Medicinal preparation for treating pulmonary fibrosis

Abstract

The invention discloses a pharmaceutical preparation for treating pulmonary fibrosis. The pharmaceutical preparation comprises an Arnt2 gene inhibitor and a pharmaceutically acceptable carrier or auxiliary material. The research finds that the Arnt2 gene is an important action target for treating pulmonary fibrosis, and the pulmonary fibrosis can be effectively treated by silencing, interfering or reducing the expression of the Arnt2 gene. The invention provides a new strategy for the treatment of pulmonary fibrosis, the screening of pulmonary fibrosis drugs and the research on the pathogenic mechanism of pulmonary fibrosis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a drug preparation for treating pulmonary fibrosis. BACKGROUND

[0002] Pulmonary fibrosis is a serious occupational disease that harms the health of coal mine workers. Long-term exposure to coal dust particles in a coal mine environment can trigger chronic inflammation and abnormal repair processes in lung tissue, ultimately leading to excessive deposition of pulmonary interstitial fibrous connective tissue, resulting in destruction of lung tissue structure and loss of function. According to statistics, the incidence of pulmonary fibrosis in coal mine workers is significantly higher than that in the general population due to long-term exposure to coal dust, which seriously affects the quality of life and labor capacity of patients, and even endangers their lives.

[0003] Currently, the main clinical treatment methods for pulmonary fibrosis caused by coal dust particles include drug therapy, oxygen therapy, and lung transplantation. Drug therapy mainly uses glucocorticoids and immunosuppressive agents, but these drugs have significant side effects and limited long-term efficacy; oxygen therapy can only alleviate the symptoms of dyspnea in patients and cannot fundamentally stop the progression of pulmonary fibrosis; lung transplantation can effectively improve lung function, but there are problems such as donor shortage and immune rejection. Therefore, there is an urgent need to develop a safe and effective new treatment method.

[0004] In recent years, with the development of molecular biology technology, gene therapy has brought new hope for the treatment of pulmonary fibrosis. However, there is currently no gene target related to pulmonary fibrosis caused by coal dust particles. SUMMARY

[0005] The purpose of the present application is to provide the application of Arnt2 gene as a target in the treatment of pulmonary fibrosis to solve the problems existing in the prior art. The present application researches and finds that Arnt2 gene is an important target for treating pulmonary fibrosis caused by coal dust carbon nanoparticles, and by silencing, interfering or reducing the expression of Arnt2 gene, pulmonary fibrosis can be effectively treated. The present application provides a new strategy for the treatment of pulmonary fibrosis, the screening of pulmonary fibrosis drugs, and the study of the pathogenesis of pulmonary fibrosis.

[0006] To achieve the above purpose, the present application provides the following scheme: The present application provides a drug preparation for treating pulmonary fibrosis, wherein the drug preparation comprises an Arnt2 gene inhibitor and a pharmaceutically acceptable carrier or excipient.

[0007] Preferably, the Arnt2 gene inhibitor comprises shRNA that silences the Arnt2 gene; the sense strand of the shRNA is shown in SEQ ID NO. 1, and the antisense strand is shown in SEQ ID NO. 2.

[0008] Preferably, the pharmaceutical preparation is any pharmaceutically acceptable dosage form suitable for a gene inhibitor. More preferably, the dosage form of the pharmaceutical preparation is an injection preparation or a nasal administration preparation. Among them, the nasal administration preparation includes nose drops, nose sprays, nose gels, nose ointments, nose powders, nose films, nose powder sprays, nose liposomes, or nose microsphere preparations. Among them, the nose drops of the present application refer to any pharmaceutical preparation form suitable for nasal instillation in the art.

[0009] The present application also provides the use of Arnt2 gene as a target in the preparation of a drug for treating pulmonary fibrosis.

[0010] The present application also provides the use of Arnt2 gene inhibitors in the preparation of a drug for treating pulmonary fibrosis.

[0011] Optionally, the Arnt2 gene inhibitor includes an agent that inhibits, interferes with, or silences the expression of Arnt2 gene.

[0012] Optionally, the agent that silences the expression of Arnt2 gene includes shRNA that silences Arnt2 gene. The sense strand of the shRNA is shown in SEQ ID NO. 1, and the antisense strand is shown in SEQ ID NO. 2.

[0013] The present application also provides the use of Arnt2 gene as a target in the preparation of a drug for treating pulmonary fibrosis.

[0014] The present application also provides a method for screening a drug for treating pulmonary fibrosis by detecting the expression amount of Arnt2 gene before and after administration to screen a drug for treating pulmonary fibrosis.

[0015] Further, the pulmonary fibrosis of the present application includes pulmonary fibrosis induced by coal dust particles.

