A method for constructing a rat model of acute lung injury

Abstract

The present application aims to develop an animal model which can focus on the core symptoms of acute lung injury and can well evaluate the drug efficacy of acute lung injury. The rat acute lung injury model constructed in the present application shows a high success rate and stability in inducing classic symptoms of acute injury (dyspnea, pulmonary edema and pulmonary fibrosis, etc.), as well as symptoms such as depression, alveolar atrophy, alveolar inflammatory infiltration, and reduced blood oxygen of acute lung injury. In the construction method of the rat acute lung injury model of the present application, the rat acute lung injury model is obtained by intraperitoneal injection of cholic acid and mixed solution of polyethylene glycol and polysorbate + heat stress + silica intake + exercise induction. This non-single factor induced acute lung injury animal model is consistent with the etiology that acute lung injury is induced by multiple factors, and can better simulate the characteristics of human acute lung injury.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of animal model construction of children diseases, and particularly relates to a method for constructing a rat acute lung injury model. BACKGROUND

[0002] Acute lung injury (ALI) and its severe form acute respiratory distress syndrome (ARDS) are non-cardiogenic respiratory failure characterized by acute onset, refractory hypoxemia, pulmonary edema and epithelial damage. The typical pathological changes are diffuse inflammatory injury, destruction of respiratory membrane and vascular tissue structure. The current clinical treatment of acute lung injury mainly focuses on mechanical ventilation, lacks specific drug treatment, and the mortality rate is as high as 35%-46%. The clinical manifestations of acute lung injury are panting, fullness, coma and fever, among which panting and fullness are more common. Most doctors classify it into the category of "sudden panting" in traditional Chinese medicine, including "panting" and "panting death". When acute lung injury occurs, acute systemic inflammatory response can cause microvascular injury, increase the permeability of lung blood vessels and epithelium, and cause non-cardiogenic pulmonary edema, alveolar-capillary reduction, and further lead to hypoxic respiratory failure and acute respiratory distress syndrome. The clinical manifestations are obvious hypoxemia, bilateral diffuse lung infiltration, pulmonary edema and decreased lung compliance. The pathological changes include increased vascular permeability caused by alveolar capillary barrier damage, alveolar hemorrhage and fibrin deposition.

[0003] Acute lung injury is a common clinical critical illness, which is caused by various pathogenic factors inside and outside the lung other than cardiogenic factors, leading to acute and progressive hypoxic respiratory failure. Clinically, it is characterized by early manifestations of acute and progressive dyspnea, respiratory distress, refractory hypoxemia and non-cardiogenic pulmonary edema. Although there has been great progress in airway management and protective mechanical ventilation strategies in recent years, the etiology and pathogenesis are complex and have not been fully elucidated. Acute lung injury has numerous pathogenic links and lacks specific treatment measures, which is extremely destructive and seriously affects the quality of life and prognosis of patients hospitalized in critical care medicine. Therefore, the mortality rate caused by acute lung injury is high, and acute lung injury has become a research hotspot and difficulty in clinical critical care medicine. Acute lung injury and more severe acute respiratory distress syndrome are the main causes of acute respiratory failure in clinical critical illness. The causes are diverse and can be classified into two categories, including lung factors and extrapulmonary factors. Lung factors include: (1) chemical factors, such as inhalation of smoke dust, nanoparticles and oxygen poisoning, etc.; (2) physical factors, such as lung contusion and radiation injury, etc.; (3) biological factors, such as severe pneumonia. Extrapulmonary factors include severe shock, septic toxicity, severe non-thoracic trauma, severe burns, massive blood transfusion, acute pancreatitis and drug poisoning, among which infection occupies a dominant position.

