CN111820188B

CN111820188B - Establishment method and application of asthma-like slow obstructive pulmonary overlapping airway inflammation mouse model

Info

Publication number: CN111820188B
Application number: CN202010771733.0A
Authority: CN
Inventors: 曾智敏; 郭禹标; 黄鑫炎; 曾宇莹; 陈凤佳; 谭卫平
Original assignee: First Affiliated Hospital of Sun Yat Sen University
Current assignee: First Affiliated Hospital of Sun Yat Sen University
Priority date: 2020-08-04
Filing date: 2020-08-04
Publication date: 2021-10-29
Anticipated expiration: 2040-08-04
Also published as: CN111820188A

Abstract

The invention discloses a method for establishing a mouse model of asthma-like slow obstructive pulmonary overlapping airway inflammation and application thereof. The compound is constructed by any one of the following methods: s1, smoking and aspergillus protease-induced ACO-like model: the mice smoke the cigarette every day from day 0 to smoke; after smoking treatment is finished for 3-7 days, aspergillus protease solution is dripped into the nose to sensitize the mouse, and an ACO-like model is constructed; s2, nano carbon black particles and aspergillus protease-induced ACO-like model: from day 0, the mice were anesthetized and the carbon black nanoparticles were added intranasally; after the nano carbon black particles are treated for 2 weeks, aspergillus protease solution is dripped into the nose to sensitize the mouse, and the ACO-like model is constructed. The constructed similar ACO model can simulate the characteristic of Chronic Obstructive Pulmonary Disease (COPD) of asthma in the later stage of the disease of a patient suffering from Chronic Obstructive Pulmonary Disease (COPD) clinically, and provides an experimental animal model for ACO pathogenesis and drug treatment research.

Description

Establishment method and application of asthma-like slow obstructive pulmonary overlapping airway inflammation mouse model

Technical Field

The invention belongs to the field of medical biological research, and particularly relates to a method for establishing a mouse model of asthma-like slow obstructive pulmonary overlapping airway inflammation and application thereof.

Background

Asthma-COPD overlap (ACO) is a disease state clinically characterized by both Asthma and Chronic Obstructive Pulmonary Disease (COPD), with an incidence of about 20% in obstructive respiratory disease. Global asthma reports^[1]It is pointed out that ACO patients have a faster decline in lung function and more frequent exacerbations than asthma alone or COPD, and have become one of the major diseases with high global disability rate and mortality rate. However, due to the heterogeneity and uncertainty of chronic airway inflammation and the lack of knowledge of ACO, most clinical studies of asthma and COPD exclude patients with ACO characteristics. In addition, most of the current ACO clinical studies are only from defining typing, analyzing disease characteristics and researching related animal modelsThe construction of the ACO model is of great significance.

At present, no recognized and stable ACO animal model is established at home and abroad, and only Genyo Ikeda and the like^[2]Mice were induced to develop both asthma and emphysema using Ovalbumin (OVA) and trypsin (PPE). However, neither OVA nor PPE are the natural causative agents of asthma and emphysema/COPD, and this model suffers from the following disadvantages: firstly, although OVA is a classic method of an asthma model, before the OVA is dripped into a mouse to stimulate the mouse intranasally, an intra-abdominal injection OVA sensitization method is adopted to successfully model, and the method is different from the pathogenesis process that an asthma patient directly inhales air sensitization; secondly, the model of the lobular emphysema totality caused by acute injury of alveoli caused by one-time instillation of elastase is inconsistent with the pathological change of the lobular central emphysema caused by the common long-term smoking stimulation of COPD patients. Therefore, the molding method does not conform to the natural morbidity process of ACO patients, so that the application of the current ACO model is extremely limited.

Furthermore, although not a few studies have focused on the mechanism of action of smoking on asthma models, there are few animal experiments that combine the consideration of long-term inhalation of smoke and asthma allergens, mainly due to the difficult resolution of the following problems: firstly, the inflammation types of asthma and emphysema/COPD are emphasized, and the two disease characteristics are easy to disappear when the mouse inhales cigarettes and sensitizers at the same time, and the simple superposition treatment is not realized; secondly, emphysema mice are weak in constitution and easy to die due to long-term smoking, and especially, the mice are easy to shoot die when the inhaled allergen is active fungal spores; and thirdly, in the experimental process, the detection of the airway hyperreactivity requires the injection of anesthetic and invasive operation, and the mouse cannot be revived after the conventional detection is finished, so that the long-term dynamic detection cannot be carried out.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a method for establishing a mouse model of asthma-like chronic obstructive pulmonary disease overlapping airway inflammation.

