Method for establishing an animal model of acute lung injury

Abstract

The application belongs to the technical field of animal models for basic scientific research, and particularly relates to a method for establishing an acute lung injury animal model, which comprises the following steps: sending a podophyllotoxin or a podophyllotoxin reagent into a rat body through oral administration or intragastric perfusion to obtain the acute lung injury animal model. The method for establishing the acute lung injury animal model provided by the application uses the podophyllotoxin as an inducer for establishing a mouse acute lung injury model, and the mouse can orally take or intragastrically perfuse the podophyllotoxin to achieve the method, without needing a special medicine injection device, so that the method is simple in medicine taking, few in experimental steps, and does not need complex surgical operations.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of animal models for basic scientific research, and particularly relates to a method for establishing an acute lung injury animal model. BACKGROUND

[0002] Acute lung injury (ALI) is caused by damage to alveolar epithelial cells and capillary endothelial cells due to various factors, and is characterized by decreased lung compliance, hypoxemia, and even progressive respiratory failure. When the chest X-ray shows bilateral diffuse exudation-like changes, and the oxygenation index (PaO2 / FiO2) is ≤300 mmHg, it can be diagnosed. The pathogenesis of ALI is complex, the disease progresses rapidly, and the mortality rate is extremely high. In order to find an effective treatment method, establishing an animal model that is similar to the human phenotype and pathogenesis, and is stable and repeatable, is one of the keys to promoting ALI research. In order to study the pathophysiological mechanisms of ALI, discover new ALI biomarkers, and explore ALI treatment intervention strategies, researchers often need to use animal models to carry out preclinical research. So far, the establishment of a rat acute lung injury model is usually based on common pathogenic factors in the clinic, which cannot simulate all the characteristics of human acute lung injury. In addition, the lungs of humans and animals are not completely the same in structure and function, and their responses to stimuli are also different. In the development and research of related drugs and the application of ALI mechanism, similar factors should be used to cause ALI models according to the actual purpose and requirements. For example, LPS-ALI models based on immune factors, smoke-induced ALI models based on environmental factors, and endotoxin-ALI models based on pathogenic factors. Drug-induced acute lung injury also occurs in ALI patients. In order to meet the research needs of ALI occurring during drug development and use, and to overcome the limitations of existing ALI animal models, an animal model of drug-induced lung injury also needs to be constructed.

[0003] Podophyllotoxin (PPT) is a natural lignan compound with extremely wide distribution, which is mainly found in plants of the genus Podophyllum, the genus Sinopodophyllum, the genus Sinopodophyllum, and the genus Sinopodophyllum. Podophyllotoxin extract has been proven to be a laxative and can also be used to treat various medical complications such as gonorrhea, tuberculosis, menstrual disorders, psoriasis, edema, cough, syphilis, and sexually transmitted diseases. In addition, podophyllotoxin compounds also have various biological properties such as cytotoxicity, insecticidal, antifungal, antiviral, anti-inflammatory, neurotoxicity, immunosuppression, anti-rheumatism, antioxidant, spasmolytic, and hypolipidemic activities. Its derivative etoposide is a commonly used antitumor drug in clinical practice. However, excessive intake of PPT can cause damage to multiple organ functions.

