Animal experiment model for simulating inhalation lung injury as well as reagent and application of animal experiment model

Abstract

The invention relates to a composition of an animal experiment model for simulating inhalation lung injury, the composition is composed of gastric acid simulation liquid and lipopolysaccharide, and the gastric acid simulation liquid is a hydrochloric acid solution with the pH value of 1-3. The invention also provides application of the composition in preparation of an animal experiment model reagent for simulating inhalation lung injury and an animal experiment model for simulating inhalation lung injury. According to the method, the gastric acid simulation liquid and the lipopolysaccharide are used in a combined mode, the inhaled lung injury pathological process under the combined action of gastric acid aspiration and bacterial infection is successfully reproduced, and the clinical correlation of an experimental model is remarkably improved. The pH value and dosage of the instillation liquid are precisely designed, the damage degree can be regulated and controlled, standardization and repeatability of the model are achieved, and the problem that in the prior art, results are unstable is solved. The obvious lung injury can be induced within 24 hours, the experimental period is greatly shortened, and time and resources are saved for pathological research and drug screening.

Description

Technical Field

[0001] The present invention relates to the interdisciplinary field of biomedical engineering and clinical medicine, and in particular to an animal experimental model and composition and application thereof for simulating inhalation lung injury, which are suitable for the study of the mechanism of respiratory diseases, drug screening and the development of treatment methods. Background Art

[0002] In the prior art, experimental models of aspiration lung injury are mainly concentrated in the following two categories:

[0003] Single factor induction model: simple hydrochloric acid inhalation model (intratracheal instillation of hydrochloric acid), sterile gastric content particle inhalation model (small nonacidic gastric particles)

[0004] ① Simple hydrochloric acid inhalation model: In this animal model, simple hydrochloric acid inhalation is used to simulate clinical aspiration. There is no fixed "optimal" dose for the concentration and infusion volume of hydrochloric acid. The pH of hydrochloric acid is usually between 1 and 4 according to the experimental needs, and the volume of hydrochloric acid infusion is also not fixed.

[0005] ② Sterile gastric content particle inhalation model: Tiny non-acidified gastric particles are used to simulate clinical aspiration. Tiny non-acidified gastric particles can generally be prepared from the gastric contents of rodents. Although this model can also cause experimental lung inflammation in experimental animals, it does not have the acute phase of chemical lung injury (such as tissue edema), which is obviously different from the common clinical symptoms of aspiration patients. Therefore, this model is not suitable as a model for clinical aspiration research.

[0006] Composite factor model: combined acid and small particles inhalation model

[0007] ① Hydrochloric acid / gastric content particle combined inhalation model: Hydrochloric acid and tiny non-acidified gastric particles were used in combination to establish an inhalation rat model. The aspiration rat model was established by mixing hydrochloric acid (pH = 1-4) with tiny non-acidified gastric particles and then dripping them into the trachea. This model combines the characteristics of the above two single-factor induced models, but ignores the pathogenic factors and cannot simulate the pathological process of clinical aspiration well.

[0008] The shortcomings or disadvantages of the existing technology are as follows:

[0009] 1. Insufficient clinical relevance: In the existing technology, the aspiration lung injury model induced by gastric acid or tiny non-acidified gastric particles alone cannot reproduce the combined pathological effects of gastric acid aspiration and bacterial infection at the same time, which is inconsistent with clinical reality. This leads to the low applicability of the model in mechanism research and drug evaluation.

[0010] 2. Poor repeatability of the model: In existing studies, the pH value and dosage of the infusion solution often lack precise control, the operation steps are complicated and rely on the experience of the experimenter, resulting in poor repeatability and consistency of the experimental results, which limits the promotion of this model in scientific research.

[0011] 3. Lack of dose-effect relationship: Current technology has not clarified the effects of different gastric acid concentrations and LPS doses on the degree of lung damage, and is unable to quantify the synergistic effect of the two. There is a lack of in-depth exploration and guidance of the pathological mechanism.

[0012] 4. Insufficient standardization: The experimental protocols of existing technologies lack standardized processes, and the methods for establishing animal models are diverse and inconsistent, making it difficult to compare or integrate research results, especially in the fields of drug screening and mechanism verification.

[0013] 5. Complex operation or reliance on expensive equipment: Some existing models require complex equipment (such as sterilization dryers, fine particle crushers, etc.) or high-cost reagents. The experimental operation is cumbersome, which increases the difficulty of the experiment and resource consumption, and is not conducive to widespread application. Summary of the invention

[0014] The purpose of the present invention is to provide an animal model for simulating inhalation lung injury and a method for constructing the same, so as to achieve efficient simulation of the combined effects of gastric acid aspiration and bacterial infection by precisely controlling the pH value and dosage of the instillation solution, and to solve the problems of poor model repeatability, insufficient clinical relevance, and unclear dose-effect relationship in the prior art.

