Method for constructing mouse pulmonary fibrosis model through exposure of triclocarban and application

Abstract

The invention discloses a method for constructing a mouse pulmonary fibrosis model, and provides application of an antibacterial agent triclocarban (TCC) in establishment of a pulmonary fibrosis animal model. Specifically, 50 mg / kg of TCC is introduced into the lung tissue of an adult mouse in a surgical airway administration mode, and the pulmonary fibrosis mouse model can be obtained after the mouse is continuously fed for 7 days and 14 days. By means of pathological section and immunohistochemical analysis of mouse lung tissue, it is found that the mouse shows typical pulmonary fibrosis pathological characteristics 7 days after TCC exposure, the pulmonary fibrosis degree is aggravated 14 days after exposure, and the characteristic that clinical pulmonary fibrosis is aggravated progressively is simulated. The animal model established by the invention is simple to operate, low in cost, high in mouse survival rate, short in modeling time and uniform in pulmonary fibrosis distribution, and can well simulate the clinical fibrosis pathogenesis process. The invention also provides application of the mouse model obtained by the method in pulmonary fibrosis pathogenesis research.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to an application and method of an antibacterial agent triclocarban in establishing a mouse pulmonary fibrosis model. Background Art

[0002] Pulmonary fibrosis is a chronic, progressive, irreversible interstitial lung disease characterized by extensive proliferation of fibroblasts and deposition of extracellular matrix, which leads to destruction of lung tissue structure, fulminant respiratory failure, gradual loss of lung function and ultimately death. [1] Therefore, it is of great significance to construct an animal model of pulmonary fibrosis and study its pathogenesis and diagnosis and treatment strategies.

[0003] At present, the main methods for constructing animal models of pulmonary fibrosis include chemical drug method (bleomycin, silicon dioxide, isothionine fluorescein, etc.), radiation induction method and gene induction method. [4][5] Each method has its own defects and shortcomings. For example, radiation and gene induction methods are expensive, take a long time to model, and have poor stability. [6] The cost of intratracheal administration of isothionine fluorescein and silica aerosol inhalation modeling is high and cannot simulate the clinical characteristics of interstitial pneumonia in pulmonary fibrosis. [7] Bleomycin is relatively cheap and is currently the most commonly used drug to induce pulmonary fibrosis. However, the bleomycin-induced pulmonary fibrosis model takes a long time to establish (more than 28 days), has poor stability, and is prone to cause death of experimental animals. [8][9] In addition, bleomycin-induced pulmonary fibrosis is self-healing, which is inconsistent with the progressive aggravation of clinical pulmonary fibrosis.

[10] Therefore, there is an urgent need to develop a method for preparing an animal model that is rapid, stable, and can simulate the clinical characteristics of clinical pulmonary fibrosis. References

[0004] King TE Jr, Pardo A, Selman M. Idiopathic pulmonary fibrosis. Lancet.2011 Dec 3; 378(9807): 1949-61. Lederer, DJ and Martinez, FJ, Idiopathic pulmonary fibrosis. NEngl J Med [J], 2018. 379(8): 797-798. Thannickal, VJ, Toews, GB, White, ES, et al., Mechanisms of pulmonary fibrosis. Annu Rev Med [J], 2004. 55: 395-417. Li R, Jia Y, Kong X, Nie Y, Deng Y, Liu Y. Novel drug deliverysystems and disease models for pulmonary fibrosis. J Control Release. 2022Aug; 348: 95-114. Li Zhihui, Yu Xueqing, Yang Shuguang, et al. Research progress on experimental models of idiopathic pulmonary fibrosis[J]. Chinese Journal of Laboratory Animal Science, 2024, 32(1): 118-127. Chua F, Gauldie J, Laurent GJ. Pulmonary fibrosis: searching for model answers. Am J Respir Cell Mol Biol. 2005 Jul; 33(1): 9-13. Moore BB, Hogaboam CM. Murine models of pulmonary fibrosis. Am JPhysiol Lung Cell Mol Physiol. 2008 Feb; 294(2): L152-60. Moeller A, Ask K, Warburton D, Gauldie J, Kolb M. The bleomycinanimal model: a useful tool to investigate treatment options for idiopathic pulmonary fibrosis? Int J Biochem Cell Biol. 2008;40(3): 362-82. Wang Bo, Song Qinghua, Tang Huimeng, et al. Research progress of animal models of bleomycin-induced pulmonary fibrosis[J]. Journal of Chinese Laboratory Animal Science, 2023, 31(12): 1617-1628. Chua F, Gauldie J, Laurent GJ. Pulmonary fibrosis: searching for model answers. Am J Respir Cell Mol Biol. 2005 Jul; 33(1): 9-13. Summary of the invention

[0005] 1. The present invention aims to solve the technical problems of the existing animal models of pulmonary fibrosis, such as high cost, long time period, poor stability, and inability to replicate clinical pathological characteristics, and aims to provide an animal model that is fast, economical, efficient, stable, and more in line with the characteristics of progressive aggravation of clinical pulmonary fibrosis pathology.

