Method for establishing idiopathic pulmonary fibrosis model through unilateral lung tissue bleomycin in-situ injection

Abstract

The invention relates to a method for establishing an idiopathic pulmonary fibrosis model through in-situ injection of bleomycin in unilateral lung tissue. According to the method, lung tissue is directly exposed in a surgical operation mode, and bleomycin solution is introduced into the lung in a direct injection mode. Wherein the difficulty of exposing the lung tissue by the surgical operation is obviously reduced compared with that of exposing the lung tissue by the traditional trachea, so that the applicability of the disease model is greatly improved. And secondly, the bleomycin solution is directly injected into the lung tissue, so that the operability is better than that of a mode of atomizing from a trachea. The death rate of animals with successful modeling is 0, vital signs such as the body weight of the animals are not abnormal, histopathologic examination shows that the bleomycin is more uniformly distributed in lungs after direct injection, and the injury degrees of all areas of the lungs are similar.

Description

Technical Field

[0001] The invention relates to the technical field of animal models, and in particular to a method for establishing an idiopathic pulmonary fibrosis model by in situ injection of bleomycin into unilateral lung tissue. Background Art

[0002] Idiopathic pulmonary fibrosis (IPF) is a serious and complex interstitial lung disease with a median survival rate of only 3-5 years after diagnosis. The mortality rate of IPF is even higher than that of most tumors, and it is called a "tumor-like disease." Despite progress in the understanding of the pathobiology of the disease, effective treatments are still limited, and currently only nintedanib and pirfenidone are conditionally recommended drugs.

[0003] IPF animal models play a vital role in studying pathological mechanisms, evaluating new treatments, and tracking disease progression. There are many different methods for constructing IPF animal models, each with its own advantages and disadvantages. Common modeling methods include drug induction such as bleomycin and cyclophosphamide, and mineral induction such as silica and asbestos. Among these modeling methods, bleomycin is widely used because of its rapid induction of disease progression. Bleomycin is a mixture of water-soluble alkaline glycopeptide antibiotics, mainly composed of bleomycin A2 (55-70%) and B2 (25-32%). It was originally widely used to treat squamous cell carcinoma of the head and neck, esophagus and genitals, and Hodgkin's lymphoma. However, in clinical use, it was found that it can cause nonspecific pneumonia, accompanied by pulmonary fibrosis in severe cases, and the combination of bleomycin and vincristine can even lead to rapid death from pulmonary fibrosis.

[0004] In the past few decades, the scientific community has widely believed that bleomycin-induced lung injury can represent the typical characteristics of IPF, and it has become the most commonly used reagent for IPF modeling. There are many ways to induce lung injury and lung repair with bleomycin, including intratracheal instillation, intraperitoneal injection, subcutaneous injection, intravenous injection, and osmotic pumps. Among them, intratracheal instillation allows bleomycin to contact lung tissue more quickly and directly and is adopted by many researchers. After entering the lung tissue, bleomycin mediates damage to alveolar epithelial cells and vascular endothelial cells in the lung tissue through multiple pathways, including oxidative stress, inflammatory response, activation of coagulation cascade reactions, and disruption of the balance of cell proliferation and apoptosis. These damage responses will initiate the process of lung tissue damage and repair, which is also considered to be the key initiation step of pulmonary fibrosis.

[0005] After the cascade reaction is initiated, the continuous damaging factors activate fibroblasts and transform them into myofibroblasts that produce extracellular matrix. The excessive deposition of collagen and other ECM components secreted by myofibroblasts will lead to changes in tissue structure. The entire repair process is further disturbed by irritating factors, which eventually leads to changes in lung tissue structure and scar formation, seriously damaging lung function.

[0006] In the 1970s, RW Fleischman et al. studied the systemic safety of bleomycin in dogs. After intravenous injection of different doses of bleomycin (5, 2.5, 1.25 and 0.625 mg / kg) of bleomycin, dogs developed varying degrees of interstitial pneumonia and pulmonary fibrosis. In another toxicology study, mice were intraperitoneally injected with bleomycin solution (0.1 mg / kg) twice a week, and the results showed that the lung lesions (perivascular edema, cell infiltration and fibrosis) were very severe. The data from these toxicology studies provided a reliable basis for bleomycin-induced IPF. Subsequently, researchers established animal models of bleomycin-induced IPF in multiple species (mice, rats, guinea pigs, hamsters, dogs and primates). Mice have become the most commonly used animals because of their wide range of sources, among which C57BL / 6J mice are more sensitive to bleomycin.