[0015] In a first aspect, the present invention provides a composition for simulating an animal experimental model of inhalation lung injury, wherein the composition is composed of a gastric acid simulation solution and lipopolysaccharide, wherein the gastric acid simulation solution is a hydrochloric acid solution with a pH value of 1-3.

[0016] As a preferred example, the hydrochloric acid solution is 1.5 μl / g.BW, and the lipopolysaccharide is 10-30 μg / g.BW.

[0017] As another preferred example, the gastric acid simulation liquid is a hydrochloric acid solution with a pH value of 1.

[0018] As another preferred example, the lipopolysaccharide is 20 μg / g.BW.

[0019] In a second aspect, the present invention provides use of the composition in preparing an animal experimental model reagent for simulating inhalation lung injury.

[0020] In a third aspect, the present invention provides an animal experimental model for simulating inhalation lung injury, wherein the animal experimental model is induced by the following method: the animal inhales the composition through a tracheal intubation.

[0021] As a preferred example, the interval between inhalation of hydrochloric acid solution and lipopolysaccharide is 1 hour.

[0022] The present invention aims to: provide an experimental model of inhalation lung injury that conforms to clinical practice, which can simultaneously reproduce the interactive pathological mechanism of chemical injury (gastric acid aspiration) and inflammatory response (LPS stimulation); by precisely controlling the pH value of the instillation solution (pH=1-3) and the concentration of LPS (20μg / g.BW,), the degree of injury can be controlled, providing support for studying different stages of the disease; provide a standardized method that is easy to operate and highly repeatable, significantly improving the reproducibility and reliability of the experimental model; and provide an efficient, economical and reliable tool for the study of the pathological mechanism of inhalation lung injury, drug screening and the development of treatment methods.

[0023] The present invention not only optimizes the animal model construction process, but also has high clinical transformation potential, and can be widely used in basic research on respiratory diseases and the development of clinical treatment strategies.

[0024] The advantages of the present invention are:

[0025] 1. Technical advantages

[0026] o Highly close to clinical practice:

[0027] The present invention successfully reproduces the pathological process of aspiration lung injury under the combined action of gastric acid aspiration and bacterial infection by combining the use of gastric acid simulated liquid and lipopolysaccharide (LPS), thereby significantly improving the clinical relevance of the experimental model.

[0028] o Experimental conditions are controllable and highly reproducible:

[0029] The pH value and dosage of the infusion solution are precisely designed to regulate the degree of injury, achieve standardization and repeatability of the model, and avoid the problem of unstable results in existing technologies.

[0030] o Fast and Efficient:

[0031] Significant lung damage can be induced within 24 hours, greatly shortening the experimental cycle and saving time and resources for pathological research and drug screening.

[0032] o Multiple disease stage simulation:

[0033] Through different gradients of pH values ​​and LPS dosages, the present invention can simulate mild, moderate and severe inhalation lung injury to meet different research needs.

[0034] 2. Social Effect

[0035] o Promoting research on the mechanisms of aspiration lung injury:

[0036] The present invention provides a reliable research tool that helps to reveal the pathogenesis of aspiration lung injury, especially the interaction between gastric acid chemical damage and bacterial inflammation, and promotes basic research in related fields.

[0037] o Promote new drug development:

[0038] This model provides a standardized platform for screening and validating anti-inflammatory drugs, antioxidants and other therapeutic options, accelerating the development of new treatments.

[0039] o Improve clinical treatment level:

[0040] Models that simulate various disease stages can be used to evaluate the effectiveness of different treatment options, provide scientific basis for clinicians, and improve patient prognosis.

[0041] 3. Economic Effect

[0042] o Reduce experimental costs:

[0043] The invention is easy to operate, the experiment takes a short time, the required equipment and reagents are economical and easy to obtain, and compared with complex in vitro models or expensive large animal experiments, the experimental cost is significantly reduced.

[0044] o Shorten the new drug development cycle:

[0045] Efficient and reliable models can be used to quickly screen candidate drugs, reduce the waste of ineffective experiments, and significantly improve the efficiency of drug development.

[0046] o Improving the efficiency of translational medicine research:

[0047] By providing standardized and reproducible animal models, we can shorten the time it takes to transform basic research results into clinical applications, creating greater economic value for the medical industry.

[0048] In summary, the present invention provides an important tool for basic research, new drug development and optimization of treatment methods for respiratory diseases by constructing an animal model of inhalation lung injury with strong clinical relevance, simple operation and reliable results. It has significant social and economic value in promoting scientific progress, improving medical standards, and reducing R&D costs, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 : Schematic diagram of experimental technical operations.