The invention also aims to provide application of the establishment method of the asthma-like slow obstructive pulmonary overlapping airway inflammation mouse model.

The purpose of the invention is realized by the following technical scheme:

a method for establishing a mouse model of asthma-like Chronic Obstructive Pulmonary Disease (COPD) airway inflammation is realized by any one of the following modes:

s1, smoking (Smoke) and Aspergillus Protease (PAM) induced ACO-like models

(1) Smoking treatment: the mice inhale cigarette smoke every day from day 0 (namely the day), 4 +/-1 cigarettes every day, 5 +/-1 days every week for 4-6 months;

(2) allergen treatment: after smoking treatment is finished for 3-7 days, anesthetizing the mouse, then dropping aspergillus Protease (PAM) solution into the nose to sensitize the mouse, treating for 1 time every 1-2 days, and 8-10 times in total to construct a mouse model (ACO model) of asthma-like slow obstructive pulmonary overlapping airway inflammation;

s2, nano carbon black particles (nCB) and Aspergillus Protease (PAM) induced ACO-like model

(3) nCB treatment: from day 0, after anesthetizing the mice, dripping the nano carbon black particles (nCB) into the nose for 2-4 times per week for 4-6 weeks;

(4) allergen treatment: nCB after finishing the treatment for 2-4 weeks (preferably, beginning at week 7, namely day 43), the mice are anesthetized, then aspergillus Protease (PAM) solution is dripped into the nose to sensitize the mice, the treatment is carried out for 1 time every 1-2 days and 8-10 times in total, and a mouse model (ACO model) of asthma-like slow obstructive pulmonary overlapping airway inflammation is constructed.

The mice in the steps (1) and (3) are C57BL/6 mice; preferably a C57BL/6 female mouse; further preferably 4-8 weeks old C57BL/6 female mice; even more preferably, the C57BL/6 female mouse is 6 weeks old.

The cigarette in the step (1) is a conventional cigarette sold in the market, preferably a Wanbao Lu cigarette; more preferably 100's ten-treasure-road cigarette.

The cigarettes smoked in step (1) are about 5 minutes per cigarette, with 10 minutes intervals between cigarettes.

The smoke of the inhaled cigarette in the step (1) is preferably: 4 pieces are taken every day, 5 days every week, and the total time is 4-6 months; more preferably: 4 pieces per day, 5 days per week for 4 months.

The smoke of the cigarette in the step (1) is realized by adopting a smoking device.

The smoking device comprises an air compressor, a miniature pressure regulator, a gas flowmeter, an electromagnetic valve, a time control valve driver, a glass tube and a box;

the air compressor is connected with a gas flowmeter through a miniature pressure regulator;

the gas flowmeter is connected with the electromagnetic valve;

the electromagnetic valve is connected with a time control valve driver;

the time control valve driver is connected with the box;

the electromagnetic valve is connected with the glass tube through the oxygen flow tube;

one end of the glass tube is connected with the box;

the box is provided with an opening for the gas circulation of the box.

Preferably, the allergen treatment in the step (2) is 5-7 days after smoking treatment is finished; more preferably 6 days after the end of the smoking treatment.

The allergen treatment in the steps (2) and (4) is preferably 1 treatment every other day.

The number of times of allergen treatments described in steps (2) and (4) is preferably 8.

The anesthesia in the steps (2) and (4) is preferably performed by using isoflurane aerosol.

The aspergillus Protease (PAM) solution in the steps (2) and (4) is prepared by the following method: aspergillus protease was dissolved in PBS buffer to prepare a 0.18mg/mL Aspergillus protease solution.

The dosage of the aspergillus Protease (PAM) in the steps (2) and (4) is 9 +/-1 mu g/mouse.

The carbon black nanoparticles dropped in step (3) are preferably a mixed solution prepared by using a PBS buffer solution, and the concentration of the mixed solution is 1 mg/mL.

