Construction and evaluation method of PM2.5 induced chronic obstructive pulmonary disease acute exacerbation mouse model

Abstract

The invention discloses a construction and evaluation method of a PM2.5 induced chronic obstructive pulmonary disease acute exacerbation mouse model, and relates to the technical field of disease animal models. The invention aims to evaluate whether PM2.5 can induce acute exacerbation of COPD through a mouse model, a COPD model is established by adopting combined treatment of smoking and lipopolysaccharide (LPS), and an infectious AECOPD model is established by using common klebsiella pneumoniae (KP) nasal drop on the basis. Meanwhile, PM2.5 processing is added on the basis of the COPD model, and the effect of PM2.5 on acute exacerbation of COPD is evaluated. Through comparison with a classical model (an infectious AECOPD model), the potential effect of PM2.5 in induction of acute exacerbation of COPD is evaluated, and an important reference is provided for future research on an AECOPD animal model induced by air pollution.

Description

Technical Field

[0001] The present invention relates to the technical field of disease animal models, and in particular to a method for constructing and evaluating a PM2.5-induced acute exacerbation mouse model of chronic obstructive pulmonary disease. Background Art

[0002] Chronic obstructive pulmonary disease (COPD) is a progressive, disabling respiratory disease characterized by persistent airflow limitation. The main causes of COPD include long-term smoking, air pollution, and genetic susceptibility. COPD patients often experience acute exacerbations (AECOPD), which not only worsen their condition but also significantly increase their hospitalization and mortality rates. The development of AECOPD is often closely related to respiratory infections, environmental pollution, and other triggers. Therefore, understanding the causes and mechanisms of AECOPD is crucial for improving the management of COPD patients.

[0003] In recent years, air pollution, particularly fine particulate matter (PM2.5), has been recognized as a significant environmental factor in the exacerbation of COPD. PM2.5, due to its small particle size, can penetrate deep into the lungs and trigger a local immune response, leading to airway inflammation and worsening lung function. Epidemiological studies have shown that long-term exposure to PM2.5 significantly increases the disease burden in COPD patients. However, whether PM2.5 can directly induce acute exacerbations of COPD, and particularly how it compares with existing COPD and AECOPD models, remains a lack of systematic experimental data and research. Summary of the Invention

[0004] The present invention aims to develop and evaluate a mouse model of acute exacerbation of chronic obstructive pulmonary disease (COPD) induced by PM2.5. The goal is to assess whether PM2.5 can induce acute exacerbations of COPD using this mouse model. To this end, the present invention established a COPD model using a combination of smoke and lipopolysaccharide (LPS) treatment. Furthermore, an infectious AECOPD model was established using nasal drops of the commonly used Klebsiella pneumoniae (KP) bacterium. Furthermore, PM2.5 treatment was added to the COPD model to assess its effect on acute exacerbations of COPD. By comparing this model with a conventional model, the potential role of PM2.5 in inducing acute exacerbations of COPD was evaluated, providing an important reference for future research on animal models of air pollution-induced AECOPD.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] The method for constructing a mouse model of acute exacerbation of chronic obstructive pulmonary disease induced by PM2.5 comprises the following steps:

[0007] Step 1, establishment of COPD mouse model:

[0008] After one week of adaptive feeding, SPF C57BL / 6 mice were placed in a fumigation chamber and smoked for 1 hour with 10 cigarettes each time for 12 consecutive weeks. Simultaneously, 20 μL (0.5 μg / μL) of lipopolysaccharide (LPS) was instilled into the nasal cavity on days 1, 15, and 29. No fumigation was performed on the day of nasal instillation.

[0009] Step 2: Establishment of PM2.5-induced COPD mouse model:

[0010] On the third day of the 13th week, a suspension containing PM2.5 (7.5 mg / kg) was instilled into the nasal cavity twice a day for 4 consecutive days; relevant indicators were tested on the seventh day of the 14th week.