[0007] Different laboratories have developed a variety of modeling methods using different bleomycin dose combinations and induction techniques. Intraperitoneal injection can be induced with a dose of 20-50 mg / kg bleomycin, while intravenous injection (10-20 mg / kg) and intratracheal injection (3-5 mg / kg) can only accept lower doses. In existing studies, researchers most often use intratracheal aerosol spray injection of bleomycin to induce pulmonary fibrosis.

[0008] Looking back at these modeling methods, high mortality and inconvenience of operation are always an unavoidable fact. In the process of single-dose bleomycin tracheal spray modeling, the animal mortality rate is sometimes as high as 50%, and the mortality rate induced by long-term low doses is as high as 30%. The reason for the high lethality is that the operation must be performed under deep anesthesia, when the animal's breathing becomes shallow, and the introduction of bleomycin can easily cause suffocation, leading to instant death of the animal. At the same time, the mouse's pharynx is narrow and the tracheal opening is extremely small. Bleomycin is easily injected into the esophagus during injection, resulting in weakened stimulation or failure of stimulation. Moreover, organ injection also requires professional equipment for assistance. These unfavorable factors make tracheal injection modeling face serious challenges in animal welfare and experimental costs. Summary of the invention

[0009] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for establishing an idiopathic pulmonary fibrosis model by in situ injection of bleomycin into unilateral lung tissue. The idiopathic pulmonary fibrosis animal model is prepared by directly injecting bleomycin solution guided by surgically visualized lung tissue. The prepared mouse model conforms to the symptoms and signs of idiopathic pulmonary fibrosis, so that it can be used for research on the mechanism and drug screening of idiopathic pulmonary fibrosis.

[0010] To achieve the above object, the technical solution adopted by the present invention is:

[0011] In a first aspect, the present invention provides a method for establishing an idiopathic pulmonary fibrosis model by in situ injection of bleomycin into unilateral lung tissue, characterized in that the method comprises the following steps:

[0012] S1. Anesthetize the mouse, disinfect it, cut the skin and muscle under its armpit, and expand the wound to expose one lung;

[0013] S2. Inject 50 μL of bleomycin solution at 5 mg / kg into one lung to obtain the idiopathic pulmonary fibrosis model.

[0014] Specifically, the method for constructing the model is to introduce bleomycin by in situ injection to induce pulmonary fibrosis symptoms in mice, and evaluate it by histopathology and molecular biology. The modeling method is as follows: first, after the animal is deeply anesthetized, iodine is used to disinfect the skin on the left rib of the animal, and surgical scissors are used to cut about 1 cm of skin along the armpit of the mouse, and the muscles under the skin are cut to expose the ribs. At this time, the pulsating left lung can be seen. After 1ml syringe draws 50μL bleomycin solution, it is pierced into the left lung at about 30 degrees, and then steadily pushed into the lung. After the injection is completed, the muscles and skin are sutured in layers and monitored until the animal is awake. The pulmonary fibrosis modeling method of the present invention successfully induces inflammation and fibrosis of lung tissue, monitors the development process of the disease, and finally uses clinical drugs to verify the model. The content of the present invention confirms that the model is successfully established and can be used for the mechanism research of pulmonary fibrosis diseases such as IPF and the development and screening of related drugs.

[0015] In some embodiments, the entire modeling time is 2-3 minutes.

[0016] In some embodiments, the animal presents diffuse extensive damage to the left lung.

[0017] In some embodiments, the animals exhibit zero mortality.

[0018] In some embodiments, the animal experiences minimal weight loss and rapid recovery.

[0019] In some embodiments, the animal is a mouse.

[0020] In a second aspect, the present invention provides the use of bleomycin in preparing the idiopathic pulmonary fibrosis model described in the first aspect.

[0021] Preferably, the pulmonary fibrosis model presents pulmonary inflammatory response and collagen fiber deposition lesions, both of which are at their peak at 0-4 weeks after modeling.