The dosage of the nano carbon black particles in the step (3) is 50 +/-2 mu g/piece.

The nCB treatment in step (3) is preferably: 3 times a week for 4 weeks.

The nano carbon black particles in the step (3) are nano carbon black particles with the particle size of 15 nm.

The method for establishing the asthma-like slow obstructive pulmonary overlapping airway inflammation mouse model is applied to screening of effective drugs for asthma slow obstructive pulmonary overlapping (ACO).

Compared with the prior art, the invention has the following advantages and effects:

(1) the invention combines the design characteristics of a smoking model and an asthma model, firstly induces the mice to have emphysema disease characteristics, and then inhales the mice into the allergen to avoid mutual offset of the ACO disease characteristics, and meanwhile, the scheme of constructing the model by using nCB can not only confirm the effect of cigarette components on the ACO development, but also greatly shorten the experimental period and avoid the death of the mice, thereby accelerating the working efficiency, improving the experimental feasibility and being beneficial to the wide application of the mouse model.

(2) The invention improves the anesthesia mode of airway hyperresponsiveness and adopts a resuscitation scheme for mice, so that the whole process of development and development of diseases can be observed and analyzed.

(3) The invention simulates the characteristic of Chronic Obstructive Pulmonary Disease (COPD) of asthma in the later stage of the disease of a patient suffering from Chronic Obstructive Pulmonary Disease (COPD) clinically, and provides an experimental animal model for research on ACO pathogenesis and drug treatment.

Drawings

FIG. 1 is a schematic diagram of the induction process of the ACO-like mouse model (Smoke + PAM).

FIG. 2 is a chest micro-CT of C57BL/6 mice modeled, differences in airway reactivity, and inflammatory cells in alveolar lavage fluid (. about.P)<0.05,**P<0.01,***P<0.005,****P<0.001); wherein A is the lung volume of mice in a micro-CT imaging quantitative analysis control group and a smoking group; b is Mean Linear Intercept (MLI) analysis of pathological sections of the mouse lung for airway wall destruction; c is pathological section of lung observed under microscope (100X/200X, HE staining); D. e is the airway resistance (R) at different cumulative concentrations of intravenous acetylcholine (Ach) and Ach of 3.2mg/kg_RS) (ii) a change; f is all cells and lymph in BALAbsolute counts of basocytes (Lym), neutrophils (Neu), eosinophils (Eos), and macrophages (Mac); negative Control in the figure: a negative control group; PAM: aspergillus protease group; smok: a smoking group; smok + PAM: smoking + aspergillus protease group.

FIG. 3 is a photograph of a pathological section of the lung observed under a microscope (100X) (PAS staining, arrows indicate mucin positive, triangles indicate inflammatory cell infiltration; Negative Control in the drawing; PAM: Aspergillus protease group; Smoke: smoking group; Smoke + PAM: smoking + Aspergillus protease group).

FIG. 4 shows the total IgE of the sera measured by ELISA (Negative Control: PAM: Aspergillus protease; Smoke: smoking group; Smoke + PAM: smoking + Aspergillus protease group) (. P <0.05,. P <0.01,. P <0.005,. P < 0.001).

FIG. 5 is a schematic representation of the induction process of an ACO-like mouse model (nCB + PAM).

FIG. 6 is a chest micro-CT of C57BL/6 mice modeled, differences in airway reactivity, and inflammatory cells in alveolar lavage fluid (. about.P)<0.05,**P<0.01,***P<0.005,****P<0.001); wherein A is micro-CT quantitative analysis of the lung volume of the mouse; b is Mean Linear Intercept (MLI) analysis of pathological sections of the mouse lung for airway wall destruction; c is pathological section of lung observed under microscope (100X/200X, HE staining); D. e is R at different cumulative concentrations of intravenous Ach and Ach of 3.2mg/kg_RS(ii) a change; f is the absolute count of all cells in BAL and of Lym, Neu, Eos and Mac; negative Control in the figure: a negative control group; PAM: aspergillus protease group; nCB: a set of nano-carbon black particles; nCB + PAM: nano carbon black particles + aspergillus protease group.

