Application of Padi2 gene as action target in preparation of medicine for treating pulmonary fibrosis

Abstract

The invention relates to the field of biological medicines, in particular to application of a Padi2 gene as an action target in preparation of a medicine for treating pulmonary fibrosis. According to the application disclosed by the invention, research finds that lung inflammatory response and pulmonary fibrosis caused by bleomycin can be effectively relieved after targeted knockout of the Padi2 gene of the macrophage. Therefore, the medicine for inhibiting the expression of the Padi2 gene can be used for treating the pulmonary fibrosis. The invention provides a new target spot for the treatment of pulmonary fibrosis, and has a wide medical application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of Padi2 gene as a target in the preparation of a drug for treating pulmonary fibrosis. Background Art

[0002] Pulmonary fibrosis is a chronic progressive lung disease that causes patients to suffer from impaired gas exchange, dyspnea, decreased quality of life, and eventually death from respiratory failure. The cause of pulmonary fibrosis is unknown and can be induced by a variety of factors, mainly causing persistent inflammation in the lungs, and ultimately causing the deposition of fibrotic tissue in the lungs. Current studies suggest that stimuli can abnormally activate macrophages and alveolar epithelial cells, leading to the release of pro-inflammatory and pro-fibrotic factors, inducing fibroblast proliferation and differentiation into myofibroblasts, thereby releasing extracellular matrix including collagen fibers to drive the progression of pulmonary fibrosis. In animal model studies, the bleomycin (BLM)-induced pulmonary fibrosis model has been widely used, widely studied, and reported in a variety of ways, but there is still a lack of effective treatments for pulmonary fibrosis. Therefore, there is an urgent need to find promising therapeutic targets to prevent or treat the progression of pulmonary fibrosis.

[0003] Protein arginine deiminase (PADI) is a post-translational modification enzyme that converts positively charged arginine into neutrally charged citrulline. Five PADI isoforms have been found in mammals, including PADI1-4 and PADI6, which have different tissue expression profiles. Among them, PADI2 is the most common enzyme and is ubiquitous in a variety of cells. Various factors can also induce the expression of PADI2. Studies have reported that smoking triggers protein citrullination by upregulating PADI2, thereby inducing airway inflammation. PADI2 can also be induced by nano-silica particles and is crucial in inflammatory and autoimmune diseases. In macrophages lacking PADI2, inflammatory cytokine secretion is inhibited under lipopolysaccharide stimulation, suggesting that inhibiting the expression of macrophage PADI2 can reduce the expression and release of inflammatory factors to alleviate disease progression. However, there are no reports on whether targeted knockout of the Padi2 gene can play a regulatory role in the process of pulmonary fibrosis. Summary of the invention

[0004] The purpose of the present invention is to provide an application of the Padi2 gene as a target in the preparation of a drug for treating pulmonary fibrosis, so as to solve the problems existing in the above-mentioned prior art. The present invention can antagonize pulmonary fibrosis by knocking out the Padi2 gene of macrophages and reducing the inflammatory response and fibrosis of the lungs of mice.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides the use of Padi2 gene as a target in the preparation of a medicine for treating pulmonary fibrosis.

[0007] Preferably, the effect of treating pulmonary fibrosis is exerted by inhibiting the expression level of the Padi2 gene in macrophages.

[0008] The present invention provides application of a Padi2 gene inhibitor in preparing a medicine for treating pulmonary fibrosis.

[0009] Preferably, the drug plays a role in treating pulmonary fibrosis by inhibiting the expression level of the Padi2 gene in macrophages.

[0010] The invention provides a medicine for treating pulmonary fibrosis, which comprises a Padi2 gene inhibitor.

[0011] Preferably, the drug plays a role in treating pulmonary fibrosis by inhibiting the expression level of the Padi2 gene in macrophages.

[0012] Preferably, the drug further comprises pharmaceutically acceptable excipients.

[0013] The present invention provides the application of Padi2 gene as a target in screening drugs for treating pulmonary fibrosis.

[0014] The present invention provides a method for screening drugs for treating pulmonary fibrosis, by detecting the expression level of Padi2 gene of a subject before and after drug administration.

