A biomarker associated with pulmonary fibrosis and uses thereof

Abstract

The present application relates to a kind of biomarker associated with pulmonary fibrosis and its application.The biomarker includes interferon regulatory factor 8.The present application utilizes the classic bleomycin induced pulmonary fibrosis model, induces pulmonary fibrosis in experimental animal mouse, carries out transcriptome level analysis, finds that the expression level of bone marrow endothelial rich interferon regulatory factor 8 in pulmonary fibrosis mouse model is reduced, further test finds that the expression level of interferon regulatory factor 8 (such as by agonist regulation etc.) in mouse model can delay the occurrence and development of pulmonary fibrosis, indicates that IRF8 can be used as the new target of pulmonary fibrosis diagnosis, treatment, provides new target, new strategy for the treatment of pulmonary fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a biomarker related to pulmonary fibrosis and its application. Background Technology

[0002] Bone is one of the most important organs in mammals, playing vital roles in locomotion, support, and protection. The bone marrow within the bone is the primary site of hematopoiesis. The bone marrow microenvironment, composed of various cells including bone marrow endothelial cells, continuously produces and maintains blood cells by maintaining and regulating the behavior of hematopoietic stem and progenitor cells, ensuring normal oxygen transport and the proper functioning of the immune system. Recent studies have suggested interactions between multi-organ diseases and bone marrow hematopoiesis or bone marrow vascularization. For example, bone marrow vascular dysfunction caused by cardiovascular disease can lead to hematopoietic abnormalities, thereby exacerbating the development and progression of cardiovascular disease.

[0003] Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and fatal lung disease characterized by alveolar wall fibrosis caused by fibroblast and myofibroblast proliferation 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 treatment focuses on slowing disease progression, relieving symptoms, and improving patients' quality of life. Common treatments include: (1) using antifibrotic drugs such as pirfenidone and nintedanib to reduce lung scarring through anti-inflammatory and antifibrotic mechanisms; (2) oxygen therapy to relieve dyspnea and hypoxemia caused by pulmonary fibrosis; and (3) lung transplantation to improve survival in patients with rapidly progressing disease or those who do not respond well to conventional treatment. However, the above treatments can only partially slow the decline in lung function, and the mortality rate remains high. Furthermore, some medications have adverse reactions, such as gastrointestinal reactions, liver damage, and weight loss caused by pirfenidone. Therefore, current clinical treatments for pulmonary fibrosis are mostly symptomatic, aiming to slow the disease progression.

[0004] In conclusion, the lack of new targets, methods, and drugs for the prevention and treatment of idiopathic pulmonary fibrosis remains one of the most pressing issues to be addressed in the field of pulmonary fibrosis treatment. Summary of the Invention

[0005] In response to the shortcomings of existing technologies and practical needs, this invention provides a biomarker related to pulmonary fibrosis and its application, revealing the molecular mechanism by which pulmonary fibrosis induces abnormalities in bone marrow vascularization and intramedullary hematopoietic function, and providing a new target for the diagnosis, prevention and treatment of pulmonary fibrosis.

[0006] To achieve this objective, the present invention adopts the following technical solution:

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

[0008] In this invention, based on a mouse model of pulmonary fibrosis, the changes in its transcriptome were analyzed in depth, and it was found that the expression level of IRF8 was reduced in mice with pulmonary fibrosis, 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 the use of the pulmonary fibrosis-related biomarkers and / or their detection reagents described in the first aspect in the preparation of products for diagnosing pulmonary fibrosis.

[0010] Thirdly, the present invention provides a diagnostic kit for pulmonary fibrosis, the kit comprising reagents for detecting the expression levels of the pulmonary fibrosis-related biomarkers described in the first aspect.

[0011] Fourthly, the present invention provides the use of the pulmonary fibrosis-related biomarkers and / or their detection reagents described in the first aspect in screening drugs or methods for the prevention and / or treatment of pulmonary fibrosis.

[0012] Preferably, 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 improve the expression level of interferon regulatory factor 8 for verification testing.

[0013] Fifthly, the present invention provides the use of the pulmonary fibrosis-related biomarkers and / or agonists described in the first aspect in the preparation of medicaments for treating pulmonary fibrosis.

[0014] It is understood that the agonist includes at least one of the following: an active drug, enzyme, or hormone that promotes the expression level of IRF8 in the body, or a reagent that enhances the expression of IRF8 in the body through genetic engineering.