[0011] As a preferred technical solution of the present invention, the steps for preparing the PM2.5-containing suspension are:

[0012] Atmospheric sampling points were set at an altitude of 70-100 m above the ground. A large-flow air sampler equipped with a PM2.5 cutter was used and the flow rate was set to 1.05 m 2 / min, collect PM2.5 onto a glass fiber filter membrane, and replace the filter membrane every 72 h; cut the sampling filter membrane into 2 cm × 2 cm size, immerse it in double-distilled water solution, and ultrasonically oscillate twice for 20 min each time to elute the particulate matter on the filter membrane. The eluate is collected and filtered through 6-8 layers of sterile gauze. The collected suspension is frozen overnight and vacuum freeze-dried to a dry powder; when used, a 7.5 mg / mL suspension is prepared in phosphate buffered saline (PBS).

[0013] The present invention further proposes a method for evaluating the PM2.5-induced acute exacerbation of chronic obstructive pulmonary disease mouse model, comprising the following steps:

[0014] Step 1, mouse observation:

[0015] Observe the mice's mental state, fur gloss, agility, reaction sensitivity, activity, breathing stability, and oral and nasal secretions;

[0016] Step 2, pulmonary function test:

[0017] General anesthesia was performed using an intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg). The trachea was isolated, and a 2 mm incision was made transversely at the cricoid cartilage for endotracheal intubation. The intubation was fixed to the trachea with 4-0 sutures to prevent air leakage. The mouse was placed supine in a plethysmography chamber, and the endotracheal tube was tightly connected to the instrument interface to prevent air leakage. The pulmonary function system was activated to measure the mouse's pulmonary function. The measurement indicators included forced vital capacity (FVC), inspiratory capacity (IC), and forced expiratory volume in 0.1 second (FEV). 0.1 ), Forced Expiratory Volume in 0.2 second (FEV 0.2 ), 0.1 second rate (FEV0.1 / FVC ratio, FEV 0.1 / FVC), FEV0.2 / FVC ratio, FEV 0.2 / FVC); the test was repeated 3 times and the average value was taken as the measured value for statistical analysis;

[0018] Step 3, blood gas analysis:

[0019] Under general anesthesia with an intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg), arterial blood samples were collected from mice using the left ventricular blood sampling method. Blood was drawn into a sodium heparin anticoagulant tube, mixed, and allowed to stand. A blood gas test card was removed from a 4°C refrigerator and placed at room temperature for at least 5 minutes. The outer packaging was opened, the test card was removed, and 100 μL of anticoagulated whole blood sample was injected into the injection port. The injection port was then sealed. The test card was inserted into the analyzer and the test results were awaited. The following parameters were measured: partial pressure of oxygen (PO2), partial pressure of carbon dioxide (PCO2), pH, and oxygen saturation (SO2).

[0020] Step 4, pathological examination:

[0021] The right lungs of mice in each group were collected, fixed, embedded, and then sectioned, stained with hematoxylin-eosin, mounted with neutral gum, and photographed.

[0022] The above test results were statistically analyzed using Prism10.3.0; normality and homogeneity of variance were tested, and ANOVA analysis was used, and the Tukey test was used to determine the specific differences between the groups.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] This study established a COPD mouse model using combined smoke and lipopolysaccharide (LPS) treatment. Two AECOPD models were also established using intranasal exposure to Klebsiella pneumoniae (KP) and PM2.5, respectively. The mice were randomly divided into four groups: a control group (referred to as the Control group), a basic model group (referred to as the COPD group), a KP-induced AECOPD group (referred to as the AECOPD group), and a group with exacerbated COPD induced by PM2.5 (referred to as the COPD+PM2.5 group). Pulmonary function tests, blood gas analysis, and histopathological evaluation were performed on each group. The results showed that compared with the Control group, lung function indicators in the COPD and AECOPD groups showed significant airflow limitation, and lung histopathology revealed typical inflammation and alveolar structural damage. In the COPD+PM2.5 group, PM2.5 exposure further exacerbated lung function impairment and alveolar destruction, with abnormally elevated lung function indicators such as forced vital capacity (FVC) and intrapulmonary embolism (IC), indicating pulmonary hyperinflation. Blood gas analysis revealed elevated PCO2 and decreased SO2 in the COPD+PM2.5 group, indicating more severe symptoms than those in the AECOPD group. Therefore, the present invention experimentally confirmed that both PM2.5 and KP can induce acute exacerbations in COPD mouse models, but with different characteristics. The successful establishment of a PM2.5-induced COPD acute exacerbation mouse model highlights the important role of PM2.5 as an environmental pollutant in COPD acute exacerbations and provides a new reference for future research on the interaction between air pollution and COPD acute exacerbations. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Figure 2 shows the body weight change trends of mice in different groups (compared with the Control group, *P<0.05).