[0016] The present application discloses the following technical effects: The present application researches and finds that coal dust particle exposure can induce epithelial mesenchymal transition (EMT) of type II alveolar epithelial cells and cause pulmonary fibrosis, cause high expression of Arnt2 gene, and Arnt2 gene can be used as a target for treating pulmonary fibrosis. The present application further finds through in vivo gene silencing experiment and in vitro gene silencing experiment that by silencing Arnt2 gene in the EMT model of type II alveolar epithelial cells and the pulmonary fibrosis mouse model, the EMT phenotype of the cells can be reversed, the pulmonary fibrosis lesion condition of the mouse lung tissue can be reversed and improved, the pulmonary fibrosis score can be degraded, and the method has a significant effect of treating pulmonary fibrosis.

[0017] The present application researches and finds that Arnt2 gene is an important role target for treating pulmonary fibrosis, and pulmonary fibrosis can be effectively treated by silencing, interfering or reducing the expression of Arnt2 gene. The present application provides a new strategy for the treatment of pulmonary fibrosis, the screening of pulmonary fibrosis drugs, and the study of the pathogenesis of pulmonary fibrosis. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0019] Figure 1 The collection of coal dust nanoparticles and the determination results of chemical components thereof; wherein, A is the appearance of KN95 particulate matter filter cotton at different underground operation times; B is a scanning electron microscope image of a representative coal dust particle sample; C is a particle size distribution graph of the representative coal dust particle sample calculated by volume; D is the weight content of chemical elements in the representative coal dust particle sample; E is the weight content ratio of oxygen to each chemical element in the representative coal dust particle sample; Figure 2 Experimental results of coal dust carbon nanoparticle exposure induced pulmonary fibrosis in mice; wherein, A is the lung appearance of each group of mice; B is the lung coefficient of each group of mice; C is a hematoxylin-eosin (HE) staining diagram of lung tissue of each group of mice; D is a Masson staining diagram of lung tissue of each group of mice; E is the expression level of pulmonary fibrosis marker molecules in lung tissue of each group of mice; F is the pulmonary fibrosis score of each group of mice according to the Szapiel scoring system; the data is expressed as mean ± standard deviation, n = 6, **P < 0.01 and ***P < 0.001; Figure 3Figure 2 shows the EMT of type II alveolar epithelial cells in each group of mice; wherein A is the expression level of EMT marker molecules in the lung tissues of each group of mice; B is the EMT of type II alveolar epithelial cells in the lung tissues of mice; C is the cluster heat map of differentially expressed genes in the control group and the type II alveolar epithelial cell EMT model group; D is the volcano plot of differentially expressed genes in the control group and the type II alveolar epithelial cell EMT model group; E is the GO analysis result of Focal adhesion; F is the KEGG analysis result of Focal adhesion; G is the GO analysis result of Cell matrix adhesion; H is the GO analysis result of Cell substrate adhesion; I is the GO analysis result of Collagen binding; J is the GO analysis result of Collagen biosynthesis; K is the GSVA analysis result based on the REACTOME database; Figure 4 Figure 3 shows the screening results of differentially expressed genes in each group of mice; wherein A is the cluster heat map of differentially expressed genes in each group of mice; B is the Wayne diagram of differentially expressed genes in each group of mice; C is the immunofluorescence staining detection result of the expression level of Arnt2 protein in the lung tissues of each group of mice; D is the immunohistochemical detection result of the expression level of Arnt2 protein in the lung tissues of each group of mice; Figure 5 Figure 4 shows the results of the experiment of silencing Arnt2 gene in vitro; wherein A is the migration ability of each group of cells; B is the invasion ability of each group of cells; C is the expression level of EMT marker molecules in each group of cells, the data is expressed as mean ± standard deviation, n = 3, **P < 0.01 and ***P < 0.001; Figure 6 Figure 5 shows the results of the experiment of silencing Arnt2 gene in vivo; wherein A is the gene expression level of Arnt2 in the lung tissues of each group of mice; B is the protein expression level of Arnt2 in the lung tissues of each group of mice; C is the hematoxylin-eosin (HE) staining diagram of the lung tissues of each group of mice; D is the Masson staining diagram of the lung tissues of each group of mice; E is the pulmonary fibrosis score of each group of mice; F is the expression level of pulmonary fibrosis marker molecules in the lung tissues of each group of mice; the data is expressed as mean ± standard deviation, n = 6, **P < 0.01 and ***P < 0.001. DETAILED DESCRIPTION

[0020] The detailed description set forth above is not intended as being a limitation on the present application, but rather a description of certain aspects, features, and embodiments of the present application. Many changes, modifications, variations, and other uses of the present application will become readily apparent to those skilled in the art, such changes, modifications, variations, and other uses being within the scope of the present application.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0026] The animals used in the following examples were 6-8 week old male C57BL / 6 mice purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. They were housed in a specific pathogen-free room with controlled temperature (22 ± 2 ℃) and circulating light, and were allowed free access to food and water. All animal experiments in this invention followed the ARRIVE guidelines and the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals (NIH Publication No. 8023, revised 1978), and were reviewed and approved by the Biomedical Research Ethics Committee of Anhui University of Science and Technology.