[0006] 2. To solve the above technical problems, the present invention provides the use of TCC in establishing an animal model of pulmonary fibrosis.

[0007] 3. The present invention also provides a method for constructing an animal model of pulmonary fibrosis, which specifically comprises the following steps: after anesthetizing mice, exposing the lung tissue of adult mice to TCC by intratracheal surgical administration, and continuing to feed for 7-14 days to obtain the animal model of pulmonary fibrosis.

[0008] 4. The present invention also provides the application of the mouse model obtained by the above method in the pathogenesis of pulmonary fibrosis and related research. Beneficial Effects

[0009] Compared with the prior art, the present invention has the following beneficial effects: 1. TCC chemical synthesis technology is mature, commercialized, low-priced, and easily available. It has shown good results in constructing a pulmonary fibrosis model in experiments, providing a new method for constructing an animal model of pulmonary fibrosis.

[0010] 2. TCC is a broad-spectrum synthetic antibacterial agent widely used in human production and life. The environmental pollution and health effects caused by it are increasingly concerned. TCC has been detected in human blood, urine, umbilical cord blood and breast milk. The present invention uses TCC to induce animal pulmonary fibrosis disease model, which can be used to study the pathogenesis of pulmonary fibrosis and evaluate the efficacy of drugs for treating pulmonary fibrosis caused by TCC pollution, and has broad application prospects.

[0011] 3. Compared with the existing animal induced models of pulmonary fibrosis, the present invention induces pulmonary fibrosis through intratracheal administration, which is simple to operate, has a low mortality rate, and is low in cost. It does not require a gas exposure device or continuous administration. At the same time, the modeling time cycle is short and the success rate is high. The constructed animal model can simulate the characteristics of progressive aggravation of clinical pulmonary fibrosis and has great application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 : TCC molecular structure formula.

[0013] Figure 2 : Schematic diagram of intratracheal surgical drug delivery in mice (A) and actual operation diagram (B).

[0014] Figure 3 : Statistical graphs of weight changes (B) and survival rate (A) of mice after TCC exposure.

[0015] Figure 4 : Pathological analysis results after 7 and 14 days of TCC exposure. Scale bar: 100 µm.

[0016] Figure 5 :α-SMA immunohistochemical staining (A) and quantitative statistical graph (B) of mouse lung tissue after TCC intratracheal administration. Scale bar: 50 µm.

[0017] Figure 6 :Masson staining (A) and statistical graph (B) of mouse lung tissue after TCC intratracheal administration. Scale bar: 50 µm. DETAILED DESCRIPTION

[0018] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the above content of the present invention will be described in detail with reference to the accompanying drawings and embodiments. The specific embodiments described herein are only part of the embodiments of the present invention and are used to explain the present invention. This should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. Other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0019] It should be noted that the mice used in the examples of the present invention were purchased from the Experimental Animal Center of Anhui Medical University and were kept in the Animal Center of Anhui University. Paraformaldehyde was purchased from Macklin Company, item number 30525-89-4; hematoxylin-eosin staining solution was purchased from Yuanye Biological Company, item number R20570-2; chloral hydrate for anesthesia was purchased from MACKLIN Company, item number 302-17-0; TCC was purchased from Aladdin, item number 101-20-2; PBS solution composition: KH2 PO4 was purchased from Aladdin Company with the item number 7778-77-0; Na2HPO4·12H2O was purchased from Shanghai Test Company with the item number 10039-32-4; ​​citric acid was purchased from Wokai with the item number 77-92-9; NaCl was purchased from White Shark Biological Company with the item number 7647-14-5; Masson staining kit was purchased from Solebold Technology Co., Ltd. with the item number G1340; a-SMA antibody was purchased from Wuhan Sanying Biotechnology with the item number 80008-1-RR; anhydrous ethanol was purchased from Shanghai Test Company with the item number 64-17-5.

[0020] The English abbreviations used in the present invention represent the following meanings: TCC: triclocarban; PBS: phosphate buffered saline; PFA: paraformaldehyde; Masson: Masson staining; α-SMA: α-smooth muscle actin.

[0021] The embodiment of the present invention provides the use of TCC in establishing a pulmonary fibrosis mouse model.

[0022] 2. An embodiment of the present invention also provides a method for constructing an animal model of pulmonary fibrosis, which comprises the following steps: anesthetizing mice by intraperitoneal injection of chloral hydrate, exposing lung tissues of adult mice to TCC by surgical intratracheal administration, and continuing to feed for 7-14 days to obtain the animal model of pulmonary fibrosis.

[0023] 3. Surgical method: TCC is injected into the airway by anesthetizing the mouse with 10% chloral hydrate, exposing the mouse's large airway tissue, inserting a syringe into the airway annular septum, and injecting 20 mg / mL of ultrasonic TCC / PBS solution into the mouse's airway. Then, the mouse is rotated and shaken to allow TCC to evenly enter all parts of the lung. The drug is administered once.

