Long-term continuous cigarette smoke exposure induced chronic obstructive pulmonary disease mouse model and construction method and application thereof

Abstract

The invention discloses a chronic obstructive pulmonary disease mouse model induced by long-term continuous cigarette smoke exposure as well as a construction method and application thereof, and the construction method comprises the following steps: introducing cigarette smoke with constant concentration into a smoke exposure device, so that a mouse is in a cigarette smoke exposure environment for a long time and maintains normal diet and drinking; performing macroscopic characterization and microcosmic index evaluation on the mouse model with the chronic obstructive pulmonary disease until clinical symptoms of the chronic obstructive pulmonary disease appear; wherein the smoke exposure device comprises a smoke mixing box, a sealed feeding exposure box and a control box, the sealed feeding exposure box provides a cigarette smoke exposure environment, and the control box maintains the stability of the cigarette smoke exposure environment. According to the method, the occurrence cause of the chronic obstructive pulmonary disease of the mouse is simulated by optimizing exposure parameter conditions, the clinical symptoms of the chronic obstructive pulmonary disease of the mouse model are evaluated in multiple dimensions, and the method accords with the symptoms and signs of the chronic obstructive pulmonary disease, is high in similarity and good in reliability, and can be used as a disease model for research of the chronic obstructive pulmonary disease.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal models, and particularly relates to a chronic obstructive pulmonary disease mouse model induced by long-term continuous cigarette smoke exposure, and a construction method and application thereof. Background Art

[0002] Chronic obstructive pulmonary disease (COPD) ranks third among the leading causes of death worldwide, and the number of deaths it causes is increasing year by year. COPD is a respiratory disease characterized by chronic inflammation of the airways and emphysema. Air pollution and smoking are common triggers of COPD. Due to the worsening of pollution and the increase in the number of smokers, the prevalence of COPD in my country and even globally is increasing year by year. Experimental animal models are important tools for studying the mechanisms of human diseases. They can be used to study the mechanisms of disease occurrence and development as well as treatment methods. Whether the disease reflected by the animal model is consistent with the clinical condition is a key factor affecting drug evaluation. At present, there have been many studies on the establishment of COPD animal models. Because the pathogenic mechanism of COPD is still unclear, there are no relatively unified standards for the animal species used in the establishment of COPD animal models, the methods for establishing the model, and the evaluation indicators. There is still a lack of reliable methods for establishing COPD animal models.

[0003] At present, the most commonly used COPD animal model is rodents, especially mice. The advantages of modeling include clear genetic background and operability, fast breeding speed, and saving manpower and material resources. The methods used to prepare COPD mouse models include inhalation of harmful gases, tracheal instillation of tissue decomposing enzymes, and genetic engineering technology, which can be used alone or in combination. Clinical statistics show that about 90% of COPD patients are smokers. The use of cigarette smoke (CS) exposure is closest to the human disease environment and can better reflect the pathogenic mechanism of COPD. The modeling methods currently used to construct COPD mouse models using CS exposure vary. The exposure methods used include local fumigation and whole-body exposure. The modeling time ranges from a few days to several months, and the cigarette brand, fumigation time, and dosage are also different. Different degrees of COPD mouse models can be obtained using different modeling methods, but the following problems also exist:

[0004] (1) Different exposure methods and smoke exposure devices are used, with different standards. The shapes, sizes, and ventilation conditions of exposure boxes are also different, and there is a lack of a relatively unified exposure box standard.

[0005] (2) COPD is a chronic disease with a complex natural course. Short-term exposure to smoke only causes acute lung symptoms. Traditional intermittent exposure to prepare a COPD model requires at least half a year. Intermittent exposure causes self-repair damage to the animal body, resulting in a long modeling cycle, unclear typical symptoms, and inconsistent industry standards.

[0006] (3) The cigarette brands, smoking times, and dosages used in the modeling differed across studies. Although the University of Kentucky in the United States introduced 3R4F and 1R6F reference cigarettes in an attempt to standardize relevant indicators, their popularity is not high. Due to the different cigarette brands used, the cigarette ingredients and concentration doses are not standardized, resulting in low model reproducibility.