[0027] Example 1: Collection and chemical composition analysis of coal dust nanoparticles KN95 particulate filter cotton used by coal miners in five large mines was collected to collect coal dust particles. It was found that the filter cotton trapped a large number of fine coal dust particles, and the longer the coal miners worked underground, the more fine coal dust particles were trapped in the filter cotton. Figure 1 A). Scanning electron microscopy (SEM) analysis of 37 coal dust particle samples collected from eluted filter cotton revealed that the particle size was all <500 nm and tended to be spherical. Figure 1 (B); Detection using a nanoparticle size potentiometer revealed that the average content of coal dust particles with a diameter <100 nm was 95.31±3.66% (B). Figure 1 (C). The above results indicate that coal dust nanoparticles are the main particulate matter exposed to by coal miners.

[0028] The weight content of chemical components in the above 37 coal dust samples was further determined by X-ray energy dispersive spectroscopy (Table 1, representative sample analysis is shown in Table 1). Figure 1 The D-values ​​showed that the average weight content of silicon was 2.88 ± 2.52%, while the carbon content was the highest, averaging 74.49 ± 12.87%. Furthermore, these coal dust particle samples all exhibited extremely low oxygen-to-carbon ratios (O2 / C2). Figure 1 The presence of E indicates that it has low wettability and is not easily soluble, properties that make it more biotoxic.

[0029] Table 1. Volume distribution (Volume %) and weight percentage (Weight %) of nano-sized coal dust particles in 37 samples. <100 nm C O Na Mg Al Si S K Ti Fe Ca N Cl 1 99.80 62.67 21.74 0.53 0.30 6.34 6.30 0.65 0.23 0.71 0.54 2 97.65 75.09 15.91 0.45 0.24 3.20 3.56 0.44 0.47 0.64 3 94.35 54.97 25.24 0.60 0.07 7.53 8.57 0.64 0.88 1.09 0.42 4 99.73 79.20 14.71 0.72 0.28 2.09 2.16 0.45 0.17 0.21 5 97.86 56.61 17.71 0.47 1.02 1.16 1.18 0.37 0.09 20.92 0.47 6 95.80 83.74 11.04 0.83 0.75 0.42 3.22 7 99.07 77.70 15.66 2.41 2.03 0.33 1.88 8 97.85 46.17 30.93 3.68 0.35 0.23 0.25 1.15 17.23 9 98.45 85.97 10.81 1.21 1.18 0.68 0.16 10 89.12 55.89 25.37 9.73 8.13 0.88 11 98.77 71.99 19.30 4.67 3.84 0.20 12 98.65 84.69 10.36 2.59 2.08 0.28 13 95.45 55.78 28.26 8.32 7.14 0.24 0.12 0.14 14 96.43 80.71 14.14 0.65 4.21 0.29 15 96.50 58.64 20.18 1.09 0.46 0.73 0.63 1.03 0.44 0.70 15.80 0.30 16 95.75 80.14 12.12 2.59 2.18 0.27 2.70 17 97.54 87.46 9.02 1.42 1.33 0.64 0.13 18 98.30 87.70 9.53 1.02 0.84 0.69 0.22 19 89.10 88.65 8.23 1.29 1.23 0.60 20 97.70 76.29 15.92 0.69 2.92 3.32 0.48 0.09 0.18 0.12 21 98.06 52.89 34.59 6.72 5.48 0.05 0.26 22 98.54 62.98 20.35 7.87 8.43 0.28 0.10 23 81.03 81.38 10.35 3.97 3.52 0.47 0.30 24 92.24 82.05 10.31 1.81 1.44 0.33 4.06 25 95.32 89.01 8.84 0.83 0.72 0.43 0.16 26 94.43 85.31 8.43 1.30 1.02 0.25 3.69 27 95.40 86.70 11.64 0.58 0.52 0.37 0.19 28 94.43 87.47 9.71 0.95 1.05 0.60 0.22 29 95.50 62.63 23.95 6.49 6.27 0.45 0.20 30 93.45 88.10 9.07 1.09 0.97 0.60 0.17 31 92.42 59.74 39.02 0.29 0.41 0.42 0.12 32 92.30 83.98 12.85 0.92 0.96 0.68 0.28 0.16 0.17 33 92.30 81.02 11.41 2.77 2.52 0.30 1.98 34 94.40 59.64 26.52 6.88 6.27 0.33 0.13 0.23 35 95.40 77.38 13.81 0.46 4.08 3.78 0.34 0.07 0.07 36 91.80 83.91 10.28 0.96 0.91 0.27 3.67 37 95.45 82.04 12.15 1.74 1.57 0.40 2.10 mean 95.31 74.49 16.47 0.63 0.86 3.06 2.88 0.44 0.25 0.43 3.36 1.72 4.55 0.85 SD 3.66 12.87 7.97 0.21 1.28 2.66 2.52 0.18 0.22 0.34 7.75 4.89 4.63 1.24 The above results indicate that coal dust nanoparticles are the main particulate matter that coal miners are exposed to, and they have the highest carbon content. Therefore, coal dust carbon nanoparticles may be another key toxic component in coal miners' pneumoconiosis and pulmonary fibrosis.

