Method for establishing non-infectious pulmonary fibrosis acute exacerbation animal model by using TritonX-100 solution

Abstract

The invention provides an application of TritonX-100 in establishing an animal model with acute exacerbation of non-infectious pulmonary fibrosis and a method for establishing the animal model with acute exacerbation of non-infectious pulmonary fibrosis by using a TritonX-100 solution, and the method comprises the following steps: inducing animal pulmonary fibrosis by using a bleomycin solution; and then applying the TritonX-100 working solution to the pulmonary fibrosis animal model, and establishing the non-infection cause pulmonary fibrosis acute exacerbation animal model. The invention also provides application of the animal model with acute exacerbation of non-infective pulmonary fibrosis in screening, verification, evaluation, evaluation or research of drugs and / or diagnostic strategies and / or treatment strategies for preventing and / or treating acute exacerbation of non-infective pulmonary fibrosis. According to the method, bleomycin (BLM) recommended by a guide is firstly used for establishing pulmonary fibrosis, a non-toxic non-infective reagent TritonX-100 is combined, a brand new non-infective animal model is successfully established, clinical practice is highly simulated, and a modeling scheme is economical, convenient, repeatable, non-toxic and worthy of popularization.

Description

Technical Field

[0001] The present invention belongs to the technical field of pulmonary fibrosis research, and particularly relates to a method for establishing an animal model of acute exacerbation of non-infectious-induced pulmonary fibrosis by using Triton X-100 solution. Background Art

[0002] In recent years, a variety of animal models of acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF) have been reported. Among them, the more common ones are to induce and establish a mouse pulmonary fibrosis model by using bleomycin (BLM) or transgenic methods, etc. On this basis, viruses, bacteria or other physical and chemical substances are used for stimulation to induce AE. However, there are many controversies in the currently published modeling methods, mainly focusing on the administration methods, administration times and frequencies of inducing AE, as well as the selection of reagents.

[0003] In terms of the administration method, currently there are nasal drip, oropharyngeal inhalation, intratracheal injection, tracheotomy administration, subcutaneous injection, etc.; for nasal drip and oropharyngeal inhalation, there may be problems such as the drug not being able to completely enter the lungs, drug loss, and inconsistent modeling effects due to large individual differences; the tracheotomy method causes great damage to mice, with high infection rates and mortality rates; subcutaneous injection may have problems with drug absorption.

[0004] In terms of the administration time and administration frequency, the main problems currently are that the administration time is too early. For example, when inducing acute exacerbation of pulmonary fibrosis (AE) by re-administering drugs less than 14 days after bleomycin administration, the mouse model is still in the acute inflammation phase at this time, and re-administering drugs cannot accurately mimic the clinical actual situation. Some protocols use the method of multiple administrations, which is cumbersome and complex, and easily leads to an increase in the mortality rate of mice, and cannot reflect that a certain factor "triggers" AE, which is different from the "sudden" and rapid progression situations when AE occurs in clinical IPF patients.

[0005] The reagents reported for inducing AE are currently also various, such as bacteria and replication-active viruses, physical and chemical reagents, etc. Bacteria and replication-active viruses have high requirements for laboratory conditions and are at risk of easy infection, while physical and chemical reagents are highly toxic and may pose safety risks to experimental personnel.

[0006] According to the published literature, it can be found that about 80% of the existing animal models use infectious factors such as viruses or bacteria, while the "non-infectious" animal models only account for about 20%. This seriously does not match the fact that "non-infectious" AE-IPF accounts for a larger proportion in clinical practice.