[0015] The present invention discloses the following technical effects:

[0016] The present invention has found through research that targeted knockout of the macrophage Padi2 gene can effectively reduce bleomycin-induced pulmonary inflammation and pulmonary fibrosis. It can be seen that drugs that inhibit the expression of the Padi2 gene can be used to treat pulmonary fibrosis. The present invention provides a new target for the treatment of pulmonary fibrosis and has broad medical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 Micro-CT observation of lung shadows of mice in each group (A) and lung fiber volume percentage statistics (B);

[0019] Figure 2The results of non-invasive detection of lung function of mice in each group by WBP system; A is the statistical result of airway stenosis index; B is minute ventilation; C is maximum respiratory flow rate; D is the statistical result of maximum inspiratory flow rate; E is the statistical result of mid-respiratory flow rate;

[0020] Figure 3 The effect of targeted macrophage Padi2 gene knockout on the pathological morphology of lung tissue in mice with pulmonary fibrosis; A is the staining results of HE and MASSON; B is the statistical result of lung fibrosis area; C is the statistical result of collagen fiber area;

[0021] Figure 4 The effect of targeted macrophage Padi2 gene knockout on the expression of CoL I and FN1 proteins in lung tissue of mice with pulmonary fibrosis; A is the result of immunoblotting, B is the statistical result of the relative expression of CoL I protein; C is the statistical result of the relative expression of FN1 protein;

[0022] Figure 5 The effect of targeted macrophage Padi2 gene knockout on the expression of TNF-α and IL-1β proteins in lung tissue of mice with pulmonary fibrosis; A is the result of immunoblotting, B is the statistical result of the relative expression of TNF-α protein; C is the statistical result of the relative expression of IL-1β protein. DETAILED DESCRIPTION

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0026] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0027] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0028] Example 1 Therapeutic effect of targeting macrophage Padi2 gene knockout on BLM-induced pulmonary fibrosis in mice

[0029] (1) Experimental materials:

[0030] 1) Experimental animals: Padi2 gene knockout targeting macrophages (Padi2 flox / flox；Lyz2-cre ) 10 female mice and 10 male mice, Padi2 flox / flox Ten female mice and 10 male mice, weighing 20 ± 5 g, were purchased from Jiangsu Saiye Biotechnology Co., Ltd. (SCXK Su 2022-0016); the mice were housed in the Barrier Laboratory of the Experimental Animal Center of North China University of Technology (SYXK Ji 2020-007), maintained in a constant temperature environment of 22 ± 2 °C, alternating day and night, with free access to food and water.

[0031] 2) Main experimental reagents:

[0032] Bleomycin (BLM) was purchased from CAYMAN, USA; hematoxylin-eosin (HE) and VG staining reagents were purchased from Beso Biotech, Zhuhai, China; CoL I antibody was purchased from Affinity, USA; FN1 antibody was purchased from Huaan Biotech, Zhejiang, China; TNF-α antibody was purchased from Genetex, USA; IL-1β antibody was purchased from Affinity, USA; βactin antibody was purchased from Abotek, Wuhan, China.

[0033] 3) Main instruments and equipment:

[0034] The micro CT was purchased from China Pingsheng Co., Ltd.; the gel imaging system was purchased from Shanghai Qinxiang Co., Ltd., China; and the whole body plethysmography (WBP) system was purchased from Buxco, Inc., USA.

[0035] (2) Experimental animal grouping and treatment:

[0036] The specific groups are as follows:

[0037] ①Control group: Padi2 flox / floxThe mice were perfused with 50 μL of saline once in the trachea and fed for 4 weeks before subsequent treatment and testing. flox / flox There were 5 male and 5 female mice;

[0038] ②Padi2 knockout group: Padi2 flox / flox；Lyz2-cre The mice were perfused with 50 μL of saline once in the trachea and fed for 4 weeks before subsequent treatment and testing. flox / flox；Lyz2-cre There were 5 male and 5 female mice;

[0039] ③BLM induction group: Padi2 flox / flox The mice were perfused with 50 μL BLM (1 mg / kg) solution once in the trachea and fed for 4 weeks before subsequent treatment and testing. flox / flox There were 5 male and 5 female mice;

[0040] ④Padi2 knockout + BLM induction group: Padi2 flox / flox；Lyz2-cre The mice were perfused with 50 μL BLM (1 mg / kg) solution once in the trachea and fed for 4 weeks before subsequent treatment and testing. flox / flox；Lyz2-cre There were 5 male and 5 female mice.