[0004] In the prior art, patent document CN 109939221 A discloses a method for establishing an animal model of acute lung injury caused by ricin, which uses ricin to cause acute lung injury, but the toxicity of ricin is too strong and it is easy to be used by illegal persons; and it needs "attack mode: adopt handheld liquid aerosol lung delivery device to immunize tracheal into lung", needs to use a special device to hit the toxin into the lung through the trachea, increases the cost of the device, and the dosage of ricin in B2 group is slightly larger than that in B1 group, that is, causes the death of mice, the toxicity of ricin is large, and the operation cost is high. Patent document CN 115813593A discloses a method for establishing an animal model of different degrees of acute lung injury induced by sepsis, which "after removing the abdominal hair of the mouse, anesthetizing with isoflurane, fixing on the operating table, disinfecting the abdomen, making a skin incision of about 2cm in the middle, then cutting the muscle layer, carefully separating the cecum from the left lower abdomen, finding the junction of the cecum and the small intestine and the large intestine, the sham group is not treated, the other four groups are ring-ligated with No. 4 silk thread, punctured with a needle at the position of the ligation segment and the end of the cecum, wherein the 50%+26G group is ligation at the position of 50% of the end of the cecum and punctured with a 26G needle, the 50%+20G group is ligation at the position of 50% of the end of the cecum and punctured with a 20G needle, the 75%+26G group is ligation at the position of 75% of the end of the cecum and punctured with a 26G needle, and the 75%+20G group is ligation at the position of 75% of the end of the cecum and punctured with a 20G needle, and attention should be paid to avoid damaging the mesenteric blood vessels during puncture, and the cecum is put back into the abdomen after extruding the appropriate feces, and the muscle layer and the skin layer are sutured in turn"; although it needs toxin to induce lung injury, the operation is complex and not easy to operate. CN 118020709 A discloses a method for constructing a rat acute lung injury model, which obtains a rat acute lung injury model by injecting bile acid into the abdominal cavity of the animal, and mixing polyethylene glycol and polysorbate + heat stress + intake of silicon dioxide + exercise induction, the induction method has many steps and a long duration. SUMMARY

[0005] The purpose of the present application is a method for establishing an acute lung injury animal model, which uses podophyllotoxin as an inducer for establishing a mouse acute lung injury model, and the mouse can be realized by oral or intragastric perfusion of podophyllotoxin, without the need for a special drug injection device, the drug is simple to use, the experimental steps are few, and complex surgical operations are not required.

[0006] In order to solve the above technical problems, the acute lung injury animal model establishment method provided by the present application is realized as follows:

[0007] A method for establishing an acute lung injury animal model, wherein podophyllotoxin or a podophyllotoxin reagent is sent into the body of a rat by oral or intragastric perfusion, thereby obtaining the acute lung injury animal model.

[0008] Optionally, the dosage of the podophyllotoxin is 20 mg / kg per day, and the administration is continuous for 4-5 days.

[0009] Optionally, the dosage of the podophyllotoxin reagent is 20 mg / kg of podophyllotoxin per day.

[0010] Optionally, the rats are 6-8 weeks old male SD rats, and the weight is 210±20 g.

[0011] Optionally, the podophyllotoxin reagent is prepared by dissolving podophyllotoxin in 2% DMSO, and then adding 0.5% sodium carboxymethyl cellulose to make the podophyllotoxin reagent.

[0012] The application also provides a detection method of an acute lung injury animal model, which comprises detecting the cytokines in the alveolar lavage fluid of the rats, detecting the lung weight of the rats, detecting the inflammatory factors in the lung tissue of the rats, and pathological detection after continuous administration of podophyllotoxin or a podophyllotoxin reagent.

[0013] Optionally, the detection of the cytokines in the alveolar lavage fluid is performed by using a full-automatic biochemical analyzer Thermo Scientic to detect the contents of lactate dehydrogenase, alkaline phosphatase, albumin and total protein.

[0014] Optionally, the detection of the inflammatory factors in the lung tissue is performed by using an ELISA method to detect the contents of IL-18, TNF-α, IL-6 and IL-1β.

[0015] Optionally, the detection of the lung weight is performed by comparing the organ coefficients of the bilateral lungs of the rats.

[0016] Optionally, the pathological detection is performed by using hematoxylin-eosin staining to observe the lung tissue.

[0017] The method for establishing an acute lung injury animal model provided by the application has small toxicity of podophyllotoxin, and is not easy to cause the death of mice, and is convenient for experiments; only oral administration or intragastric perfusion is needed, and no special tools are needed for operation, and the method is simple, and the experimental cost is reduced; and the application only needs oral administration or intragastric perfusion, and no complex operation is needed for the rats, and no injection of drugs + heat stress + movement induction and other complex experimental steps are needed, and the experimental method is simple to operate.

