Biomarker related to pulmonary fibrosis and application thereof

Abstract

The invention relates to a biological marker related to pulmonary fibrosis and application of the biological marker. The biomarker comprises an interferon regulatory factor 8. A classical bleomycin-induced pulmonary fibrosis model is utilized to induce pulmonary fibrosis in an experimental animal mouse, transcriptome level analysis is carried out, and it is found that the expression level of the interferon regulatory factor 8 enriched with bone marrow endothelium in the pulmonary fibrosis mouse model is reduced. Further tests find that the occurrence and development of the pulmonary fibrosis can be delayed by improving the expression level of the interferon regulatory factor 8 in a mouse model (such as regulation and control by an agonist and the like), which indicates that the IRF8 can be used as a new target for diagnosis and treatment of the pulmonary fibrosis, and a new target and a new strategy are provided for treatment of the pulmonary fibrosis.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a biomarker related to pulmonary fibrosis and an application thereof. Background Art

[0002] Bone is one of the most important organs of mammals, with important functions of movement, support and protection of the body. The bone marrow inside the bone is the most important site for hematopoiesis. The bone marrow microenvironment composed of various cells such as bone marrow endothelial cells can maintain and regulate the behavior of hematopoietic stem and progenitor cells, continuously produce and maintain blood cells, and ensure the normal transportation of oxygen and the performance of immune function. In recent years, many studies have shown that there is an interaction between multi-organ diseases and bone marrow hematopoiesis or bone marrow blood vessels. For example, bone marrow vascular dysfunction caused by cardiovascular disease can cause hematopoietic abnormalities, which in turn aggravates the occurrence and development of cardiovascular disease.

[0003] Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and fatal lung disease characterized by alveolar wall fibrosis caused by proliferation of fibroblasts and myofibroblasts and progressive destruction of lung tissue due to excessive deposition of extracellular matrix (ECM). Its clinical manifestations mainly include progressive dyspnea, dry cough and chest pain. Since pulmonary fibrosis is an irreversible disease, current treatments are mainly focused on slowing down the progression of the disease, relieving symptoms and improving the quality of life of patients. Common treatments include: (1) the use of antifibrotic drugs such as pirfenidone and nintedanib to reduce scarring of lung tissue through anti-inflammatory and anti-fibrotic mechanisms; (2) oxygen therapy to relieve dyspnea and hypoxemia caused by pulmonary fibrosis; (3) lung transplantation to improve survival for patients with rapid disease progression or poor response to conventional treatment. However, the above treatments can only partially alleviate the rate of decline in lung function in patients, and the mortality rate of the disease remains high. In addition, some drugs are accompanied by adverse reactions, such as gastrointestinal reactions caused by pirfenidone, liver damage, weight loss, etc. It can be seen that the current clinical treatments for pulmonary fibrosis are mostly symptomatic treatments to delay the course of the disease.

[0004] In summary, the lack of new targets, new methods and new drugs for the prevention and treatment of idiopathic pulmonary fibrosis remains one of the urgent problems to be solved in the field of pulmonary fibrosis treatment. Summary of the invention

[0005] In view of the deficiencies of the existing technology and actual needs, the present invention provides a biomarker related to pulmonary fibrosis and its application, revealing the molecular mechanism by which pulmonary fibrosis induces abnormalities in bone marrow blood vessels and intramedullary hematopoietic function, and providing a new target for the diagnosis, prevention and treatment of pulmonary fibrosis.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a biomarker associated with pulmonary fibrosis, wherein the biomarker comprises interferon regulatory factor 8 (IRF8).

[0008] In the present invention, based on the pulmonary fibrosis mouse model, an in-depth analysis of the changes in its transcriptome level was conducted, and it was found that the expression level of IRF8 was reduced in pulmonary fibrosis mice, indicating that IRF8 has the potential to serve as a biomarker for pulmonary fibrosis, providing a new target for the diagnosis, prevention and treatment of pulmonary fibrosis.

[0009] In a second aspect, the present invention provides use of the pulmonary fibrosis-related biomarkers and / or detection reagents thereof described in the first aspect in the preparation of products for diagnosing pulmonary fibrosis diseases.

[0010] In a third aspect, the present invention provides a diagnostic kit for pulmonary fibrosis, the kit comprising a reagent for detecting the expression level of the biomarker associated with pulmonary fibrosis described in the first aspect.

[0011] In a fourth aspect, the present invention provides use of the pulmonary fibrosis-related biomarkers and / or detection reagents thereof described in the first aspect in screening drugs or methods for preventing and / or treating pulmonary fibrosis.

[0012] Preferably, the screening comprises: detecting the effect of candidate drugs or methods on the expression level of interferon regulatory factor 8 in the body, and selecting candidate drugs or methods that can increase the expression level of interferon regulatory factor 8 for verification testing.

