A method for constructing a combined induction and exacerbation asthma animal model

Abstract

The application discloses a kind of construction methods of combined induction aggravation asthma animal model.The method includes: abdominal cavity injection egg white protein or house dust mite extract is sensitized;Allergen is inhaled by atomization and is exposed to airway;In aggravation stage, give pseudomonas aeruginosa, lung fumagine or respiratory syncytial virus and expose to 500 μg / m 3 PM2.5 aerosol;Subsequently, lung tissue and bronchoalveolar lavage fluid are detected.The model shows that airway resistance is significantly enhanced and continues, the rate of mucus plug formation is greater than 50%, smooth muscle is significantly thickened, accompanied by eosinophil and neutrophil infiltration and high expression of various inflammatory factors.The model of the application has good stability, strong repeatability, can truly simulate clinical asthma acute exacerbation and steroid-insensitive characteristics, and provides a platform for asthma mechanism research and new drug screening.

Description

Technical Field

[0001] This invention relates to the technical field of medicine, and in particular to a method for constructing a combined induced asthma exacerbation animal model. Background Technology

[0002] Asthma is a chronic inflammatory airway disease involving multiple inflammatory cells and mediators. Its clinical manifestations include recurrent episodes of wheezing, shortness of breath, chest tightness, and cough. Some patients are prone to asthma exacerbations after exposure to factors such as viral infections, air pollution, or allergen re-exposure, characterized by escalating inflammation, increased airway hyperresponsiveness, and decreased sensitivity to glucocorticoid therapy.

[0003] Currently, most commonly used animal models of asthma use a single allergen (such as ovalbumin or house dust mite) for sensitization and provocation, which can simulate the basic inflammatory state of asthma, but it is difficult to truly reproduce the acute exacerbation situation under the combined effect of multiple factors in clinical practice. These traditional models have the following shortcomings: (1) They can only induce typical Th2-type inflammatory responses and lack neutrophil inflammation and Th17 responses associated with clinical severe asthma; (2) The models have poor stability and short duration of exacerbation; (3) They lack typical pathological features of severe asthma such as excessive airway mucus secretion and smooth muscle thickening.

[0004] Therefore, there is an urgent need for an animal model that can comprehensively simulate the effects of multiple factors such as allergens, pathogenic microbial infections, and environmental pollution, and can induce typical pathological manifestations of severe clinical asthma, in order to meet the needs of asthma exacerbation mechanism research and new drug screening. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a method for constructing a combined induced asthma exacerbation animal model.

[0006] A method for constructing a combined induced asthma exacerbation animal model includes the following steps:

[0007] (1) Sensitization stage: Experimental animals were selected and sensitized by intraperitoneal injection of allergen solution on day 1 and day 14;

[0008] (2) Airway provocation phase: After sensitization, the airway is re-exposed by nebulized inhalation of the allergen solution from day 21 to day 27.

[0009] (3) Aggravation induction phase: On days 24 to 27, pathogenic microorganisms were administered into the airway, and PM2.5 aerosol exposure was carried out daily.

[0010] (4) Model evaluation stage: On day 28, lung tissue, bronchoalveolar lavage fluid and serum were collected for testing.

[0011] Preferably, the allergen is ovalbumin or house dust mite extract.

[0012] Preferably, the pathogenic microorganism is selected from one or more of bacteria, fungi, or viruses.

[0013] Preferably, the pathogenic microorganism is selected from one or more of Pseudomonas aeruginosa, Aspergillus pulmonaryis, or respiratory syncytial virus.

[0014] Preferably, the concentration of the PM2.5 aerosol is 500 μg / m³. 3 The exposure time was 20 minutes.

[0015] Preferably, the airway hyperresponsiveness of the model is characterized by an airway resistance apnea value (Penh) greater than 5.0 after methacholine stimulation.

[0016] Preferably, the pathological characteristics of the model include a bronchial mucus plug formation rate of greater than 50% and a smooth muscle thickness to basement membrane perimeter ratio of not less than 0.12.

[0017] Preferably, the detection indicators include the differential count of eosinophils, neutrophils and macrophages in bronchoalveolar lavage fluid, and the mRNA or protein expression levels of IL-4, IL-5, IL-13, IL-17A and TNF-α in lung tissue.