[0026] Figure 2 The lung function FVC, IC, FEV of mice in different groups 0.1 , FEV 0.2 , FEV 0.1 / FVC, FEV 0.2 / FVC measurements (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

[0027] Figure 3 Arterial blood gas PO2, PCO2, pH, and SO2 measurements of mice in different groups (*P < 0.05; **P < 0.01).

[0028] Figure 4 HE staining images of lung tissues of mice in different groups. DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with embodiments and drawings.

[0030] This study aims to establish a mouse model of acute exacerbation of COPD induced by PM2.5, providing a reliable animal model for subsequent research. The specific steps are as follows:

[0031] 1 Materials and Methods

[0032] 1.1 Materials

[0033] 1.1.1 Experimental Animals

[0034] SPF C57BL / 6 mice, weighing 18–22 g, were purchased from Hangzhou Qizhen Experimental Animal Technology Co., Ltd. [Production License No. SCXK (Zhejiang) 2022-0005]. They were housed in the animal room of Anhui University of Traditional Chinese Medicine (temperature 21–24°C, relative humidity 55%–60%) and provided with adequate water and feed daily for 1 week of acclimatization. All experimental procedures strictly adhered to animal ethics regulations (Ethics No. 2024-008).

[0035] 1.1.2 Main instruments and reagents:

[0036] High-flow air sampler (model: XA-1000, Qingdao Xinao), freeze dryer (model: CoolSafe™55-9L, labogene, Denmark), small animal spirometer (model: fexiVent, SCIREQ, Canada), handheld hematology analyzer (model: i-STAT 300-G, Abbott), blood gas and biochemistry test card (model: G3+, Abbott), microtome (model: KD-3358-VI, Cody), microscope (model: BX53, Olympus), electronic balance (model: PL-203, Mettler-Toledo), hematoxylin (catalog number: B600020, Mitaka), eosin stain (Sigma), 4% paraformaldehyde neutral tissue fixative (Google Bio), Hongtashan cigarettes (nicotine content 1.0 mg, tar content 10 mg, carbon monoxide content 11 mg, smoke ... mg, Hongta Tobacco Group), lipopolysaccharide (Sigma), Klebsiella pneumoniae (No.: CCTCCAB 2010163, China Center for Type Culture Collection).

[0037] 1.2 Methods

[0038] 1.2.1 Preparation of PM2.5 suspension

[0039] From November 2023 to February 2024, a sampling point was set up on the 26th floor of a residential building in Yaohai District, Hefei City (about 75m above the ground). A large-flow air sampler equipped with a PM2.5 cutter was used, and the flow rate was set at 1.05 m 2PM2.5 was collected onto glass fiber filters at a rate of 100 μg / min. The filters were replaced every 72 hours. The filters were cut into 2 cm × 2 cm pieces and immersed in double-distilled water. Ultrasonic vibration was performed twice for 20 minutes each time to elute the particles from the filters. The eluate was collected and filtered through 6–8 layers of sterile gauze. The collected suspension was frozen overnight and freeze-dried under vacuum to a dry powder. A 7.5 mg / mL suspension was prepared in phosphate-buffered saline (PBS) for use.

[0040] 1.2.2 Animal grouping and treatment

[0041] Twenty-four mice were randomly divided into Control group, COPD group, AECOPD group, and COPD+PM2.5 group, with 6 mice in each group, and adaptively fed for 1 week.

[0042] COPD models were established in the COPD group, AECOPD group, and COPD+PM2.5 group from 1 to 12 weeks:

[0043] The mice were placed in a 60 cm × 40 cm × 40 cm homemade smoking box and smoked for 1 h each time with 10 Hongtashan cigarettes for 12 consecutive weeks. On the 1st, 15th, and 29th days, 20 μL of lipopolysaccharide (LPS) (0.5 μg / μL) was instilled into the nasal cavity for 3 times. No smoking was performed on the same day. The blank group received nasal instillation of an equal volume of normal saline.

