阅读说明：本技术 一种用于比格犬哮喘模型造模的组合物和哮喘模型造模方法 (Composition for beagle asthma model modeling and asthma model modeling method ) 是由 李时悦 陈迪非 罗钰龙 苏柱泉 姚利红 刘明 刘经纬 于 2021-06-25 设计创作，主要内容包括：本发明公开了一种基于豚草抗原及屋尘螨抗原构建比格犬哮喘动物模型的方法。本发明提供了一种含有豚草粉抗原提取液和屋尘螨抗原提取液的用于比格犬哮喘模型造模的组合物。并基于该组合物构建了雾化结合注射的比格犬哮喘模型构建方法。本发明的造模方法相对容易操作、造模成功率高。更重要的是本方法构建的动物模型,其哮喘症状明显且可长期维持,具有非常好的优势和应用价值。(The invention discloses a method for constructing a beagle asthma animal model based on ragweed antigen and house dust mite antigen. The invention provides a composition containing a grass powder antigen extracting solution and a dermatophagoides pteronyssinus antigen extracting solution and used for beagle dog asthma model modeling. And a construction method of a beagle asthma model by combining atomization and injection is constructed on the basis of the composition. The molding method of the invention is relatively easy to operate and has high molding success rate. More importantly, the animal model constructed by the method has obvious asthma symptoms and can be maintained for a long time, and has very good advantages and application values.)

1. A composition for beagle asthma model modeling is characterized by comprising a ragweed pollen antigen extracting solution and a dust mite antigen extracting solution, wherein the total protein concentration of the ragweed pollen antigen extracting solution is not lower than 0.1mg/mL, and the total protein concentration of the dust mite antigen extracting solution is not lower than 0.05 mg/mL.

2. The composition of claim 1, wherein the ragweed pollen antigen extract has a total protein concentration of not less than 0.5mg/mL and the dust mite antigen extract has a total protein concentration of not less than 0.1 mg/mL.

3. A kit for constructing a beagle asthma model, comprising:

(1) antigen solution for injection: the injection comprises ragweed pollen antigen extracting solution, dust mite antigen extracting solution and an alum adjuvant, wherein the working concentration of total protein of the ragweed pollen antigen extracting solution is 0.25-3 mg/mL, the working concentration of total protein of the dust mite antigen extracting solution is 0.08-0.12 mg/mL, and the alum adjuvant accounts for 30-50% of the volume ratio of the antigen solution for injection;

(2) ragweed pollen atomized liquid: the ragweed pollen antigen extracting solution is contained, and the working concentration of total protein is 0.25-5 mg/mL;

(3) mixing atomized liquid: the ragweed pollen antigen extract liquid comprises a ragweed pollen antigen extract liquid and a dust mite antigen extract liquid, wherein the total protein working concentration of the ragweed pollen antigen extract liquid is 0.25-5 mg/mL; the working concentration of the total protein of the dust mite antigen extracting solution is 0.08-0.15 mg/mL.

4. The kit according to claim 3, wherein the antigen solution for injection comprises both an antigen solution for intraperitoneal injection and an antigen solution for subcutaneous injection,

wherein, in the antigen liquid for intraperitoneal injection: the working concentration of total protein of the ragweed pollen antigen extracting solution is 2-3 mg/mL, the working concentration of total protein of the dust mite antigen extracting solution is 0.08-0.2 mg/mL, and the alum adjuvant accounts for 30-50% of the volume ratio of the antigen solution for intraperitoneal injection;

in the antigen liquid for subcutaneous injection, the working concentration of total protein of ragweed pollen antigen extract is 0.4-0.6 mg/mL, the working concentration of total protein of dust mite antigen extract is 0.08-0.2 mg/mL, and alum adjuvant accounts for 30-50% of the antigen liquid for subcutaneous injection in volume ratio.

5. The kit of claim 3, wherein the ragweed pollen atomized liquid has a total protein working concentration of the ragweed pollen antigen extract of 2-3 mg/mL.

6. The kit of claim 3, wherein the working concentration of total protein of the ragweed pollen antigen extract in the mixed atomized liquid is 2-3 mg/mL; the working concentration of the total protein of the dust mite antigen extracting solution is 0.08-0.15 mg/mL.