[0007] Existing "non-infectious" animal models still have many limitations. First, in some animal models, polyhexamethylene guanidine (PHMG) is used for treatment. After establishing a pulmonary fibrosis model, cadmium chloride (CdCl2) is used for stimulation multiple times (on the 3rd, 6th, 9th, and 12th days) to establish a model of acute exacerbation of pulmonary fibrosis. The main defects of such animal models are that the reagent is toxic, PHMG is not a commonly used reagent recommended by the guidelines for establishing pulmonary fibrosis, and the administration on the 3rd, 6th, 9th, and 12th days is too early. At this time, pulmonary fibrosis has not been fully formed, and multiple administrations increase the difficulty and complexity of the experiment. In addition, although some animal models use bleomycin (BLM), a reagent recommended by the guidelines, to establish a pulmonary fibrosis model, nickel chloride is selected as the reagent to induce acute exacerbation, and it is administered simultaneously with BLM (on the 0th day). Not only is nickel chloride toxic, but the administration time is too early. At this time, pulmonary fibrosis has not been fully formed, which does not conform to the clinical reality. There is also an animal model established by using BLM twice on the 0th day and the 21st day, and the modeling results are evaluated on the 35th day; or an animal model is established by administering BLM once every two weeks for a total of 6 times (for up to 12 weeks). Although such models select BLM to establish animal models according to the guidelines, the duration of these studies is too long, ranging from 5 weeks to 12 weeks, which seriously reduces the research efficiency. In addition, there is an animal model established by continuously subcutaneously pumping BLM. Each mouse requires 1 micropump, which has a high usage cost, complex operation, and is invasive, and is not suitable for large-scale popularization and use.

[0008] Therefore, on the basis of using BLM recommended by the guidelines to establish pulmonary fibrosis, how to overcome the technical problems existing in the above-mentioned solutions and effectively establish an animal model of acute exacerbation of "non-infectious" induced pulmonary fibrosis urgently needs to be studied. Summary of the Invention

[0009] To solve the above-mentioned technical problems existing in the prior art, the present invention provides a method for establishing an animal model of acute exacerbation of "non-infectious" induced pulmonary fibrosis by using Triton X-100 solution.

[0010] The technical solution of the present invention is as follows:

[0011] The first object of the present invention is to provide the application of Triton X-100 in establishing an animal model of acute exacerbation of "non-infectious" induced pulmonary fibrosis.

[0012] Further, the application is to use Triton X-100 to induce acute exacerbation of "non-infectious" induced pulmonary fibrosis on the basis of a pulmonary fibrosis animal model.

[0013] Further, a pulmonary fibrosis animal model is established by using bleomycin.

[0014] The second object of the present invention is to provide a method for establishing an animal model of acute exacerbation of non-infectious induced pulmonary fibrosis, comprising the following steps:

[0015] S1: Induce pulmonary fibrosis in animals with bleomycin solution to obtain an animal model of pulmonary fibrosis;

[0016] S2: Administer Triton X-100 working solution to the animal model of pulmonary fibrosis obtained in S1 to establish an animal model of acute exacerbation of non-infectious induced pulmonary fibrosis.

[0017] Further, the concentration of the bleomycin solution in S1 is 2 μg / μl, and the solvent is physiological saline.

[0018] Further, the bleomycin solution in S1 is injected intratracheally, and the injection dose is 3 μg / g.

[0019] Further, the treatment days in S1 are 14 days.

[0020] Further, the concentration of the Triton X-100 working solution in S2 is 0.1%, and the solvent is physiological saline.

[0021] Further, the Triton X-100 working solution in S2 is injected intratracheally.

[0022] Preferably, when the model animal is a mouse, the injection dose of the Triton X-100 working solution is 50-100 μl / animal.

[0023] Further, the treatment days in S2 are 7 days.

[0024] The third object of the present invention is to provide an animal model of acute exacerbation of non-infectious induced pulmonary fibrosis constructed by the aforementioned method.

[0025] Further, the animal is a mouse, rat, rabbit, pig, dog, sheep.

[0026] The fourth object of the present invention is to provide the application of the aforementioned animal model of acute exacerbation of non-infectious induced pulmonary fibrosis in screening, verifying, evaluating, assessing or studying drugs and / or diagnostic strategies and / or treatment strategies for preventing and / or treating acute exacerbation of non-infectious induced pulmonary fibrosis.

