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

Abstract

The invention discloses application of SCTR as an action target in preparation of a medicine for treating pulmonary fibrosis, and belongs to the field of biological medicine. According to the invention, the secretin receptor SCTR is used as an action target for the first time and is applied to preparation of the medicine for treating pulmonary fibrosis. By studying the pathological characteristics of a targeted macrophage Sctr gene knockout transgenic mouse after bleomycin induces idiopathic pulmonary fibrosis, it is found that targeted macrophage Sctr gene knockout can relieve epithelial cell senescence of an idiopathic fibrosis model, improve lung functions and antagonize pulmonary fibrosis progress, and the targeted macrophage Sctr gene knockout has a remarkable effect. The invention provides a new target spot SCTR for the treatment of pulmonary fibrosis, also provides a new thought for searching an action target spot of pulmonary fibrosis and medicine research, 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 SCTR as a target in the preparation of medicines for treating pulmonary fibrosis. Background Art

[0002] Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial fibrosis disease of unknown cause. Its main feature is the interaction between macrophages, epithelial cells and fibroblasts, which leads to excessive deposition of extracellular matrix, affecting the patient's pulmonary ventilation and gas exchange function damage, and ultimately causing respiratory failure and death. At present, the pathogenesis of IPF is increasingly studied. In the early stage of the disease, it is generally believed that macrophages and epithelial cells interact to cause the senescent phenotype of epithelial cells. Although a variety of drugs have been shown to reduce the progression of pulmonary fibrosis, it is difficult to improve lung function. Therefore, finding promising therapeutic targets and effective drugs is the current focus and difficulty.

[0003] Secretin receptor (SCTR) is a G protein-coupled receptor that usually interacts with its ligand secretin (SCT). It is widely present in a variety of diseases and has multiple physiological functions in different organs. Among them, its gene expression regulation function has been widely reported in various organ diseases. Studies have shown that the SCT-SCTR axis is abnormally expressed in liver fibrosis models, and targeted knockout of Sct or Sctr to disrupt the transmission of the SCT-SCTR axis can improve biliary aging and liver fibrosis. However, there are no reports on whether inhibiting the expression of Sctr gene or SCTR protein 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 SCTR 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. Down-regulating the expression level of the Sctr gene can significantly improve lung function and antagonize the progression of pulmonary fibrosis.

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

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

[0007] The SCTR targeted by the present invention is a secretin receptor, which is a G protein-coupled receptor, whose ligand is secretin (secretin, SCT, also known as secretin, secretin), and the coding gene of SCTR is Sctr.

[0008] Preferably, the drug plays a role in treating pulmonary fibrosis by downregulating the expression level of SCTR.

[0009] The present invention provides an application of a SCTR inhibitor in preparing a medicine for treating pulmonary fibrosis.

[0010] Preferably, the SCTR inhibitor is a substance that reduces the SCTR protein content or a substance that inhibits the expression of the Sctr gene.

[0011] Preferably, the SCTR inhibitor down-regulates the expression level of SCTR to play a role in treating pulmonary fibrosis.

[0012] Preferably, the pulmonary fibrosis is idiopathic pulmonary fibrosis.

[0013] Preferably, the idiopathic pulmonary fibrosis is induced by bleomycin.

[0014] The present invention also provides a medicine for treating pulmonary fibrosis, which uses the SCTR inhibitor as the only active ingredient.

[0015] Preferably, the SCTR inhibitor is a substance that reduces the SCTR protein content or a substance that inhibits the expression of the Sctr gene.

[0016] Preferably, a pharmaceutically acceptable excipient is also included.

[0017] The present invention discloses the following technical effects:

[0018] The present invention proposes for the first time to use the secretin receptor SCTR as a target for the preparation of drugs for the treatment of pulmonary fibrosis. By studying the pathological characteristics of transgenic mice with targeted macrophage Sctr gene knockout after bleomycin-induced idiopathic pulmonary fibrosis, it was found that targeted macrophage Sctr gene knockout can reduce epithelial cell senescence in the idiopathic fibrosis model, improve lung function, and antagonize the progression of pulmonary fibrosis, with significant effects. The present invention provides SCTR as a new target for the treatment of pulmonary fibrosis, and also provides a new idea for finding targets and drug research for pulmonary fibrosis, with broad prospects for medical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] 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.

[0020] Figure 1The following are the results of in vivo imaging of lung tissue of mice in each group; A is the shadow result of mouse lung tissue observed by micro-CT; B is the statistical result of lung fibrosis volume;

[0021] Figure 2 This is a statistical diagram showing the effect of macrophage-targeted Sctr gene knockout on the lung function of IPF mice;

[0022] Figure 3 The results of the macrophage-targeted Sctr gene knockout on the pathological morphology of lung tissue in IPF mice are shown in Figure 1. A shows the HE and VG staining results of lung tissue; B shows the statistical results of lung fibrosis area and collagen fiber area;

[0023] Figure 4 The results of the targeted knockout of Sctr gene in macrophages on the expression of FN1 and α-SMA proteins in the lung tissue of IPF mice are shown in Figure 1. A is the result of immunoblotting; B is the statistical result of protein expression;