[0018] Compared with the prior art, the beneficial effects of this application are mainly reflected in the following aspects:

[0019] First, this application employs a combined induction method involving allergens, pathogenic microorganisms, and PM2.5, rather than a single or two-factor combination. Comparative experimental results show that the traditional OVA model alone mainly exhibits a typical Th2 inflammatory response. While the OVA+Af or OVA+PM2.5 models may show some degree of neutrophilic inflammation or airway mucus secretion, the inflammatory type is incomplete, airway remodeling is insufficient, and the duration is limited. In contrast, the three-factor combined model of this invention can stably induce a comprehensive pathological phenotype, including mixed Th2 / Th17 inflammation, dual infiltration of eosinophils and neutrophils, an airway mucus plug formation rate exceeding 50%, and significant smooth muscle thickening, demonstrating a qualitative difference rather than a quantitative additive effect.

[0020] Secondly, regarding pharmacological response, the model in this application showed significant insensitivity to glucocorticoids. Experimental results showed that after dexamethasone intervention, the expression levels of IL-17A and TNF-α decreased by more than 40% in the OVA or two-factor model, while the decrease was less than 20% in the three-factor model, suggesting that this model can reproduce the typical characteristics of clinically steroid-resistant asthma. This is something that existing univariate or two-factor models cannot stably achieve.

[0021] Furthermore, the stability and reproducibility of the model in this invention are significantly improved. Statistical data shows that over 95% of the mice modeled using the three-factor combination in the examples exhibited consistent pathological and inflammatory phenotypes, while this proportion was only about 40%–50% in the two-factor model. Therefore, this invention not only shortens the modeling cycle but also enables the acquisition of consistent and controllable severe asthma phenotypes in different experimental animals.

[0022] Finally, the simulated trigger combination (allergen exposure + respiratory infection + air pollution) in this invention highly matches the real-world scenario of acute asthma exacerbations, making the pathological phenotype obtained from the model more closely correlated with human diseases. This model not only helps to deeply analyze the pathogenesis of acute asthma exacerbations and steroid resistance, but can also be widely applied to the pharmacodynamic evaluation and efficacy verification of novel anti-inflammatory drugs, immunomodulators, and cell therapy methods, providing a reliable platform for clinical translational research. Attached Figure Description

[0023] Figure 1 The flowchart shows the construction process of the combined induced exacerbation asthma animal model of Example 1 and Comparative Examples 1-4;

[0024] Figure 2 The graph shows the total number of white blood cells in BALF in Example 1, Comparative Example 1, and Comparative Example 2.

[0025] Figure 3 The graph shows the number of neutrophils in BALF in Example 1, Comparative Example 1, and Comparative Example 2.

[0026] Figure 4 This is a statistical graph showing the number of eosinophils in BALF from Example 1, Comparative Example 1, and Comparative Example 2.

[0027] Figure 5 This is a statistical chart of the number of macrophages in BALF of Example 1, Comparative Example 1, and Comparative Example 2;

[0028] Figure 6 The curves showing the changes in airway hyperresponsiveness (Penh value) in mice after methacholine stimulation in Examples 1, 1, and 2 are shown.

[0029] Figure 7The graph shows the statistical levels of IL-4 expression in the lung tissues of Example 1, Comparative Example 1, and Comparative Example 2.

[0030] Figure 8 Statistical graphs of IL-5 expression levels in lung tissues of Example 1, Comparative Example 1, and Comparative Example 2;

[0031] Figure 9 This is a statistical graph showing the expression levels of IL-13 in the lung tissues of Example 1, Comparative Example 1, and Comparative Example 2.

[0032] Figure 10 Graph showing the expression levels of IL-17A in lung tissues of Example 1, Comparative Example 1, and Comparative Example 2;

[0033] Figure 11 PAS staining image of lung tissue in Comparative Example 1;

[0034] Figure 12 PAS staining image of lung tissue in Comparative Example 2;

[0035] Figure 13 PAS staining image of lung tissue from Example 1;

[0036] Figure 14 This is a statistical graph showing the positive rate of airway epithelial goblet cells in Example 1, Comparative Example 1, and Comparative Example 2. Detailed Implementation

[0037] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0038] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0039] The animal experimental models involved in this invention were constructed in accordance with the Animal Protection Law of the People's Republic of China and the Regulations on the Administration of Laboratory Animals, among other relevant laws and regulations. All experiments were conducted after review and approval by the ethics committee and in accordance with the relevant regulations on the use and management of laboratory animals.