[0030] Example 2: Coal dust carbon nanoparticle exposure induces pulmonary fibrosis To verify the effect of coal dust carbon nanoparticles on pulmonary fibrosis, this invention attempted to construct a mouse model of pneumoconiosis pulmonary fibrosis using coal dust carbon nanoparticles. The construction process of the mouse model of pneumoconiosis pulmonary fibrosis is as follows: Carbon nanoparticles were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., item number 633100-25G; coal dust nanoparticles were obtained by elution from KN95 particulate filter cotton collected in Example 1.

[0031] According to the "Occupational Health Standard of the People's Republic of China - Occupational Exposure Limits for Hazardous Factors in the Workplace - Part 1: Chemical Hazardous Factors" (GBZ2.1-2019), the concentration exposure limit for respirable coal dust (free SiO2 < 10%) is 2.5 mg / m³. 3Assuming an average adult weight of 60 kg, the daily inhalation volume is approximately 23 m³. 3 [Pleil JD, ArielGeer Wallace M, Davis MD, Matty CM. The physics of human breathing: flow, timing, volume, and pressure parameters for normal, on-demand, and ventilator respiration. J Breath Res. 2021;15(4):10.] The national statutory working day is 8 hours per day. Based on this, the maximum daily coal dust exposure dose for coal miners is estimated to be approximately 2.5 mg / m³. 3 × 23 m 3 × 8 h / 24 h / 60 kg = 319 µg / kg. Considering the filtering and interception function of the protective device in actual situations, the maximum dosage of coal dust nanoparticles in this experiment was 300 μg / kg / d, lower than the calculated maximum dosage. Based on the weight percentage of carbon in the coal dust nanoparticles, the dosages of carbon nanoparticles in this experiment were 200 μg / kg / d (CNPs-H group), 20 μg / kg / d (CNPs-M group), and 2 μg / kg / d (CNPs-L group), while the dosage of coal dust nanoparticles was 300 μg / kg / d (CD-NPs group). A control group (Control) was also set up, administering an equal amount of blank solvent. Finally, when mice were anesthetized by nasal inhalation and entered a deep coma, these particulate suspensions were administered to the mice via nasal drip for 3 months to establish mouse pneumoconiosis models with different exposure doses. After 3 months, pulmonary fibrosis-related indicators were detected, as follows: (1) Observe the appearance of the lungs and calculate the lung coefficient. Mice were euthanized, and their lung tissue was removed via thoracic dissection. Surrounding adipose tissue and interstitium were removed, and the lung tissue was observed and photographed. The bronchi were then removed, and the lungs were weighed using an electronic balance to calculate the lung coefficient. The formula for calculating the lung coefficient is as follows: Lung organ coefficient = lung tissue mass (g) / mouse body weight (g) × 100%.

[0032] (2) Hematoxylin-eosin staining and Masson staining were used to detect the pathological morphology and collagen formation of mouse lung tissue. Fresh mouse lung tissue was fixed in fixative for 24 h and then sequentially immersed in 70%, 80%, 90%, 95%, and 100% ethanol for 40 min each for dehydration. The tissue blocks were then cleared with xylene, embedded in paraffin, and sectioned. The sections were flattened in a 45 ℃ slide box, mounted on glass slides, and dried in a 45 ℃ incubator. The paraffin sections were then dewaxed and hydrated. Finally, routine hematoxylin-eosin and Masson staining were performed, and the sections were mounted with neutral resin and observed and photographed under an optical microscope.

[0033] (3) Score and classification of pulmonary fibrosis The degree of pulmonary fibrosis was assessed according to the scoring system published by Szapiel et al. [Szapiel SV, Elson NA, Fulmer JD, Hunninghake GW, Crystal RG. Bleomycin-induced interstitial pulmonary disease in the nude, athymic mouse. Am Rev Respir Dis. 1979;120(4):893-9.]. The degree of fibrosis was graded using the following criteria: none (0), no fibrosis; mild (1+), focal area of ​​fibrosis with some distortion of alveolar structure; moderate (2+), more extensive fibrosis and fibrosis still in focus; severe (3+), extensive fibrosis, confluent lesions, and extensive dysregulation of parenchymal structure.