[0007] (4) Model evaluation indicators are not unified. Different studies evaluate COPD animal models based on relatively rough indicators such as lung function, pathological morphology, body weight, and inflammatory factors. This is not objective and comprehensive enough. There is an urgent need to establish an objective, scientific, and comprehensive model evaluation system. Summary of the Invention

[0008] Based on this, the main purpose of the present invention is to provide a method for constructing a COPD mouse model induced by long-term continuous cigarette smoke exposure. A smoke exposure device is used to induce long-term continuous cigarette smoke exposure in mice, and the simulation conditions are optimized to construct a COPD mouse model.

[0009] Another object of the present invention is to provide a chronic obstructive pulmonary disease mouse model induced by long-term continuous cigarette smoke exposure. The COPD mouse model constructed by the above-mentioned construction method and optimized simulation conditions conforms to the symptoms and signs of COPD and can be used for subsequent research on COPD.

[0010] Another object of the present invention is to provide a use of the COPD mouse model induced by long-term continuous cigarette smoke exposure in preparing a disease model of COPD.

[0011] To achieve the above object, the present invention adopts the following technical solutions:

[0012] In a first aspect, the present invention provides a method for constructing a mouse model of chronic obstructive pulmonary disease induced by long-term continuous cigarette smoke exposure, comprising the following steps:

[0013] (1) A constant concentration of cigarette smoke was introduced into the smoke exposure apparatus, and mice were exposed to cigarette smoke for a long time, while maintaining normal diet and water intake, until clinical symptoms of chronic obstructive pulmonary disease were induced in the mice, thereby obtaining a chronic obstructive pulmonary disease mouse model;

[0014] (2) performing macroscopic characterization and microscopic index evaluation on the COPD mouse model;

[0015] The smoke exposure device includes a smoke mixing box, a sealed breeding exposure box and a control box, wherein the sealed breeding exposure box provides a cigarette smoke exposure environment and is connected to the smoke mixing box through a silicone tube, and the control box maintains the stability of the cigarette smoke exposure environment in the sealed breeding exposure box.

[0016] Preferably, the modeling time is 10 weeks.

[0017] Preferably, the cigarette brand used is Huangguoshu brand cigarettes, in which the tar content is 10 mg, the nicotine content is 0.9 mg, the carbon monoxide content is 12 mg, and the PM2.5 concentration of cigarette smoke particles is 400.

[0018] Preferably, the mice are exposed to smoke for 6 hours every day and are kept in the exposure chamber of the smoke exposure device with normal diet and water intake.

[0019] Preferably, the mouse is a male mouse.

[0020] Preferably, the clinical symptoms of chronic obstructive pulmonary disease include macroscopic characteristics and microscopic indicators, wherein the macroscopic characteristics include weight gain characteristics and pathological morphology; the microscopic indicators include alveolar linear intercept, the percentage of low attenuation area volume to the total lung volume of both lung tissues and physiological and biochemical molecular parameters.

[0021] Preferably, the evaluation indicators of the chronic obstructive pulmonary disease mouse model include: slow weight gain, macroscopic manifestations of inflammation and emphysema in pathological morphology; increased alveolar mean linear intercept, increased percentage of low attenuation area volume to total lung volume of both lung tissues, and microscopic indicators of airway wall thickening.

[0022] In a second aspect, the present invention further provides a COPD mouse model induced by long-term continuous cigarette smoke exposure, which is obtained by any of the aforementioned methods for constructing a COPD mouse model induced by long-term continuous cigarette smoke exposure.

[0023] The third aspect of the present invention further provides use of the COPD mouse model induced by long-term continuous cigarette smoke exposure in preparing a disease model for studying COPD.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The present invention utilizes mouse cages as smoke exposure sites, preserving the mice's daily breeding environment while exposing them to smoke. This can reduce the impact of the smoke exposure device on the construction of a long-term smoke exposure animal model and provide a reference for the subsequent construction of a batch smoke exposure animal model.

[0026] 2. The present invention evaluates the COPD mouse model by combining the clinical symptoms and physiological and biochemical indicators of COPD, which can effectively prove that the long-term smoke exposure animal model of mice constructed by the present invention is successful and feasible, and can be used for subsequent research on COPD mouse models. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1This is a physical picture of the COPD mouse model in the examples.

[0028] Figure 2 The graph of the weight gain of experimental mice in the experimental group and the control group in the embodiment; the control group (n=5) and the model group (n=5), compared with the control group, ** P<0.01.