[0050] Figure 2 : Determination of the optimal perfusion volume.

[0051] Figure 3 : Determination of hydrochloric acid concentration for intratracheal instillation.

[0052] Figure 4 : Second strike interval assessment.

[0053] Figure 5 :Validation of LPS concentration optimization.

[0054] Figure 6 : Effects of hydrochloric acid combined with LPS on lung function.

[0055] Figure 7 : Changes and time course from 24 hours to 7 days after modeling. DETAILED DESCRIPTION

[0056] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0057] Example 1

[0058] The present invention provides an animal model for simulating aspiration lung injury by endotracheal intubation, aiming to accurately reproduce the pathological process of lung injury under the combined effects of gastric acid aspiration and bacterial infection in clinical practice. Figure 1 .

[0059] 1. Methods

[0060] Infusion solution configuration: Gastric acid simulation solution: hydrochloric acid solution with a pH value of 1-3, a dose of 1.5 μl / g.BW. G-bacterial inflammatory effect simulation: LPS solution: concentration of 20 μg / g.BW.

[0061] Dosage method: Use endotracheal intubation to drip the prepared solution directly into the trachea of ​​the experimental rat to ensure uniform distribution of the drug in the lungs. The drip solution is injected in batches to ensure that the model is successfully induced without causing mechanical damage. The interval between two doses is 1 hour to ensure that the drugs do not affect each other and achieve the best modeling effect.

[0062] Experimental animals: Animal selection: Healthy adult male rats, BW range 140-200g.

[0063] Rearing conditions: standard laboratory environment, temperature 22-25℃, humidity 50-60%, free access to food and water.

[0064] Verification of experimental results: Time point: Observe lung injury 24-72 hours after instillation.

[0065] Main Outcome Measures: Lung tissue pathology (such as H&E staining), inflammatory factor levels (such as TNF-α and IL-6), lung function, etc.

[0066] 2. Experimental results and result verification:

[0067] Verification result 1: See Figure 2 .

[0068] Determination of the optimal perfusion volume (A) Representative images of respiratory flow rate curves and tidal volume curves of rats in the basal state and after instillation of different volumes of normal saline; Quantitative analysis of lung function indicators in the basal state and after instillation of different volumes of normal saline, n = 6: (B) tidal volume; (C) respiratory rate; (D) minute ventilation; (E) peak inspiratory flow rate; (F) inspiratory time; (G) inspiratory flow rate at 50% of maximum inspiratory volume (EF50); (H) Appearance of the lungs after inhalation of different volumes of Evans blue solution. Data are presented as mean ± standard error (SEM), and each point represents an independent experiment. *P < 0.05, significantly different from the corresponding group.

[0069] Final result: The optimal inhalation volume is 1.5 μl / g.BW. This volume will not cause suffocation and respiratory impairment in animals, and can allow the droplets to reach as deep as possible.

[0070] Verification result 2: See Figure 3

[0071] Determination of the concentration of hydrochloric acid for intratracheal instillation (A) Appearance of the lungs and HE staining results after inhalation of normal saline and different concentrations of hydrochloric acid; (B) Lung injury score; (C) Protein concentration in bronchoalveolar lavage fluid (BALF); (D) Heat map of inflammatory factor concentrations; (EG) Levels of inflammatory factors in bronchoalveolar lavage fluid (BALF): IL-6 (E), TNF-α (F), and IL-1β (G).

[0072] Final results: Infusion of hydrochloric acid at pH = 1 can cause the most significant lung damage.

[0073] Verification result 3: See Figure 4 .

[0074] Evaluation of the interval between two blows: (A) tidal volume after saline and hydrochloric acid instillation; (B) inspiratory flow rate at 50% of maximum inspiratory volume (EF50); (C) respiratory rate; (D) minute ventilation; (E) peak inspiratory flow rate; (F) end-expiratory pause time; (G) ratio of expiratory time to inspiratory time;

[0075] Pulmonary and vascular permeability were assessed by FITC-dextran leakage: (H) representative images; (I) fluorescence quantification; (J) pH measurement of bronchoalveolar lavage fluid (BALF).

[0076] Final result: Instillation of hydrochloric acid at pH = 1 can cause chemical pneumonia. One hour interval is the best interval between the second injections, at which LPS is not easily hydrolyzed and has the least effect on respiratory function.

[0077] Verification result 4: See Figure 5 .