[0040] The present invention will now be described in further detail with reference to the accompanying drawings:

[0041] Reference Figure 1 The modeling flowchart is set up with the following examples and comparative examples:

[0042] Example 1: This example discloses a method for constructing a combined induced exacerbation asthma animal model, including the following steps:

[0043] Female BALB / c mice aged 6–8 weeks were selected, with 10 mice per group, and housed in an SPF-grade animal facility at a temperature of 22±2℃ and humidity of 50%–60%, with free access to food and water. The modeling steps are as follows:

[0044] (1) Sensitization phase: Mice were sensitized on day 1 and day 14 by intraperitoneal injection of a mixture of ovalbumin (OVA, 100 μg / mouse, dissolved in 200 μL PBS) and aluminum hydroxide adjuvant (2 mg / mouse). The injection volume was 200 μL / mouse.

[0045] (2) Airway provocation phase: After sensitization, mice were given 1% OVA solution nebulized by a vibrating mesh nebulizer daily from day 21 to day 27. The median mass diameter of the nebulized particles was 2.0 μm, the geometric standard deviation was 2.0, the nebulization output rate was 0.3 mL / min, and the exposure was 25 minutes at a temperature of 22℃ and a relative humidity of 50%, once a day.

[0046] (3) Intensive induction phase: On day 24, under mild anesthesia, a suspension of Aspergillus fumigatus ATCC204305 spores (1×10⁻⁶) was administered via pharyngeal drip. 6 Spores / animal, 50 μL / animal), simulating fungal infection; and from day 24 to 27, daily exposure to PM2.5 aerosols (derived from environmental particulate matter collected using a high-flow-rate air sampler in urban Hangzhou, filtered by ultrasonic elution, lyophilized, and fractionated to obtain particles ≤2.5 μm) at a concentration of 500 μg / m³. 3 The exposure time was 20 minutes, which caused a partial overlap between pathogen infection and contamination exposure on day 24.

[0047] (4) Model evaluation stage: Mice were sacrificed on day 28, and bronchoalveolar lavage fluid (BALF), lung tissue and serum were collected. BALF eosinophils, neutrophils and macrophages were classified and counted. Airway hyperresponsiveness was detected after acetylcholine provocation, and the airway resistance apnea value (Penh) was greater than 5.0. The mRNA or protein levels of IL-4, IL-5, IL-13, IL-17A and TNF-α in lung tissue were detected. PAS staining was used to observe the mucus plug formation rate in the bronchial lumen and the airway smooth muscle thickness / basement membrane perimeter ratio.

[0048] Example 2: This example discloses a method for constructing a combined induced exacerbation asthma animal model, including the following steps:

[0049] Female BALB / c mice aged 6–8 weeks were selected, with 10 mice per group, and housed in an SPF-grade animal facility at a temperature of 22±2℃ and humidity of 50%–60%, with free access to food and water. The modeling steps are as follows:

[0050] (1) Sensitization phase: Mice were sensitized on day 1 and day 14 by intraperitoneal injection of house dust mite extract solution (1 mg / mL, total protein, 200 μL / mouse) combined with aluminum hydroxide adjuvant (2 mg / mouse).

[0051] (2) Airway provocation phase: From day 21 to 27, mice were given a nebulizer with a vibrating mesh to nebulize HDM solution (1 mg / mL) daily. The median diameter of the nebulized particles was 2.0 μm, the geometric standard deviation was 2.0, the output rate was 0.3 mL / min, and the mice were exposed to 22℃ and 50% humidity for 25 minutes once a day.

[0052] (3) Intensive induction phase: On day 24, under mild anesthesia, a suspension of Pseudomonas aeruginosa strain ATCC27853 (1×10⁻⁶) was administered via intranasal instillation. 7 CFU / animal, 50 μL / animal; and PM2.5 aerosol exposure (500 μg / m³) was administered daily after HDM nebulization from day 24 to 27. 3 (20 minutes).

[0053] (4) Model evaluation phase: Mice were sacrificed on day 28, and BALF, lung tissue, and serum were collected. The results showed:

[0054] The total cell count, neutrophils, and eosinophils in BALF were all significantly increased;

[0055] Penh value greater than 5.0;

[0056] The levels of IL-4, IL-17A, and TNF-α in lung tissue were significantly increased, indicating mixed-type inflammation.