[0027] The beneficial effects of the present invention are as follows:

[0028] The present invention first uses bleomycin (BLM) recommended by the guidelines to establish pulmonary fibrosis, and intratracheally injects a non-toxic "non-infectious" reagent, Triton X-100 working solution, on the 14th day to induce acute exacerbation of pulmonary fibrosis. It not only successfully establishes a new "non-infectious" animal model, but also highly simulates the clinical reality. The modeling scheme is economical, convenient, repeatable and non-toxic, and is worthy of promotion. Brief Description of the Drawings

[0029] Figure 1 . Flow chart for establishing the model of acute exacerbation of pulmonary fibrosis

[0030] Figure 2 . Detection of mouse lung tissue on the 21st day, where:

[0031] Figure 2 A Gross specimen and CT image of mouse lung tissue

[0032] Figure 2 B Detection result of hydroxyproline content in lung tissue

[0033] Figure 2 C H.E. and Masson staining diagrams of lung tissue

[0034] Figure 3 . Detection of the expression level of Collagen I in mouse lungs by immunoblotting

[0035] Figure 4 . Detection of the apoptosis level in mouse lungs, where:

[0036] Figure 4 A Diagram of detecting the apoptosis level by TUNEL

[0037] Figure 4 B Immunofluorescence detection diagram of the type II alveolar epithelial cell marker SPC

[0038] Figure 5 . Giemsa staining diagram of mouse bronchoalveolar lavage fluid (BALF)

[0039] Figure 6 H.E. staining and Masson staining results of lung tissue in the 0.05% Triton working solution treatment group

[0040] Figure 7 . Detection of mouse lung tissue on the 21st day, where:

[0041] Figure 7 A H.E. and Masson staining diagrams of mouse lung tissue

[0042] Figure 7 B Detection result of hydroxyproline content in lung tissue

[0043] Figure 7 C Immunoblotting profile of Collagen I Detailed Implementation Modes

[0044] The present invention will be further explained below in conjunction with embodiments, but the embodiments do not impose any form of limitation on the present invention.

[0045] Example 1

[0046] 1. Equipment, reagents, and mouse preparation:

[0047] 1.1 Mice:

[0048] Male C57BL / 6J mice aged 6 - 8 weeks were selected.

[0049] 1.2 Instruments:

[0050] Ear tags, adjustable mouse boards, fixators (rubber bands, tapes), blunt-tipped forceps, mouse laryngoscopes, and intratracheal atomization drug delivery devices for mice.

[0051] 1.3 Reagents:

[0052] Isoflurane, bleomycin powder, TritonX-100 stock solution, and normal saline.

[0053] 2. Preparation of bleomycin solution and TritonX-100 working solution:

[0054] 2.1 Preparation of bleomycin solution:

[0055] The drug specification is 15 mg / vial.

[0056] In a sterile operating table, dissolve 15 mg of bleomycin in 7.5 ml of normal saline and shake well (the concentration of the bleomycin solution is 2 μg / μl).

[0057] The dosage of bleomycin is 3 μg / g. According to the body weight of the mice, aspirate the corresponding volume of the solution.

[0058] 2.3 Preparation of TritonX-100 working solution:

[0059] The specification of TritonX-100 is 100 ml / vial. Dilute 1 μl of TritonX-100 to 1000 μl to obtain a 0.1% TritonX-100 working solution.

[0060] The dosage for each mouse is 50 μl of 0.1% TritonX-100 working solution, which is injected intratracheally.

[0061] 3. Modeling process

[0062] 3.1 Induce pulmonary fibrosis in mice by intratracheal injection of bleomycin:

[0063] 1. On day 0, weigh the mice and mark them with ear tags; anesthetize the mice by aerosol inhalation of isoflurane, and pay attention to keeping them warm;

[0064] 2. After successful anesthesia of the mice, place them in a supine position on the mouse board, fix the upper incisors with wire, and fix the limbs with tape;

[0065] 3. Adjust the mouse board to an appropriate angle with the mouse's head facing upward. Gently pull out the mouse's tongue to one side with forceps.