[0018] And, the prepared acute lung injury animal model of the application, detecting the cytokine of the rat alveolar lavage fluid, taking out the rat lung to detect the comparison of lung weight, detecting the rat lung tissue to detect the inflammatory factor and the pathology detection, determines that the lung tissue appears obvious damage, inflammatory cell infiltration and capillary expansion congestion, and the acute lung injury is indeed formed. The prepared rat acute lung injury model of the application appears the typical characteristics similar to the human acute lung injury, is an effective method for establishing the acute lung injury, and can be used for exploring the target and drug research of the mechanism of the acute lung injury.

[0019] The application successfully establishes the rat acute lung injury model by using the podophyllotoxin, lays a good foundation for establishing a stable acute lung injury model for basic research. The modeling method of the application is simple, the podophyllotoxin is given by gavage or oral administration for 4-5 days, the acute lung injury patient is simulated through the alveolar lavage fluid biochemical detection, lung tissue inflammatory factor level detection and lung pathology observation, the rat lung tissue appears obvious damage, inflammatory cell infiltration, alveolar wall capillary expansion congestion and other symptoms. The model index has good stability and can be repeated, and the application provides a reliable method for the construction of the acute lung injury. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is the phenotype damage graph of the rat after the podophyllotoxin is given in the application;

[0021] Figure 2 is the determination of the lung injury index in the alveolar lavage fluid of the rat after the podophyllotoxin is given in the application;

[0022] Figure 3 is the lung inflammatory factor level of the rat after the podophyllotoxin is given in the application;

[0023] Figure 4 is the HE staining graph of the lung of the rat after the podophyllotoxin is given in the application. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical scheme and advantages of the application more clear, the following examples further illustrate the application. It should be understood that the specific examples described herein are only used to explain the application, and are not used to limit the application.

[0025] The experimental methods in the following examples are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents and the like used in the following examples can be obtained from commercial channels, unless otherwise specified.

[0026] The experiment of the present application is repeated at least 3 times with independent samples, and the count data is shown by mean ± standard deviation (x ± s), and the SPSS 26.0 software is used for statistical processing. If the population obeys normal distribution, independent sample t test is used when variances are equal; Mann-Whitney U test is used when variances are not equal. P < 0.05 represents that the difference has statistical significance.

[0027] Example one: Construction of podophyllotoxin acute lung injury model

[0028] 1. Experimental animals

[0029] 30 SPF male SD rats were purchased from Fuvitong Lihua Company, weighing 210 ± 20 g, and were raised in the animal experiment center of the first affiliated hospital of Henan University of Science and Technology, with 12 h light and dark alternation light, environmental temperature of 23 ± 2 ℃, environmental humidity of 35 ± 5%, without restriction of food and water, and adaptive feeding for 1 day. After 1 d, the rats were randomly divided into PPT group and control group, 22 in PPT group and 20 in control group. The animal study was approved by the ethics committee (animal experiment ethics subcommittee of medical ethics committee of the first affiliated hospital of Henan University of Science and Technology). All experimental procedures were carried out in accordance with the national legislation and local guidelines of China.

[0030] 2. Drug configuration

[0031] 2.1 Main drug: podophyllotoxin (Shanghai Yuanye Bio-Technology Co., Ltd., S24887), dimethyl sulfoxide (DMSO, Dimethyl Sulfoxide) (Shanghai Macklin Biochemical Technology Co., Ltd., D806647), sodium carboxymethyl cellulose (Shanghai Hushi Laboratory Equipment Co., Ltd., 20120928)

[0032] 2.2 Taking the drug dose of 20 mg / kg / d as an example: taking the SD rat with a body weight of 200 g as an example, i.e. 4 mg / d is needed for each. 22 rats need to be given drugs for 4 days, a total of 352 mg of podophyllotoxin is needed; the amount of drug given to each rat is 2 mL according to the ratio of body weight: dose = 200 g: 2 mL, i.e. a total of 176 mL of solution volume needs to be configured.