[0004] Many studies have assessed the efficacy of various treatments, but the establishment of a reasonable and effective animal model of acute lung injury is the premise for the effective development of these studies. The establishment of an in vivo model is an important means of studying the development of the disease, the mechanism of the disease, the effect of prevention and treatment, and the prognosis of the disease. The in vivo model of acute lung injury is designed based on common pathogenic factors in clinical practice, and most of them have one or more characteristics of human acute lung injury. Secondly, the in vivo model of acute lung injury is established in mammals, including rats, rabbits, and primates. Among them, rodents such as mice or rats are widely used because of their fast reproduction, low price, and easy source. However, the lungs of humans and animals are not completely the same in terms of anatomical structure and physiological function, and their responses to acute injury stimuli are also different, which greatly affects the evaluation of lung injury. The establishment of an acute lung injury model has many ways and influencing factors, so the current in vivo model has not been able to completely replicate all the characteristics of human acute lung injury. The current commonly used modeling methods include: endotoxin method, which is easy to observe the changes of inflammatory response, easy to repeat, but the changes of alveolar capillary permeability are not obvious. Oleic acid method, easy to repeat, suitable for simulating lung injury caused by fat embolism, but cannot simulate lung injury caused by sepsis, and is more difficult to operate. High oxygen method, easy to repeat, but difficult to operate. Mechanical ventilation method, easy to observe the changes of lung ventilation function, but difficult to operate with clinical significance. Pathogen method, easy to observe the changes of inflammatory response, more targeted, easy to repeat, but with large species differences. Smoke method, easy to repeat, need appropriate experimental equipment, but difficult to operate. Cecum ligation and puncture method, easy to observe the changes of inflammatory response and alveolar capillary permeability, but need appropriate experimental equipment, difficult to operate.

[0005] The choice of animal is an important factor in determining the success of animal experiments in pathophysiology experiments. Generally, for experimental purposes, the choice of experimental animal species is based on the biological characteristics of the experimental animal and the experience of replicating animal disease models. According to the use of experimental animals in China and the commonly used experimental animals reported in foreign literature, the most commonly used experimental animal species are: mice, rats, hamsters, guinea pigs, rabbits, dogs, etc. Among them, mice are the most commonly used experimental animal species. They have many strains, a wide range of options, a short reproductive cycle, strong fertility, low cost, and a good understanding of their biological characteristics. Genetic research is in-depth and extensive, and experimental data is abundant, so they are popular among researchers. In the process of studying the pathogenesis of lung injury and developing drugs for the clinical treatment of lung injury, ethical and research funding issues have greatly limited research in patients or large mammals. However, rats are often used as experimental animals when researchers construct or modify lung injury models. Therefore, successfully constructing a mouse model of lung injury is crucial for lung injury-related research. Previous animal models of lung injury were mainly divided into invasive and non-invasive methods. Invasive methods involve surgically exposing the trachea, such as the method used by Wang Ting et al. to construct an acute lung injury model by isolating and exposing the trachea, intubating, and then instilling endotoxin solution through the bronchus. Specifically, adult SD rats were divided into normal, tracheal instillation of LPS (LPS-IT), and sham operation (SHAM) groups, with ten rats in each group. The normal group did not undergo any intervention. The LPS-IT group was anesthetized with intraperitoneal injection of chloral hydrate (8%, 1 mL), fixed, tracheal exposure, intubation, and slow instillation of endotoxin solution (5 mg / kg, 100 μL) through the bronchus, and the wound was sutured. The SHAM group was intubated as in the LPS-IT group, and 100 μL of normal saline was instilled into the trachea. Twenty-four hours after modeling, blood was taken from the abdominal aorta, the entire lung was removed, the right lung was ligated, and subsequent analysis was performed. However, for mouse models, invasive methods have the following disadvantages: (1) They are invasive, with a high risk of infection during surgery, and require deep anesthesia of the mouse, resulting in a high mortality rate during and after surgery; (2) Instilling particulate suspensions can easily block the trachea and cause animals to suffocate; (3) Instilling solutions can cause uneven distribution of liquid in the lungs. Therefore, using mice for modeling has a high mortality rate and low success rate, which affects the repeatability of the experiment. Some researchers use the method of instilling drugs through the nose to construct models (such as Chinese patent CN201910793141.6), but this method involves nasal administration, and some literature reports that most drugs will remain in the nasal cavity, resulting in a significant reduction in the amount of drug entering the lungs, which affects the strength of the drug's effect on lung injury. Moreover, the amount of drug entering the lungs is not stable between samples, which affects the stability of the experiment. The above methods still have the disadvantages of unstable modeling, low success rate, etc. Therefore, we propose a simple and easy-to-control method for constructing a rat model of acute lung injury that is reliable, stable, and cost-effective. SUMMARY