[0044] Establishment of AECOPD model: On the third day of the 13th week, 20 μL of bacterial solution (6×10 14 CFU / mL), twice a day for 4 consecutive days;

[0045] Establishment of the COPD+PM2.5 model: On the third day of the 13th week, PM2.5 suspension (7.5 mg / kg) was instilled into the nasal cavity twice a day for 4 consecutive days;

[0046] The control group and COPD group were given equal amounts of PBS via nasal instillation during the same period.

[0047] Relevant indicators were tested on the 7th day of the 14th week.

[0048] 1.2.3 Pulmonary function tests

[0049] Three mice were randomly selected from each group and anesthetized with 1% sodium pentobarbital (50 mg / kg) by intraperitoneal injection. The trachea was isolated, and a 2 mm incision was made transversely at the cricoid cartilage for endotracheal intubation. The intubation tube was fixed to the trachea with 4-0 sutures to prevent air leakage. The mice were placed supine in a plethysmography chamber, and the endotracheal tube was tightly connected to the instrument interface to prevent air leakage. The pulmonary function system was activated to measure the lung function of the mice. The measurement indicators included forced vital capacity (FVC), inspiratory capacity (IC), and forced expiratory volume in 0.1 second (FEV). 0.1 ), Forced Expiratory Volume in 0.2second (FEV 0.2 ), 0.1 second rate (FEV0.1 / FVC ratio, FEV 0.1 / FVC), FEV0.2 / FVC ratio, FEV 0.2 The test was repeated 3 times and the average value was taken as the measured value for statistical analysis.

[0050] 1.2.4 Blood gas analysis

[0051] Three additional mice in each group were anesthetized with an intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg). Arterial blood samples were collected from the mice using the left ventricular blood sampling method. Blood was drawn into a sodium heparin anticoagulation tube, mixed, and allowed to stand. A blood gas test card was removed from a 4°C refrigerator and allowed to stand at room temperature for at least 5 minutes. The outer packaging was opened, the test card was removed, and 100 μL of anticoagulated whole blood was injected into the injection port. The injection port was then sealed. The test card was inserted into the analyzer and the test results were awaited. Measurements included partial pressure of oxygen (PO2), partial pressure of carbon dioxide (PCO2), pH, and oxygen saturation (SO2).

[0052] 1.2.5 HE staining

[0053] The right lungs of mice in each group were collected, fixed and embedded, and then sliced, stained with hematoxylin-eosin, sealed with neutral gum, and photographed for observation.

[0054] 1.2.6 Statistical analysis

[0055] The results were statistically analyzed using Prism 10.3.0. After normality and homogeneity of variance were tested, ANOVA analysis was performed, followed by Tukey's test to determine specific differences between the groups.

[0056] 2 Results

[0057] 2.1 General

[0058] The mice in the control group were in good spirits, with shiny fur, agile movements, sensitive reactions, and stable breathing. After the COPD modeling was completed, the mice in each pathological model group showed lethargy, slow reactions, reduced activity, rapid breathing, increased oral and nasal secretions, dry and flaky hair, reduced food and water intake, and significantly lower body weight than the control group ( Figure 1 After exposure to KP and PM2.5, the above symptoms in the AECOPD group and the COPD+PM2.5 group were further aggravated. Among them, the oral and nasal secretions in the AECOPD group increased significantly, while the shortness of breath and reduced activity level in the COPD+PM2.5 group were more serious.

[0059] 2.2 Lung function

[0060] FVC, IC, and FEV of COPD group mice 0.1 , FEV 0.2 Significantly higher than the Control group, AECOPD, COPD + PM2.5 group FVC, FEV 0.1 , FEV 0.2 The IC values ​​in the COPD+PM2.5 group were significantly higher than those in the COPD group, while there was no significant difference between the AECOPD group and the COPD group. 0.1 / FVC, FEV 0.2 / FVC showed a decrease in all pathological model groups, and COPD group > AECOPD group > COPD+PM2.5 group, with significant differences among the groups ( Figure 2 shown).