7. The kit of any one of claims 3 to 6, comprising:

(1) antigen liquid for intraperitoneal injection: the injection comprises ragweed pollen antigen extracting solution, dust mite antigen extracting solution and an alum adjuvant, wherein the working concentration of total protein of the ragweed pollen antigen extracting solution is 2-3 mg/mL, the working concentration of total protein of the dust mite antigen extracting solution is 0.08-0.2 mg/mL, and the alum adjuvant accounts for 30-50% of the volume ratio of the antigen solution for injection;

(2) antigen liquid for subcutaneous injection: the antigen solution comprises ragweed pollen antigen extracting solution, dust mite antigen extracting solution and alum adjuvant, wherein the working concentration of total protein of the ragweed pollen antigen extracting solution is 0.4-0.6 mg/mL, the working concentration of total protein of the dust mite antigen extracting solution is 0.08-0.2 mg/mL, and the alum adjuvant accounts for 30-50% of the volume ratio of the antigen solution for injection;

(3) ragweed pollen atomized liquid: the ragweed pollen antigen extract comprises ragweed pollen antigen extract and normal saline, wherein the total protein working concentration of the ragweed pollen antigen extract is 2-3 mg/mL.

(4) Mixing atomized liquid: the ragweed pollen antigen extract liquid comprises ragweed pollen antigen extract liquid, dust mite antigen extract liquid and normal saline, wherein the total protein concentration of the ragweed pollen antigen extract liquid is 2-3 mg/mL; the total protein concentration of the dust mite antigen extracting solution is 0.08-0.15 mg/mL.

8. A method for constructing a beagle asthma model is characterized by comprising the following steps:

(1) intraperitoneally injecting the antigen liquid for intraperitoneal injection according to any one of claims 4 to 5 within 24 hours after birth of a beagle dog;

(2) subcutaneously injecting the antigen solution for subcutaneous injection according to any one of claims 4 to 5 once every 1, 4 or 8 weeks after birth of beagle dogs;

(3) meanwhile, atomizing and inhaling the beagle dog by using the ragweed pollen atomized liquid as claimed in any one of claims 3-7 2-7 times per week during 1-12 weeks after the birth of the beagle dog;

and simultaneously, replacing the ragweed pollen atomized liquid with the mixed atomized liquid of any one of claims 3-7 once during the 1 st, 2 nd, 3 rd, 4 th, 8 th and 12 th weeks;

(4) nebulizing beagle dogs for inhalation once a week using the ragweed pollen nebulization solution of any one of claims 3-7 during 13-36 weeks after birth of the beagle dogs.

9. The method of claim 8, wherein the intraperitoneal injection dosage of the antigen solution for intraperitoneal injection in the step (1) is 150 to 250 μ L; the injection dosage of the antigen liquid for subcutaneous injection in the step (2) is 80-120 mu L.

10. The method of claim 9, wherein in steps (3) and (4), the amount of the ragweed pollen atomized liquid inhaled per time is 6-8 mL, and the amount of the mixed atomized liquid inhaled per time is 6-8 mL.

Technical Field

The invention belongs to the technical field of disease animal models, and particularly relates to a composition for beagle asthma model molding and an asthma model molding method.

Background

Asthma is a common and chronic respiratory disease, and accounts for 1-18% of different countries. Allergen exposure induced airway hyperresponsiveness and chronic airway inflammation are important physiological and pathological features thereof, and thus, sensitization and priming of animals by using allergens (e.g., ovalbumin, house dust mite, ragweed pollen) is currently the most commonly used method for asthma modeling. At present, a plurality of preclinical animal models for asthma research exist, wherein small animal models established by using mice and guinea pigs have low cost and are widely applied to research of asthma pathogenesis and therapeutic drugs; the beagle dog asthma model is a large animal model established by using beagle dogs, monkeys and the like, wherein the beagle dogs are moderate in body size, fast in breeding and mild in temperament, are the most common large experimental animals, and can be induced to form typical characteristics of airway inflammatory infiltration, airway hyperresponsiveness and the like by using the ragweed pollen antigen to sensitize and excite the established beagle dog asthma model at present.

However, the existing animal asthma model has short symptom maintenance time and a self-healing trend, and is difficult to observe long-term treatment effect; and because the animal size is small, the animal is difficult to be used for preclinical research of non-drug asthma treatment technologies such as respiratory intervention and the like. Research reports (http:// dx. doi. org/10.1080/08958370304474) show that the existing biggee asthma model has the defects of low modeling success rate (50-60%), long modeling period (8 months are needed), low change amplitude of airway hyperreactivity (the airway resistance is only increased by about 1 time under the stimulation of histamine of 3 mg/ml), and correspondingly, the cost of the animal model is too high.

