Preparation and detection method of mouse cough model induced by diesel tail gas particulate matters

Abstract

The invention discloses a preparation and detection method of a mouse cough model induced by diesel tail gas particulate matters, and belongs to the field of biological medicines. Comprising the following steps: preparing a diesel tail gas particulate matter solution DEP; the method comprises the following steps: anesthetizing a mouse, suspending the mouse in a dissecting plate after the mouse is anesthetized, opening an airway by using tweezers, pulling open a tongue, pinching a nose, observing the breath of the mouse, placing a hose needle into the airway, and injecting a diesel tail gas particulate matter solution DEP into a rear airway of the mouse in a tracheal instillation manner when a gas phase is to be inhaled; observing the breath of the mouse and whether the lung of the mouse has wet rale or not, wherein the injection is successful if the wet rale appears; and putting the mouse back into the cage after resuscitation. The prepared and constructed mouse model can effectively simulate the cough symptom caused by diesel exhaust particles, the mouse model can show a stable cough model after DEP induction, and the mouse model is suitable for clarification of a cough mechanism induced by exposure of real particulate matters.

Description

Technical Field

[0001] This invention relates to a method for preparing and detecting a mouse cough model induced by diesel exhaust particulate matter, belonging to the field of biomedicine. Background Technology

[0002] Diesel exhaust particles (DEPs) are a significant component of air pollutants, primarily composed of carbon nuclei, adsorbed organic matter (such as polycyclic aromatic hydrocarbons), and metallic particles. Long-term exposure to DEPs is closely associated with respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD), and cough. Studies have shown that DEPs can exacerbate the cough reflex by inducing airway inflammation, oxidative stress, and altered nerve sensitivity. However, the lack of standardized animal models to investigate the mechanisms by which DEPs induce cough limits the progress in related drug development and toxicity assessment.

[0003] Existing cough studies mostly use guinea pig models, but guinea pigs are expensive, complex to raise, and sensitive to experimental environments (e.g., prone to death due to stress), making them unsuitable for large-scale drug screening. Furthermore, existing models often use capsaicin or ammonia nebulization to induce cough, which, while effectively eliciting the reflex, differs significantly from the DEP exposure mechanisms in real-world environments. The complex toxicity of DEPs (e.g., particulate matter deposition, oxidative damage) cannot be simulated by a single chemical stimulus, resulting in insufficient correlation between the model's pathological mechanisms and actual exposure. Summary of the Invention

[0004] Purpose of the invention: To address the problem that the combined toxicity of diesel exhaust particles (DEPs) (such as particulate matter deposition and oxidative damage) cannot be simulated by a single chemical stimulus, resulting in insufficient correlation between the pathological mechanism of the model and actual exposure, this invention provides a method for preparing a mouse cough model induced by diesel exhaust particles. The mouse model constructed using this method can effectively simulate cough symptoms induced by diesel exhaust particles in the real environment. After induction by diesel exhaust particles, the mouse model can exhibit a stable cough model, which is suitable for elucidating the mechanism of cough induced by diesel exhaust particles.

[0005] Technical solution: To achieve the above objectives, the technical solution adopted by this invention is as follows:

[0006] A method for preparing a mouse cough model induced by diesel exhaust particulate matter includes the following steps:

[0007] Step 1: Preparation of diesel exhaust particulate solution (DEP).

[0008] A certain weight of diesel exhaust particulate matter was obtained, and dimethyl sulfoxide (DMSO) was added as a solvent to aid dissolution. Then, PBS buffer was added to obtain a diesel exhaust particulate matter solution (DEP). The DEP solution was ultrasonically mixed before each use.

[0009] Step 2, mouse selection.

[0010] Select mice of similar weight and acclimatize them for a period of time.

[0011] Step 3: Anesthetize the mice.

[0012] The mice were anesthetized and then suspended in a dissecting board.

[0013] Step 4: Diesel exhaust particulate solution (DEP) dripping.

[0014] For the mouse suspended in the dissecting board in step 3, use forceps to open the airway, pull back the tongue, and pinch the nose to observe the mouse's breathing. Insert a soft tube into the airway, and during the inspiratory phase, administer diesel exhaust particulate matter solution (DEP) into the mouse's posterior airway via intratracheal drip. Observe the mouse's breathing and listen for moist rales in the lungs; the presence of moist rales indicates successful injection. After resuscitation, return the mouse to its cage.

[0015] Preferred: The modeling time is more than one week in total, and diesel exhaust particulate matter solution (DEP) is dripped into the gas tube more than once a week.