[0033] 1) Precisely weigh 352 mg of solid powder podophyllotoxin with an analytical balance;

[0034] 2) Precisely remove 3.52 mL of DMSO needed by 176 mL*2% into a 5 mL EP tube with a pipette;

[0035] 3) Dissolve the weighed podophyllotoxin in DMSO, mix, shake and centrifuge;

[0036] 4) Transfer the above reagents to a clean reagent bottle, and make up to the required volume with 176mL - 3.52mL = 172.48mL of 0.5% sodium carboxymethyl cellulose, and shake to mix.

[0037] 3. Experimental Methods

[0038] Administer the drug at 10:00 AM daily. Weigh the rats and administer the drug via gavage at a ratio of body weight:dose = 200g:2mL (the specific dosage needs to be determined based on body weight). Repeat the above procedure for 4 days, and collect samples on the 5th day.

[0039] Example 2: Detection Indicators and Methods

[0040] 4.1 Observation of general animal morphology

[0041] Changes in rats were observed after administration of drugs in each group, including gastrointestinal reactions such as diarrhea and its severity, physical changes such as gait changes, activity frequency, and tremors, as well as general phenotypic observations such as changes in appearance, changes in hair luster, and bleeding from the mouth, nose, and limbs. The weight, diet, and water intake of rats in each group were assessed and recorded every 12 hours.

[0042] General phenotype: After appropriate treatment in each group, the healthy control group showed no significant changes. For example... Figure 1 As shown, on day 3 after drug administration, rats in the PPT intervention group exhibited varying degrees of surface ecchymosis and petechiae, and even bleeding, including on the face, mouth, nose, and fore and hind paws. Gait scoring analysis showed that the PPT group scored significantly higher than the control group, suggesting that PPT exposure led to impaired motor function. Compared to the control group, the PPT group showed a trend of increasing lung organ index scores, but the difference did not reach statistical significance. Figure 1 As shown, the PPT group experienced a significant decrease in body weight compared to the control group two days after drug administration. Figure 1 CON is the control group.

[0043] 4.2 Pathological analysis of the lungs

[0044] After treating rats, lung tissue was harvested and fixed in 4% paraformaldehyde solution at room temperature for 48 hours. Then, it was dehydrated, embedded, sectioned, stained, and mounted. The sections were examined under a microscope, and the tissue sections were examined in detail at different magnifications. Basic pathological changes such as congestion, ecchymosis, hemorrhage, edema, degeneration, necrosis, hyperplasia, fibrosis, organization, granulation tissue, and inflammatory changes were carefully observed to assess the pathological damage to the lungs.

[0045] Results of bronchoalveolar lavage fluid lung injury marker tests:

[0046] After anesthetizing the rats, the abdominal cavity was opened, and the rats were euthanized by exsanguination through the abdominal aorta. The thoracic cavity was then opened, the lungs were dissected, the trachea was exposed, and the left lung was ligated. A small "V"-shaped incision was made at the main trachea, and an irrigation needle was inserted and secured. The right lung was irrigated with 0.9% sodium chloride injection at 37°C. The 0.9% sodium chloride injection was slowly injected into the lung while gently massaging the chest wall, and then the irrigation fluid was slowly aspirated. Irrigation was performed twice. For the first irrigation, 6 mL was injected, and 3 mL was collected. For the second irrigation, 3 mL was injected, aspirated twice, mixed, and 3 mL was collected. The bronchoalveolar lavage fluid was centrifuged at 2500 rpm for 15 min, and the supernatant was collected. Lung injury markers were detected using a Thermo Scientific fully automated biochemical analyzer. Figure 2 As shown, the levels of alkaline phosphatase (ALP), albumin (ALB), lactate dehydrogenase (LDH), and total protein (TP) in the bronchoalveolar lavage fluid of the PPT group were significantly higher than those of the healthy control group, indicating that lung tissue was damaged.