[0066] 4. Hold the mouse laryngoscope in the left hand and insert it into the mouse's throat. Press down on the root of the tongue and gently lift it to fully expose the glottis, where regular opening and closing of the glottis can be seen. Gently insert the nebulized drug delivery device into the mouse's lungs through the glottis and inject the corresponding volume of bleomycin solution.

[0067] 5. After modeling, it can be observed that the mouse's respiratory rate increases, the chest of the mouse rises and falls significantly, and moist rales can be heard when the mouse is moved close to the ear.

[0068] 6. The assistant releases the fixation, places the mouse in the prone position in the cage, and observes the state of the mouse after waking up.

[0069] 7. Observe the mouse's activities, respiratory rate, food intake, hair color changes, and survival status every day, and measure the mouse's weight every other day, and make records.

[0070] 3.2 Induce acute exacerbation of pulmonary fibrosis in mice by intratracheal injection of Triton X-100 working solution:

[0071] 1. On the 14th day after bleomycin treatment, nebulize and inhale isoflurane again to fully anesthetize the mouse, and observe the anesthetic effect and any adverse reactions.

[0072] 2. After the mouse is successfully anesthetized, inject 50 μl of Triton X-100 working solution into the trachea using the same method as in procedure 3.1.

[0073] 3. After modeling, it can be observed that the mouse's respiratory rate increases, the chest of the mouse rises and falls significantly, and moist rales can be heard when the mouse is moved close to the ear.

[0074] 4. The assistant releases the fixation, places the mouse in the prone position in the cage, and observes the state of the mouse after waking up.

[0075] 5. Observe the mouse's activities, respiratory rate, food intake, hair color changes, and survival status every day, and measure the mouse's weight every other day, and make records.

[0076] 6. On the 21st day, perform low-dose chest CT examination; euthanize the mouse by cervical spinal cord transection, and collect samples such as blood, lung tissue, and bronchoalveolar lavage fluid to evaluate the modeling effect.

[0077] The modeling process of Example 1 is as Figure 1 shown. At the same time, set up a normal saline treatment group (NS), a bleomycin treatment group (BLM), and a Triton X-100 treatment group (Triton X) as controls. Except for the difference in experimental drugs, the rest of each control group is the same as steps 3.1 and 3.2.

[0078] On the 21st day, the results of mouse lung tissue and low-dose chest CT examinations showed that the degree of lung injury in the BLM+Triton group was significantly stronger than that in the BLM group and the other groups ( Figure 2 A); Detection of hydroxyproline content in lung tissue indicated that the content in the BLM+Triton group was significantly higher than that in the BLM group and the other groups ( Figure 2 B); H.E. and Masson staining of lung tissue showed that reusing Triton on the basis of bleomycin could cause obvious inflammatory reactions and more significant collagen deposition ( Figure 2 C).

[0079] Immunoblotting was used to detect the expression level of Collagen I in mouse lungs. The results showed that the level of type I collagen in the BLM+Triton group was significantly higher than that in the BLM group and the other groups ( Figure 3 ).

[0080] TUNEL was used to detect the level of cell apoptosis. It was found that the level of cell apoptosis in the lungs of mice in the BLM+Triton group was significantly higher than that in the BLM group and the other groups ( Figure 4 A); Immunofluorescence detection of the type 2 alveolar epithelial cell marker SPC indicated that the death of a large number of type 2 alveolar epithelial cells in the lungs of mice in the BLM+Triton group was significantly stronger than that in the BLM group and the other groups ( Figure 4 B).

[0081] Giemsa staining of mouse bronchoalveolar lavage fluid (BALF) was performed. It was found that a large number of macrophages and neutrophils aggregated in the BALF of mice in the BLM+Triton group, indicating that the level of lung inflammation was significantly higher than that in the BLM group and the other groups ( Figure 5 ).