[0057] HE staining revealed significant airway inflammatory cell infiltration, while PAS staining showed markedly increased mucus secretion and thickened smooth muscle.

[0058] Example 3: This example discloses a method for constructing a combined induced exacerbation asthma animal model, including the following steps:

[0059] Female BALB / c mice aged 6–8 weeks were selected, with 10 mice per group, and housed in an SPF-grade animal facility at a temperature of 22±2℃ and humidity of 50%–60%, with free access to food and water. The modeling steps are as follows:

[0060] (1) Sensitization stage: Mice were sensitized on day 1 and day 14 by intraperitoneal injection of 1% OVA solution (200 μL / mouse) combined with aluminum hydroxide adjuvant (2 mg / mouse).

[0061] (2) Airway stimulation phase: From day 21 to day 27, mice were given a 1% OVA solution via a vibrating mesh nebulizer. The median diameter of the nebulized particles was 2.0 μm, the geometric standard deviation was 2.0, and the output rate was 0.3 mL / min. The mice were exposed to 22℃ and 50% humidity for 25 minutes once a day.

[0062] (3) Severity induction phase: On day 24, respiratory syncytial virus ATCCVR-26 strain suspension (1×10⁻⁶) was administered via intranasal instillation under mild anesthesia. 6 TCID 50 / animal, 50 μL / animal); and from day 24 to 27, PM2.5 aerosol exposure (500 μg / m³) was administered daily after OVA nebulization. 3 (20 minutes).

[0063] (4) Model evaluation phase: Mice were sacrificed on day 28, and BALF, lung tissue, and serum were collected. The results showed:

[0064] The number of neutrophils was significantly increased in BALF, and the number of eosinophils was also elevated;

[0065] Penh value greater than 5.0;

[0066] Significantly elevated expression of IL-17A and TNF-α indicates a steroid-resistant phenotype;

[0067] PAS staining showed that the airway mucus plug formation rate was about 55%, indicating a high degree of airway remodeling.

[0068] Comparative Example 1: Control Group

[0069] (1) Sensitization phase: Mice were injected intraperitoneally with an equal volume of PBS solution (200 μL / mouse) on day 1 and day 14, without ovalbumin sensitization.

[0070] (2) Airway stimulation phase: From day 21 to day 27, PBS solution was nebulized daily using a vibrating mesh nebulizer with a median mass diameter of 2 μm, a geometric standard deviation of 2.0, and an output rate of 0.3 mL / min. The exposure was carried out at 22°C and 50% humidity for 25 minutes.

[0071] (3) Intensification induction phase: On days 24 to 27, no pathogenic microorganism infusion or PM2.5 exposure is performed, only air exposure is performed.

[0072] (4) Model evaluation stage: Mice were sacrificed on day 28, and BALF, lung tissue and serum were collected. The results showed that all the test indicators were within the normal range, and no obvious inflammatory response and pathological changes were observed.

[0073] Comparative Example 2: OVA Group

[0074] (1) Sensitization stage: Mice were sensitized by intraperitoneal injection of 1% OVA solution (200 μL / mouse) on day 1 and day 14, combined with aluminum hydroxide adjuvant (2 mg / mouse).

[0075] (2) Airway stimulation phase: From day 21 to day 27, a 1% OVA solution was atomized daily using a vibrating mesh nebulizer. The median diameter of the atomized particles was 2 μm, the geometric standard deviation was 2.0, the output rate was 0.3 mL / min, and the exposure was carried out at 22℃ and 50% humidity for 25 minutes.

[0076] (3) Intensification induction phase: No pathogenic microorganism infusion or PM2.5 exposure is performed on days 24 to 27.

[0077] (4) Model evaluation stage: Mice were sacrificed on day 28, and BALF, lung tissue and serum were collected. The results showed that the proportion of eosinophils was increased, and the expression of IL-4, IL-5 and IL-13 was significantly increased, showing typical Th2-type inflammatory characteristics, but no obvious neutrophil inflammation or airway remodeling was observed.

[0078] Comparative Example 3: OVA + Af group

[0079] (1) Sensitization stage: Mice were sensitized by intraperitoneal injection of 1% OVA solution (200 μL / mouse) on day 1 and day 14, combined with aluminum hydroxide adjuvant (2 mg / mouse).