Disclosure of Invention

The invention aims to solve the technical problem of improving the molding success rate of the beagle asthma model. . The molding method of the invention is relatively easy to operate and has high molding success rate. The constructed animal model has obvious asthma symptoms and can be maintained for a long time.

The invention aims to provide a composition for beagle asthma model modeling.

The invention also aims to provide a kit for beagle asthma model modeling.

Another object of the present invention is to provide a method for constructing a beagle asthma model.

In order to achieve the purpose, the invention provides the following technical scheme:

a composition for modeling a beagle asthma model comprises ragweed pollen antigen extract and dust mite antigen extract, wherein the total protein concentration of the ragweed pollen antigen extract is not less than 0.1mg/mL, and the total protein concentration of the dust mite antigen extract is not less than 0.05 mg/mL.

Preferably, the total protein concentration of the ragweed pollen antigen extracting solution is not lower than 0.5mg/mL, and the total protein concentration of the dust mite antigen extracting solution is not lower than 0.1 mg/mL.

Specifically, as a preferred optional embodiment, the total protein concentration of the ragweed pollen antigen extracting solution is 0.5-10 mg/mL, and the total protein concentration of the dust mite antigen extracting solution is 0.1-5 mg/mL.

More preferably, the total protein concentration of the ragweed pollen antigen extracting solution is 0.5-5 mg/mL, and the total protein concentration of the dust mite antigen extracting solution is 0.1-1.5 mg/mL.

A kit for constructing a beagle asthma model comprising:

(1) antigen solution for injection: the injection comprises ragweed pollen antigen extracting solution, dust mite antigen extracting solution and an alum adjuvant, wherein the working concentration of total protein of the ragweed pollen antigen extracting solution is 0.25-3 mg/mL, the working concentration of total protein of the dust mite antigen extracting solution is 0.08-0.12 mg/mL, and the alum adjuvant accounts for 30-50% of the volume ratio of the antigen solution for injection;

(2) ragweed pollen atomized liquid: the ragweed pollen antigen extracting solution is contained, and the working concentration of total protein is 0.25-5 mg/mL;

(3) mixing atomized liquid: the ragweed pollen antigen extract liquid comprises a ragweed pollen antigen extract liquid and a dust mite antigen extract liquid, wherein the total protein working concentration of the ragweed pollen antigen extract liquid is 0.25-5 mg/mL; the working concentration of the total protein of the dust mite antigen extracting solution is 0.08-0.15 mg/mL.

As a specific embodiment, preferably, the antigen liquid for injection includes two types of antigen liquid for intraperitoneal injection and antigen liquid for subcutaneous injection:

wherein, in the antigen liquid for intraperitoneal injection: the working concentration of total protein of the ragweed pollen antigen extracting solution is 2-3 mg/mL, the working concentration of total protein of the dust mite antigen extracting solution is 0.08-0.2 mg/mL, and the alum adjuvant accounts for 30-50% of the volume ratio of the antigen solution for intraperitoneal injection;

in the antigen liquid for subcutaneous injection, the working concentration of total protein of ragweed pollen antigen extract is 0.4-0.6 mg/mL, the working concentration of total protein of dust mite antigen extract is 0.08-0.2 mg/mL, and alum adjuvant accounts for 30-50% of the antigen liquid for subcutaneous injection in volume ratio.

In addition, preferably, in the ragweed pollen atomized liquid, the total protein working concentration of the ragweed pollen antigen extracting solution is 2-3 mg/mL.

Preferably, in the mixed atomized liquid, the working concentration of total protein of the ragweed pollen antigen extracting solution is 2-3 mg/mL; the working concentration of the total protein of the dust mite antigen extracting solution is 0.08-0.15 mg/mL.

Particularly preferably, the kit for constructing the beagle asthma model comprises:

(1) antigen liquid for intraperitoneal injection: the injection comprises ragweed pollen antigen extracting solution, dust mite antigen extracting solution and an alum adjuvant, wherein the working concentration of total protein of the ragweed pollen antigen extracting solution is 2-3 mg/mL, the working concentration of total protein of the dust mite antigen extracting solution is 0.08-0.2 mg/mL, and the alum adjuvant accounts for 30-50% of the volume ratio of the antigen solution for injection;

(2) antigen liquid for subcutaneous injection: the antigen solution comprises ragweed pollen antigen extracting solution, dust mite antigen extracting solution and alum adjuvant, wherein the working concentration of total protein of the ragweed pollen antigen extracting solution is 0.4-0.6 mg/mL, the working concentration of total protein of the dust mite antigen extracting solution is 0.08-0.2 mg/mL, and the alum adjuvant accounts for 30-50% of the volume ratio of the antigen solution for injection;

(3) ragweed pollen atomized liquid: the ragweed pollen antigen extract comprises ragweed pollen antigen extract and normal saline, wherein the total protein working concentration of the ragweed pollen antigen extract is 2-3 mg/mL.