[0061] 2.3 Blood gas analysis

[0062] The PO2 and SO2 of mice in the COPD group were significantly lower than those in the Control group, and PCO2 was significantly higher. Compared with the COPD and AECOPD groups, the PCO2 of the COPD+PM2.5 group was significantly higher, and SO2 was significantly lower. There was no significant change in any of the indicators in the AECOPD group compared with the COPD group ( Figure 3 shown).

[0063] 2.4 Pathology

[0064] Control group: alveolar size was basically uniform, alveolar wall structure was relatively intact, alveolar collapse was not obvious, alveolar wall rupture and fusion were occasionally observed, and alveolar cavity and alveolar wall infiltration was less; COPD group: alveolar size was basically uniform, alveolar collapse was minor, and alveolar cavity and alveolar wall infiltration was minor; AECOPD group: alveolar arrangement was chaotic, with a large number of collapses, no connection between alveolar ducts, and a large number of inflammatory cells between pulmonary epithelial cells compared with COPD group; COPD+PM2.5 group: alveolar arrangement was chaotic, with a large number of collapses, no connection between alveolar ducts, and a large number of inflammatory cells between pulmonary epithelial cells ( Figure 4 shown).

[0065] 3 Discussion

[0066] Based on a COPD mouse model, this study established two AECOPD mouse models by treating them with Klebsiella pneumoniae (KP) and PM2.5, respectively. Results showed that PM2.5 significantly exacerbated COPD pathological features and functional impairment, demonstrating a specific induction of AECOPD. This study provides an experimental basis for exploring the potential mechanism by which PM2.5 may contribute to AECOPD and its differences from infection-induced AECOPD.

[0067] The results showed that PM2.5 treatment significantly aggravated lung dysfunction and blood gas abnormalities compared with the COPD group, especially in FEV 0.1 / FVC, FEV 0.2 The decline in indicators such as FVC / FVC was more obvious, suggesting that PM2.5 can further worsen the airflow limitation of COPD. At the same time, the FVC and IC indicators increased abnormally, indicating lung hyperinflation, which may be due to gas retention and decreased alveolar elasticity caused by airway obstruction. This phenomenon is a typical pathophysiological manifestation in COPD and AECOPD, especially in the PM2.5 exposure group, where the characteristics are more significant. In addition, pathological results showed that PM2.5 treatment caused obvious alveolar collapse and inflammatory cell infiltration, further confirming that PM2.5 caused the aggravation of COPD pathological characteristics.

[0068] Although both PM2.5 and KP treatment induced acute exacerbations in the COPD model, the two treatments exhibited differences in some indicators. For example, oral and nasal secretions in the AECOPD group increased significantly, suggesting that infectious AECOPD may exacerbate the disease primarily through pathogenic irritation of the respiratory mucosa. Mice in the PM2.5 group, on the other hand, exhibited shortness of breath and a significant decrease in activity, potentially indicating deeper airway damage. Combined with blood gas analysis, the PM2.5 group showed a significant increase in PCO2 and a decrease in SO2. This characteristic may reflect that PM2.5 further affects lower respiratory tract function through particle deposition and gas exchange impairment.

[0069] Air pollution is one of the risk factors that cannot be ignored for the occurrence and development of COPD. Previous animal studies have shown that PM2.5 can cause acute lung damage, or long-term exposure can cause COPD. The results of the present invention show that on the basis of COPD, short-term exposure to PM2.5 will lead to acute exacerbation of COPD, providing experimental evidence for the potential role of PM2.5 in inducing acute exacerbation of COPD. By comparison with infectious AECOPD, it is revealed that PM2.5 is an independent cause of acute exacerbation of COPD. Although the mechanism of PM2.5 exposure is not yet fully understood, it may aggravate the COPD condition by inducing oxidative stress, enhancing local immune response, etc. The PM2.5-induced AECOPD model provides an ideal experimental platform for further studying the association mechanism between air pollution and acute exacerbation of COPD. It provides a scientific basis for further exploring the mechanism and intervention strategy of PM2.5-related AECOPD.