[0006] The present application aims to develop an animal model which can focus on the core symptoms of acute lung injury and can well evaluate the drug efficacy of acute lung injury. The rat acute lung injury model constructed in the present application shows a high success rate and stability in inducing classic symptoms of acute injury (dyspnea, pulmonary edema and pulmonary fibrosis, etc.), as well as symptoms accompanying acute lung injury such as depression, alveolar atrophy, alveolar inflammatory infiltration, and reduced blood oxygen. The present research can provide a reliable animal model for the evaluation of acute lung injury drug efficacy, which has the advantages of low cost, stable model, simple operation, and non-invasive, etc.

[0007] The present application is realized by the following technical solutions:

[0008] A construction method of a rat acute lung injury model, characterized in that it comprises the following steps:

[0009] (1) Intraperitoneal injection: 4-week-old SD rats are selected, and are raised under SPF conditions. After adaptive feeding for two weeks, modeling is started. The total modeling time is four weeks. After the start of modeling, the rats are intraperitoneally injected with cholic acid and a mixed solution of polyethylene glycol and polysorbate, once every two days. At each injection, the rats are first intraperitoneally injected with cholic acid, and then intraperitoneally injected with the mixed solution of polyethylene glycol and polysorbate 30 minutes later. The cholic acid is dissolved with DMSO and then diluted with physiological saline to a final concentration of 1 mmol / L. The injection dose of cholic acid is 0.01 mmol / kg of body weight each time. The mixed solution of polyethylene glycol and polysorbate is prepared by dissolving polyethylene glycol and polysorbate in physiological saline. The injection doses of polyethylene glycol and polysorbate are 80-100 μg / kg and 100-120 μg / kg of body weight, respectively.

[0010] (2) Heat stress: One week after the start of modeling, the rats are placed in a high-temperature incubator at 38±1℃ for 4 hours every day, for a total of 7 days.

[0011] (3) Silicon dioxide intake: Two weeks after the start of modeling, silicon dioxide is added to the regular feed of the rats, and the addition is continued until the end of modeling. The added amount of silicon dioxide is 3% of the weight of the regular feed.

[0012] (4) Exercise induction: Three weeks after the start of modeling, the rats are made to exercise at least 2 hours a day using a pet running wheel toy. The pet running wheel toy uses food as an inducement, and the exercise is continued for one week, thereby obtaining a rat acute lung injury model.

[0013] After four weeks of modeling, the model is evaluated by observing the general signs of the rats, HE staining of lung tissue, measuring the wet / dry weight ratio of lung tissue, detecting the content of inflammatory factors and oxidative free radical related indicators in lung tissue by ELISA, lung function detection, chest X-ray, observing the general changes of lung tissue, detecting arterial blood oxygen partial pressure and oxygenation index, and detecting the expression of AQP4 and p-NF-κB, Na+-K+-ATPase α1 protein in lung tissue, etc. The results show that the rats have typical symptoms of lung injury, which meets the reliability and effectiveness of the animal model of acute lung injury, that is, the cause of the disease, the symptoms, and the pathophysiology. According to the construction method of the present application, the rat model of acute lung injury can be successfully obtained.

[0014] The technical scheme of the present application has the following advantages:

[0015] 1. In the construction method of the rat model of acute lung injury, the rat model of acute lung injury is obtained by intraperitoneal injection of cholic acid, and a mixture of polyethylene glycol and polysorbate + heat stress + intake of silicon dioxide + exercise induction. This non-single factor induced acute lung injury animal model is consistent with the view that acute lung injury is induced by multiple factors in etiology, and can better simulate the characteristics of human acute lung injury. The non-single factor induced acute lung injury animal model of the present application basically meets three effectivenesses, whether from the perspective of pathophysiology theory or from the perspective of simulating typical clinical symptoms and treatment prediction, it is a relatively ideal animal model, which provides support for the multiple factor induced pathogenesis of acute lung injury, and provides a theoretical basis for developing new schemes for treating acute lung injury.