(4) Mixing atomized liquid: the ragweed pollen antigen extract liquid comprises ragweed pollen antigen extract liquid, dust mite antigen extract liquid and normal saline, wherein the total protein concentration of the ragweed pollen antigen extract liquid is 2-3 mg/mL; the total protein concentration of the dust mite antigen extracting solution is 0.08-0.15 mg/mL.

Most preferably, the kit for constructing a beagle asthma model comprises:

(1) antigen liquid for intraperitoneal injection: the ragweed pollen antigen extract liquid with the total protein concentration of 5mg/mL, the alum adjuvant and the dust mite antigen extract liquid with the total protein concentration of 1.2 mg/mL; and the volume ratio of the three is 6: 5: 1;

(2) antigen liquid for subcutaneous injection: consists of ragweed pollen antigen extract with total protein concentration of 1mg/mL, alum adjuvant and dust mite antigen extract with total protein concentration of 1.2 mg/mL; and the volume ratio of the three is 6: 5: 1;

(3) mixing atomized liquid: the ragweed pollen antigen extract with the total protein concentration of 5mg/mL, the dust mite antigen extract with the total protein concentration of 1mg/mL and normal saline constitute the raw materials; and the volume ratio of the three is 3.75: 0.9: 2.85;

(4) ragweed pollen atomized liquid: the ragweed pollen antigen extract comprises ragweed pollen antigen extract with total protein concentration of 5mg/mL and physiological saline, wherein the volume ratio of the ragweed pollen antigen extract to the physiological saline is 1: 1.

in addition, the invention also provides a method for constructing the beagle asthma model, which comprises the following steps:

(1) injecting the antigen solution for intraperitoneal injection into the abdominal cavity within 24 hours after birth of the beagle;

(2) injecting the antigen solution for subcutaneous injection once every 1, 4 and 8 weeks after birth of beagle dogs;

(3) meanwhile, the ragweed pollen atomized liquid is used for inhaling the beagle dogs 2-7 times per week during 1-12 weeks after the birth of the beagle dogs;

meanwhile, in the 1 st, 2 nd, 3 rd, 4 th, 8 th and 12 th weeks, the mixed atomized liquid is used for replacing ragweed pollen atomized liquid to enable the beagle dogs to be atomized and inhaled once;

(4) and (3) inhaling the beagle dog by using the ragweed pollen atomized liquid once a week during 13-36 weeks after the beagle dog is born.

Wherein, the injection dosage of the antigen liquid for intraperitoneal injection in the step (1) is 150-250 mu L; the injection dosage of the antigen liquid for subcutaneous injection in the step (2) is 80-250 muL.

Specifically, in the step (3) and the step (4), the inhalation dose of the ragweed pollen atomized liquid is 6-8 mL each time, and the inhalation dose of the mixed atomized liquid is 6-8 mL each time.

The invention has the following beneficial effects:

the invention provides a composition for modeling a beagle asthma model, and provides a modeling method constructed by using the composition, which is easy to operate. . And the asthma model has high modeling success rate and obvious asthma symptoms and can be maintained for a long time.

Drawings

Fig. 1 is a scene of atomizing antigen liquid inhaled by beagle dogs.

Figure 2 is a graph of maximum change in airway resistance-methacholine at week 36 in beagle dogs.

Figure 3 is a graph of maximum change in airway resistance versus methacholine for beagle dogs who stopped challenge for 12 weeks.

FIG. 4 is a pathological section of beagle dogs in daily nebulization group, which shows significant inflammatory cell infiltration under tracheal mucosa

FIG. 5 is a pathological section of beagle dogs in alternate-day nebulization group, which shows the proliferation of tracheal mucosa goblet cells.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Example 1 kit for constructing asthma model

1. Preparing ragweed pollen antigen extracting solution:

placing 1g of ragweed pollen and 10mL of Tris-NaCl solution in a 50mL centrifuge tube, and slowly shaking for 48 hours at the temperature of 4 ℃ by using a shaking table. After completion of shaking, the mixture was centrifuged at 1500G at 4 ℃ for 60 minutes, and the supernatant was collected. After measuring the protein concentration of the supernatant using a Bradford protein concentration measurement kit, the supernatant was diluted to a total protein concentration of 5mg/mL and dispensed at 1 mL/tube, and stored at-80 ℃.