[0047] 5.3 Results of lung tissue inflammatory factor level detection

[0048] After anesthetizing rats, the abdominal cavity was opened, the lungs were dissected, and 3mg tissue blocks were removed from the lung tissue. These blocks were placed in physiological saline and rinsed three times to remove blood. The surface moisture of the tissue blocks was then wiped dry with filter paper. The tissue blocks were weighed and placed in a beaker. Physiological saline at 4°C was pipetted into the beaker, with the total volume of saline to the weight of the lung tissue block being 9:1, immersing the tissue block in the saline solution. Under ice-water bath conditions, the immersed tissue block was rapidly shredded with ophthalmic scissors and further ultrasonically pulverized to prepare a 10% lung tissue homogenate. Inflammatory factors were detected using enzyme-linked immunosorbent assay (ELISA). The detection steps were as follows: Standard wells and sample wells were set up, with 50μL of different concentrations of standard added to each standard well; blank wells (blank control wells without sample or enzyme-labeled reagent, all other steps were the same) and sample wells were also set up. Add 40 μl of sample diluent to the wells of the enzyme-labeled plate, and then add 10 μl of the sample to be tested (the final sample dilution is 5 times). Add the sample to the bottom of the wells of the ELISA plate, avoiding contact with the well walls as much as possible, and gently shake to mix. Add 100 μl of enzyme-labeled reagent to each well, except for the blank wells. Seal the plate with sealing film and incubate at 37°C for 60 minutes. Dilute the 20-fold concentrated washing buffer 20 times with distilled water and set aside. Carefully remove the sealing film, discard the liquid, and shake dry. Fill each well with washing buffer, let stand for 30 seconds, then discard. Repeat this process 5 times, and pat dry. Add 50 μl of chromogenic reagent A to each well, followed by 50 μl of chromogenic reagent B, and gently shake to mix. Incubate at 37°C in the dark for 15 minutes. Add 50 μl of stop solution to each well to stop the reaction (the blue color will immediately turn yellow). Zero the plate using the blank well and measure the absorbance (OD value) of each well sequentially at a wavelength of 450 nm. Measurements should be performed within 15 minutes after adding the stop solution. Figure 3As shown, inflammatory factors such as IL-1β, IL-6, IL-18, and TNF-α were significantly elevated in the lung tissue of the PPT group. In the lung tissue, the oxidative stress markers malondialdehyde (MDA) and reduced glutathione (GSH) were significantly elevated. Figure 2 ).

[0049] 5.2 Pathological results of HE staining of lung tissue

[0050] Fresh lung tissue was immediately dissected and immersed in fixative. After removal from the fixative, the tissue was trimmed and smoothed in a fume hood using a scalpel. The trimmed tissue and corresponding labels were placed in an embedding frame. The dehydration box was placed in a dehydrator for sequential dehydration with alcohol: 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, anhydrous ethanol I for 30 minutes, anhydrous ethanol II for 30 minutes, benzene for 5-10 minutes, xylene I for 5-10 minutes, xylene II for 5-10 minutes, 65°C melted paraffin I for 1 hour, 65°C melted paraffin II for 1 hour, and 65°C melted paraffin III for 1 hour. The paraffin-impregnated tissue was then embedded in an embedding machine. First, the melted paraffin was placed in the embedding frame. Before the paraffin solidified, the tissue was removed from the dehydration box, placed in the embedding frame according to the embedding surface requirements, and labeled accordingly. Cool at -20°C on a freezing stage. After the wax solidifies, remove the wax block from the embedding frame and trim it. Place the trimmed wax block on a paraffin microtome and section it to a thickness of 4μm. Float the section on 40°C warm water in a slide spreader to flatten the tissue. Remove the tissue from the slide and bake it in a 60°C oven. After the wax melts in the oven, remove it and store it at room temperature for later use. Sequentially, immerse the sections in environmentally friendly dewaxing solution I for 20 min, environmentally friendly dewaxing solution II for 20 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and 75% ethanol for 5 min, then rinse with tap water. Pre-treat the sections with high-resolution constant staining solution for 1 min. Stain the sections with hematoxylin solution for 3-5 min, rinse with tap water, differentiate with differentiation solution, rinse with tap water, re-blue with blue solution, and rinse with running water. Dehydrate the sections with 95% ethanol for 1 min, then stain with eosin solution for 15 s. The sections were sequentially immersed in anhydrous ethanol I for 2 min, then anhydrous ethanol II for 2 min, then anhydrous ethanol III for 2 min, then n-butanol I for 2 min, then n-butanol II for 2 min, then xylene I for 2 min, and finally xylene II for 2 min. After clearing, the sections were mounted with neutral resin. The sections were then examined under a microscope, and images were acquired and analyzed. Figure 4 As shown, no obvious congestion or inflammatory cell infiltration was observed in the CON control group; lymphocyte infiltration (black arrow), congestion (red arrow), dilation and congestion of alveolar wall capillaries, and a small number of red blood cells were observed in the PPT intervention group.