[0016] 2. The acute lung injury animal model constructed by the present application has stable effect and small individual difference; the multiple factors synergize with each other, and the constructed disease model is more accurate. The model shows a high success rate and stability in inducing classic symptoms of acute lung injury (dyspnea, pulmonary edema and pulmonary fibrosis, etc.), as well as symptoms such as depression, alveolar atrophy, alveolar inflammatory infiltration, and reduced blood oxygen.

[0017] 3. The acute lung injury animal model of the present application is a non-single factor induced acute lung injury animal model, which provides support for the multiple factor induced pathogenesis of acute lung injury, and better simulates the advantages of human acute lung injury, thereby providing an important theoretical basis for developing a new drug preparation for effectively treating acute lung injury. Not only can it be used to explore the scientific connotation of multiple factors on acute lung injury, but also can provide an animal model reflecting the essence of the disease for screening new drugs.

[0018] 4. This invention employs intraperitoneal injection of bile acids and an induction method involving a mixture of polyethylene glycol (PEG) and polysorbate. Bile acids are important pro-inflammatory factors, and intraperitoneal injection can induce lung injury in animals to a certain extent. The PEG and polysorbate mixture can enhance the expression of phosphorylated signal transducers and transcription activator 3 (T3), and can exacerbate bile acid-induced inflammatory damage by regulating JAK2 / STAT3 signaling. Injecting the PEG and polysorbate mixture half an hour after intraperitoneal injection of bile acids allows the PEG and polysorbate to synergistically assist bile acids in inducing lung injury in rats. The combination of these three reagents significantly optimizes the induction of lung injury in rats, facilitating the establishment of an acute lung injury model in rats. One week after the start of intraperitoneal injection, rats were subjected to a week-long heat stress to further stimulate the lungs, greatly consolidating the induction effect of intraperitoneal injection and also further aggravating the pathological changes in the rat lungs.

[0019] 5. This invention employs an induction method involving the addition of silica to the rats' regular diet. Adding an appropriate amount of silica to the rats' regular diet during the later stages of modeling can, to a certain extent, exacerbate the inflammatory response in the rats' lungs. Silica is an exogenous inflammatory inducer that can enhance the inflammatory response in the lungs and aggravate inflammatory symptoms; it also exacerbates the inflammatory response by promoting the release of inflammatory mediators, such as inflammatory cytokines and interleukins. In this invention, exercise induction was performed on the rats during the last week of modeling. After silica aggravated the inflammatory response in the lungs, the subsequent exercise induction significantly increased the sensitivity of the rats' lungs and exacerbated the pathological response in the lungs, further inducing the development of pulmonary edema and fibrosis. The exercise induction during the last week of modeling played a crucial role in this study, ensuring a stable acute lung injury animal model.

[0020] This study provides strong theoretical support and a model reference for a deeper understanding of the pulmonary immune mechanisms following acute lung injury (ALI) and for evaluating new treatment methods. The research team has repeatedly validated the model, demonstrating its reliability. The model has also been used to evaluate the efficacy of various drugs, validating its application value. This animal model exhibits typical characteristics of ALI: dyspnea, pulmonary edema, and pulmonary fibrosis. This model can be used for basic research in the field of ALI, laying the experimental animal foundation for exploring the pathogenesis of ALI. This study provides a reliable animal model for evaluating the efficacy of drugs in ALI, offering advantages such as low cost, model stability, ease of operation, and non-invasiveness. Detailed Implementation

[0021] A method for constructing a rat acute lung injury model, characterized by comprising the following steps:

[0022] (1) Intraperitoneal injection: 4-week-old SD rats were selected and fed under SPF conditions. After adaptive feeding for two weeks, modeling was started. The total modeling time was four weeks. After the start of modeling, the rats were intraperitoneally injected with cholic acid and a mixture of polyethylene glycol and polysorbate. The injection was performed every two days. At each injection, the rats were first intraperitoneally injected with cholic acid, and then intraperitoneally injected with a mixture of polyethylene glycol and polysorbate 30 minutes later. The cholic acid was dissolved in DMSO and then diluted with physiological saline to a final concentration of 1 mmol / L. The injection dose of cholic acid was 0.01 mmol / kg of body weight each time. The mixture of polyethylene glycol and polysorbate was prepared by dissolving polyethylene glycol and polysorbate in physiological saline. The injection doses of polyethylene glycol and polysorbate were 80-100 μg / kg and 100-120 μg / kg of body weight, respectively.