[0013] In a fifth aspect, the present invention provides use of the pulmonary fibrosis-related biomarkers and / or their agonists described in the first aspect in the preparation of drugs for treating pulmonary fibrosis diseases.

[0014] It can be understood that the agonist includes at least one of an active drug, enzyme or hormone that promotes the increase in the expression level of IRF8 in the body, or an agent that increases the expression of IRF8 in the body based on genetic engineering means.

[0015] Preferably, the genetic engineering means comprises at least one of plasmid transfection, lentiviral transfection or engineered strain transplantation.

[0016] In the present invention, the agonist may be at least one of interferon-γ (IFN-γ), rheic acid or interleukin 10 (IL-10).

[0017] In a sixth aspect, the present invention provides a pharmaceutical composition, comprising an agonist of a biomarker associated with pulmonary fibrosis as described in the first aspect.

[0018] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0019] The auxiliary materials include any one or a combination of at least two of carriers, diluents, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, coating materials, colorants, pH regulators, antioxidants, antibacterial agents or buffers.

[0020] Compared with the prior art, the present invention has at least the following beneficial effects:

[0021] The present invention utilizes the classic bleomycin-induced pulmonary fibrosis model to induce pulmonary fibrosis in experimental mice, and performs transcriptome-level analysis, and finds that the expression level of interferon regulatory factor 8 enriched in the bone marrow endothelium is reduced in the pulmonary fibrosis mouse model. Further testing finds that increasing the expression level of interferon regulatory factor 8 in the mouse model (such as through agonist regulation, etc.) can delay the occurrence and development of pulmonary fibrosis, indicating that IRF8 can be used as a new target for the diagnosis and treatment of pulmonary fibrosis, and provides a new target and new strategy for the treatment of pulmonary fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Masson and Sirius red staining of lung tissue in the mouse bleomycin-induced pulmonary fibrosis model;

[0023] Figure 2A This is the result of single-cell transcriptome sequencing of mouse multi-organ endothelial cells;

[0024] Figure 2B This is the result of sequencing the Irf8 gene in mouse multi-organ endothelial cells;

[0025] Figure 3 This is the result of gene sequencing of Irf8 in lung and bone marrow endothelium of mice before and after pulmonary fibrosis;

[0026] Figure 4 Schematic diagram of the strategy for constructing endothelial-specific Irf8 knockout mice;

[0027] Figure 5A Sirius red staining of lung tissues in control and endothelial-specific Irf8 knockout mice after induction of pulmonary fibrosis;

[0028] Figure 5B Immunohistochemical fluorescence staining of lung tissues in control and endothelial-specific Irf8 knockout mice after induction of pulmonary fibrosis;

[0029] Figure 6This is a graph showing changes in endothelial Irf8 gene mRNA expression after treatment with the IRF8 agonist IFN-γ;

[0030] Figure 7 Schematic diagram of IRF8 agonist administration;

[0031] Fig. 8A This is the hematoxylin and eosin and Sirius red staining of lung tissues of wild-type mice after administration of IRF8 agonists;

[0032] Figure 8B This is an immunohistofluorescence staining image of lung tissue after administration of IRF8 agonist to wild-type mice;

[0033] Fig. 9A This is the hematoxylin and eosin and Sirius red staining of lung tissues in endothelial-specific Irf8 knockout mice after administration of IRF8 agonists;

[0034] Fig. 9B This is an immunohistofluorescence staining image of lung tissue in endothelial-specific Irf8 knockout mice after administration of IRF8 agonist. DETAILED DESCRIPTION

[0035] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0036] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0037] In response to the current lack of new targets for the prevention and treatment of idiopathic pulmonary fibrosis, the present invention constructs a pulmonary fibrosis mouse model and conducts in-depth analysis to explore new targets for the prevention and treatment of idiopathic pulmonary fibrosis, and further develops new methods and new drugs for the prevention and treatment of idiopathic pulmonary fibrosis.

[0038] Example 1

[0039] The pulmonary fibrosis model (BLM) was induced by a single airway injection of bleomycin. The specific process included: 6 SPF-grade C57BL / 6J male mice (8 weeks old, purchased from Jicui Yaokang) were taken, and they were allowed to eat and drink freely under normal temperature and oxygen. The model was established after one week of adaptive feeding. The mice were anesthetized by intraperitoneal injection of 2.5% tribromoethanol (400 mg / kg), the neck hair was shaved and disinfected with alcohol, and they were fixed on the operating table in a supine position. The neck skin was cut with ophthalmic scissors, and the muscle tissue was bluntly separated to expose the trachea. A 1.5 mg / kg bleomycin solution was injected into the trachea with an insulin injection needle. The animals were placed upright for 5 minutes to allow the mice to fully inhale the solution, and the wound was quickly ligated. Then the mice were placed on a 37°C constant temperature pad to wait for awakening. They were raised under normal temperature and oxygen conditions for 21 days. The mice injected with an equal amount of 0.9% saline were used as the control group and cultured under the same conditions.