[0041] (3) Test indicators and test results

[0042] Step (2) After 4 weeks of treatment and before sampling, micro-CT tomography was used for lung tissue imaging observation; WBP system was used to non-invasively and dynamically monitor the lung function of mice; then the mice were sampled, 1% sodium pentobarbital was injected into the peritoneal cavity to anesthetize the mice, the mice were fixed on the operating board, the mice were cut open along the midline of the abdomen, the lungs were removed, the left lung was fixed with 4% paraformaldehyde solution, then routinely paraffin-embedded, sectioned, HE and VG stained, and morphologically observed; the remaining lung lobe tissues were quickly frozen in liquid nitrogen, stored in a -80°C refrigerator, and subjected to immunoblotting experiments, as follows:

[0043] 1) Micro-CT imaging examination: The NEMO-II NMC-20 device was used to capture lung tomography images of live mice. The specific steps were as follows: the mice were placed in a prone position in a small animal chamber, and the mice were continuously anesthetized with isoflurane, and the body temperature was maintained at 34±2°C. During this period, the status of the mice was continuously observed using PadCam video monitoring. Cruiser 1.7.4.0 software was used to collect data, recon daemon 1.7.4.0 software was used to reconstruct data offline, and avatar31.7.4.0 software was used to analyze the data and reconstruct lung images. The results are shown in the figure. Figure 1As shown in the figure. The micro-CT results showed that compared with the control group, the lung texture was disordered in the BLM-induced group, and multiple high-density shadows of varying sizes were found in the lung field; compared with the BLM-induced group, the volume of high-density shadows in the Padi2 knockout + BLM-induced group was reduced; compared with the control group, there was no significant difference in the Padi2 knockout group. The above results indicate that Padi2 gene knockout can reduce lung damage in mice with pulmonary fibrosis.

[0044] 2) Pulmonary function test: Place the mice in the WBP system mouse chamber, and after they are stable, formally test the lung function, including the adaptation period (8 minutes), nebulization period (no nebulization treatment, i.e. set to 1 second), reaction period (5 minutes) and recovery period (1 minute). After the test is completed, the data is exported to the Excel spreadsheet. The main lung function indicators include: airway stenosis index, ventilation volume per minute, maximum expiratory flow rate, maximum inspiratory flow rate and mid-expiratory flow rate. The test results are as follows: Figure 2 As shown. The results of pulmonary function showed that compared with the control group, the pulmonary function indicators such as airway stenosis index, minute ventilation, maximum expiratory flow rate, maximum inspiratory flow rate and mid-expiratory flow rate in the BLM-induced group were significantly upregulated; compared with the BLM-induced group, the pulmonary function indicators such as airway stenosis index, minute ventilation, maximum expiratory flow rate, maximum inspiratory flow rate and mid-expiratory flow rate in the Padi2 knockout + BLM-induced group were significantly downregulated; compared with the control group, there was no significant difference in the Padi2 knockout group. The above results indicate that targeting the macrophage Padi2 gene knockout can improve the lung function of mice with pulmonary fibrosis.

[0045] 3) Dyeing

[0046] 3.1) HE staining: The paraffin sections of lung tissue were dewaxed to water by xylene I and II for 15 min each, anhydrous ethanol I and II for 10 min each, 90%, 80%, 70% ethanol for 5 min each, and tap water for 15 min. Then, the nuclei were stained with hematoxylin for 3 min, the excess stain was washed off with running water, differentiated with 1% hydrochloric acid alcohol for 5 s, blued in water for 15 min, eosin stained with cytoplasm for 1 min, dehydrated with gradient alcohol, transparentized with xylene, and sealed with neutral gum.

