Acute lung injury animal model building method

Abstract

The invention 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 is characterized in that podophyllotoxin or a podophyllotoxin reagent is fed into a rat body through oral administration or intragastric perfusion to obtain the acute lung injury animal model. According to the method for establishing the acute lung injury animal model, podophyllotoxin is used for establishing the model for inducing the acute lung injury of the mouse, the mouse can orally take podophyllotoxin or perfuse podophyllotoxin into the stomach of the mouse, a special medicine injecting device is not needed, medicine application is simple, the number of experimental steps is small, and complex surgical operation is not needed.

Description

Technical Field

[0001] The present invention relates to the technical field of animal models for basic scientific research, and in particular to a method for establishing an acute lung injury animal model. Background Art

[0002] Acute lung injury (ALI) is an injury to alveolar epithelial cells and capillary endothelial cells caused by multiple factors, manifested as decreased lung compliance, hypoxemia, and even progressive respiratory failure. It can be diagnosed when the patient's chest X-ray shows bilateral diffuse exudative changes and the oxygenation index (PaO2 / FiO2) is ≤300 mmHg. The pathogenesis of ALI is complex, the course of the disease progresses rapidly, and it has an extremely high mortality rate. In order to find effective treatments, establishing an animal model that is similar to the human phenotype and pathogenesis and is stable and reproducible is one of the keys to promoting ALI research. In order to study the pathophysiological mechanism of ALI, discover new ALI biomarkers, and explore ALI treatment intervention strategies, researchers often need to use animal models to conduct preclinical studies. So far, the establishment of rat acute lung injury models is often based on the common pathogenic factors in clinical practice, which cannot simulate all the characteristics of human acute lung injury. In addition, the lungs of humans and animals are not exactly the same in structure and function, and their responses to stimuli are also different. In the development of related drugs and the study of the mechanism of ALI, ALI models caused by similar factors should be used as much as possible according to actual purposes and requirements. For example, the LPS-ALI model constructed based on immune factors, the ALI model constructed by the smoke method based on environmental factors, and the endotoxin-ALI model constructed by pathogen factors. ALI patients also have drug-induced acute lung injury. In order to meet the research needs of ALI occurring during drug development and use and overcome the limitations of existing ALI animal models, it is also necessary to construct an animal model of drug-induced lung injury.

[0003] Podophyllotoxin (PPT) is a widely distributed natural lignan compound, mostly found in plants of the Berberidaceae family Podophyllum, Podophyllum, Podophyllum and Podophyllum in North America. Podophyllotoxin extracts have been shown 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 a variety of biological properties, such as cytotoxicity, insecticide, antifungal, antiviral, anti-inflammatory, neurotoxic, immunosuppressive, anti-rheumatic, antioxidant, antispasmodic and hypolipidemic activities. Its derivative, etoposide, is a commonly used anti-tumor drug in clinical practice. However, excessive intake of PPT can lead to multi-organ damage.

[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 toxin, which uses ricin toxin to cause acute lung injury, but ricin toxin is too toxic and can be easily exploited by criminals; and it requires "drug attack method: using a handheld liquid aerosol lung delivery device to immunize through the trachea into the lungs", and a special device is needed to inject the toxin into the lungs through the trachea, which increases the cost of the device, and the amount of ricin used in group B2 is slightly greater than that in group B1, which causes the death of mice. Ricin toxin is highly toxic and has high operating costs. Patent document CN 115813593A discloses a method for establishing an animal model of acute lung injury induced by sepsis to different degrees, which includes "removing the hair from the abdomen of the mouse, anesthetizing it with isoflurane, fixing it on the operating table, routinely disinfecting the abdomen, making a midline skin incision of about 2 cm, then cutting the muscle layer, carefully separating the cecum from the left lower abdomen, and finding the junction between the cecum and the small intestine and the large intestine. The sham group was not treated, and the other four groups were ligated with No. 4 silk thread in a circular manner. The cecum was punctured with a needle at the position of the ligated segment at the end of the cecum. The 50%+26G group was punctured at 50% of the distance from the end of the cecum. The 50%+20G group was ligated at 50% of the distance from the end of the cecum and punctured with a 26G needle. The 75%+26G group was ligated at 75% of the distance from the end of the cecum and punctured with a 26G needle. The 75%+20G group was ligated at 75% of the distance from the end of the cecum and punctured with a 20G needle. During puncture, care was taken to avoid damaging the mesenteric blood vessels. After squeezing out an appropriate amount of feces, the cecum was put back into the abdomen, and the muscle layer and skin layer were sutured in sequence. Although it is different from the method that requires toxins to induce lung injury, the operation is complicated and difficult to operate. CN 118020709 A discloses a method for constructing a rat acute lung injury model, which is induced by intraperitoneal injection of bile acid, a mixture of polyethylene glycol and polysorbate + heat stress + silica intake + exercise to obtain a rat acute lung injury model. The induction method has many steps and lasts for a long time. Summary of the invention