[0024] Figure 5 The results of targeting macrophage Sctr gene knockout on the expression of p53 and p21 proteins in the lung tissue of IPF mice are shown in Figure 1; A is the immunoblotting result; B is the statistical result of protein expression. DETAILED DESCRIPTION

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] Example 1

[0031] 1. Materials and methods:

[0032] 1.1 Experimental animals

[0033] Targeted macrophage Sctr gene knockout (Sctr flox / flox；Lyz2-cre ) mouse, Sctr flox / flox Mice were used as the control group. 8-week-old SPF-grade mice, 20 female and 20 male mice, a total of 40 mice (n = 10 per group), weighing 20 ± 5 g, were purchased from Jiangsu Saiye Biotechnology Co., Ltd. (SCXK Su 2022-0016). They were raised in the Barrier Laboratory of the Experimental Animal Center of North China University of Technology (SYXK Ji 2020-007), maintained at a constant temperature of 22 ± 2 ° C, alternating day and night, and free access to food and water.

[0034] 1.2 Main experimental reagents

[0035] Bleomycin (BLM) was purchased from CAYMAN, USA; hematoxylin-eosin (HE) and VG staining reagents were purchased from Beso Biotech, Zhuhai, China; FN1 antibody was purchased from Huaan Biotech, Zhejiang, China; α-SMA antibody was purchased from Huaan Biotech, China; p53 antibody was purchased from Arigo, China; p21 antibody was purchased from BD Biotech, USA; and αTub antibody was purchased from Affinity, USA.

[0036] 1.3 Main instruments and equipment

[0037] The micro CT was purchased from Pingsheng Co., Ltd. in China; the electrophoresis / electrotransfer instrument was purchased from Junyi Co., Ltd. in Beijing, China; the gel imaging system was purchased from Qinxiang Co., Ltd. in Shanghai, China; and the whole body plethysmography (WBP) system was purchased from Buxco Co., Ltd. in the United States.

[0038] 1.4 Experimental Animal Grouping and Treatment

[0039] The specific groups are:

[0040] Control group: Sctr flox / floxThe mice were perfused with 50 μL of saline once in the trachea until 4 weeks of treatment;

[0041] Sctr knockout group: Sctr flox / flox；Lyz2-cre The mice were perfused with 50 μL of saline once in the trachea until 4 weeks of treatment;

[0042] BLM-induced group: Sctr flox / flox The mice were perfused once with 50 μL of BLM (1 mg / kg) solution through the trachea until 4 weeks of treatment;

[0043] Sctr knockout + BLM induction group: Sctr flox / flox；Lyz2-cre The mice were perfused once with 50 μL of BLM (1 mg / kg) solution through their trachea for up to 4 weeks of treatment.

[0044] 2. Experimental Methods

[0045] Four weeks after the mouse model was established, the WBP system was used to non-invasively and dynamically monitor the lung function of the mice before sampling. Micro-CT tomography was used for lung tissue imaging observation. Subsequently, the mice were sampled and anesthetized by intraperitoneal injection of 1% sodium pentobarbital. The mice were fixed on the surgical board and cut along the midline of the mouse abdomen with surgical scissors to remove the lungs from the chest cavity. The left lung was fixed with 4% paraformaldehyde solution, then routinely paraffin-embedded, sectioned, and HE and VG stained for morphological observation. The remaining lung lobe tissues were quickly frozen in liquid nitrogen and stored in a -80°C refrigerator for immunoblotting experiments.

[0046] 2.1 micro-CT imaging examination

[0047] The NEMO-II NMC-20 device was used to capture images of live mouse lungs. The mice were placed in a prone position in a small animal chamber and continuously anesthetized with isoflurane, with body temperature 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.

[0048] 2.2 Pulmonary function test

[0049] The mice were placed in the mouse chamber of the WBP system. After they were stable, the lung function was formally tested, including the adaptation period (8 minutes), the nebulization period (no nebulization treatment, i.e. set to 1 second), the reaction period (5 minutes) and the recovery period (1 minute). After the test, the data were exported in an Excel spreadsheet. The main lung function indicators included: respiratory rate, tidal volume, airway stenosis index, maximum inspiratory flow rate, maximum expiratory flow rate and mid-expiratory flow rate.

[0050] 2.3 Staining observation

[0051] 2.3.1 HE staining

[0052] The paraffin sections of lung tissue were dewaxed to water by xylene I and II for 15 minutes each, anhydrous ethanol I and II for 10 minutes each, 90%, 80%, and 70% ethanol for 5 minutes each, and tap water for 15 minutes. Then, the tissue was covered with hematoxylin stain for 3 minutes, the excess stain was washed off with tap water, differentiated with 1% hydrochloric acid alcohol for 3 seconds, blued in tap water for 15 minutes, covered with eosin stain for 1 minute, dehydrated with gradient alcohol, transparentized with xylene, and sealed with neutral gum.