[0082] Comparative Example 1

[0083] We further tried a lower concentration of Triton solution (0.05% Triton working solution) and evaluated its modeling effect. Except that the concentration of the Triton working solution was replaced with 0.05% Triton, the other operations were the same as those in Example 1. The results of H.E. staining and Masson staining of lung tissue showed that compared with BLM mice, there was no significant increase in lung tissue inflammation and fibrosis in the BLM+0.05% Triton group ( Figure 6 ), which was significantly different from the effects of lung tissue inflammation and fibrosis obtained by treating with 0.1% TritonX-100 working solution in Example 1 ( Figure 2 C).

[0084] Comparative Example 2

[0085] We further compared the present invention with a comparative experiment using a higher concentration of Triton solution (0.3% Triton working solution). Except that the concentration of the Triton working solution was replaced with 0.3% Triton, the operations were the same as those in Example 1.

[0086] Meanwhile, a comparative experiment using BLM for modeling twice was also set up. Except that the Triton working solution was replaced with BLM, the operations were the same as those in Example 1.

[0087] The pathological results, the determination of hydroxyproline content, and the results of Collagen I immunoblotting showed that: in the 0.3% Triton working solution treatment group, the blue-stained area shown by Masson staining was significantly smaller than that in the 0.1% Triton working solution treatment group, and both the hydroxyproline content and the expression level of Collagen I were significantly lower than those in the 0.1% Triton working solution treatment group; in the group treated with BLM twice, the blue-stained area shown by Masson staining was significantly smaller than that in the 0.1% Triton working solution treatment group, and the hydroxyproline content and the expression level of Collagen I were also significantly lower than those in the 0.1% Triton working solution treatment group ( Figure 7 ), and the above results indicate that the effect after modeling with the 0.1% Triton working solution is significantly better than these two schemes.

[0088] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Application of Triton X-100 in establishing an animal model of acute exacerbation of non-infectious-induced pulmonary fibrosis.

2. The application according to claim 1, characterized in that The application is to use Triton X-100 to induce acute exacerbation of non-infectious-induced pulmonary fibrosis on the basis of an animal model of pulmonary fibrosis.

3. The application according to claim 2, characterized in that, Use bleomycin to establish an animal model of pulmonary fibrosis.

4. A method for establishing an animal model of acute exacerbation of non-infectious induced pulmonary fibrosis, characterized in that, It includes the following steps: S1: Induce animal pulmonary fibrosis with a bleomycin solution to obtain an animal model of pulmonary fibrosis; S2: Administer a Triton X-100 working solution to the animal model of pulmonary fibrosis obtained in S1 to establish an animal model of acute exacerbation of non-infectious-induced pulmonary fibrosis.

5. The method according to claim 4, wherein The concentration of the Triton X-100 working solution described in S2 is 0.1%, and the solvent is physiological saline.

6. The method according to claim 4, wherein The Triton X-100 working solution described in S2 is administered by intratracheal injection; Preferably, when the model animal is a mouse, the injection dose of the Triton X-100 working solution is 50 - 100 μl / animal.

7. The method according to claim 4, wherein The treatment days in S2 are 7 days.

8. An animal model of acute exacerbation of non-infectious cause pulmonary fibrosis, characterized in that, Constructed by the method described in any one of claims 4 to 7.

9. The animal model of acute exacerbation of non-infectious-induced pulmonary fibrosis according to claim 8, characterized in that, The animal is a mouse, rat, rabbit, pig, dog, sheep.

10. Application of the animal model of acute exacerbation of non-infectious-induced pulmonary fibrosis described in claim 8 in screening, validating, evaluating, assessing or studying drugs and / or diagnostic strategies and / or treatment strategies for preventing and / or treating acute exacerbation of non-infectious-induced pulmonary fibrosis.

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