[0016] Preferably, the concentration of the diesel exhaust particulate matter solution (DEP) is 0.3-0.7 mg / mL.

[0017] Preferably, the ultrasonic mixing time is 10-20 minutes.

[0018] Preferred method: Use aphrodisiac mice.

[0019] Another objective of this invention is to provide a method for detecting the sensitivity of mice to cough. This method is used to detect a mouse cough model induced by diesel exhaust particulate matter. The mouse cough model includes: placing mice in a sealed glass tank using a whole-body volume plethysmography (WBP) system; administering atomized citric acid or capsaicin to induce coughing, given a specific airflow velocity in the WBP system; and recording the number of coughs occurring within a certain period after the start of nebulization using a sensor. During observation, any respiratory behavior that results in a change in intra-tank air pressure greater than a given unit is recorded as a cough. If the number of coughs occurring within a certain period after the start of nebulization is significantly different from that of the control group, it is considered a significant increase in cough frequency, indicating successful mouse cough modeling.

[0020] Preferred concentration: citric acid or capsaicin concentration is 0.2-0.4 M per 1 mL.

[0021] Preferred: The sensor records the number of coughs that occur within 5-10 minutes after the nebulization begins.

[0022] Preferably, the flow rate of the biased airflow in the whole-body volume plethysmography system is 0.1-0.3 mL / min.

[0023] Preferably, the given unit is 38-42 units.

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

[0025] 1. The mouse model constructed using the preparation method of the present invention can effectively simulate the cough symptoms induced by diesel exhaust particles. After DEP induction, the mouse model can exhibit a stable cough model, which is suitable for elucidating the mechanism of cough induced by real particulate matter exposure.

[0026] 2. The C57BL / 6 mice used in this model have a clear genetic background and are less expensive than guinea pig models. Attached Figure Description

[0027] Figure 1 DEP exposure timeline;

[0028] Figure 2 This is a schematic diagram of endotracheal infusion.

[0029] Figure 3 Changes in body weight before and after the experiment;

[0030] Figure 4 Changes in cough sensitivity in mice;

[0031] Figure 5 To detect the cough sensitivity of mice induced by citric acid cough;

[0032] Figure 6 To detect cough sensitivity in mice induced by capsaicin cough;

[0033] Figure 7 This represents the total number of Balf cells in mice.

[0034] Figure 8 Pathological sections of mouse lungs and evaluation of inflammation (20X); black dots represent DEP. Detailed Implementation

[0035] The present invention will be further illustrated below with reference to the accompanying drawings and specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention. After reading this invention, any modifications of the invention in various equivalent forms by those skilled in the art will fall within the scope defined by the appended claims.

[0036] Example 1

[0037] Existing cough studies mostly use guinea pig models, but guinea pigs are expensive, complex to raise, and sensitive to experimental environments (e.g., prone to death due to stress), making them unsuitable for large-scale drug screening. Furthermore, existing models often use capsaicin or ammonia nebulization to induce cough, which effectively triggers the reflex, but differs significantly from the DEP (distilled particulate matter) exposure mechanisms in real-world environments. The complex toxicity of DEPs (e.g., particulate matter deposition, oxidative damage) cannot be simulated by a single chemical stimulus, resulting in insufficient correlation between the model's pathological mechanisms and actual exposure. Therefore, this embodiment provides a method for preparing a mouse cough model induced by diesel exhaust particulate matter, including the following steps:

[0038] Step 1: Preparation of diesel exhaust particulate solution (DEP).

[0039] Weigh 10 mg of DEP using a 15 mL centrifuge tube. Add 100 μL of dimethyl sulfoxide (DMSO) to aid dissolution. Add 10 mL of PBS buffer. Sonicate for 15 minutes to obtain a diesel exhaust particulate matter (DEP) solution with a concentration of 0.5 mg / mL. Sonicate for 15 minutes before each use. In another embodiment, by adjusting the diesel exhaust particulate matter content, a DEP concentration of 0.3 mg / mL can be obtained. In another embodiment, by adjusting the diesel exhaust particulate matter content, a DEP concentration of 0.7 mg / mL can be obtained. In another embodiment, sonicate for 10 minutes before each use; in another embodiment, sonicate for 20 minutes before each use.

[0040] Step 2, mouse selection.

[0041] C57BL / 6 mice with similar body weight were selected and randomly divided into a blank control group and a cough detection group (divided into two groups: 30μg DEP group and 60μg DEP group) after 3 days of adaptive feeding, with 8 mice in each group.

[0042] Step 3: Anesthetize the mice.