[0034] (4) Western blot assay to detect the expression levels of EMT marker molecules in mouse pulmonary fibrosis. Mice were euthanized, and lung tissue was harvested via thoracic dissection. Surrounding adipose tissue, interstitium, and bronchi were removed, and the tissue was weighed using an electronic balance. 1 mL of RIPA lysis buffer was added per 100 mg of lung tissue, and the tissue was ground on ice until no tissue fragments remained. The RIPA lysis buffer was pre-supplemented with a protease phosphatase inhibitor mixture at a 1:50 ratio. The tissue was then centrifuged at 15,000 rpm for 15 min at 4°C to collect lung tissue proteins, and protein concentration was determined using a BCA kit according to the manufacturer's instructions. Next, the proteins were separated by electrophoresis on an SDS-PAGE separating gel and transferred to a PVDF membrane. The PVDF membrane was blocked in 5% skim milk at room temperature for 1 h. The membrane was then incubated overnight at 4°C with primary antibodies against pulmonary fibrosis markers (COL-1 and COL-3), primary antibodies against EMT markers (N-cadherin, E-cadherin, Vimentin), and β-actin working solution. The primary antibodies were recovered, and the membrane was washed three times with TBST. The membrane was incubated with the secondary antibody working solution at room temperature for 1 hour and washed three times with TBST. Finally, it was developed using ECL luminescent solution.

[0035] (5) Immunohistochemical detection of the expression levels of pulmonary fibrosis and EMT marker molecules in mice. Paraffin sections were dewaxed three times in xylene, hydrated twice in anhydrous ethanol, and hydrated once each in 90%, 80%, 70%, and 50% ethanol. They were then rinsed with tap water and distilled water for 5 min each. The sections were completely immersed in 3% hydrogen peroxide and blocked at room temperature in the dark for 30 min, followed by rinsing with PBS three times. An appropriate amount of sodium citrate antigen retrieval solution was added, and the sections were autoclaved for 10 min, followed by rinsing with PBS three times. The sections were then blocked for 10 min in a 37°C digital display constant temperature water bath with normal sheep serum working solution. Primary antibody working solution was added, and the sections were incubated overnight at 4°C. The primary antibody was recovered, and the sections were rinsed with PBS. Biotin-labeled secondary antibody was added, and the sections were incubated at 37°C for 30 min. The secondary antibody was recovered, and the sections were rinsed with PBS. Horseradish peroxidase-labeled streptomycin ovalbumin working solution was added, and the sections were incubated at 37°C for 30 min, followed by rinsing with PBS. Stain with DAB solution and observe under an inverted microscope to determine the optimal staining intensity. Rinse with tap water to stop the staining process. Counterstain with hematoxylin for 15 seconds, rinse with tap water for 2 minutes, and then rinse with PBS. Dehydrate paraffin sections with 75%, 85%, 90%, and 95% ethanol for 2 minutes each, followed by dehydration twice with anhydrous ethanol for 5 minutes each time. Clear the sections twice with xylene for 15 minutes each time. Finally, mount with neutral resin and observe and photograph under an optical microscope.

[0036] (6) Immunofluorescence co-localization analysis of the co-expression of EMT markers (Vimentin and N-cadherin), pulmonary fibrosis markers (COL-1 and COL-3), and type II alveolar epithelial cell markers (SP-A). Dewaxing paraffin sections: Immerse sections sequentially in xylene I and II for 15 min each, anhydrous ethanol I and II for 5 min each, and 85% and 75% ethanol for 5 min each, finally rinsing with distilled water. Antigen retrieval: Place tissue sections in a retrieval box filled with EDTA antigen retrieval buffer and microwave on medium heat for 8 min, turn off for 8 min, and then on medium-low heat for 7 min to perform antigen retrieval. Circle drawing: After slightly drying the sections, draw circles around the tissue using a histochemical pen. Blocking: Add bovine serum albumin to the circle and incubate for 30 min. Add primary antibody: Gently shake off the blocking solution, add primary antibody working solution to the section, and incubate overnight at 4 °C in a humidified chamber. Add fluorescent secondary antibody: Place the slide in PBS and wash three times on a decolorizing shaker, 5 min each time. After slightly drying the sections, add fluorescent secondary antibody working solution corresponding to the species marker of the primary antibody to the circle to cover the tissue, and incubate at room temperature for 50 min. Repeat the incubation with primary antibody and fluorescent secondary antibody sequentially until all target antibody incubation is complete. DAPI counterstaining of cell nuclei: After slightly drying the sections, add DAPI staining solution to the circle and incubate at room temperature in the dark for 10 min. Autofluorescence quenching: After slightly drying the sections, add autofluorescence quencher to the circle for 5 min, and rinse with running water for 10 min. Mounting: Mount the slides with antifluorescence quenching mounting medium. Microscopic examination and photography: Place the slides in a 3DHISTECH slide scanner to acquire images.