FIG. 7 is a photograph of pathological section of lung observed under a microscope (100X) (PAS staining; arrows indicate mucin positive and triangles indicate inflammatory cell infiltration; Negative Control in the drawing: Negative Control; PAM: Aspergillus protease group; nCB: carbon black nanoparticle group; nCB + PAM: carbon black nanoparticle + Aspergillus protease group).

FIG. 8 shows the total IgE of the sera measured by ELISA (Negative Control: PAM: Aspergillus protease group; nCB: carbon nanoparticle group; nCB + PAM: carbon nanoparticle + Aspergillus protease group) (. P <0.05,. P <0.01,. P <0.005,. P < 0.001).

Figure 9 is a schematic view of a smoking device.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, reagents and starting materials for use in the present invention are commercially available.

Direct contact of aspergillus spore protease in air and long-term exposure of cigarettes are important pathogenesis factors causing allergic asthma and COPD early stage-lobular central emphysema respectively, so a mouse model is constructed by inhaling cigarette smoke/main pathogenic components thereof and aspergillus protease to simulate disease characteristics of clinical ACO patients.

Example 1

1.1 construction model (smoking and PAM induced ACO model)

A. Laboratory animals and groups

20 SPF grade C56BL/6 female mice are 4-6 weeks old and 16-20 g in weight, and are raised in an SPF grade animal house. The mice are randomly divided into 4 groups (n is 5) which are respectively a Negative Control group (NC group), a simple asthma group (PAM group), a simple emphysema group (Smoke group) and an overlapping group (Smoke + PAM group), and the specific operation steps are as follows:

smoking (Smoke) and aspergillus Protease (PAM) -induced ACO-like models:

(a) ACO-like group (cook + PAM) (as shown in figure 1):

smoking treatment: mice inhaled 4 cigarettes/cigarette smoke daily (about 5 minutes/cigarette, 10 minutes apart per cigarette) from day 0 for 5 days per week (2 days left per week without smoking treatment, normal rearing was sufficient), for a total of 120 days. To simulate the human active smoking process, the smoking device (fig. 9) sets each smoking cycle to 3 seconds of pressurized airflow smoking (4L/min) plus 20 seconds of air inhalation; wherein, the smoking is got through the equipment, and the equipment that involves is current conventional equipment:

the smoking device comprises an air compressor, a miniature pressure regulator, a gas flowmeter, an electromagnetic valve, a time control valve driver, a glass tube and a box; the air compressor is connected with a gas flowmeter through a miniature pressure regulator; the gas flowmeter is connected with the electromagnetic valve; the electromagnetic valve is connected with a time control valve driver; the time control valve driver is connected with the box; the electromagnetic valve is connected with the glass tube through the oxygen flow tube; one end of the glass tube is connected with the box; the box is provided with an opening for the gas circulation of the box. When in work: an air compressor (TIMETER ARidyne 2000air compressor) is connected with a gas flowmeter (Model #2MFA2001) through a micro pressure regulator (Norgren R07-100-RGKA), the working pressure of the micro pressure regulator is 5000kPa, the flow rate of the gas flowmeter during working is regulated to 4L/min, the gas flowmeter is connected with OUT of a solenoid valve (Humphrey 310direct acting solenoid valve, 310-12VDC), and the solenoid valve is connected with a time control valve driver (12VDC valve-driver), wherein when the time control valve driver is IN operation, OFF time is controlled at 3 seconds, ON time is controlled at 20 seconds, IN and EXIT of the electromagnetic valve are connected with the glass tube through the oxygen flow tube, wherein IN is connected with one end for placing cigarettes, EXIT is connected with the other end, one side of the glass tube for placing cigarettes is connected with the side face of the box with 8 liters, and the other side face of the box is provided with a hole for the gas circulation of the box.

② allergen treatment: mice were sensitized by intranasal instillation of 50. mu.L of PAM (9. mu.g/mouse) 8 times every other day from day 126.

(b) Asthma group (PAM): normally feeding for the first 125 days, and performing short-term allergen stimulation (the same as the step (a) on the previous step) on the 126 th day without smoking.

(c) Emphysema group (cook): the smoking treatment was the same as in the above step (a), and no allergen treatment was performed (the same amount of PBS buffer solution was administered from day 126).