[0023] (2) Heat stress: One week after the start of modeling, the rats were placed in a high-temperature incubator at 38±1℃ for 4 hours every day for a total of 7 days.

[0024] (3) Silicon dioxide intake: Two weeks after the start of modeling, silicon dioxide was added to the regular feed of the rats, and this was continued until the end of modeling. The amount of silicon dioxide added was 3% of the weight of the regular feed.

[0025] (4) Exercise induction: Three weeks after the start of modeling, the rats were made to exercise at least 2 hours a day using a pet wheel toy. The pet wheel toy used food as an inducement, and the exercise was continued for one week, resulting in an acute lung injury model in rats.

[0026] After four weeks of modeling, the model was evaluated by observing the general signs of the rats, performing HE staining of the lung tissue, measuring the wet-to-dry weight ratio of the lung tissue, detecting the content of inflammatory factors and oxidative free radical-related indicators in the lung tissue by ELISA, performing lung function detection, observing the general changes in the lung tissue, detecting the partial pressure of arterial oxygen and oxygenation index, and detecting the expression of AQP4 and p-NF-κB, Na+-K+-ATPase α1 proteins in the lung tissue. The blank control group was injected with physiological saline and fed regularly. The results were as follows:

[0027] 1. General observation:

[0028] The rats in the blank control group had shiny fur, good spirits, frequent activity, normal appetite, and no huddling. After modeling, the modeled rats gradually showed abnormal breathing, wheezing, high mental depression, body tremors, poor appetite, and huddling.

[0029] 2. HE staining of lung tissue

[0030] The lung tissue structure of the blank control group was normal, the alveolar septum thickness was normal, and no obvious inflammatory infiltration was observed. The alveolar cavity was not filled with red blood cells and inflammatory cells, and there was no edema. The alveoli were regular and clear, and the lung interstitium was uniform in thickness. The lung tissue structure of the model group was destroyed, the alveolar septum was obviously widened, the alveoli were atrophic, the alveolar wall was edematous and congested, and there was a large amount of inflammatory infiltration.

[0031] 3. Measurement of lung wet / dry weight ratio

[0032] After the surface moisture of the lung tissue was carefully wiped with filter paper, the lung wet weight was measured and the lung tissue was placed in an oven at 80°C. After 2 days, the dried lung tissue was taken out again to measure the lung dry weight, and the wet / dry weight ratio was calculated. Compared with the blank control group, the lung wet / dry weight ratio of the model group was significantly increased, indicating that pulmonary edema occurred in the model group.

[0033] 4. ELISA detection of lung tissue inflammatory factor content and oxidative free radical related indicators

[0034] The lung alveolar lavage fluid was taken, and the expression of IL-10, IL-1β, IL-6 and TNF-α was detected by double antibody sandwich method. Compared with the blank group, the mass concentration of TNF-α, IL-6 and IL-1β in the model group was significantly up-regulated, and the expression level of IL-10 was significantly down-regulated (P<0.01).

[0035] The concentration of malondialdehyde (MDA) and superoxide dismutase (SOD) in the lung tissue was detected by ELISA. Compared with the blank control group, the lung MDA mass concentration of the model group was significantly up-regulated (P<0.01), and the SOD mass concentration was significantly down-regulated (P<0.01).

[0036] 5. Lung function detection

[0037] The forced vital capacity (FVC), forced expiratory volume in 0.1 second (FEV0.1), forced expiratory volume in 0.1 second to forced vital capacity ratio (FEV0.1 / FVC), forced expiratory volume in 0.3 second (FEV0.3), and forced expiratory volume in 0.3 second to forced vital capacity ratio (FEV0.3 / FVC) were detected by connecting the respirator and the signal conditioner with a catheter and using lung function detection software. Compared with the blank control group, the FEV0.1, FEV0.3, FEV0.1 / FVC and FEV0.3 / FVC of the model group decreased significantly (P<0.01), and the difference in FVC was not significant.