[0029] Figure 3 Comparison of IL-6 and TNF-α levels in serum and BALF of mice in the control group and model group in the examples; A) Comparison of IL-6 concentration in mouse serum; B) Comparison of IL-6 concentration in BALF; C) Comparison of serum TNF-α concentration; D) Comparison of TNF-α concentration in BALF, 3 mice in each group; **P<0.05.

[0030] Figure 4 Figures 2 and 3 show the morphological changes of lung tissues in the control and model groups of experimental mice in the examples; A) HE staining of alveoli (×200); B) HE staining of bronchus (×100); C) Comparison of MLI values ​​between the control and model groups, **P<0.01.

[0031] Figure 5 This is a graph showing changes in collagen in the lung tissues of experimental mice in the control group and model group (×400).

[0032] Figure 6 These are the CT image analysis results of the experimental mice in the control group and model group in the examples. DETAILED DESCRIPTION

[0033] In order to more fully understand and demonstrate the technical solutions, objectives, and advantages of the present invention, the following is a further detailed and complete description of the technical effects produced by the present invention in conjunction with the accompanying drawings and specific embodiments. Obviously, the described embodiments are only some of the embodiments of the present invention, and not all of them. It should be pointed out that for those skilled in the art, other embodiments obtained without departing from the concept of the present invention are all within the scope of protection of the present invention.

[0034] Example 1

[0035] 1. Mouse Model Preparation

[0036] 1. Experimental Animals

[0037] Twenty 6-week-old SPF male C57BL / 6J mice were purchased from Zhejiang Weitonglihua Laboratory Animal Technology Co., Ltd. (License: SCXK(Zhe)2024-0001, Quality Certificate Number: 20240530ABC c0019000299). After 1 week of adaptive feeding, the 20 experimental mice were randomly divided into a normal group (n = 10) and a model group (n = 10), with 5 mice per cage.

[0038] 2. Smoke exposure device

[0039] The smoke exposure device used in this embodiment is recorded in patent No. ZL2022227374713, and consists of a smoke mixing box, a sealed breeding exposure box and a control box. The sealed breeding exposure box provides a smoke exposure environment and is connected to the smoke mixing box by a silicone tube. The control box controls and maintains the stability of the smoke environment in the sealed breeding exposure box, so that the living environment of the mice is a poisoned environment, thereby enabling animal poisoning experiments to be carried out continuously for a long time to simulate real conditions.

[0040] 3. Cigarettes

[0041] Huangguoshu brand cigarettes, tar content 10mg, nicotine content 0.9mg, carbon monoxide content 12mg, commercially available.

[0042] 4. Modeling

[0043] A long-term smoke exposure experiment was conducted on mice using a smoke exposure apparatus to establish a mouse model. The model group was exposed to smoke six times daily for one hour each time. The exposure environment maintained a PM2.5 concentration of 400 and a normal oxygen partial pressure. The mice maintained a normal diet and water intake and remained in the exposure chamber. The control group was housed normally and received no other treatments for 10 consecutive weeks.

[0044] 2. Model mouse testing

[0045] 1. Weight check

[0046] During the modeling period, the body weight of the experimental animals was measured and recorded every 2 weeks.

[0047] 2. Histopathological examination

[0048] Mice were sacrificed by cervical dislocation, and lung tissue was collected. The left lung was removed and fixed in fixative for 24 hours. The sections were then dehydrated, embedded, and sectioned to produce paraffin sections. After dewaxing, the sections were stained with hematoxylin and eosin (HE) and Masson's staining. HE staining was used to observe and record pathological changes in the mouse lung tissue, while Masson's staining was used to observe fibrin deposition in the lung parenchyma and airways, which was used to compare lung pathological changes and assess the severity of fibrosis.

[0049] 3. Imaging testing

[0050] After modeling, mice underwent in vivo microCT imaging using a Bruker Skyscan 1276. Scanning parameters were 70 kV, 144 μA, and 18 μm resolution. Emphysema and tracheal parameters were measured on the Micro-CT images. Emphysema parameters included lung density and the percentage of air volume to total lung volume (LAA %-928); tracheal parameters included bronchial wall thickness and lumen diameter. Lung density and LAA %-928 were measured using the Micro-CT post-processing software Image Process Language (IPL).

[0051] Lung density measurement method: Five slices were randomly selected in each lobe of the lung to measure lung density, and the average value represented the lung density of each lobe.

[0052] LAA%-928 measurement method: Use software to manually outline the lung boundaries and remove non-lung tissue components such as the trachea and blood vessels. Then set the air threshold to -928HU. The software automatically calculates the volume of the part with density ≤-928HU (i.e., air volume) as a percentage of the total lung volume.