(d) Negative control group: no allergen and smoking treatment (normal feeding was performed on the first 125 days and the same amount of PBS buffer was administered on day 126).

B. Induction of a model

The smoking stage adopts cigarettes of Wanbao Lu (100's cigarettes, Wanbao Lu company, USA); in the priming stage, mice are subjected to isoflurane aerosol anesthesia (the induction concentration is 3-4% (v/v), the oxygen flow meter of a pattern vehicle analyzer (80-100 mL/min) and then are subjected to nasal drip priming, 50 mu L of 0.18mg/mL PAM (Sigma company in the United states) solution prepared by PBS is given to the PAM group and the Smoke + PAM group during priming, and the NC group and the Smoke group are given to the PBS buffer solution with the same amount.

1.2 identification method

A. Characterization of emphysema

a. Chest micro-CT detection mouse model lung volume

CT imaging technology based on human body and micro-CT imaging method^[3]And detecting the severity of emphysema of the mice. Anesthetized mice were injected intraperitoneally with etomidate (Amidate, 2mg/mL) at an injection dose (mL) equal to the weight (g) x 16 of the mice, followed by placement in an animal CT scanner and acquisition of a complete chest image. The three-dimensional image of the lungs is processed and analyzed using AMIRA3.1.1 software.

b. Analysis of lung morphology

Pathological section (HE) staining to observe the change of airway structure and Mean Linear Intercept (MLI) of alveoli: the morphological structure of mouse lung tissue was evaluated by measuring its MLI. At 25cm H₂Mice lungs were perfused with 10% (v/v) formalin under O pressure and embedded in paraffin. Pathological sections were 5 μm, HE stained, and 10 fields were randomly selected and photographed in the left lung lobe (excluding large airways and blood vessels) double-blind. MLI values were calculated using ImageJ software to place an average of 5 parallel lines per field: MLI is the length of the line x the number of lines/number of cuts.

B. Identification of airway hyperreactivity

Flexivent small animal pulmonary function apparatus detects airway hyperresponsiveness (tail vein injection acetylcholine challenge): anesthetized mice were intraperitoneally injected with etomidate at an injection dose (ml) equal to the mouse weight (g) × 16, followed by airway intubation, mice ventilated with 100% oxygen (Air), and lung function was determined. Changes in bronchial pressure and changes in flow were then quantified continuously at 70% tidal volume (body weight (g) × 9) to determine a stable baseline for total lung Resistance (RL), and cumulative doses (0mg/kg,0.032mg/kg,0.1mg/kg,0.32mg/kg,1mg/kg,3.2mg/kg) of Acetylcholine (ACh) were injected via an indwelling tail vein catheter. Airway hyperreactivity is indicated by a dose-response curve. After the detection is finished, the tail intravenous injection needle of the mouse is pulled out to stop bleeding, the airway cannula is reserved, the mouse is moved to a hypoxia incubator (25-35 ℃), the mouse is observed to breathe until the mouse spontaneously revives and expectorates the airway cannula, and the mouse can be put back into a rearing cage.

Bal cell classification

HEMA3 staining (Fisher corporation, HEMA 3Stat Pack) classified eosinophils, neutrophils, macrophages, lymphocytes and other inflammatory cells in Bronchoalveolar lavage fluid (BAL): perfused and recovered 2 times in the endotracheal tube with 0.8mL of physiological saline, BAL was collected and cells were counted under a microscope. Then about 10⁵Individual cells were centrifuged onto slides and air dried overnight at room temperature before staining with HEMA3 kit. The 100 or 200 cells were counted under microscope for sorting and the percentage and absolute number (percentage x total number of cells) of all cells in BAL as well as lymphocytes (Lym), neutrophils (Neu), eosinophils (Eos) and macrophages (Mac) were calculated.

Determination of mucin

PAS staining (Sigma-Aldrich, PAS staining kit) expression of mucin in lung tissue was observed: soaking the glass slide in a Periodic Acid (Periodic Acid) solution for 5 minutes, and flushing the glass slide for 1 minute by running water; soaking the glass slide in Schiff's reagent for 15 minutes, and flushing with running water for 5 minutes; soaking in a hematoxylin solution for 45-60 seconds, and washing for 1 minute with running water; soaking in 85% ethanol for 10 times; quickly soaking in 95% ethanol for 10 times; quickly soaking in 100% ethanol for 10 times; soaking in dimethylbenzene for 15 minutes; and (4) observing under a microscope.