[0005] The purpose of the present invention is to provide a method for establishing an acute lung injury animal model. Podophyllotoxin is used as an induction for establishing an acute lung injury model in mice. The model can be achieved by oral administration or intragastric perfusion of podophyllotoxin in mice. No special drug injection device is required, the medication is simple, the experimental steps are few, and no complicated surgical operations are required.

[0006] In order to solve the above technical problems, the method for establishing an acute lung injury animal model provided by the present invention is implemented as follows:

[0007] A method for establishing an acute lung injury animal model comprises delivering podophyllotoxin or a podophyllotoxin reagent into a rat by oral administration or intragastric perfusion to obtain the acute lung injury animal model.

[0008] Optionally, the dosage of podophyllotoxin is 20 mg / kg per day, for 4 to 5 consecutive days;

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

[0010] Optionally, the rats are male SD rats aged 6 to 8 weeks, weighing 210±20 g.

[0011] Optionally, the podophyllotoxin reagent is prepared by dissolving podophyllotoxin in 2% DMSO and fixing the volume with 0.5% sodium carboxymethylcellulose to prepare the podophyllotoxin reagent.

[0012] The present invention also provides a method for detecting an acute lung injury animal model. After continuous administration of podophyllotoxin or podophyllotoxin reagent, cytokines in rat bronchoalveolar lavage fluid are detected, lungs of rats are removed for detection and comparison of lung weights, inflammatory factors in rat lung tissues are detected, and pathological tests are performed.

[0013] Optionally, the detection of cytokines in bronchoalveolar lavage fluid is: using a fully automatic biochemical analyzer Thermo Scientic to detect lactate dehydrogenase, alkaline phosphatase, albumin and total protein content.

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

[0015] Optionally, the detection and comparison of lung weight is: comparing the organ coefficients of bilateral lungs of rats.

[0016] Optionally, the pathological test is: performing pathological observation on lung tissue using hematoxylin-eosin staining.

[0017] The method for establishing an acute lung injury animal model provided by the present invention uses podophyllotoxin with low toxicity, is not likely to cause death of mice, and is convenient for experiments; it only needs oral administration or intragastric infusion, does not require special tools for operation, and is simple to use, thereby reducing experimental costs; and the present invention only needs oral administration or intragastric infusion, does not require complex surgery on rats, and does not require complex experimental steps such as injection of drugs+heat stress+exercise induction, and the experimental method is simple to operate.

[0018] Moreover, the animal model of acute lung injury prepared by the present invention detects cytokines in rat bronchoalveolar lavage fluid, removes rat lungs to detect and compare lung weight, detects inflammatory factors in rat lung tissue, and conducts pathological tests to determine that the lung tissue is significantly damaged, inflammatory cells infiltrate, and capillary dilation and congestion occur, and acute lung injury is indeed formed. The rat acute lung injury model prepared by the present invention has typical characteristics similar to human acute lung injury, is an effective method for establishing acute lung injury, and can be used for target and drug research to explore the mechanism of action of acute lung injury.

[0019] The present invention successfully established a rat acute lung injury model using podophyllotoxin, laying a good foundation for establishing a stable acute lung injury model for basic research. The modeling method of the present invention is simple, and podophyllotoxin is administered orally for 4 to 5 days. Through biochemical detection of alveolar lavage fluid, detection of inflammatory factor levels in lung tissue, and lung pathological observation, acute lung injury patients are simulated, and it is found that rat lung tissue is significantly damaged, with symptoms such as inflammatory cell infiltration, alveolar wall capillary dilation and congestion. This model has good index stability and strong repeatability, providing a reliable method for the construction of acute lung injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a diagram of phenotypic damage to rats after administration of podophyllotoxin according to the present invention;

[0021] Figure 2 The present invention is the determination of lung injury indexes in bronchoalveolar lavage fluid of rats after administration of podophyllotoxin;

[0022] Figure 3 The level of inflammatory factors in the lungs of rats after administration of podophyllotoxin according to the present invention;

[0023] Figure 4 This is a HE staining picture of rat lung after administration of podophyllotoxin according to the present invention. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail in the following embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

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

[0026] The experiment involved in the present invention was repeated at least 3 times with independent samples, and the counting data were expressed as mean ± standard deviation (x ± s), and SPSS26.0 software was used for statistical processing. If the population obeyed the normal distribution, the independent sample t test was used, and the Mann-Whitney U test was used for unequal variances. P < 0.05 represented that the difference was statistically significant.