[0022] In a third aspect, the present invention provides an evaluation method for the idiopathic pulmonary fibrosis model described in the first aspect, characterized in that it comprises the following steps: evaluating from the macroscopic phenotype and microscopic indicators, and treating based on the clinically recommended drug nintedanib to disprove the rationality of the model construction method; the macroscopic phenotype includes the animal's coat condition, weight changes, lung tissue appearance and texture; the microscopic indicators include histopathological HE and Maoosn staining observations, and qPCR inspection indicators.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) In the model construction method of the present invention, lung tissue is directly visualized by surgical operation, and bleomycin solution is introduced into the lung by direct injection. Compared with the traditional method of visualizing lung tissue in the trachea, the difficulty of surgical operation is significantly reduced, which will greatly improve the applicability of the disease model. Secondly, directly injecting bleomycin solution into lung tissue is more quantitative than atomizing from the trachea. The mortality rate of animals with successful modeling was 0, and there were no abnormalities in the vital signs of the animals, such as weight. Histopathological examination showed that the distribution of bleomycin in the lungs after direct injection was more uniform, and the degree of damage in various areas of the lungs was similar.

[0025] (2) The method of evaluating the pulmonary fibrosis animal model of the present invention is to observe typical clinical symptoms such as survival status, behavioral status, weight changes, and histopathology to systematically score bleomycin-induced inflammation and collagen fiber deposition for comprehensive evaluation, and at the same time, further verify whether the creation of the model animal is successful by evaluating the therapeutic effect of the clinically recommended drug nintedanib on the pulmonary fibrosis model. Finally, the cumulative quantification of various evaluation indicators proves that the idiopathic pulmonary fibrosis model created by the present invention is successful and feasible, and can be used for subsequent research related to idiopathic pulmonary fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Comparison of the lung distribution of the same volume indicator in the orthotopic injection group and the tracheal injection group;

[0027] Figure 2 The body weight changes and lung / body weight coefficient after in situ and tracheal injection of 5 mg / kg bleomycin were recorded;

[0028] Figure 3Hydroxyproline content in lung tissue after in situ and tracheal injection of 5 mg / kg bleomycin;

[0029] Figure 4 The lung lesion area after in situ and tracheal injection of 5 mg / kg bleomycin;

[0030] Figure 5 The manifestations of lung tissue damage at different times after in situ injection of bleomycin;

[0031] Figure 6 HE and Masson staining at different times after bleomycin in situ injection modeling;

[0032] Figure 7 The inflammation and fibrosis scores of lung tissue at different times after in situ injection of bleomycin were calculated;

[0033] Figure 8 The expression of inflammatory and fibrotic genes in the left and right lungs at different times after in situ injection of bleomycin;

[0034] Fig. 9 The weight changes and lung / body weight coefficients of animals after nintedanib treatment;

[0035] Fig.10 HE and Masson staining after nintedanib treatment;

[0036] Fig.11 To score tissue inflammation and fibrosis after nintedanib treatment;

[0037] Fig.12 Figure 2 is the expression of inflammatory and fibrotic genes after nintedanib treatment. DETAILED DESCRIPTION

[0038] Many specific details are set forth in the following description to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art can make similar modifications without violating the connotation of the present invention, so the present invention is not limited to the specific implementation disclosed below.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0040] Example 1 Comparison of unilateral in situ injection of bleomycin and tracheal injection

[0041] 6-8 week old male C57BL / 6J mice (Sichuan Weitong Lihua Experimental Animal Technology Co., Ltd.) were selected and injected with 50 μL of cobalt blue solution in situ and trachea after surgery to visualize lung tissue. The animals were immediately euthanized and the distribution of the dye was observed. The distribution of the dye after in situ injection of the same volume was more uniform. Figure 1 Further, the model was established by introducing bleomycin at a dose of 5 mg / kg and 50 μL. The weight recovery rate of the animals after in situ injection was faster than that after tracheal injection. The lung / body weight coefficient was also significantly increased compared with normal animals 17 days after model establishment (*, P < 0.05). Figure 2 Further lung hydroxyproline detection showed an increasing trend after in situ injection modeling. Figure 3 Histopathological examination with HE and Masson staining showed that after in situ modeling, necrosis and collagen fiber deposition were distributed in large areas of lung tissue, while tracheal induction was scattered in small areas in the main bronchi. Figure 4 The above results show that in situ injection of bleomycin is better than tracheal injection in inducing lung injury. "*" represents the comparison between the model group and the control group.