2. Preparing a dust mite antigen extracting solution:

1g of dust mite and 20mL of PBS solution were placed in a 50mL centrifuge tube and shaken slowly at 4 ℃ for 8 hours using a shaker. After completion of shaking, the mixture was centrifuged at 6000rpm at 4 ℃ for 30 minutes, and the supernatant was collected and filtered using a 0.22 μm filter. After measuring the protein concentration of the supernatant using a Bradford protein concentration measurement kit, the supernatant was diluted to a total protein concentration of 1.2mg/mL and dispensed at 1 mL/piece, and stored at-80 ℃.

3. The kit for constructing the asthma model comprises antigen liquid for intraperitoneal injection, antigen liquid for subcutaneous injection, mixed atomized liquid and ragweed pollen atomized liquid:

(1) preparing an antigen liquid for intraperitoneal injection: placing 600 mu L of ragweed pollen antigen extract with the concentration of 5mg/mL, 500 mu L of alum adjuvant and 100 mu L of dust mite antigen extract with the concentration of 1.2mg/mL in an EP tube, fully mixing, and placing in a shaking table to shake for 30 minutes at the temperature of 40 ℃.

(2) Preparation of antigen solution for subcutaneous injection: placing 300 mul of ragweed pollen antigen extract diluted to 1mg/mL, 250 mul of alum adjuvant and 50 mul of dust mite antigen extract diluted to 1.2mg/mL into an EP tube, fully mixing, and placing in a shaking table to shake for 30 minutes at 40 ℃.

(3) Preparing mixed atomized liquid: 3.75mL of ragweed pollen antigen extract with the concentration of 5mg/mL, 0.9mL of dust mite antigen extract with the concentration of 1mg/mL and 2.85mL of normal saline are put into a centrifuge tube and mixed evenly.

(4) Preparing ragweed pollen atomized liquid: 3.75mL of ragweed pollen antigen extract with the concentration of 5mg/mL and 3.75mL of normal saline are put into a centrifuge tube and are fully and evenly mixed.

Example 2 asthma model construction

1. Experiment grouping

6 healthy newborn beagle dogs were selected and randomly divided into a normal control group, a daily nebulization group and an alternate-day nebulization group (n-2).

2. The asthma model construction time is 48 weeks, and the specific operations are as follows:

the normal control beagle dogs did not receive any treatment for this 48 week period. The daily and alternate-day nebulization groups were treated as follows:

(1) 200. mu.L of each of the intraperitoneal injection antigen solutions of example 1 was intraperitoneally injected into beagle dogs in the daily atomization group and the alternate-day atomization group within 24 hours after birth;

(2) at 1, 4, and 8 weeks after birth, 100. mu.L of each of the antigen solutions for subcutaneous injection of example 1 was subcutaneously injected to perform sensitization;

(3) meanwhile, the beagle dogs in the daily atomized group are excited by the ragweed pollen atomized liquid in the example 1 time a day during the 1 st to 12 th weeks after birth, and are atomized and inhaled for half an hour each time;

and the beagle dogs in the alternate-day nebulization group are excited by the ragweed pollen nebulization liquid for nebulization in the example 12 times a week during the 1 st to 12 th weeks after birth, and are inhaled for half an hour in each nebulization;

moreover, beagle dogs in the daily atomization group and the alternate-day atomization group are subjected to atomization inhalation by replacing ragweed pollen atomization liquid with the mixed atomization liquid of example 1 once in weeks 1, 2, 3, 4, 8 and 12;

(4) after 12 weeks, namely 13-36 weeks after birth of the beagle dogs, the beagle dogs in the daily atomization group and the alternate-day atomization group are excited once every week by using the ragweed pollen atomization liquid and are continuously aged for 36 weeks.

In the method, the conscious beagle dog is put into an atomization box with the volume of 1m by 1m during atomization activation, an atomization input port is arranged at the top of the box, and a plurality of vent holes are arranged at the periphery of the box (figure 1). The antigen liquid is atomized by using an atomizer and then filled into the box from an atomization input port, and the beagle dog in the box automatically inhales the atomized substance so as to realize antigen inhalation excitation.