[0027] Example 1: Construction of podophyllotoxin-induced acute lung injury model

[0028] 1. Experimental Animals

[0029] Thirty SPF male SD rats were purchased from Weitong Lihua Company, weighing 210±20g, and were kept in the Animal Experiment Center of the First Affiliated Hospital of Henan University of Science and Technology, with 12h light and dark alternating light, ambient temperature of 23±2℃, ambient humidity of 35±5%, and no restriction on food and water, and adaptive feeding for 1 day. After 1d, the rats were randomly divided into PPT group and control group, with 22 rats in PPT group and 20 rats in control group. This animal study was approved by the Ethics Committee (Animal Experiment Ethics Subcommittee of the Medical Ethics Committee of the First Affiliated Hospital of Henan University of Science and Technology). All experimental procedures were carried out in accordance with Chinese national legislation and local guidelines.

[0030] 2. Drug configuration

[0031] 2.1 Main drugs: Podophyllotoxin (Shanghai Yuanye Biotechnology Co., Ltd., S24887), Dimethyl Sulfoxide (DMSO) (Shanghai McLean Biochemical Technology Co., Ltd., D806647), Sodium Carboxymethyl Cellulose (Shanghai Hushi Laboratory Equipment Co., Ltd., 20120928)

[0032] 2.2 Take the preparation of 20mg / kg / d dosage as an example: By default, SD rats with a body weight of 200g are taken as an example, that is, each rat needs 4mg / d. 22 rats need to be administered for 4 days, and a total of 4mg*22*4=352mg of podophyllotoxin is required; the dosage for each rat is based on the ratio of body weight: dosage = 200g: 2mL, that is, a total of 2mL*22*4=176mL of solution needs to be prepared.

[0033] 1) 352 mg of solid powdered podophyllotoxin was accurately weighed using an analytical balance;

[0034] 2) Use a pipette to precisely transfer the required 176 mL*2%=3.52 mL of DMSO into a 5 mL EP tube;

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

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

[0037] 3. Experimental methods

[0038] The drug was administered at 10:00 am every day. The rats were weighed and gavage was performed according to the ratio of body weight: dose = 200 g: 2 mL (the specific dose should be determined according to body weight). The above operation was repeated for 4 days, and samples were collected on the 5th day.

[0039] Example 2: Detection indicators and methods

[0040] 4.1 Observation of general morphology of animals

[0041] After administration, the changes in rats in each group were observed, including gastrointestinal reactions such as diarrhea and the degree of diarrhea, body changes such as gait changes, activity frequency, tremor, etc., and general phenotypic observations such as changes in appearance, changes in hair gloss, bleeding in the mouth, nose and limbs, etc. The body weight, diet and drinking water of each group of rats were evaluated and recorded every 12 hours.

[0042] General phenotype: After each group was given corresponding treatment, there was no significant change in the healthy control group. Figure 1 As shown in the figure, after the third day of administration, rats in the PPT intervention group developed varying degrees of congestion and ecchymosis on the body surface, and even bleeding, including on the face, mouth, nose, front and back paws, etc. Gait score analysis showed that the score of the PPT group was significantly higher than that of the control group, indicating that PPT exposure led to impaired motor ability. Compared with the lung organ index of the control group, the score of the PPT group tended to increase, but the difference did not reach statistical significance. Figure 1 As shown, the body weight of the PPT group decreased significantly compared with the control group after 2 days of administration. Figure 1 CON was the control group.

[0043] 4.2 Lung pathology analysis

[0044] After the rats were treated, the lung tissues were removed and fixed in 4% paraformaldehyde solution at room temperature for 48 hours, then dehydrated, embedded, sliced, stained, and sealed. The slices were viewed under a microscope, and the tissue slices were examined in detail at different magnifications. Basic pathological changes such as congestion, congestion, hemorrhage, edema, degeneration, necrosis, hyperplasia, fibrosis, organization, granulation tissue, and inflammatory changes were carefully observed to evaluate the pathological damage to the lungs.

