 2 to 4 in the preparation of a medicament for treating asthma.

6. Use of any one of claims 2 to 4 in the preparation of a drug for reducing peri-airway inflammatory cell infiltration and Th2 inflammatory response.

7. Use of any one of claims 2 to 4 in the preparation of a medicament for reducing the degree of goblet cell metaplasia in bronchial epithelium of lung tissue.

8. The use of any one of claims 2 to 4 in the preparation of a drug for reducing collagen deposition in the pulmonary interstitium.

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