[0070] In summary, the present invention experimentally demonstrates that both PM2.5 and bacterial infection can directly induce acute exacerbations of COPD, but their effects differ from those described above. Furthermore, the present invention provides a reliable PM2.5-induced AECOPD mouse model, laying the foundation for studying the mechanisms and interventions for the exacerbation of COPD by air pollution.

Claims

1. A method for constructing a mouse model of acute exacerbation of chronic obstructive pulmonary disease induced by PM2.5, characterized in that: The steps include: Step 1, establishment of COPD mouse model: After one week of adaptive feeding, SPF C57BL / 6 mice were placed in a fumigation chamber and smoked for 1 hour with 10 cigarettes each time for 12 consecutive weeks. Simultaneously, 20 μL (0.5 μg / μL) of lipopolysaccharide (LPS) was instilled into the nasal cavity on days 1, 15, and 29. No fumigation was performed on the day of nasal instillation. Step 2: Establishment of PM2.5-induced COPD mouse model: On the third day of the 13th week, a suspension containing PM2.5 (7.5 mg / kg) was instilled into the nasal cavity twice a day for 4 consecutive days; relevant indicators were tested on the seventh day of the 14th week.

2. The construction method according to claim 1, wherein The steps for preparing the PM2.5-containing suspension are as follows: Atmospheric sampling points were set at an altitude of 70-100 m above the ground. A large-flow air sampler equipped with a PM2.5 cutter was used and the flow rate was set to 1.05 m 2 / min, collect PM2.5 onto a glass fiber filter membrane, and replace the filter membrane every 72 h; cut the sampling filter membrane into 2 cm × 2 cm size, immerse it in double-distilled water solution, and ultrasonically oscillate twice for 20 min each time to elute the particulate matter on the filter membrane. The eluate is collected and filtered through 6-8 layers of sterile gauze. The collected suspension is frozen overnight and vacuum freeze-dried to a dry powder; when used, a 7.5 mg / mL suspension is prepared in phosphate buffered saline (PBS).

3. The method for evaluating the PM2.5-induced acute exacerbation of chronic obstructive pulmonary disease mouse model according to claim 1, wherein: The steps include: Step 1, mouse observation: Observe the mice's mental state, fur gloss, agility, reaction sensitivity, activity, breathing stability, and oral and nasal secretions; Step 2, pulmonary function test: General anesthesia was performed using an intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg). The trachea was isolated, and a 2 mm incision was made transversely at the cricoid cartilage for endotracheal intubation. The intubation was fixed to the trachea with 4-0 sutures to prevent air leakage. The mouse was placed supine in a plethysmography chamber, and the endotracheal tube was tightly connected to the instrument interface to prevent air leakage. The pulmonary function system was activated to measure the mouse's lung function. The measurement indicators included forced vital capacity (FVC), inspiratory capacity (IC), and forced expiratory volume in 0.1 second (FEV). 0.1 ), Forced Expiratory Volume in 0.2 second (FEV 0.2 ), 0.1 second rate (FEV0.1 / FVC ratio, FEV 0.1 / FVC), FEV0.2 / FVC ratio, FEV 0.2 / FVC); the test was repeated 3 times and the average value was taken as the measured value for statistical analysis; Step 3, blood gas analysis: Under general anesthesia with an intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg), arterial blood samples were collected from mice using the left ventricular blood sampling method. Blood was drawn into a sodium heparin anticoagulant tube, mixed, and allowed to stand. A blood gas test card was removed from a 4°C refrigerator and placed at room temperature for at least 5 minutes. The outer packaging was opened, the test card was removed, and 100 μL of anticoagulated whole blood sample was injected into the injection port. The injection port was then sealed. The test card was inserted into the analyzer and the test results were awaited. Measured indicators included partial pressure of oxygen (PO2), partial pressure of carbon dioxide (PCO2), pH, and oxygen saturation (SO2). Step 4, pathological examination: The right lungs of mice in each group were collected, fixed, embedded, and then sectioned, stained with hematoxylin-eosin, mounted with neutral gum, and photographed. The above test results were statistically analyzed using Prism10.3.0; normality and homogeneity of variance were tested, and ANOVA analysis was used, and the Tukey test was used to determine the specific differences between the groups.

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