[0045] Results of lung injury indicators in bronchoalveolar lavage fluid:

[0046] After the rats were anesthetized, the abdominal cavity was opened. After the rats were bled out from the abdominal aorta, the chest cavity was opened, the lungs were stripped, the trachea was exposed, the left lung was ligated, a small "V"-shaped incision was made at the main trachea, the lavage needle was inserted, and the ligature was fixed. The right lung was lavaged with 37°C, 0.9% sodium chloride injection as the lavage fluid. The 0.9% sodium chloride injection was slowly injected into the lungs while gently massaging the chest wall, and then the lavage fluid was slowly withdrawn. Lavage was performed twice. The first time, 6 mL was injected and 3 mL was collected. The second time, 3 mL was injected, and the mixture was aspirated twice to mix and 3 mL was collected. The alveolar lavage fluid was centrifuged at 2500 r / min for 15 minutes, and the supernatant was collected. The lung injury indicators were detected using the fully automatic biochemical analyzer Thermo Scientic. Figure 2 As shown in the figure, 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 the lung tissue was damaged.

[0047] 5.3 Results of inflammatory factor levels in lung tissue

[0048] After anesthesia, the rats were opened in the abdominal cavity, the lungs were dissected, and a 3 mg block of tissue was taken from the lung tissue and placed in saline. The tissue was rinsed repeatedly for 3 times to remove the blood in the tissue block, and the surface moisture of the tissue block was wiped with filter paper; the tissue block was weighed and placed in a beaker, and 4°C saline was measured in the beaker with a pipette, and the ratio of the total volume of saline to the weight of the lung tissue block was 9:1, so that the tissue block was immersed in saline; in an ice water bath, the soaked tissue block was quickly cut into pieces with ophthalmic scissors, and further ultrasonically crushed to prepare a 10% lung tissue homogenate, and the enzyme-linked immunosorbent assay (ELISA) method was used to detect inflammatory factors. The detection steps are as follows: set standard wells and sample wells, add 50 μL of different concentrations of standard wells to each standard well; set blank wells (blank control wells without samples and enzyme-labeled reagents, and the rest of the steps are the same) and sample wells to be tested. First add 40 μl of sample diluent to the sample well to be tested on the enzyme-labeled plate, and then add 10 μl of the sample to be tested (the final dilution of the sample is 5 times). Add the sample to the bottom of the ELISA plate well, try not to touch the well wall, and shake gently to mix; add 100μl of ELISA reagent to each well, except for the blank well; seal the plate with a sealing film and incubate at 37℃ for 60 minutes; dilute the 20-fold concentrated washing solution with 20-fold distilled water for later use; carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard, repeat this 5 times, and pat dry; first add 50μl of color developer A to each well, then add 50μl of color developer B, gently shake to mix, and color at 37℃ in the dark for 15 minutes; add 50μl of stop solution to each well to stop the reaction (the blue color will turn yellow immediately at this time); adjust the blank well to zero, and measure the absorbance (OD value) of each well in sequence at a wavelength of 450nm. The measurement should be performed within 15 minutes after adding the stop solution. Figure 3As shown in the figure, inflammatory factors such as IL-1β, IL-6, IL-18, and TNF-α were significantly increased in the lung tissue of the PPT group. In the lung tissue, the oxidative stress marker malondialdehyde (MDA) was significantly increased, and reduced glutathione (GSH) was significantly increased ( Figure 2 ).

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

[0050] Fresh lung tissue was immediately put into tissue fixative, the tissue was taken out of the fixative, and the target part of the tissue was trimmed with a scalpel in the fume hood, and the trimmed tissue and the corresponding label were placed in the embedding frame. The dehydration box was placed in the dehydrator for dehydration in gradient 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, alcohol benzene for 5-10 minutes, xylene I for 5-10 minutes, xylene II for 5-10 minutes, 65° melted paraffin I for 1 hour, 65° melted paraffin II for 1 hour, 65° melted paraffin III for 1 hour. The tissue soaked in wax was embedded in the embedding machine. First, the melted wax was placed in the embedding frame, and before the wax solidified, the tissue was taken out of the dehydration box and placed in the embedding frame according to the requirements of the embedding surface and labeled accordingly. Cool in a -20° freezer. After the wax solidifies, remove the wax block from the embedding frame and trim the wax block. Place the trimmed wax block on a paraffin slicer and slice it to a thickness of 4μm. Float the slices on the 40°C warm water of the spreader to flatten the tissue, pick up the tissue with a slide, and bake the slices in a 60°C oven. After the wax is baked dry, take it out and store it at room temperature for later use. Put the slices into environmentally friendly dewaxing liquid I for 20min-environmentally friendly dewaxing liquid II for 20min-anhydrous ethanol I for 5min-anhydrous ethanol II for 5min-75% alcohol for 5min, and wash with tap water. The slices are treated in high-definition constant staining pretreatment solution for 1min. Slices are stained in hematoxylin staining solution for 3-5min, washed with tap water, differentiated with differentiation solution, washed with tap water, blued with blue return solution, and rinsed with running water. Dehydrate the slices in 95% alcohol for 1min and stain them in eosin staining solution for 15s. The slices were placed in anhydrous ethanol I for 2 min, anhydrous ethanol II for 2 min, anhydrous ethanol III for 2 min, n-butanol I for 2 min, n-butanol II for 2 min, xylene I for 2 min, xylene II for 2 min, and then sealed with neutral gum. Microscope examination and image acquisition and analysis were performed. Figure 4 As shown, no obvious congestion and inflammatory cell infiltration were observed in the CON control group; in the PPT intervention group, lymphocyte infiltration (black arrow), congestion (red arrow), dilation and congestion of alveolar wall capillaries, and a small amount of red blood cells in the alveolar cavity were observed.