[0037] The results showed that, compared with the control group, the lungs of mice exposed to coal dust nanoparticles and carbon nanoparticles were slightly harder, with a large number of fine coal deposits and a moderate number of nodules; the number of coal deposits and nodules in the lungs of mice in the medium-dose and low-dose carbon nanoparticle groups decreased sequentially. Figure 2 Further analysis revealed that, compared to the control group, the lung coefficient of mice exposed to carbon nanoparticles was significantly increased, and this increase was dose-dependent. Figure 2 B). Hematoxylin-eosin and Masson staining results showed that, compared with the control group, the coal dust nanoparticle and carbon nanoparticle treatment groups exhibited similar interstitial lung lesions, such as alveolar space widening, inflammatory cell infiltration, and alveolar structural damage. Both groups also formed a large number of small nodules and blue collagen, and the lesions tended towards pulmonary fibrosis, showing a dose-dependent relationship with carbon nanoparticle exposure. Figure 2 (C and D). Subsequently, immunohistochemical results showed that, compared with the control group, the expression levels of pulmonary fibrosis marker molecules in the lung tissue of mice exposed to carbon nanoparticles were significantly increased, and this also showed a dose-dependent relationship with carbon nanoparticle exposure (C and D). Figure 2Finally, the pulmonary fibrosis scores showed that, compared with the control group, the pulmonary fibrosis scores of mice in the carbon nanoparticle exposure group were significantly increased, still exhibiting a dose-dependent effect of carbon nanoparticle exposure (E). Figure 2 (F).

[0038] Type II alveolar epithelial cell EMT is a crucial pathological process in pulmonary fibrosis. Therefore, further examination of type II alveolar epithelial cell EMT in the lung tissues of mice in each group was conducted. Figure 3 As shown in Figure A, compared with the control group, the levels of pro-EMT marker molecules (N-cadherin and Vimentin) in the lung tissue of mice exposed to carbon nanoparticles were significantly increased in a dose-dependent manner. Furthermore, immunofluorescence co-localization staining revealed a high degree of co-localization between pulmonary fibrosis marker molecules (COL-1 and COL-3) and pro-EMT marker molecules (N-cadherin and Vimentin) and type II alveolar epithelial cell marker molecules (SP-A) in the lung tissue of the carbon nanoparticle-exposed group. Figure 3 B).

[0039] Next, using type II alveolar epithelial cells as the experimental subject, a type II alveolar epithelial cell EMT model was constructed using carbon nanoparticles. The construction process is as follows: The maximum daily total exposure dose of coal dust to coal miners is approximately 2.5 mg / m³. 3 ×23 m 3×8 h / 24 h = 19.17 mg. The average total lung volume of a normal adult is approximately 4.67 L [Cheng T, Li Y, Pang S, Wan H, Shi G, Cheng Q, Li Q, Pan Z, Huang S. Normal lung attenuation distribution and lung volume on computed tomography in a Chinese population. Int J Chron ObstructPulmon Dis. 2019;14:1657-1668.]. Based on this, the maximum coal dust exposure concentration for alveolar epithelial cells was calculated to be approximately 19.17 mg / 4.67 L = 4.1 µg / mL. In this experiment, a type II alveolar epithelial cell EMT model was constructed using a carbon nanoparticle exposure concentration of 2 µg / mL. The specific steps are as follows: Type II alveolar epithelial cells (generation 0, P0) obtained from the first digestion and passage culture after resuscitation were treated daily with 2 µg / mL of carbon nanoparticles. After 5 consecutive days of treatment, the cells were digested and passaged for 24 h (generation 1 type II alveolar epithelial cells adhered to the culture vessel for 24 h), and the resulting cells were named generation 1 type II alveolar epithelial cells (P1). After P1 cells adhered to the culture vessel for 24 h, they were treated again with 2 µg / mL of nanoparticles for 5 consecutive days. The resulting cells were then digested and passaged again, and this cycle was repeated 20 times (P20) to construct the type II alveolar epithelial cell EMT model. A control group (Control) cells were also set up. Finally, transcriptome sequencing combined with bioinformatics analysis was used to analyze the EMT model cells and the control group cells.

[0040] The results showed that, compared with the control group, genomic expression changes occurred in EMT model cells, including 947 upregulated and 977 downregulated genes. Figure 3 (C and D), these differentially expressed genes are mainly enriched in lesion adhesion reduction ( Figure 3 E and F), inhibition of cell matrix adhesion ( Figure 3 Signaling pathways such as G and H) and collagen binding inhibition (G and H) Figure 4 (of IK).

[0041] The above results indicate that coal dust carbon nanoparticles can induce pulmonary fibrosis, and type II alveolar epithelial cell EMT is an important pathological process in coal dust carbon nanoparticle-induced pulmonary fibrosis.

[0042] Example 3: Differentially expressed genes in type II alveolar epithelial cell EMT and pulmonary fibrosis induced by coal dust and carbon nanoparticles. The mechanisms of EMT encompass multiple levels, including signal transduction pathway activation, transcription factor regulation, and epigenetic regulation. Identifying the upstream transcriptional activators of the main driving signals of EMT is crucial for elucidating the mechanism of carbon nanoparticle-induced pulmonary fibrosis. Therefore, after determining that type II alveolar epithelial cell EMT is an important pathological process in coal dust carbon nanoparticle-induced pulmonary fibrosis, this embodiment conducted a series of preliminary experiments to identify the upstream transcriptional activators of the main driving signals of EMT.