[0043] Except for the blank control group, all mice were anesthetized with 40 uL (considering loss) of aphthylamine. After anesthesia, the mice were suspended in a dissecting plate.

[0044] Step 4: Diesel exhaust particulate solution (DEP) dripping.

[0045] Gently open the airway of the mouse suspended in the dissecting board in step 3 using forceps, pull back the tongue, and pinch the nose. Observe the mouse's breathing. Insert a soft tube into the airway, and during the inspiratory phase, administer 30 μg and 60 μg of diesel exhaust particulate matter solution (DEP) into the mouse's posterior airway via intratracheal drip. Observe the mouse's breathing and listen for moist rales in the lungs. The presence of moist rales indicates successful injection. After resuscitation, return the mouse to its cage.

[0046] The mouse cough model was established over a period of two weeks, with DEP administered via intratracheal infusion once a week at a dose of 30 or 60 μg. Cough detection and sampling were performed at the end of the two weeks. The overall DEP exposure timeline is shown below. Figure 1 DEP infusion method see Figure 2 During the two-week rearing period, mice were given free access to food and water, and their weight was recorded daily. The number of coughs was also recorded on days 1, 4, 7, 11, and 14. In another embodiment, the modeling period lasted one week, with DEP (diesel exhaust particulate matter) solution administered via tracheal infusion twice per week. In yet another embodiment, the modeling period lasted three weeks, with DEP solution administered via tracheal infusion once per week.

[0047] 1. This invention employs DEP intratracheal instillation: Intratracheal instillation allows for the direct, quantitative injection of DEP into the posterior airway, ensuring a consistent deposition of particulate matter in the lungs, and emphasizing that the primary pathogenic effects of DEP are concentrated in the lungs (e.g., oxidative damage, inflammatory response). Compared to nasal inhalation, intratracheal instillation avoids dosage deviations caused by differences in respiratory rate in mice or nasal filtration mechanisms (e.g., nasal cilia or mucus barrier), thus more realistically simulating particulate matter deposition patterns in the lungs.

[0048] 2. The DEP cough model of this invention is applicable to cough induced by real particulate matter exposure:

[0049] ①DEP contains various chemical components, including polycyclic aromatic hydrocarbons, heavy metals, and organic carbon, which are also commonly found in real particulate matter exposure. The particle size distribution of DEP is also similar to that of real particulate matter, including PM2.5 and ultrafine particles. These fine particles can penetrate deep into the respiratory tract and even the alveoli, coming into contact with more respiratory tissues and triggering coughing.

[0050] ② DEP infusion can induce pulmonary oxidative stress (elevated ROS and RNS levels) and inflammatory responses (IL-6 and TNF-α release), both of which are important factors leading to cough. The DEP cough model simulates long-term pulmonary exposure to particulate matter through intratracheal infusion (once a week for two weeks), effectively allowing for the observation of dynamic changes in mouse pathological sections and pulmonary inflammation. This model shows a high degree of consistency with the long-term exposure effects of human nasal inhalation of DEP in presenting particulate matter deposition and the resulting pathological mechanisms.

[0051] ③ The DEP cough model of this invention can precisely control parameters such as particulate matter concentration and exposure time under experimental conditions, which is helpful for studying the dose-response and time-response relationships of particulate matter-induced cough. Compared with complex real particulate matter exposure models, the construction of the DEP cough model is relatively simple, and the required equipment and material costs are lower. In addition, in the DEP cough model, a variety of physiological and biochemical indicators can be easily measured, making it highly operable.

[0052] 3. Limitations of the nasal inhalation DEP cough model: When using nasal inhalation of DEP, individual differences in respiratory rate and depth lead to significant variations in DEP exposure among mice. Furthermore, nasal inhalation of DEP may induce nasal mucosal inflammation, resulting in a cough response, which can obscure lung-specific analyses of the DEP-induced cough mechanism.

[0053] 4. The differences between the DEP cough model of the present invention and the general capsaicin cough model:

[0054] ①Different mechanisms triggering cough:

[0055] The DEP cough model of the present invention: DEP contains a large number of pro-oxidative components, which mainly activate the cough reflex through oxidative stress (increased ROS and RNS levels) and inflammatory response (release of IL-6 and TNF-α), involving the MAPK cascade signaling pathway.

[0056] Capsaicin cough model: Capsaicin can directly stimulate TRPV1 receptors, depolarize nerve cells, activate the vagus nerve pathway, and lead to the release of neuropeptides (such as SP and NKA), which increases vascular permeability, causing local tissue edema and triggering a cough reflex.