[0042] Example 2 Progression of lung lesions after unilateral in situ injection of bleomycin

[0043] Example 1 confirms that unilateral in situ injection of bleomycin into the lung is an appropriate modeling method. 6-8 week old male C57BL / 6J mice (Sichuan Weitong Lihua Experimental Animal Technology Co., Ltd.) were selected and injected with bleomycin in situ at a dose of 5 mg / kg and 50 μL for modeling. The development process of the lung disease was then studied to determine the window period of the model. The mice were euthanized on the 7th, 14th, 21st, and 28th day of modeling. Obvious damage (bruises and changes in volume) was visible in the left lung on the 7th, 14th, and 21st days, and gradually returned to normal on the 28th day. The results are as follows: Figure 5 HE and Masson staining were used to examine the inflammatory cell infiltration and collagen fiber deposition at different times. The inflammatory period was mainly in the first 3 weeks, and fibrosis lasted for 4 weeks. The inflammation and fibrosis of the model group were significantly higher than those of the control group (****, P < 0.0001 and *, P < 0.05). The scores gradually decreased with time. Figure 6 , 7 The expression of genes related to inflammation and fibrosis in the left and right lungs was studied in terms of mechanism. The results of qPCR were similar to the phenotype and histopathology. Both inflammatory and fibrotic genes were highly expressed, and the left lung changed first before the right lung. The results are as follows Figure 8 "*" represents the comparison between the model group and the control group.

[0044] Example 3 Nintedanib counter-evidence unilateral in situ injection of bleomycin model

[0045] Examples 1 and 2 determined that the method of unilateral in situ injection is a new way to model IPF, and severe lung inflammation and fibrosis in the model animals occurred within 3 weeks after modeling. The IPF mouse model was then evaluated for its therapeutic effect with nintedanib. During the 2-week treatment, the weight and other vital signs of the animals were stable. At the end of the treatment, the lung / body weight coefficient of the model animals increased significantly (####, P < 0.0001), and nintedanib significantly reduced the lung / body weight coefficient of the animals (*, P < 0.05). The results are as follows Fig. 9 At the same time, histopathological examination also showed that the fibroblasts (arrows), inflammatory cells and collagen fiber deposition were improved after treatment. The scores showed that the model group animals had significant inflammation and fibrosis (####, P < 0.0001), and the scores of these two indicators were significantly reduced after treatment (**, P < 0.01). Fig.10 , 11 The qPCR results at the mechanism level also confirmed the therapeutic effect of nintedanib. Fig.12 "#" represents the comparison between the model group and the control group, and "*" represents the comparison between the drug-treated group and the model group.

[0046] In summary, the method of establishing an idiopathic pulmonary fibrosis (IPF) model by in situ injection of bleomycin in unilateral lung tissue described in the present invention is feasible. The method has the characteristics of low difficulty in operation, small animal weight loss and low mortality, which is suitable for use by a large number of researchers. The present invention describes the occurrence and development process of the model in detail and clarifies the window period for treatment. The therapeutic effect of the clinically recommended drug nintedanib is used to confirm that the modeling method has a controllable degree of damage to the lungs and is suitable for drug screening. This series of studies show that the IPF model created by the present invention is successful and feasible, and can be used for subsequent studies related to IPF.

[0047] The present invention has been introduced in detail above, and the preferred embodiments have been specifically applied to explain the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core ideas, including the best mode, and also enable any technician in the field to practice the present invention, including manufacturing and use, and implementing any combined method. It should be pointed out that for ordinary technicians in this technical field, without departing from the principle of the present invention, the present invention can also be improved and modified, and these improvements and modifications also fall within the scope of protection of the claims of the present invention. The scope of patent protection of the present invention is defined by the claims and may include other embodiments that can be thought of by those skilled in the art. If these other embodiments have structural elements that are not different from the textual expression of the claims, or if they include equivalent structural elements that are not substantially different from the textual expression of the claims, then these other embodiments should also be included in the scope of the claims.

Claims

1. A method for establishing an idiopathic pulmonary fibrosis model by in situ injection of bleomycin into unilateral lung tissue, characterized in that: The following steps are involved: S1. Anesthetize the mouse, disinfect it, cut the skin and muscle under its armpit, and expand the wound to expose one lung; S2. Inject 50 μL of bleomycin solution at 5 mg / kg into one lung to obtain the idiopathic pulmonary fibrosis model.

2. Use of bleomycin in preparing the idiopathic pulmonary fibrosis model according to claim 1.

3. The use according to claim 2, characterized in that: The pulmonary fibrosis model presents pulmonary inflammatory response and collagen fiber deposition lesions, which are at their peak at 0-4 weeks after modeling.

4. The method for evaluating an idiopathic pulmonary fibrosis model according to claim 1, wherein: The following steps are involved: Evaluation was conducted based on macroscopic phenotypes and microscopic indicators, and treatment was performed based on the clinically recommended drug nintedanib to disprove the rationality of the model construction method.

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