[0043] First, transcriptome analysis was used to examine the mice in each group in Example 2. The results showed that 95 genes were simultaneously highly expressed in the lung tissues of mice exposed to coal dust nanoparticles and different doses of carbon nanoparticles, among which only Arnt2 and Bhlhe41 could act as transcription activators. Figure 4 (A and B). Next, prediction using the JASPAR database revealed that Bhlhe41 only has a strong binding site with YAP, a key node molecule in the main EMT driving signal, while Arnt2 has strong binding sites with Smad2 / 3, Wnt3A, YAP, and Notch1, all key node molecules in the main EMT driving signal (Table 2). Arnt2 is a basic cyclic helical transcription factor that can directly transcribe target genes under various environmental and physiological stimuli. Subsequently, immunofluorescence co-localization detection revealed that, compared with the control group, Arnt2 and the type II alveolar epithelial cell marker molecule SP-A were significantly highly expressed in the lung tissue of the carbon nanoparticle exposure group, and showed high co-localization expression ( Figure 5 The results of transcriptome analysis in the mouse pneumoconiosis model (C and D) were verified, indicating that the elevated Arnt2 level may originate from type II alveolar epithelial cells. Furthermore, immunohistochemical experiments further confirmed the high expression of Arnt2 in mouse lung tissue.

[0044] Table 2. JASPAR analysis results of Bhlhe41 and Arnt2 binding sites in the promoter regions of Smad2 / 3, Wnt3A, YAP, and Notch1 genes. Example 4: Application of Arnt2 in the regulation of EMT and pulmonary fibrosis in type II alveolar epithelial cells 1. In vitro Arnt2 gene silencing experiment To preliminarily analyze the role of Arnt2 in regulating type II alveolar epithelial cell EMT in carbon nanoparticle-induced pulmonary fibrosis, this invention first analyzed changes in cellular EMT by silencing Arnt2 in vitro. The construction method of the type II alveolar epithelial cell EMT model (CNPs-H) was the same as in Example 2, with a control group (Control). The method for silencing Arnt2 in vitro is as follows: Healthy type II alveolar epithelial cells (EMT) model cells were seeded in 24-well plates at a density of 20%-25% and cultured overnight at 37°C until the density reached 30%-50%. Cells were divided into a treatment group (CNPs-H+shArnt2) and a control group (CNPs-H+shControl). Before infection, the virus containing shRNA was thawed on ice. The original culture medium in the treatment group was removed, and half a volume (500 μL) of fresh culture medium containing the original virus solution was added. The amount of virus added was calculated based on the MOI value (MOI=50) of type II alveolar epithelial cells (virus amount per well μL = MOI * cell number / virus titer (TU / mL) * 1000). After 4 h, the total culture volume (500 μL) was replenished. The control group used an empty vector plasmid and followed the same procedure. 24 h post-infection, the virus-containing culture medium was removed, replaced with fresh complete culture medium, and cultured at 37°C. Forty-eight hours post-infection, stable transduced cell lines were screened using fresh complete culture medium containing 2 μg / mL puromycin.

[0045] The shRNA sequence of the treatment group (CNPs-H+shArnt2) is as follows: Arnt2-F: 5'-CCGGGCAGGTGGTTAAGCTGAAAGGCTCGAGCCTTTCAGCTTAACCACCTGCTTTTTTG-3' (SEQ ID NO. 1); Arnt2-R: 5'-AATTCAAAAAAGCAGGTGGTTAAGCTGAAAGGCTCGAGCCTTTCAGCTTAACCACCTGC-3' (SEQ ID NO. 2).

[0046] After successfully silencing Arnt2 in vitro, the migration and invasion abilities of cells in each group were assessed using scratch healing and Transwell assays. The expression levels of EMT marker molecules in each group were detected using Western blotting. The results showed that, compared to the Control group, CNPs-H group, and CNPs-H+shControl group, silencing Arnt2 in carbon nanoparticle-induced EMT model cells could reverse the EMT phenotype, including cell migration. Figure 5 A), invasion ( Figure 5 The expression of B) and EMT-labeled molecules ( Figure 6 (C).