[0057] ② Differences in pathological characteristics:

[0058] The DEP cough model of this invention simulates a long-term process that may be accompanied by alveolar structural disorder, decreased antioxidant enzyme activity and chronic inflammation, and is closer to chronic cough induced by environmental pollutants.

[0059] Capsaicin cough model: mainly characterized by acute cough with mild pathological changes, it can be used for screening antitussive drugs.

[0060] ③ Differences in applicable research scope:

[0061] The DEP cough model of this invention is suitable for environmental toxicology studies to explore the comprehensive effects of long-term particulate matter exposure on the respiratory system (such as oxidative stress and immune regulation).

[0062] Capsaicin cough model: suitable for studying the neuromodulation mechanism of cough and evaluating the efficacy of antitussive drugs.

[0063] Example 2

[0064] This embodiment provides a method for detecting the susceptibility of mice to cough, used to detect a mouse cough model induced by diesel exhaust particulate matter, and includes the following steps:

[0065] 1. Changes in mouse body weight before and after experiments

[0066] See details Figure 3 As the dosage increased (from saline to 30 μg DEP and then to 60 μg DEP), the average weight change gradually decreased, and there was no statistically significant difference between the 60 μg group and the 30 μg group (p = 0.572). However, the weight change in the 60 μg group was significantly different from that in the Saline group.

[0067] 2. Changes in cough sensitivity in mice

[0068] See details Figure 4 The number of coughs in mice was recorded during the experiment. As the experiment progressed, the number of coughs in the 60 μg group gradually increased, especially on day 14, when the number of coughs in the 60 μg group was significantly higher than in the other two groups. The increase in the number of coughs in the Saline and 30 μg groups was not significant. Increasing the experimental treatment dose may enhance the cough sensitivity of mice.

[0069] 3. Cough sensitivity test in mice

[0070] The mouse cough susceptibility testing protocol utilizes a whole-body volume plethysmography (WBP) system. Mice are placed in a 1000 ml transparent, sealed glass container and allowed to acclimatize for 1 minute. Coughing is induced by nebulized 0.3 M 1 mL of citric acid or capsaicin. In another embodiment, the concentration of citric acid or capsaicin is 0.2 M 1 mL; in yet another embodiment, the concentration is 0.4 M 1 mL. The number of coughs occurring within 6 minutes of nebulization initiation is recorded by a sensor. In another embodiment, the number of coughs occurring within 5 minutes of nebulization initiation is recorded by the sensor; and in yet another embodiment, the number of coughs occurring within 10 minutes of nebulization initiation is recorded by the sensor. The deflection flow rate of the WBP system is 0.2 mL / min. In another embodiment, the deflection flow rate is 0.1 mL / min. In yet another embodiment, the deflection flow rate is 0.3 mL / min. During the observation period, all respiratory behaviors that resulted in intracytoplasmic pressure changes greater than 40 units were recorded as coughs. In another embodiment, all respiratory behaviors that resulted in intracytoplasmic pressure changes greater than 38 units were recorded as coughs. In yet another embodiment, all respiratory behaviors that resulted in intracytoplasmic pressure changes greater than 42 units were recorded as coughs. A significant difference in the number of coughs produced within 6 minutes of nebulization compared to the Saline group was considered a significant increase in cough frequency, indicating successful cough modeling in mice.

[0071] 1) Citric acid triggers cough

[0072] See details Figure 5 With increasing dosage (from Saline to 30 μg DEP and then to 60 μg DEP), the number of coughs in mice stimulated by citric acid increased significantly. The 60 μg group showed a statistically significant difference in coughing under citric acid stimulation compared to the Saline group.

[0073] 2) Capsaicin triggers cough

[0074] See details Figure 6 The difference between the 30 μg group and the Saline group was not statistically significant (p = 0.323). The differences between the 60 μg group and both the Saline and 30 μg groups were statistically significant. With increasing dosage (from Saline to 30 μg and then to 60 μg), the number of coughs in mice stimulated by capsaicin gradually increased, especially in the 60 μg group, where the number of coughs was significantly higher than in the other two groups.

[0075] 4. Total number of BALF cells in mice

[0076] See details Figure 7Airway and lung lavage fluid were collected from mice to observe the total number of cells in the bronchoalveolar lavage fluid (BALF). There was no statistically significant difference between the 30 μg group and the Saline group (p = 0.947). Statistically significant differences were observed between the 60 μg group and both the Saline and 30 μg groups. With increasing dosage (from Saline to 30 μg and then to 60 μg), the total number of cells in the mouse BALF significantly increased. The experimental dosage increased the degree of airway inflammation, leading to more inflammatory cells entering the alveolar space.