[0024] 4. The mice are of C57 / BL-6 strain, 8-10 weeks old adult mice.

[0025] 5. The present invention also provides the application of the animal model obtained by the above method in the study of the pathogenesis and therapeutic drugs of pulmonary fibrosis. Example

[0026] A method for establishing a triclocarban exposure-induced pulmonary fibrosis model, the method comprising surgical intra-airway TCC administration, and the specific steps of the operation process are as follows: Prepare 20 mg / mL TCC / PBS solution and perform ultrasonic pretreatment on the TCC / PBS solution. The molecular structure of TCC is shown in the attached figure. Figure 1 shown.

[0027] Adult mice of the C57 / BL-6 strain aged 8 to 10 weeks were weighed using a precision balance. The weight of the mice used was about 25 grams.

[0028] The surface of the mouse was disinfected with 75% ethanol, and the mouse was anesthetized by intraperitoneal injection of 10% chloral hydrate with an injection volume of 75 mL / mouse.

[0029] Use surgical scissors to carefully cut open the neck of the mouse and expose the large airway tissue (attached Figure 2 AB).

[0030] Insert a sterile syringe (1 mL) into the annular septum of the large airway trachea, and inject the ultrasound-pretreated TCC / PBS solution when there is no resistance to suction. The injection dose is 50 mg / kg mouse body weight, and the total volume is 150 mL / mouse.

[0031] Grab the mouse by the tail and rotate it to make TCC enter all parts of the lungs evenly. Put the mouse on a 37℃ warming table. After waking up, move it to the breeding center for normal breeding. Observe and record the weight and survival rate of the mouse. The results showed that 50 mg / kg TCC exposure had no significant effect on the survival rate and weight of the mice (see Appendix). Figure 3 AB).

[0032] The mice were killed 7 and 14 days after TCC surgical intra-airway exposure, and the lung tissues were obtained and fixed in 4% PFA solution at 4 ℃ for 16 hours, embedded in paraffin and sectioned. The samples were stained with hematoxylin-eosin, Masson's staining, and a-SMA immunohistochemical staining. The pathological changes of lung tissues were observed under an optical microscope.

[0033] Analysis of the staining results of lung tissue pathology sections. Figure 4 As shown in the figure, after 7 days of intratracheal exposure to 50 mg / kg TCC, obvious fibrotic lesions were observed in the lung tissue of mice, and the degree of fibrosis increased significantly after 14 days. After low-dose TCC exposure (25 mg / kg TCC), no obvious lung fibrosis was produced after 7 and 14 days of exposure.

[0034] The lung tissue samples injected with 50 mg / kg TCC were subjected to immunohistochemical staining with a-SMA antibody. The results showed that the expression of a-SMA in the lung tissue of mice increased significantly after 7 days of TCC treatment, and further increased after 14 days of TCC treatment ( Figure 5 AB), indicating that TCC exposure leads to progressive aggravation of pulmonary fibrosis.

[0035] The lung samples injected with 50 mg / kg TCC were analyzed by Masson staining, and it was found that the Masson staining positive area increased significantly after 7 days of TCC treatment, and further increased after 14 days of TCC treatment ( Figure 4 AB).

[0036] In summary, the present invention has established a method for constructing an animal model of pulmonary fibrosis via triclocarban (TCC). The method is simple to operate, has a high survival rate of mice, and a short modeling period. Moreover, the established animal model of pulmonary fibrosis can replicate the characteristics of progressive aggravation of clinical fibrotic diseases, and has good value for promotion and application.

[0037] 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. An application of an antibacterial agent triclocarban (TCC) in inducing a mouse pulmonary fibrosis model and a construction method, characterized in that: The following steps are involved: Step 1: Anesthetize the mice. The specific method is to anesthetize the mice with 10% chloral hydrate intraperitoneally, with an injection volume of 75 μL / mouse. The genetic background of the selected mice is C57 / BL6, the age is 8-10 weeks, and the weight is about 25 g; Step 2: fix the mouse, disinfect the mouse neck with 75% ethanol, and surgically expose the mouse airway; Step 3: administer TCC intratracheally, the dosage of which is 50 mg / kg body weight, TCC is dissolved in PBS solution at 20 mg / mL, and ultrasonic pretreatment is performed 2 hours before use, the total volume is 150 μL / mouse, and the number of intratracheal administration is once; Step 4: After continuing to feed for 7-14 days, the pulmonary fibrosis mouse model is obtained.

2. Use of the method for constructing pulmonary fibrosis by TCC exposure as described in claim 1 in constructing an animal pulmonary fibrosis model.

3. Application of the pulmonary fibrosis animal model constructed by the mouse pulmonary fibrosis method as described in claim 1 in pulmonary fibrosis research.

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