[0051] The above results show that after PPT treatment, the lung tissue was significantly damaged, inflammatory cells infiltrated, capillary dilation and congestion; 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 increased; inflammatory factors such as IL-1β, IL-6, IL-18, TNF-α in the lung tissue of the PPT group were significantly increased, and the oxidative stress markers malondialdehyde (MDA) and reduced glutathione (GSH) were significantly increased. It is generally believed that the occurrence of ALI is caused by multiple factors (including but not limited to infection, trauma, poisoning, shock, etc.), which first trigger uncontrolled systemic inflammation, eventually leading to vascular endothelial damage and infiltration of multiple inflammatory cells, accompanied by mild alveolar damage. PPT may cause lung pathological changes by destroying cell integrity and inducing pulmonary oxidative stress response, resulting in irreversible damage and leading to the occurrence of ALI.

[0052] In summary, the modeling method of the present invention is an effective method for establishing acute lung injury through oral administration of podophyllotoxin, and the experimental group shows typical characteristics similar to human acute lung injury. It can be used for exploring targets and drug research on 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 in the protection scope of the present invention.

Claims

1. A method for establishing an acute lung injury animal model, characterized in that: Podophyllotoxin or podophyllotoxin reagent is delivered into rats by oral administration or intragastric perfusion to obtain the acute lung injury animal model.

2. The method for establishing an acute lung injury animal model according to claim 1, characterized in that: The dosage of podophyllotoxin is 20 mg / kg per day, for 4 to 5 consecutive days; The dosage of the podophyllotoxin reagent is: a reagent containing 20 mg / kg podophyllotoxin per day.

3. The method for establishing an acute lung injury animal model according to claim 2, characterized in that: The rats were male SD rats aged 6 to 8 weeks, weighing 210±20 g.

4. The method for establishing an acute lung injury animal model according to any one of claims 1 to 3, characterized in that: The podophyllotoxin reagent is prepared by dissolving podophyllotoxin in 2% DMSO and then fixing the volume with 0.5% sodium carboxymethyl cellulose to prepare the podophyllotoxin reagent.

5. The method for detecting the acute lung injury animal model according to claim 1, characterized in that: After continuous administration of podophyllotoxin or podophyllotoxin reagent, cytokines in bronchoalveolar lavage fluid of rats were detected, lungs of rats were removed to detect and compare lung weights, and inflammatory factors and pathological damage were detected in lung tissues.

6. The method for detecting an acute lung injury animal model according to claim 5, characterized in that: The detection of cytokines in bronchoalveolar lavage fluid was as follows: the lung injury indicators lactate dehydrogenase, alkaline phosphatase, albumin and total protein content were detected using a fully automatic biochemical analyzer Thermo Scientic.

7. The method for detecting an acute lung injury animal model according to claim 5, characterized in that: The detection of lung tissue inflammatory factors is: using enzyme-linked immunosorbent assay (ELISA) method to detect the content of IL-18, TNF-α, IL-6 and IL-1β.

8. The method for detecting an acute lung injury animal model according to claim 5, characterized in that: The detection and comparison of lung weight is to compare the organ coefficients of the bilateral lungs of rats.

9. The method for detecting an acute lung injury animal model according to claim 5, characterized in that: The pathological detection is: using hematoxylin-eosin staining method to perform pathological observation on lung tissue.

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