[0077] 5. Pathological sections and inflammation evaluation of mouse lungs

[0078] See pathology slides for details. Figure 8 Saline group: Lung tissue structure was relatively normal, with no obvious inflammatory cell infiltration. Alveolar structure was clear, without obvious destruction or signs of inflammation. 30μg DEP group: Some inflammatory cell infiltration began to appear in the lung tissue, and slight destruction of alveolar structure was observed in some areas. 60μg DEP group: Inflammatory cell infiltration in the lung tissue increased significantly, and alveolar structure destruction was more obvious, showing a more severe inflammatory response. With increasing dosage (from Saline to 30μg and then to 60μg), the inflammatory response in the mouse lungs gradually worsened. With increasing experimental treatment dosage, the degree of lung inflammation also increased accordingly, leading to more inflammatory cell infiltration and alveolar structure destruction. At the same time, DEP accumulation was more obvious in the lung tissue of the 60μg group, and the distribution of DEP was correlated with the severity of the inflammatory response.

[0079] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a mouse cough model induced by diesel exhaust particulate matter, characterized in that, Includes the following steps: Step 1, Preparation of Diesel Exhaust Particulate Solution (DEP); A certain weight of diesel exhaust particulate matter was obtained, and dimethyl sulfoxide (DMSO) was added as a solvent to aid dissolution. Then, PBS buffer was added to obtain a diesel exhaust particulate matter solution (DEP). The diesel exhaust particulate matter solution (DEP) was ultrasonically mixed before use. Step 2, Mouse selection; Select mice of similar weight and acclimatize them for a period of time. Step 3, anesthetize the mice; The mice were anesthetized and then suspended in a dissecting board. Step 4, Diesel Exhaust Particulate Solution (DEP) dripping; In step 3, use forceps to open the airway of the mouse suspended in the dissection board, pull open the tongue and pinch the nose at the same time, observe the mouse's breathing, insert a soft tube into the airway, and when the inspiratory phase is reached, inject diesel exhaust particulate matter solution (DEP) into the mouse's posterior airway by endotracheal drip; observe the mouse's breathing and listen for moist rales in the mouse's lungs. The presence of moist rales indicates successful injection; after the mouse is revived, put it back in the cage.

2. The method for preparing a mouse cough model induced by diesel exhaust particulate matter according to claim 1, characterized in that: The modeling process takes more than one week, with diesel exhaust particulate matter solution (DEP) administered via infusion at least once a week.

3. The method for preparing a mouse cough model induced by diesel exhaust particulate matter according to claim 2, characterized in that: The concentration of the diesel exhaust particulate solution (DEP) is between 0.3 and 0.7 mg / mL.

4. The method for preparing a mouse cough model induced by diesel exhaust particulate matter according to claim 3, characterized in that: The ultrasonic mixing time is 10-20 minutes.

5. The method for preparing a mouse cough model induced by diesel exhaust particulate matter according to claim 4, characterized in that: Mice were anesthetized with Avdin.

6. A method for detecting the cough sensitivity of a mouse cough model obtained by the method for preparing a diesel exhaust particulate matter-induced mouse cough model according to claim 1, characterized in that, include: Using a whole-body volume plethysmography system, mice were placed in a sealed glass tank. The system was given a deflection airflow rate, and nebulized citric acid or capsaicin was administered to induce coughing. The number of coughs generated within a certain period after the nebulization began was recorded by a sensor. During the observation process, all respiratory behaviors that produced changes in intracavitary air pressure greater than a given unit were recorded as coughs. If the number of coughs produced within a certain period after the start of nebulization was significantly different from that of the control group, it was judged that the cough frequency had increased significantly, indicating that the mouse cough model was successful.

7. The method for detecting mouse cough sensitivity according to claim 6, characterized in that: The concentration of citric acid or capsaicin is 0.2-0.4M per 1mL.

8. The method for preparing a mouse cough model induced by diesel exhaust particulate matter according to claim 7, characterized in that: The sensor records the number of coughs that occur within 5-10 minutes after the nebulization begins.

9. The method for preparing a mouse cough model induced by diesel exhaust particulate matter according to claim 8, characterized in that: The flow rate of the biased gas flow in the whole-body volume plethysmography system was 0.1-0.3 mL / min.

10. The method for preparing a mouse cough model induced by diesel exhaust particulate matter according to claim 9, characterized in that: The given units are 38-42 units.

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