[0080] (2) Airway stimulation phase: From day 21 to day 27, a 1% OVA solution was atomized daily using a vibrating mesh nebulizer. The median diameter of the atomized particles was 2 μm, the geometric standard deviation was 2.0, the output rate was 0.3 mL / min, and the exposure was carried out at 22℃ and 50% humidity for 25 minutes.

[0081] (3) Intensive induction phase: On day 24, spore suspension of Aspergillus fumigatus strain ATCC204305 (1×10^6 spores / animal, 50μL / animal) was administered via pharyngeal drip under mild anesthesia; no PM2.5 exposure was performed.

[0082] (4) Model evaluation phase: Mice were sacrificed on day 28, and BALF, lung tissue and serum were collected. The results showed that the number of neutrophils increased and the expression level of IL-17 was increased, but the degree of airway smooth muscle thickening and mucus secretion was lower than that in Example 1.

[0083] Comparative Example 4: OVA + PM2.5 Group

[0084] (1) Sensitization stage: Mice were sensitized by intraperitoneal injection of 1% OVA solution (200 μL / mouse) on day 1 and day 14, combined with aluminum hydroxide adjuvant (2 mg / mouse).

[0085] (2) Airway stimulation phase: From day 21 to day 27, a 1% OVA solution was atomized daily using a vibrating mesh nebulizer. The median diameter of the atomized particles was 2 μm, the geometric standard deviation was 2.0, the output rate was 0.3 mL / min, and the exposure was carried out at 22℃ and 50% humidity for 25 minutes.

[0086] (3) Intensification induction phase: On days 24–27, after daily OVA nebulization, the patient was placed in an exposure device to inhale PM2.5 aerosol (500 μg / m³). 3 (20 minutes); do not administer pathogenic microorganism infusions.

[0087] (4) Model evaluation phase: Mice were sacrificed on day 28, and BALF, lung tissue and serum were collected. The results showed an increase in the total number of inflammatory cells and the proportion of eosinophils, and mild airway mucus secretion, but the proportion of neutrophils and IL-17 expression were lower than in Example 1, and airway remodeling was not obvious.

[0088] Detection methods

[0089] 1. BALF inflammatory cell differential count

[0090] After euthanizing mice, bronchoalveolar lavage fluid was collected, washed with PBS, and centrifuged to collect cells. Dead cells were removed from the cell suspension by trypan blue staining, and the total cell count was determined. The cells were then prepared, stained with Wright-Giemsa, and neutrophils, eosinophils, macrophages, etc., were counted under a microscope, and their proportions and absolute numbers were calculated. The test results are as follows: Figures 2-5 As shown.

[0091] Conclusion: Compared with the blank control group (Comparative Example 1), the total number of white blood cells, neutrophils, eosinophils and macrophages in BALF of each model group were significantly increased (p < 0.001); compared with the OVA alone model group (Comparative Example 2), the total number of white blood cells, neutrophils, eosinophils and macrophages in BALF of the OVA+Af+PM2.5 combined model group (Example 1) were significantly increased (p < 0.001).

[0092] 2. Airway hyperresponsiveness (AHR) detection

[0093] Mice were placed in a non-invasive respiratory monitoring system and inhaled different concentrations of methacholine nebulized solution in stages. Changes in the airway resistance index, Penh, were recorded. Dose-response curves were used to compare the differences in airway responsiveness among the groups of mice. The test results are as follows: Figure 6 As shown.

[0094] Conclusion: Compared with the blank control group, the airway resistance Penh value of each model group was significantly increased after acetylcholine (Mch) stimulation (p<0.001); compared with the OVA alone model group, the airway resistance Penh value of the OVA+Af+PM2.5 combined model group was significantly increased (p<0.001).

[0095] 3. Detection of inflammatory factors in lung tissue

[0096] After euthanizing the animals, lung tissue was harvested. A portion was used for RNA extraction and qPCR detection of inflammatory cytokine (IL-4, IL-5, IL-13, IL-17A, etc.) mRNA levels; the other portion was homogenized, and ELISA kits were used to detect the concentrations of corresponding inflammatory cytokine proteins. The test results are as follows: Figures 7-10 As shown.