[0078] Validation of LPS concentration optimization: (A) Lung appearance, HE staining pathological analysis, and Evans blue evaluation results after inhalation of normal saline and hydrochloric acid combined with different concentrations of LPS; (B) Lung injury score; (C) Survival curve; (D) Evans blue level; (E) Protein level in bronchoalveolar lavage fluid (BALF); (F) Western-blot detection results of cell junction proteins ZO-1 and Occludin; (G) Lung ultrastructure observed by electron microscopy; (H) Blood oxygen partial pressure (PaO2); (I) Carbon dioxide partial pressure (PaCO2); (J) Ratio of blood oxygen partial pressure to inspired oxygen concentration (PaO2 / FIO2).

[0079] Final results: Instillation of lps = 10μg / g.BW, 20μg / g.BW, and 30μg / g.BW after hydrochloric acid instillation can cause different degrees of lung damage in a concentration-dependent manner.

[0080] Verification result 5: See Figure 6 .

[0081] Effects of hydrochloric acid combined with LPS on lung function (A) tidal volume; (B) minute ventilation; (C) respiratory rate; (D) rapid shallow breathing index (RSBI); (E) ratio of expiratory time to inspiratory time; (F) inspiratory flow rate when reaching 50% of maximum inspiratory volume (EF50); (G) enhanced pause (Penh); (H) end-expiratory pause time and peak inspiratory flow rate.

[0082] Final result: HCl (pH=1) + 20μg / g.BW LPS can lead to significant impairment of lung function.

[0083] The model changes and time course from 24 hours to 7 days: see Figure 7 .

[0084] (A) Lung appearance, HE staining pathological results and Evans blue assessment at different time points (24 hours to 7 days) after inhalation of normal saline and hydrochloric acid combined with LPS; (B) lung injury score; (C) relative level of Evans blue; (D) protein level in bronchoalveolar lavage fluid (BALF); (E) body weight monitoring; (F) blood oxygen partial pressure (PaO2); (G) carbon dioxide partial pressure (PaCO2); (H) ratio of blood oxygen partial pressure to inspired oxygen concentration (PaO2 / FIO2); (I) tidal volume; (J) rapid shallow breathing index (RSBI); (K) minute ventilation.

[0085] Final result: The model reaches the maximum damage in 24-48 hours and enters the recovery stage in about 7 days.

[0086] Advantages of the invention

[0087] ① Higher clinical relevance: Compared with the prior art, the present invention realistically reproduces the lung injury process under the combined action of gastric acid aspiration and Gram-negative bacterial infection by instilling gastric acid simulation liquid and LPS. It achieves the synergistic simulation of gastric acid chemical injury and bacterial infection inflammatory response, rather than a single etiology-induced model.

[0088] ②Precisely control experimental conditions: Use gastric acid solutions with different pH values ​​(1-3) to simulate clinical aspiration situations of different severity. Through precise dosage control (1.5μl / g.BW of hydrochloric acid solution, 20μg / g.BW of LPS), the dose-effect relationship of the degree of injury can be studied.

[0089] ③Easy to operate and highly reproducible: The amount and concentration of the instillation solution are optimized to avoid model instability or complicated operation. The standardized experimental operation steps can significantly improve the reproducibility of the model and the reliability of the experimental results.

[0090] ④ The results are more significant and rapid: significant inhalation lung injury can be induced within 24 hours. The model is rapid and suitable for comparison of multiple groups of experiments in a short period of time.

[0091] The animal model constructed by the present invention can be widely used in:

[0092] Study on the mechanism of aspiration lung injury;

[0093] Screening of new anti-inflammatory drugs and antioxidants;

[0094] Preliminary verification of clinical treatment methods;

[0095] Translational medicine research on diseases related to gastric acid aspiration.

[0096] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. A composition for simulating an animal experimental model of inhalation lung injury, characterized in that: The composition consists of gastric acid simulation liquid and lipopolysaccharide, wherein the gastric acid simulation liquid is a hydrochloric acid solution with a pH value of 1-3.

2. The composition according to claim 1, characterized in that The hydrochloric acid solution is 1.5 μl / g.BW, and the lipopolysaccharide is 10-30 μg / g.BW.

3. The composition according to claim 1, characterized in that The gastric acid simulation liquid is a hydrochloric acid solution with a pH value of 1.

4. The composition according to claim 2, characterized in that The lipopolysaccharide described is 20 μg / g.BW.

5. Use of the composition according to any one of claims 1 to 4 in preparing an animal experimental model reagent for simulating inhalation lung injury.

6. An animal experimental model for simulating inhalation lung injury, characterized in that: The animal experimental model is induced by the following method: the animal inhales the composition according to any one of claims 1 to 4 through a tracheal intubation.

7. The animal experimental model for simulating inhalation lung injury according to claim 6, characterized in that: The interval between inhalation of hydrochloric acid solution and lipopolysaccharide was 1 hour.

Images