[0040] The constructed mouse model was tested and analyzed. Through Masson staining and Sirius Red staining of frozen sections of lung tissue, the changes in lung tissue were observed. Figure 1 The significant fibrotic lesions shown in the figure are as follows: (1) increased collagen deposition in the alveolar septum and around the bronchus in histochemical staining; (2) thickening of the alveolar wall and collapse and shrinkage of the alveolar cavity in histochemical staining, showing a typical "honeycomb" structure, indicating that the mouse model of pulmonary fibrosis was successfully established.

[0041] After collecting the single-cell transcriptome sequencing data of multi-organ endothelial cells, the dimensionality reduction clustering of the gene expression matrix and the differentially expressed gene analysis of multi-organ endothelial cells showed that the bone marrow endothelium (BM) in mice had significant differences from other organ endothelial cells at the transcriptome level ( Figure 2A ), by comparing the expression levels of transcription factors in the vascular endothelium of multiple organs, it was found that the transcription factor IRF8 was enriched and highly expressed in the bone marrow vascular endothelium, but was almost not expressed in the vascular endothelium of other organs ( Figure 2B ).

[0042] Furthermore, bone marrow single cell transcriptome sequencing was performed on the pulmonary fibrosis mouse model. Six mouse models were analyzed and six healthy mice were analyzed as controls (Ctrl). The results are as follows: Figure 3As shown in the figure, the size of the solid circle represents the expression rate of Irf8 and other genes in the endothelium of different lung and bone marrow treatments. The larger the number, the higher the proportion of cells expressing Irf8 and other genes in each bone marrow endothelial subpopulation; the color of the solid circle represents the average expression of Irf8 and other genes in different subtypes of bone marrow endothelium. The larger the number, the higher the expression of Irf8 gene; the genes Cdh5, Pecam1 and Kdr shown in the figure are all endothelial-specific genes, Scn7a and Hpgd are pulmonary vascular endothelial-specific genes, and Vcam1, Stab2 and Irf8 are bone marrow endothelial-specific genes. The results show that compared with healthy mice, in the mouse model of bleomycin-induced pulmonary fibrosis, pulmonary fibrosis leads to a decrease in the expression level of Irf8 gene in bone marrow blood vessels, which has the potential to be a diagnostic / therapeutic target for pulmonary fibrosis.

[0043] Furthermore, in endothelial-specific Irf8 knockout mice (Irf8 iΔEC ) to construct a pulmonary fibrosis model, such as Figure 4 Endothelial-specific Irf8 knockout mice were obtained by mating Cdh5-CreERT2 with Irf8-flox, and the control mice were Irf8 flox / flox The endothelial-specific Irf8 knockout mouse is Cdh5-CreERT2 / Irf8 flox / flox Six pairs of 8-week-old mice were selected and 1 mg of tamoxifen was intraperitoneally injected to induce endothelial Irf8 gene knockout, and then the pulmonary fibrosis model was constructed according to the above-mentioned bleomycin induction method. Sirius red staining of frozen sections of lung tissue revealed that compared with non-knockout control mice (Ctrl), Irf8 gene knockout resulted in severe lung collagen deposition, significant thickening of alveolar walls, and collapse and atrophy of alveolar cavities after bleomycin-induced lung injury ( Figure 5A ). Tissue immunochemical staining revealed that knockout of the endothelial Irf8 gene resulted in a decrease in the number of SFTPC-positive AT2 epithelial cells in the lungs, and a significant increase in the interval between RAGE-positive AT1 lung epithelial cells and CD31-positive pulmonary blood vessels, indicating a disruption of the continuity of the air-blood barrier ( Figure 5B ), indicating that knocking out the bone marrow endothelial-enriched gene Irf8 would accelerate the course of pulmonary fibrosis.

[0044] Example 2

[0045] Testing the effects of IRF8 agonist treatment.

[0046] The agonistic effect of IRF8 agonists was tested. 20 ng / mL of IFN-γ recombinant protein (MEC, HY-P7071) was added to the mouse brain microvascular endothelial cell line (bEnd.3) cultured in vitro. After 12 hours of culture, a significant increase in the expression of Irf8 gene in endothelial cells was observed ( Figure 6).