Assay for IgE

ELISA assay serum total IgE levels: serum was prepared by rapidly cutting off the abdominal aorta and collecting whole blood at the time of sacrifice, and serum total IgE was measured. Serum diluted 1:20 was added to a 96-well plate precoated with an Anti-IgE antibody (BD Pharmingen, Purified Rat Anti-Mouse IgE), and the plate was washed after incubation at 37 ℃ for 2 hours; then, 1:250 diluted Biotin-labeled Anti-IgE (BD Pharmingen, Biotin Rat Anti-Mouse IgE) was added to each well, and the plate was washed after incubation at 37 ℃ for 2 hours; streptavidin-conjugated alkaline phosphatase (Sav-HRP) was added to the reaction mixture, and the mixture was incubated at room temperature for 30 minutes and Substrate (Invitrogen, 1X TMB Substrate Solution) for 15 to 30 minutes to develop color, and the reaction was terminated with 2M sulfuric acid. Relative concentrations were read in a standard spectrophotometer and expressed as OD 405.

1.3 statistical methods

Statistical differences were present with P <0.05, analyzed using Graphpad Prism 7 software. When the measured data obeys normal distribution, the experimental data is expressed by mean +/-standard error, and the t test and One-Way ANOVA/Two-Way ANOVA are adopted for comparative analysis.

1.4 results

A. Differences in mouse airway reactivity after modeling (results are shown in figures 2D and 2E):

compared with the NC group and the Smoke group, the Smoke + PAM group mice have airway resistance (R) when the tail vein injection of the Smoke + PAM group stimulates the drug acetylcholine_RS) Increase, but not as significant as PAM set increase.

B. Changes in mouse lungs after modeling

a. Chest micro-CT analysis lung volume (results see fig. 2A):

micro-CT shows that the lung volume is increased compared with NC group and PAM group after mice of Smok group and Smok + PAM group Smoke continuously for 4 months, namely emphysema in the pre-COPD period caused by long-term smoking appears.

b. Lung morphological analysis (results see fig. 2B and 2C):

it can be observed from pathological sections of the lungs (HE staining) that mice in the Smoke and Smoke + PAM groups developed long-term smoking, compared to the NC and PAM groups, resulting in central emphysema, airway wall destruction of COPD.

C. Inflammatory cells in mouse alveolar lavage fluid after modeling (see results in fig. 2F):

the mice in the Smoke + PAM group had significantly increased numbers of lymphocytes and eosinophils in BAL compared with those in the NC and Smoke groups, and had a similar type composition of BAL inflammatory cells to those in the PAM group.

D. Mice tracheal epithelial mucin secretion and inflammatory cytosis after modeling (results are shown in figure 3):

from pathological sections of the lung (PAS staining), it was observed that mice in the PAM group and the Smoke + PAM group exhibited a significant increase in airway epithelial cell mucin secretion and a large accumulation of inflammatory cells around the airways, as compared to the NC group and the Smoke group.

E. Differences in total IgE levels in peripheral blood of mice after modeling (results are shown in fig. 4):

the serum of mice in the PAM group and the Smoke + PAM group showed high levels of total IgE, while the total IgE in the serum of mice in the NC group and the Smoke group was in a low level state.

(5) Conclusion

The results show that: when the mouse inhales cigarette smoke and aspergillus protease simultaneously, the mouse model can have stable expression of asthma such as emphysema and airway hyperresponsiveness, namely has ACO characteristics.