After the 36-week excitation was completed in the daily and alternate-day nebulization groups, nebulization excitation was stopped for 12 weeks.

Example 3 airway inflammation test, airway reactivity test and lung pathology section test.

Collecting the bronchoalveolar lavage fluid of beagle dogs in a normal control group, a daily atomization group and an alternate-day atomization group respectively at 36 weeks after excitation and 12 weeks after excitation stopping, separating cells to prepare cell smears, carrying out HE staining, carrying out classification counting on white blood cells of BALF lavage fluid under a microscope, and calculating the proportion of macrophages, neutrophils, eosinophils and lymphocytes.

At week 36, all beagle dogs underwent a 1-way acetylcholine (Mch) challenge test to detect airway reactivity. First, the beagle dogs were anesthetized with 3% sodium pentobarbital by intramuscular injection at 0.5mL/kg + quick dormancy at 0.02 mL/kg. Fixing an animal in a supine position, then inserting an air tube and an esophagus into the trachea cannula, connecting the air flow rate sensor, a differential pressure sensor and a biopac physiological recorder, during an excitation test, sequentially atomizing and sucking a beagle dog into PBS, 0.1mg/mL, 0.3mg/mL, 1mg/mL and 3mg/mL Mch for 10 breaths respectively through the trachea cannula, immediately recording the respiratory flow rate and transpulmonary pressure (the esophageal pressure is approximate to replace the transpulmonary pressure) within 5min after the inhalation of each excitation concentration is finished, finally calculating the airway resistance by using the function of the physiological recorder with software, and representing the airway reaction intensity corresponding to the excitation concentration by using the maximum airway resistance value within 5 min.

After all the tests were completed, all dogs were euthanized and the lungs were removed for pathological section testing.

As a result: the results of the differential count of bronchoalveolar lavage fluid cells in 36 th week of beagle dogs in the normal control group, the daily nebulization group and the alternate-day nebulization group are shown in table 1, and the results of the differential count of bronchoalveolar lavage fluid cells in 12 weeks after cessation of stimulation in beagle dogs in the normal control group, the daily nebulization group and the alternate-day nebulization group are shown in table 2. The result of the bronchoalveolar lavage fluid cell classification counting shows that the beagle airway after modeling is neutrophilic granulocyte and eosinophilic granulocyte inflammation, and the asthma model obtained by the modeling method is mixed asthma.

TABLE 1 results of differential cell counts of bronchoalveolar lavage fluid at week 36

	NEU％	MAC％	EOS％	LYM％
					Normal control 1	41	58	0.25	0.75
Normal control 2	42.75	54.5	0.25	2.5
					Alternate-day atomization 1	58	35.5	3.75	2.75
Alternate-day atomization 2	75	19	3	3
					Daily atomization 1	61	29	1.5	8.5
Daily atomization 2	67.5	28	3	1.5

TABLE 2 differential counts of tracheal alveolar lavage fluid cells after 12 weeks of cessation of challenge

	NEU％	MAC％	EOS％	LYM％
					Normal control 1	43.25	56	0	0.75
Normal control 2	42.75	54	0.25	3
					Alternate-day atomization 1	55	38.5	4	2.5
Alternate-day atomization 2	42.5	36.75	8.25	12.5
					Daily atomization 1	59.5	20	15.25	5.25
Daily atomization 2	49	39.5	7.25	4.25

As can be seen from lung function detection (fig. 2 and fig. 3), compared with a normal control group, the asthma modeling beagle dogs in the daily atomization group and the alternate-day atomization group have obviously increased whole airway resistance, and simultaneously show a very obvious trend of increasing the airway resistance under the stimulation of a small dose of Mch, the airway resistance can be increased by more than 200% only through the stimulation of 0.1mg/ml Mch, and the airway responsiveness is obviously increased.

Pathological section results of lung tissues show that significant inflammatory cell infiltration phenomenon exists in the air passage tissues of beagle dogs in the daily atomization group and the alternate-day atomization group (figure 4), the inflammatory cell infiltration phenomenon is consistent with the result of alveolar lavage fluid, and meanwhile goblet cell hyperplasia is seen in the airway epithelium (figure 5).

The results show that after 36 weeks of challenge, beagle dogs in the daily and alternate-day nebulization groups showed significant signs of asthma, while the challenge was discontinued for 3 months, and asthma-related signs remained and tended to be more severe.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.