[0053] 3. Evaluation of the Long-term Smoke Exposure Mouse Model

[0054] After modeling, the mouse model was evaluated according to clinical diagnostic criteria and biochemical markers of COPD. A COPD animal model was constructed by exposing mice to long-term cigarette smoke using a smoke exposure device to simulate the etiology of COPD patients who smoke for a long time. The model mice exhibited macroscopic manifestations such as slow weight gain, pathological morphology showing inflammation and emphysema, as well as microscopic pathophysiological features such as increased alveolar mean linear intercept and increased low attenuation area volume as a percentage of total lung volume, consistent with the pathology of COPD.

[0055] 1. Weight gain

[0056] Figure 2 Figure 3 shows the changes in weight gain of experimental mice in the model group and the normal group. During the modeling period, the weight gain of the model group was slow and significantly lower than that of the experimental mice in the normal group.

[0057] 2. Comparison of serum and BALF inflammatory factor levels

[0058] The serum IL-6 concentration of mice in the model group was (27.51±0.59) / (pg·mL-1), and the serum IL-6 concentration of mice in the control group was (21.63±0.50) / (pg·mL-1). The serum IL-6 concentration of mice in the model group increased by 27.2%. The IL-6 concentration in the bronchoalveolar lavage fluid (BALF) of mice in the model group was (4.56±0.40) / (pg·mL-1), and the IL-6 concentration in the BALF of mice in the control group was (1.90±0.20) / (pg·mL-1). The IL-6 concentration in the BALF of mice in the model group increased by 140.0%. The results were significantly different (P<0.01). The serum TNF-α concentration of the mice in the model group was (722.72±26.39) / (pg·mL-1), and the serum TNF-α concentration of the mice in the control group was (619.40±17.43) / (pg·mL-1). The serum TNF-α concentration of the mice in the model group increased by 16.7%. The TNF-α concentration in the BALF of the mice in the model group was (208.05±25.58) / (pg·mL-1), and the TNF-α concentration in the BALF of the mice in the control group was (174.44±14.34) / (pg·mL-1). The TNF-α concentration in the BALF of the mice in the model group increased by 19.3%. There was a significant difference in the serum TNF-α level between the model group and the control group (P<0.01), but there was no significant difference in the TNF-α level in the BALF between the model group and the control group (P>0.05). Figure 3 shown.

[0059] 3. HE staining results of lung tissue pathology

[0060] The morphological changes of bronchi in the model group and the control group were as follows Figure 4 As shown in the figure, the alveoli of the mice in the normal group were uniform in size, with clear and complete structures, and no large number of inflammatory cells were found in the alveolar interstitium. Compared with the normal group, the mice in the model group showed the hallmark pathological changes of COPD lung tissue, with disordered alveolar structure, uneven size, thinning of alveolar walls, loss and rupture of multiple alveolar septa, expansion and fusion into bullae, obvious pulmonary interstitial hyperplasia and a large number of inflammatory cells gathering ( Figure 4 A) The trachea structure of mice in the normal group was normal, with no obvious hyperplasia of the tracheal wall and evenly distributed smooth muscle ( Figure 4 B). Compared with the normal group, the model group mice showed hallmark pathological changes in COPD lung tissue, including destruction of the tracheal structure of the lung tissue, significant thickening of the airway wall, rupture of smooth muscle, adhesion and loss of columnar ciliated epithelium, severe inflammatory infiltration of the tracheal wall and surrounding areas, extensive metaplasia of goblet cells, necrosis of many epithelial cells, and the presence of more mucus and cell debris in the tracheal cavity. By calculating the average alveolar intercept of mice, it was found that the average alveolar intercept of mice in the model group was greater than that of mice in the control group, and the results were significantly different (P<0.05) ( Figure 4 C).

[0061] 4. Changes in lung tissue collagen

[0062] Changes of collagen fibers in lung tissue of experimental mice in the experimental and control groups Figure 5 As shown in the figure, the lung tissues of mice in the model group showed obvious fibrosis in the pulmonary interstitium and peribronchial tissues, obvious thickening of the bronchial epithelium, and obvious collagen deposition, while no similar phenomenon was found in the normal group.