Example 2

2.1 construction of the model (nCB and PAM-induced ACO model)

A. Laboratory animals and groups

20 SPF grade C57BL/6 female mice are 6-8 weeks old and 16-20 g in weight, and are bred in an SPF grade animal house. The mice were randomly divided into 4 groups (n is 5), which were respectively a negative control group (NC group), a simple asthma group (PAM group), a simple emphysema group (nCB group) and an overlapping group (nCB + PAM group), and the specific operation steps were as follows:

15nm carbon nanoparticles (Nanoparticulate carbon black, nCB) (Cabot corporation, Monarch 1100^[4]And PAM-induced ACO-like model:

(a) ACO-like group (nCB + PAM) (as shown in fig. 5):

nCB: mice were titrated intranasally nCB (50 μ g/mouse) 3 times a week every other day from day 0 for 4 weeks, followed by rest for 4 weeks;

② allergen treatment: mice were sensitized with PAM (9 μ g/mouse) intranasally dropped every other day starting on day 43 (allergen treatment started two weeks after rest) for a total of 8 times (of which the first 7 times were every other day treatment and the 8 th time was every 2 days).

(b) Asthma group (PAM): the first 42 days were normally maintained, and short-term stimulation with allergen was started on day 43 (the same procedure as in step (a) above), and no nCB treatment was performed.

(c) Emphysema group (nCB): nCB treatment was performed in the same manner as in the above step (a), and no allergen treatment was performed (on day 43, the same amount of PBS buffer was administered).

(d) Negative control group: no allergen and nCB treatment (normal rearing was performed on the first 42 days and the same amount of PBS buffer was administered on day 43).

B. Induction of a model

nCB the treatment stage comprises aerosol anesthesia of isoflurane (induced concentration 3-4% (v/v), oxygen flow meter of Patterson vethesia vaporator (80-100 mL/min), intranasal dripping nCB, 50 μ L of 1mg/mL mixed solution prepared by PBS in nCB group, nasal stimulation of PAM group and Smoke + PAM group, 50 μ L of 0.18mg/mL PAM solution prepared by PBS in nCB group, and equal amount of PBS buffer solution in NC group and Smoke group.

2.2 identification method

A. Characterization of emphysema

a. Chest micro-CT detection mouse model lung volume

The severity of emphysema of mice was measured by micro-CT imaging (same procedure as in example 1).

b. Analysis of lung morphology

Pathological section (HE) was stained to observe changes in airway structure and alveolar Mean Linear Intercept (MLI) (same method as in example 1).

B. Identification of airway hyperreactivity

The Flexivent small animal pulmonary function apparatus detects airway hyperresponsiveness (tail vein injection acetylcholine challenge) (method same as example 1).

Bal cell classification

HEMA3 staining classified inflammatory cells in BAL (method same as example 1).

Assay for IgE

Serum total IgE levels were determined by ELISA (same procedure as in example 1).

2.3 statistical methods

2.4 results

A. Differences in airway reactivity of mice after modeling (results are shown in FIGS. 6D and 6E)

In comparison with the NC group and the nCB group, mice in the nCB + PAM group had airway resistance (R) when they were administered with acetylcholine challenge via tail vein injection_RS) Increase, but not as significant as PAM set increase.

B. Changes in mouse lungs after modeling

a. Chest micro-CT analysis lung volume (results see fig. 6A):

micro-CT of the nCB and nCB + PAM mice showed an increase in lung volume compared to the NC and PAM groups, i.e. central emphysema profile with increased lung volume with rupture of alveolar walls also appeared.

b. Lung morphological analysis (results see fig. 6B and 6C):

it can be observed from lung pathology sections (HE staining) that mice of nCB group and nCB + PAM group caused central emphysema, airway wall destruction of COPD similar to long-term smoking compared to NC group and PAM group.

C. Inflammatory cells in mouse alveolar lavage fluid after modeling (results see fig. 6F):

nCB + PAM and PAM groups mice had significantly increased numbers of eosinophils (Eos) in BAL.

D. Mice tracheal epithelial mucin secretion and inflammatory cytosis after modeling (results are shown in figure 7):

from pathological sections of the lung (PAS staining), it was observed that mice in the PAM group and nCB + PAM group exhibited significantly increased airway epithelial cell mucin secretion and a large accumulation of inflammatory cells around the airways, compared to the NC group and nCB group.

E. Differences in total IgE levels in peripheral blood of mice after modeling (results are shown in fig. 8):

the total IgE secretion was increased in the mice of PAM group and nCB + PAM group, while the total IgE in the sera of NC group and nCB group mice was in a low level state.

(5) Conclusion

The results further confirmed that cigarette is an important causative agent of ACO emphysema, and it was clarified that nCB and aspergillus protease also induced a mouse model that mimics ACO.