[0051] The above results indicate that PPT treatment resulted in significant damage to lung tissue, including inflammatory cell infiltration, capillary dilation, and congestion. The levels of alkaline phosphatase (ALP), albumin (ALB), lactate dehydrogenase (LDH), and total protein (TP) in the bronchoalveolar lavage fluid were significantly elevated in the PPT group. Inflammatory factors such as IL-1β, IL-6, IL-18, and TNF-α were also significantly elevated in the lung tissue of the PPT group, along with significantly elevated levels of oxidative stress markers malondialdehyde (MDA) and reduced glutathione (GSH). It is generally believed that ALI is caused by a variety of factors (including but not limited to infection, trauma, poisoning, and shock). These factors initially trigger uncontrolled systemic inflammation, ultimately leading to vascular endothelial damage and infiltration of various inflammatory cells, accompanied by relatively mild alveolar damage. PPT may induce pulmonary oxidative stress by disrupting cellular integrity, causing irreversible damage and leading to ALI.

[0052] In summary, the modeling method of the present invention, through treatment with podophyllotoxin via gavage, resulted in the experimental group exhibiting typical characteristics similar to human acute lung injury. This method is an effective way to establish acute lung injury and can be used for target and drug research to explore the mechanism of action of acute lung injury.

[0053] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for establishing an animal model of acute lung injury, characterized in that, The acute lung injury animal model was obtained by administering podophyllotoxin or podophyllotoxin reagent to rats orally or via gastric infusion. The dosage of podophyllotoxin was 20 mg / kg daily for 4-5 consecutive days. The dosage of podophyllotoxin reagent was 20 mg / kg of podophyllotoxin daily. The rats were male SD rats aged 6-8 weeks, weighing 210 ± 20 g. The podophyllotoxin reagent was prepared by dissolving podophyllotoxin in 2% DMSO and then diluting it with 0.5% sodium carboxymethyl cellulose.

2. The method for establishing an animal model of acute lung injury as described in claim 1, characterized in that, After continuous administration of podophyllotoxin or podophyllotoxin reagent, cytokines in rat bronchoalveolar lavage fluid were detected, rat lungs were removed and their weights were compared, and inflammatory factors and pathological damage were detected in lung tissue.

3. The method for establishing an animal model of acute lung injury according to claim 2, characterized in that, The detection of cytokines in bronchoalveolar lavage fluid was performed using the Thermo Scientific fully automated biochemical analyzer to detect lung injury indicators such as lactate dehydrogenase, alkaline phosphatase, albumin, and total protein levels.

4. The method for establishing an animal model of acute lung injury according to claim 2, characterized in that, The detection of inflammatory factors in lung tissue was performed by using enzyme-linked immunosorbent assay (ELISA) to detect the levels of IL-18, TNF-α, IL-6, and IL-1β.

5. The method for establishing an animal model of acute lung injury according to claim 2, characterized in that, The lung weight comparison test involves comparing the organ coefficients of both lungs in rats.

6. The method for establishing an animal model of acute lung injury according to claim 2, characterized in that, The pathological examination involved observing the lung tissue using hematoxylin-eosin staining.

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