[0038] 6. Chest X-ray film

[0039] Compared with the blank control group, the lung texture of the model group increased, the lung field transparency decreased, and the lung showed ground glass-like changes, but there was no enlargement of the heart shadow and bronchial inflation.

[0040] 7. Observation of gross changes in lung tissue

[0041] After the ex vivo lung tissue was flushed with PBS solution, the lung lobe hemorrhage, edema, tissue surface plaque, color were observed and photographed. The lung surface of the blank control rats had no focal hemorrhage, and the lung surface of the model group had extensive hemorrhage.

[0042] 8. Arterial blood oxygen partial pressure (PaO2) and oxygenation index (OI) detection

[0043] 0.5 mL of arterial blood was extracted from the abdominal aorta by using a heparin injector for blood gas analysis to determine PaO2, and to calculate the oxygenation index (OI=PaO2 / FiO2, FiO2 was 21%). Compared with the blank control group, the PaO2 and OI of the abdominal aorta blood of the LPS group were significantly reduced (P<0.05).

[0044] 9. AQP4 and p-NF-κB, Na+-K+-ATPase α1 protein expression in lung tissue

[0045] Western blotting was used for detection. Compared with the blank control group, the expression levels of AQP4 and p-NF-κB p65 proteins in the lung tissue of the model group were significantly increased, and the expression of Na+-K+-ATPase α1 protein was significantly reduced (P<0.01).

[0046] After repeated experiments, the experimental groups all showed the same results. It can be seen that the rats in the experimental groups showed typical symptoms of acute lung injury, which meets the reliability and effectiveness of the determination of the animal model of acute lung injury, i.e. the cause of the disease, the symptoms, and the pathophysiology, and the rat acute lung injury model can be successfully obtained according to the construction method of the present application.

Claims

1. A method for constructing a rat acute lung injury model, characterized in that, Includes the following steps: (1) Intraperitoneal injection: Four-week-old SD rats were selected and raised under SPF conditions. Modeling was started after two weeks of acclimatization. The total modeling time was four weeks. After the modeling started, bile acid and a mixture of polyethylene glycol and polysorbate were injected into the rats intraperitoneally every two days. Each time, bile acid was injected into the rats intraperitoneally first, followed by the mixture of polyethylene glycol and polysorbate 30 minutes later. The bile acid was dissolved in DMSO and then diluted with physiological saline to a final concentration of 1 mmol / L. The bile acid injection dose was 0.01 mmol / kg body weight. The mixture of polyethylene glycol and polysorbate was prepared by dissolving polyethylene glycol and polysorbate in physiological saline. The injection doses of polyethylene glycol and polysorbate were 80-100 μg / kg and 100-120 μg / kg body weight, respectively. (2) Heat stress: One week after the start of the modeling, the rats were placed in a high-temperature incubator at 38±1℃ for 4 hours every day for a total of 7 days; (3) Intake of silica: Two weeks after the start of modeling, silica was added to the rats’ regular diet and continued until the end of modeling; the amount of silica added was 3% of the weight of the regular diet. (4) Exercise induction: Three weeks after the start of modeling, the rats were exercised for at least 2 hours a day using a pet running wheel toy. The pet running wheel toy was induced by food. After one week of continuous exercise, the rat acute lung injury model was obtained.

2. The method for constructing a rat acute lung injury model according to claim 1, characterized in that, After four weeks of modeling, the model was evaluated by observing general signs of rats, HE staining of lung tissue, measuring the wet-to-dry weight ratio of lung tissue, detecting the content of inflammatory factors and oxidative free radical related indicators in lung tissue by ELISA, pulmonary function tests, chest X-ray, observing gross changes in lung tissue, detecting arterial blood oxygen partial pressure and oxygenation index, and the expression of AQP4, p-NF-κB, and Na+-K+-ATPaseα1 proteins in lung tissue. The results showed that the rats exhibited typical symptoms of lung injury, which met the criteria for the reliability and effectiveness of the acute lung injury animal model.