[0015] Preferably, the genetic engineering method includes at least one of plasmid transfection, lentivirus transfection, or transplantation of engineered strains.

[0016] In this invention, the agonist may be at least one of interferon-gamma (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 the pulmonary fibrosis-related biomarkers described in the first aspect.

[0018] Preferably, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0019] The excipients include any one or a combination of at least two of the following: carrier, diluent, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

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

[0021] This invention utilizes the classic bleomycin-induced pulmonary fibrosis model to induce pulmonary fibrosis in experimental mice. Transcriptomic analysis revealed that the expression level of interferon regulatory factor 8 (IRF8), which is enriched in bone marrow endothelial cells, was reduced in the pulmonary fibrosis mouse model. Further testing showed that increasing the expression level of IRF8 in the mouse model (e.g., through agonist regulation) can delay the occurrence and development of pulmonary fibrosis, indicating that IRF8 can serve as a new target for the diagnosis and treatment of pulmonary fibrosis, providing a new target and strategy for the treatment of pulmonary fibrosis. Attached Figure Description

[0022] Figure 1 Image of lung tissue stained with Masson's Red and Sirius Red in a mouse bleomycin-induced pulmonary fibrosis model.

[0023] Figure 2A Figure 1 shows the results of single-cell transcriptome sequencing of mouse multi-organ endothelial endothelium.

[0024] Figure 2B Image showing the sequencing results of the Irf8 gene in the endothelial cells of multiple organs in mice;

[0025] Figure 3 Image showing sequencing results of the Irf8 gene in the lung and bone marrow endothelial lining of mice before and after pulmonary fibrosis;

[0026] Figure 4 A schematic diagram illustrating the strategy for constructing endothelial-specific Irf8 gene knockout mice;

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

[0028] Figure 5B Immunohistochemical staining images of lung tissue after pulmonary fibrosis in control and endothelial-specific Irf8 gene knockout mice were induced.

[0029] Figure 6Figure showing the 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] Figure 8A Images of lung tissue stained with hematoxylin and eosin and Sirius red after administration of an IRF8 agonist to wild-type mice;

[0032] Figure 8B Immunohistochemical staining of lung tissue in wild-type mice after administration of an IRF8 agonist;

[0033] Figure 9A Hematoxylin-eosin and Sirius red staining images of lung tissue in endothelial-specific Irf8 gene knockout mice after administration of IRF8 agonist;

[0034] Figure 9B Immunohistochemical staining of lung tissue in endothelial-specific Irf8 gene knockout mice after administration of an IRF8 agonist. Detailed Implementation

[0035] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely 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 is determined by the claims.

[0036] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0037] This invention addresses the current lack of novel targets for the prevention and treatment of idiopathic pulmonary fibrosis by constructing a mouse model of pulmonary fibrosis and conducting in-depth analysis to explore new targets for the prevention and treatment of idiopathic pulmonary fibrosis, and further develop new methods and drugs for the prevention and treatment of idiopathic pulmonary fibrosis.

[0038] Example 1

[0039] A pulmonary fibrosis (BLM) model was induced by a single intratracheal injection of bleomycin. The specific procedure included: Six SPF-grade C57BL / 6J male mice (8 weeks old, purchased from Jicui Pharmaceutical) were used and allowed free access to food and water under normal temperature and oxygen conditions. After one week of acclimatization, the model was initiated. The mice were anesthetized by intraperitoneal injection of 2.5% tribromoethanol (400 mg / kg), the neck hair was shaved and disinfected with alcohol, and the mice were fixed in a supine position on the operating table. The neck skin was cut with ophthalmic scissors, and the muscle tissue was bluntly dissected to expose the trachea. A 1.5 mg / kg bleomycin solution was injected into the trachea using an insulin injection needle. The animals were held upright for 5 minutes to allow the mice to fully inhale the solution. The wound was quickly ligated, and the mice were then placed on a 37°C constant temperature mat to await awakening. They were then raised under normal temperature and oxygen conditions for 21 days. A control group was injected with an equal volume of 0.9% saline and cultured under the same conditions.

[0040] The constructed mouse model was analyzed, and Masson's staining and Sirius Red staining of frozen lung tissue sections revealed lung tissue abnormalities such as... Figure 1 The significant fibrotic lesions shown are specifically manifested as: (1) increased collagen deposition in the alveolar septa and peribronchial regions in histochemical staining; and (2) thickening of the alveolar walls and collapse and atrophy of the alveolar cavities in histochemical staining, presenting a typical "honeycomb" structure. This indicates that a mouse model of pulmonary fibrosis has been successfully constructed.