[0097] Conclusion: Compared with the blank control group, the expression levels of inflammatory factors mRNA such as IL-4, IL-5, IL-13, and IL-17A in the lung tissue of each model group were significantly increased (p < 0.001); compared with the OVA alone model group, the expression levels of inflammatory factors mRNA such as IL-4, IL-5, IL-13, and IL-17A in the lung tissue of the OVA+Af+PM2.5 combined model group were significantly increased (p < 0.001).

[0098] 4. PAS staining of lung tissue

[0099] Lung tissue was fixed, embedded in paraffin, sectioned, and stained with PAS. After staining, goblet cell metaplasia and mucus secretion in the airway epithelium were observed under a microscope. The degree of airway mucus secretion was reflected by counting the number of positive cells or the stained area. The test results are as follows: Figures 11-14 As shown.

[0100] Conclusions: Compared with the Control group, mice in all model groups showed extensive eosinophil infiltration in the airways and perivascular areas of the lung tissue, as well as abundant goblet epithelial cell metaplasia and mucus secretion within the airway lumen (p < 0.001). Compared with the OVA-only model group, the OVA+Af+PM2.5 combined model group showed significantly increased airway smooth muscle thickness and mucus secretion in the lung tissue (p < 0.001).

[0101] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing a combined induction-aggravated asthma animal model, characterized in that, The method comprises the following steps: (1) a sensitization stage: selecting experimental animals, and sensitizing the animals by intraperitoneal injection of an allergen solution on day 1 and day 14; the allergen is ovalbumin, which is prepared at a concentration of 1-3% (w / v) in a saline solution, or is a house dust mite extract, which is prepared at a concentration of 0.3-3 mg / mL in terms of total protein; the experimental animals are mice; (2) an airway challenge stage: after the sensitization, the experimental animals are exposed to an allergen solution atomized by a vibrating mesh nebulizer through nasal targeting from day 21 to day 27, the mass median diameter of the atomized particles is 1-3 μm, the geometric standard deviation is 1.8-2.5, the atomization output rate is 0.2-0.4 mL / min, the exposure conditions are a temperature of 20-24 ℃ and a relative humidity of 40-60%, and the exposure time is 20-30 minutes, once a day; (3) a challenge induction stage: on day 24, a pathogenic microorganism is instilled into the airway, and PM2.5 aerosol exposure is performed every day from day 24 to day 27, so that the pathogenic infection and the pollution exposure partially overlap on day 24; the pathogenic microorganism is selected from one or more of Pseudomonas aeruginosa, Aspergillus fumigatus, and respiratory syncytial virus; (4) a model evaluation stage: on day 28, lung tissue, bronchoalveolar lavage fluid, and serum are collected for detection.

2. The method of constructing a combined induction-aggravated asthma animal model according to claim 1, wherein The pathogenic microorganism is selected from one of the following: (1) P. aeruginosa ATCC 27853 strain was used, which was cultured and administered at a dose of 1 x 10 7 CFU / each by intranasal instillation; (2) Aspergillus fumigatus ATCC 204305 was used, and after culture, 1 x 10 6 spores / animal were administered by intranasal instillation; (3) Respiratory syncytial virus was used at a dose of 1 x 10 5 TCID 50 / animal, administered intranasally, using the ATCC VR-26 strain, which was expanded prior to use. 6 TCID 50 / animal, administered intranasally, using the ATCC VR-26 strain, which was expanded prior to use.

3. The method of constructing a combined induction-aggravated asthma animal model according to claim 1, wherein The concentration of the PM2.5 aerosol was 500 μg / m 3 with an exposure time of 20 minutes.

4. The method of constructing a combined induction-aggravated asthma animal model according to claim 1, wherein The airway hyperresponsiveness of the model is characterized in that the enhanced pause value of airway resistance after acetylcholine challenge is greater than 5.

0.

5. The method of constructing a combined induction-aggravated asthma animal model according to claim 1, wherein The pathological features of the model include a mucus plug formation rate in the bronchial lumen of greater than 50% and a ratio of smooth muscle thickness to basement membrane circumference of not less than 0.

12.

6. The method of constructing a combined induction-aggravated asthma animal model according to claim 1, wherein The detection indexes include the differential count of eosinophils, neutrophils, and macrophages in the bronchoalveolar lavage fluid, and the mRNA or protein expression levels of IL-4, IL-5, IL-13, IL-17A, and TNF-α in the lung tissue.

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