[0047] 2. In vivo Arnt2 gene silencing experiment To further analyze the role of Arnt2 in regulating type II alveolar epithelial cell EMT in carbon nanoparticle-induced pulmonary fibrosis, this invention analyzes changes in pulmonary fibrosis by silencing Arnt2 in vivo. The method for silencing the Arnt2 gene in vivo is as follows: The Arnt2 gene was silenced by nasal instillation of a viral vector containing shRNA directly into the lungs of mice via the respiratory tract. Eighteen male C57BL / 6 mice were selected and divided into three groups of six each: a complete control group (no treatment), a pulmonary fibrosis model group (CNPs-H), and a group receiving lentivirus with the best in vitro knockout effect combined with nasal instillation of carbon nanoparticles (CNPs-H+shArnt2). Mice in the CNPs-H and CNPs-H+shArnt2 groups received 40 μL of coal dust carbon nanoparticles via nasal instillation once daily. Simultaneously, the CNPs-H+shArnt2 group received 40 μL of the same virus (as described in "1. In vitro Arnt2 gene silencing experiment") via nasal instillation every three days for eight consecutive months. After three months, the mice were euthanized by inhaling an overdose of isoflurane, and lung tissue was collected for specimen collection. After RNA extraction, qRT-PCR was used to detect Arnt2 gene expression. Simultaneously, immunohistochemical analysis was performed on mouse lung tissue to observe the knockout efficiency of the Arnt2 gene. Hematoxylin-eosin (HE) staining was used to detect lung tissue pathological morphology, and Masson staining was used to detect collagen formation in lung tissue. Pulmonary fibrosis was scored according to the scoring system published by Szapiel et al. [Szapiel SV, Elson NA, Fulmer JD, Hunninghake GW, Crystal RG. Bleomycin-induced interstitial pulmonary disease in the nude, athymic mouse. Am RevRespir Dis. 1979;120(4):893-9.].

[0048] The qRT-PCR primer sequences are as follows: Arnt2-F: 5'-CGTCACCCCTGTTCTGAACC-3' (SEQ ID NO.3); Arnt2-R: 5'-CGGCCTGTCATTGAGTTTTCT-3' (SEQ ID NO. 4).

[0049] like Figure 6As shown in Figures A and B, compared with the pulmonary fibrosis model group, the Arnt2 gene expression level in the CNPs-H+shArnt2 group mice was significantly reduced, reaching the same level as the control group. Hematoxylin-eosin staining results showed that, compared with the pulmonary fibrosis model group, silencing Arnt2 effectively reversed carbon nanoparticle-induced interstitial lung disease, such as reduced alveolar space widening, inflammatory cell infiltration, and decreased alveolar structural damage. Figure 6 C). Masson staining results showed that, compared with the pulmonary fibrosis model group, silencing Arnt2 effectively reversed the formation of a large number of small nodules and blue collagen induced by carbon nanoparticles. Figure 6 The results of pulmonary fibrosis scoring showed that, compared with the pulmonary fibrosis model group, silencing Arnt2 effectively reversed the increase in pulmonary fibrosis score induced by carbon nanoparticles in mouse lung tissue (D). Figure 6 Immunohistochemical results showed that, compared with the pulmonary fibrosis model group, silencing Arnt2 effectively reversed the increased expression levels of pulmonary fibrosis marker molecules in mouse lung tissue induced by carbon nanoparticles. ​ (F).

[0050] The above results indicate that the Arnt2 gene is an important target for treating pulmonary fibrosis caused by coal dust and carbon nanoparticles. By silencing, interfering with, or reducing Arnt2 gene expression, the pulmonary fibrosis phenotype can be effectively reversed, providing a new strategy for the treatment of pulmonary fibrosis.

[0051] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A pharmaceutical preparation for treating pulmonary fibrosis, characterized in that, The pharmaceutical formulation contains an Arnt2 gene inhibitor and a pharmaceutically acceptable carrier or excipient.

2. The pharmaceutical preparation according to claim 1, wherein, The Arnt2 gene inhibitor includes shRNA that silences the Arnt2 gene; The sense strand of the shRNA is shown in SEQ ID NO.1, and the antisense strand is shown in SEQ ID NO.

2.

3. The pharmaceutical preparation according to claim 1, wherein, The dosage form of the pharmaceutical preparation is an injectable preparation or a nasal administration preparation.

4. Application of Arnt2 gene as a target in the preparation of drugs for the treatment of pulmonary fibrosis.

5. Application of Arnt2 gene inhibitors in the preparation of drugs for treating pulmonary fibrosis.

6. The application according to claim 5, characterized in that, The Arnt2 gene inhibitors include reagents that inhibit, interfere with, or silence Arnt2 gene expression.

7. The application according to claim 6, characterized in that, The reagent for silencing Arnt2 gene expression includes shRNA that silences the Arnt2 gene; The sense strand of the shRNA is shown in SEQ ID NO.1, and the antisense strand is shown in SEQ ID NO.

2.

8. Application of Arnt2 gene as a target in screening drugs for the treatment of pulmonary fibrosis.

9. A method for screening drugs for treating pulmonary fibrosis, characterized in that, Drugs for treating pulmonary fibrosis were screened by detecting the expression level of the Arnt2 gene before and after drug administration.

10. The application according to any one of claims 4, 5, and 8, characterized in that, The pulmonary fibrosis includes pulmonary fibrosis induced by coal dust particles.

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