[0053] 2.3.2 VG staining

[0054] The sections were routinely dewaxed to water (same steps as HE staining in 2.3.1), iron hematoxylin A and hematoxylin B dyes were mixed in equal proportions and covered with the tissue for 5 minutes, differentiated with 1% hydrochloric acid alcohol for 3 seconds, the excess dye was washed off with tap water, and hydrated for 2 minutes, the tissue was covered with VG dye for 10 minutes, then dehydrated with 90% ethanol and anhydrous ethanol, transparentized with xylene, and sealed with neutral gum.

[0055] 2.4 Immunoblotting

[0056] 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 10 μg / lane protein. Primary antibodies FN1, α-SMA, p53, and p21 (diluted 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 (αTub) was used as the relative expression of the protein.

[0057] 3. Experimental results

[0058] 3.1 micro-CT imaging examination

[0059] like Figure 1 As shown in A and B, the micro-CT results showed that compared with the control group, the BLM-induced group had multiple plaque-like high-density shadows of varying sizes in the lungs; compared with the BLM-induced group, the volume of high-density shadows in the Sctr knockout + BLM-induced group was reduced; compared with the control group, there was no significant difference in the Sctr knockout group. This indicates that targeting macrophage Sctr gene knockout can alleviate pulmonary fibrosis in mice.

[0060] 3.2 Pulmonary function test

[0061] like Figure 2As shown in the figure, compared with the control group, the respiratory rate, airway stenosis index, maximum inspiratory flow rate, maximum expiratory flow rate and mid-expiratory flow rate in the BLM-induced group were significantly upregulated, and the tidal volume was significantly downregulated; compared with the BLM-induced group, the respiratory rate, airway stenosis index, maximum inspiratory flow rate, maximum expiratory flow rate and mid-expiratory flow rate in the Sctr knockout + BLM-induced group were significantly downregulated, and the tidal volume was significantly upregulated; compared with the control group, there was no significant difference in the Sctr knockout group. This shows that targeted macrophage Sctr gene knockout can improve the lung function of mice with pulmonary fibrosis.

[0062] 3.3 HE staining and VG staining results

[0063] like Figure 3 As shown in A and B, HE staining results showed that compared with the control group, the lung tissue structure in the BLM-induced group was disordered and typical fibrosis was formed; Figure 3 As shown in B, compared with the BLM-induced group, the fibrosis area in the Sctr knockout + BLM-induced group was reduced; compared with the control group, there was no significant difference in the Sctr knockout group. This indicates that targeting macrophage Sctr gene knockout can inhibit lung damage in mice with pulmonary fibrosis.

[0064] like Figure 3 As shown in A and B, the VG staining results showed that compared with the control group, the collagen deposition in the lung tissue increased in the BLM-induced group; compared with the BLM-induced group, the collagen deposition in the Sctr knockout + BLM-induced group decreased; compared with the control group, there was no significant difference in the Sctr knockout group. This indicates that targeting macrophage Sctr gene knockout can reduce collagen deposition in mice with pulmonary fibrosis.

[0065] 3.4 Immunoblotting

[0066] like Figure 4 As shown in A and B, the results of immunoblotting showed that compared with the control group, the expression levels of FN1 and αSMA proteins in the BLM-induced group were significantly increased; compared with the BLM-induced group, the expression levels of FN1 and α-SMA proteins in the Sctr knockout + BLM-induced group were significantly decreased; compared with the control group, there was no significant difference in the Sctr knockout group.

[0067] like Figure 5 As shown in A and B, the results of immunoblotting showed that compared with the control group, the expression levels of p53 and p21 proteins in the BLM-induced group were significantly increased; compared with the BLM-induced group, the expression levels of p53 and p21 proteins in the Sctr knockout + BLM-induced group were significantly decreased; compared with the control group, there was no significant difference in the Sctr knockout group.

[0068] The above results indicate that targeted knockout of the Sctr gene in macrophages can antagonize pulmonary fibrosis by inhibiting lung aging and improving lung function in mice.

[0069] 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 SCTR as a target in the preparation of drugs for the treatment of pulmonary fibrosis.

2. The use according to claim 1, characterized in that The drug plays a role in treating pulmonary fibrosis by downregulating the expression level of SCTR.

3. Use of a SCTR inhibitor in the preparation of a drug for treating pulmonary fibrosis.

4. The use according to claim 3, characterized in that The SCTR inhibitor is a substance that reduces the SCTR protein content or inhibits the expression of the SCTR gene.

5. The use according to claim 3, characterized in that The SCTR inhibitor downregulates the expression level of SCTR, thereby playing a role in treating pulmonary fibrosis.

6. The use according to any one of claims 1 to 5, characterized in that: The pulmonary fibrosis is idiopathic pulmonary fibrosis.

7. The use according to claim 6, characterized in that The idiopathic pulmonary fibrosis is induced by bleomycin.

8. A drug for treating pulmonary fibrosis, characterized in that: The only active ingredient is SCTR inhibitor.

9. The drug according to claim 8, characterized in that The SCTR inhibitor is a substance that reduces the SCTR protein content or inhibits the expression of the SCTR gene.

10. The drug according to claim 8 or 9, characterized in that Pharmaceutically acceptable excipients are also included.

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