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.

Reference to the literature

1.Global Initiative for Asthma(GINA).Global Strategy for Asthma Management and Prevention updated 2018.http://www.ginasthma.org.

2.IKEDA G,MIYAHARA N,KOGA H,et al.Effect of a cysteinyl leukotriene receptor antagonist on experimental emphysema and asthma combined with emphysema[J].Am J Respir Cell Mol Biol,2014,50(1):18-29.

3.Yuan X,Shan M,You R,et al.Activation of C3a receptor is required in cigarette smoke-mediated emphysema[J].Mucosal Immunology,2015,8(4):874-885.

4.Ran You,Wen Lu,Ming Shan,et al.Nanoparticulate carbon black in cigarette smoke induces DNA cleavage and Th17-mediated emphysema[J].Elife,2015,4:e09623.

Claims

1. The method for establishing the mouse model of asthma-like slow obstructive pulmonary overlapping airway inflammation is characterized by comprising the following steps of:

s1, smoking and Aspergillus protease-induced ACO-like model

(1) Smoking treatment: the mice inhale cigarette smoke every day from day 0, 4 +/-1 cigarettes every day, 5 +/-1 days every week for 4-6 months;

(2) allergen treatment: after smoking treatment is finished for 3-7 days, the mice are anesthetized, then aspergillus protease solution is dripped into the nose to sensitize the mice, the mice are treated for 1 time at intervals of 1-2 days, and 8-10 times in total, and a mouse model of asthma-like slow obstructive pulmonary overlapping airway inflammation is constructed;

s2, Nano carbon Black particles and ACO-like model induced by Aspergillus protease

(3) nCB treatment: after anesthetizing the mice from day 0, dripping the nano carbon black particles into the nose for 2-4 times per week for 4-6 weeks;

(4) allergen treatment: nCB, after finishing the treatment for 2-4 weeks, firstly anesthetizing the mouse, then dripping aspergillus protease solution into the nose to sensitize the mouse, treating for 1 time every 1-2 days, and totally 8-10 times to construct a mouse model of asthma-like slow obstructive pulmonary overlapping airway inflammation;

the dosage of the aspergillus protease in the steps (2) and (4) is 9 +/-1 mu g/mouse;

the aspergillus protease solution in the steps (2) and (4) is prepared by the following method: dissolving aspergillus protease in PBS buffer solution to prepare aspergillus protease solution with the concentration of 0.18 mg/mL;

the dosage of the nano carbon black particles in the step (3) is 50 +/-2 mu g/particle;

2. The method for establishing the mouse model of asthma-like slow obstructive pulmonary overlapping airway inflammation according to claim 1, wherein:

the smoke of the cigarette inhaled in the step (1) is as follows: 4 pieces are taken every day, 5 days every week, and the total time is 4-6 months;

the nCB treatment in the step (3) is as follows: 3 times a week for 4 weeks.

3. The method for establishing the mouse model of asthma-like slow obstructive pulmonary overlapping airway inflammation according to claim 1, wherein:

the allergen treatment in the steps (1) and (4) is 1 time of treatment every other day;

the number of times of allergen treatments described in steps (1) and (4) was 8.

4. The method for establishing the mouse model of asthma-like slow obstructive pulmonary overlapping airway inflammation according to claim 1, wherein:

the nano carbon black particles dripped in the step (3) are mixed solution prepared by PBS buffer solution, and the concentration of the mixed solution is 1 mg/mL.

5. The method for establishing the mouse model of asthma-like slow obstructive pulmonary overlapping airway inflammation according to claim 1, wherein:

the mice in the steps (1) and (3) are C57BL/6 mice;

the cigarette in the step (1) is a Wanbao Lu cigarette.

6. The method for establishing the mouse model of asthma-like slow obstructive pulmonary overlapping airway inflammation according to claim 1, wherein:

the anesthesia in the steps (2) and (4) is performed by adopting isoflurane aerosol.

7. The method for establishing the asthma-like slow obstructive pulmonary overlapping airway inflammation mouse model of any one of claims 1 to 6, and the application of the method in screening of drugs effective for asthma slow obstructive pulmonary overlapping.