[0047] 3.2) MASSON staining: The sections were routinely dewaxed to water (same steps as HE staining), and iron hematoxylin A and hematoxylin B dye solutions were mixed in equal proportions and incubated with the tissue for 5 min, differentiated in 1% hydrochloric acid alcohol for 5 s, hydrated in tap water for 15 min, and then dripped with Ponceau red dye solution on the tissue surface for staining for 5 min, dripped with phosphomolybdic acid dye solution on the tissue for 1 min, dripped with aniline blue dye solution on the tissue for 2 min, washed with weak acid dye solution for 1 min, then dehydrated with 90% ethanol and anhydrous ethanol, transparentized with xylene, and sealed with neutral gum.

[0048] The results of HE staining and MASSON staining are shown in Figure 3 As shown. HE staining results showed that compared with the control group, the lung tissue structure of the BLM-induced group was disordered, with typical fibrosis formation; compared with the BLM-induced group, the fibrosis area in the Padi2 knockout + BLM-induced group was reduced; compared with the control group, there was no significant difference in the Padi2 knockout group. The above results indicate that Padi2 gene knockout can inhibit lung damage in mice with pulmonary fibrosis. MASSON staining results showed that compared with the control group, the collagen fibers in the lung tissue of the BLM-induced group increased; compared with the BLM-induced group, the collagen fibers in the Padi2 knockout + BLM-induced group decreased; compared with the control group, there was no significant difference in the Padi2 knockout group. The above shows that Padi2 gene knockout can inhibit collagen deposition in mice with pulmonary fibrosis.

[0049] 4) Immunoblotting: Mouse lung tissue protein was extracted using a total protein extraction kit, and the protein concentration was detected using a BCA protein assay kit. Then, conventional electrophoresis and electrotransfer were performed with 15 μg / lane protein. Primary antibodies CoL I, FN1, TNF-α, and IL-1β (diluted by volume 1:1000) were incubated overnight at 4°C. The next day, goat anti-rabbit or anti-mouse secondary antibodies were incubated at room temperature for 40 minutes, and ultrasensitive ECL chemiluminescence was used for development. The ratio of the target protein to the internal reference protein (βactin) was used as the relative expression of the protein. The results are shown in Figure 2. Figure 4 and Figure 5 shown.

[0050] The results of immunoblotting showed that compared with the control group, the expression levels of CoL I and FN1 proteins in the BLM-induced group were significantly increased; compared with the BLM-induced group, the expression levels of CoL I and FN1 proteins in the Padi2 knockout + BLM-induced group were significantly decreased; compared with the control group, there was no significant difference in the Padi2 knockout group ( Figure 4 ).

[0051] At the same time, the results of immunoblotting also showed that compared with the control group, the expression levels of TNF-α and IL-1β proteins in the BLM-induced group were significantly increased; compared with the BLM-induced group, the expression levels of TNF-α and IL-1β proteins in the Padi2 knockout + BLM-induced group were significantly reduced; compared with the control group, there was no significant difference in the Padi2 knockout group ( Figure 5 ).

[0052] The above results show that the Padi2 gene is involved in the occurrence of pulmonary fibrosis and is an important mechanism of pulmonary fibrosis. Knocking out the Padi2 gene in macrophages can antagonize pulmonary fibrosis by reducing the inflammatory response and fibrosis in the lungs of mice, revealing that it can be used as a drug target for the prevention or treatment of pulmonary fibrosis.

[0053] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of Padi2 gene as a target in the preparation of drugs for the treatment of pulmonary fibrosis.

2. The use according to claim 1, characterized in that: It plays a role in treating pulmonary fibrosis by inhibiting the expression level of Padi2 gene in macrophages.

3. Application of Padi2 gene inhibitors in the preparation of drugs for the treatment of pulmonary fibrosis.

4. The drug according to claim 3, characterized in that The drug plays a role in treating pulmonary fibrosis by inhibiting the expression level of the Padi2 gene of macrophages.

5. A drug for treating pulmonary fibrosis, characterized in that: The drugs include Padi2 gene inhibitors.

6. The drug according to claim 5, characterized in that The drug plays a role in treating pulmonary fibrosis by inhibiting the expression level of the Padi2 gene of macrophages.

7. The drug according to claim 5, characterized in that The medicine also includes pharmaceutically acceptable auxiliary materials.

8. Application of Padi2 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: By detecting the expression level of Padi2 gene in the subjects before and after administration.

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