[0047] The therapeutic effect of IRF8 agonist was tested. The test flow diagram is shown in Figure 7 As shown, a pulmonary fibrosis model was induced by a single airway injection of bleomycin. Two days before the injection of bleomycin (D1-D2) and on the day of injection (D0), 0.9% saline (control group, Ctrl) or IRF8 agonist (treatment group, IRF8 agonist) was injected, and the symptoms of pulmonary fibrosis in the control group and the treatment group were compared. The IRF8 agonist used in this experiment was IFN-γ, with a single injection dose of 40 μg / kg, injected once every 24h, for a total of 3 injections. Ten days after the injection of bleomycin, the control and treatment group mice were collected for analysis.

[0048] The lung tissues of the control and treatment groups were collected and frozen sections were made. Sirius red staining of the frozen sections showed that the morphological structure of the lung tissues of the treated group mice was more similar to that of the normal mice, as shown by (1) the alleviation of alveolar wall thickening and alveolar cavity collapse and atrophy; (2) the weakening of collagen deposition in the alveolar septum and around the bronchus ( Fig. 8A ). Tissue immunochemical staining revealed that administration of IRF8 agonists resulted in the following changes compared with control mice: Figure 8B The results show that the number of SFTPC-positive AT2 epithelial cells in the lungs increased significantly, and the air-blood barrier between RAGE-positive AT1 lung epithelial cells and CD31-positive pulmonary vessels was restored, indicating that IRF8 agonists can alleviate the occurrence and development of pulmonary fibrosis.

[0049] We further constructed a pulmonary fibrosis model in endothelial-specific Irf8 knockout mice and simultaneously injected an IRF8 agonist. Figure 9A-9B As shown, Fig. 9A Hematoxylin-eosin and Sirius red staining of frozen sections of lung tissues of control and Irf8 knockout mice. Fig. 9B Immunofluorescence staining of frozen sections of lung tissue of control and Irf8 gene knockout mice. Green represents RAGE-positive AT1 alveolar epithelium, red represents CD31-expressing pulmonary blood vessels, and blue represents SFTPC-positive AT2 lung epithelial cells. It was found that compared with the Irf8 gene non-knockout mouse model group, the number of AT2 lung epithelial cells increased and the endothelial-epithelial interval decreased, indicating that the alleviating effect of IRF8 agonists on the occurrence and development of lung tissue was significantly inhibited, further indicating that IRF8 agonists can alleviate the occurrence and development of pulmonary fibrosis by increasing the expression level of IRF8.

[0050] In summary, the present invention aims at the current situation that there is a lack of targeted drug treatment or alleviation of the progress of pulmonary fibrosis in the existing clinical treatment methods for pulmonary fibrosis. Pulmonary fibrosis is induced in experimental animals, mice, and bone marrow endothelial IRF8 is found to be a new target for the treatment of pulmonary fibrosis. IRF8 agonists are found to be candidate drugs for the treatment of pulmonary fibrosis, providing new targets and new strategies for the treatment of pulmonary fibrosis.

[0051] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A biomarker associated with pulmonary fibrosis, characterized in that: The biomarkers include interferon regulatory factor 8.

2. Use of the pulmonary fibrosis-related biomarkers and / or their detection reagents as described in claim 1 in the preparation of products for diagnosing pulmonary fibrosis diseases.

3. A diagnostic kit for pulmonary fibrosis, characterized in that: The kit comprises a reagent for detecting the expression level of the pulmonary fibrosis-related biomarker according to claim 1.

4. Use of the pulmonary fibrosis-related biomarker and / or its detection reagent according to claim 1 in screening drugs or methods for preventing and / or treating pulmonary fibrosis.

5. The use according to claim 4, characterized in that: The screening includes: detecting the effect of candidate drugs or methods on the expression level of interferon regulatory factor 8 in the body, and selecting candidate drugs or methods that can increase the expression level of interferon regulatory factor 8 for verification testing.

6. Use of the pulmonary fibrosis-related biomarker and / or its agonist according to claim 1 in the preparation of a drug for treating pulmonary fibrosis.

7. The use according to claim 6, characterized in that: The agonist includes at least one of an active drug, an enzyme or a hormone that promotes the increase of the expression level of IRF8 in the body, or an agent that increases the expression of IRF8 in the body based on genetic engineering methods; Preferably, the genetic engineering means comprises at least one of plasmid transfection, lentiviral transfection or engineered strain transplantation.

8. The use according to claim 6 or 7, characterized in that: The agonist comprises at least one of interferon-γ, rhein or interleukin-10.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the agonist of the pulmonary fibrosis-related biomarker according to claim 1.

10. The pharmaceutical composition according to claim 9, characterized in that The pharmaceutical composition further comprises a pharmaceutically acceptable excipient; Preferably, the auxiliary material includes any one or a combination of at least two of a carrier, a diluent, an emulsifier, a cosolvent, a solubilizer, an osmotic pressure regulator, a coating material, a colorant, a pH regulator, an antioxidant, an antibacterial agent or a buffer.

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