[0063] 5. Imaging manifestations

[0064] like Figure 6 As shown, in vivo micro-CT imaging of lung tissue in mice revealed no obvious abnormal attenuation in the control group. However, in the model group, micro-CT images showed scattered or confluent low-density areas within the lungs (CT values ​​< -900 HU), indicating alveolar wall destruction, increased lung volume, flattening or downward displacement of the diaphragm, and widening of the intercostal spaces—typical symptoms of emphysema. Bronchial walls were thickened, with airway wall obscuration or a "double-track sign" (chronic inflammation leading to submucosal hyperplasia), and focal high-density shadows (e.g., mucus retention in the small airways). Ground-glass opacity (GGO) is characterized by focal, mildly increased attenuation (inflammatory cell infiltration or interstitial edema), inflammatory infiltration, and small nodules (macrophage / lymphocyte aggregation) distributed along the bronchi. Furthermore, reticular or linear high-density shadows (Masson staining corresponds to collagen deposition) suggest a tendency toward fibrosis.

[0065] In summary, the present invention successfully induced COPD symptoms and signs consistent with COPD in mice by exposing them to cigarette smoke for a long time using a smoke exposure device, simulating the etiology of COPD in long-term smoking patients. The mice were also successfully induced to develop lung tissue inflammation, increased collagen fibers, emphysema, and other symptoms consistent with COPD. Furthermore, the mice showed changes in a series of physiological and biochemical indicators, including slow weight gain, increased alveolar mean linear intercept, and increased percentage of low attenuation zone volume to total lung volume of both lungs. This demonstrates that the COPD mouse model constructed by the present invention is successful and feasible, with high similarity and reliability, and can be used for subsequent research on COPD.

[0066] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for constructing a mouse model of chronic obstructive pulmonary disease induced by long-term continuous cigarette smoke exposure, characterized in that: The following steps are involved: (1) A constant concentration of cigarette smoke was introduced into the smoke exposure apparatus, and mice were exposed to cigarette smoke for a long time, while maintaining normal diet and water intake, until clinical symptoms of chronic obstructive pulmonary disease were induced in the mice, thereby obtaining a chronic obstructive pulmonary disease mouse model; (2) performing macroscopic characterization and microscopic index evaluation on the COPD mouse model; The smoke exposure device includes a smoke mixing box, a sealed breeding exposure box and a control box, wherein the sealed breeding exposure box provides a cigarette smoke exposure environment and is connected to the smoke mixing box through a silicone tube, and the control box maintains the stability of the cigarette smoke exposure environment in the sealed breeding exposure box.

2. The method for constructing a COPD mouse model according to claim 1, characterized in that: The modeling time is 10 weeks.

3. The method for constructing a COPD mouse model according to claim 1, characterized in that: The cigarette brand used was Huangguoshu brand cigarettes, which contained 10mg of tar, 0.9mg of nicotine, 12mg of carbon monoxide, and a PM2.5 concentration of 400 particles in cigarette smoke.

4. The method for constructing a COPD mouse model according to claim 1, characterized in that: The mice were exposed to smoke for 6 hours every day and maintained with normal diet and water intake in the exposure chamber of the smoke exposure device.

5. The method for constructing a COPD mouse model according to claim 1, characterized in that: The mice are male mice.

6. The method for constructing a COPD mouse model according to claim 1, characterized in that: The clinical symptoms of chronic obstructive pulmonary disease include macroscopic manifestations and microscopic indicators, among which: The macroscopic manifestations include weight gain manifestations and pathological morphology; The microscopic indicators include alveolar linear intercept, percentage of low attenuation area volume to total lung volume of both lung tissues, and physiological and biochemical molecular parameters.

7. The method for constructing a COPD mouse model according to claim 1, characterized in that: The evaluation indicators of the COPD mouse model include: slow weight gain, pathological morphology showing macroscopic manifestations of inflammation and emphysema; and microscopic indicators such as increased mean alveolar linear intercept, increased percentage of low attenuation area volume to total lung volume of both lung tissues, and thickening of airway walls.

8. A mouse model of chronic obstructive pulmonary disease induced by long-term continuous cigarette smoke exposure, characterized in that: The method for constructing a chronic obstructive pulmonary disease mouse model induced by long-term continuous cigarette smoke exposure according to any one of claims 1 to 7 is obtained.

9. Use of the COPD mouse model induced by long-term continuous cigarette smoke exposure according to claim 8 in preparing a disease model for studying COPD.

Images