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

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

[0043] Further, in endothelial-specific Irf8 gene knockout mice (Irf8 iΔEC Constructing a pulmonary fibrosis model in, such as Figure 4 The endothelial-specific Irf8 gene knockout mice shown were obtained by mating Cdh5-CreERT2 with Irf8-flox, while the control mice were Irf8. flox / flox Endothelial-specific Irf8 gene knockout mice are Cdh5-CreERT2 / Irf8. flox / flox Six pairs of 8-week-old mice were used. Endothelial Irf8 gene knockout was induced by intraperitoneal injection of 1 mg tamoxifen, followed by bleomycin induction to establish a pulmonary fibrosis model. Sirius red staining of frozen lung tissue sections revealed that, compared with the non-knockout control mice (Ctrl), Irf8 gene knockout led to severe pulmonary collagen deposition, significant alveolar wall thickening, and alveolar collapse and atrophy after bleomycin-induced lung injury. Figure 5A Immunochemical staining revealed that knockout of the endothelial Irf8 gene led to a decrease in the number of SFTPC-positive AT2 type lung epithelial cells and a significant increase in the septum between RAGE-positive AT1 type lung epithelial cells and CD31-positive lung vessels, indicating a disruption of the air-blood barrier continuity. Figure 5B This indicates that knocking out the bone marrow vascular endothelial enrichment gene Irf8 leads to an accelerated progression of pulmonary fibrosis.

[0044] Example 2

[0045] To test the therapeutic effect of IRF8 agonists.

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

[0047] The efficacy of IRF8 agonist therapy was tested, and the test procedure is shown in the diagram below. Figure 7 As shown, a pulmonary fibrosis model was induced by a single intratracheal injection of bleomycin. Two days before (D1-D2) and on the day of injection (D0), mice were injected with either 0.9% saline (control group, Ctrl) or an IRF8 agonist (treatment group, IRF8 agonist). The symptoms of pulmonary fibrosis were then compared between the control and treatment groups. The IRF8 agonist used in this experiment was IFN-γ, with a single injection dose of 40 μg / kg, administered every 24 hours for a total of three injections. Ten days after bleomycin injection, mice from both the control and treatment groups were collected for analysis.

[0048] Lung tissues from control and treatment mice were collected and frozen sections were prepared. By staining the frozen sections with Sirius red, it was observed that the lung tissue morphology and structure of the treatment group mice were more similar to that of normal mice compared with the control group mice, showing (1) relief of alveolar wall thickening and alveolar cavity collapse and atrophy; (2) reduced collagen deposition in alveolar septa and peribronchial fossa. Figure 8A Immunochemical staining revealed that, compared to control mice, administration of the IRF8 agonist resulted in... Figure 8B The study showed a significant increase in the number of SFTPC-positive AT2 type lung epithelial cells and the restoration of the air-blood barrier between RAGE-positive AT1 type lung epithelial cells and CD31-positive lung vessels, indicating that IRF8 agonists can alleviate the occurrence and development of pulmonary fibrosis.

[0049] Further, a lung fibrosis model was established in endothelial-specific Irf8 gene knockout mice, and IRF8 agonists were injected simultaneously. The results were as follows: Figures 9A-9B As shown, Figure 9A Hematoxylin-eosin staining and Sirius red staining of frozen lung tissue sections from control and Irf8 gene knockout mice. Figure 9B Immunofluorescence staining of frozen lung tissue sections from control and Irf8 gene knockout mice was performed. Green represents RAGE-positive AT1 alveolar epithelium, red represents CD31-expressing pulmonary vessels, and blue represents SFTPC-positive AT2 lung epithelial cells. Compared with the Irf8 gene non-knockout mouse model group, the number of AT2 lung epithelial cells increased and the endothelial-epithelial septum decreased, indicating that the alleviating effect of IRF8 agonists on lung tissue development was significantly inhibited. This further demonstrates that IRF8 agonists can alleviate the occurrence and development of pulmonary fibrosis by increasing IRF8 expression levels.

[0050] In summary, this invention addresses the current lack of targeted drug therapy or methods to alleviate the progression of pulmonary fibrosis in existing clinical treatments. By inducing pulmonary fibrosis in experimental mice, this invention identifies bone marrow endothelial IRF8 as a novel target for pulmonary fibrosis treatment and IRF8 agonists as candidate drugs for pulmonary fibrosis treatment, thus providing new targets and strategies for the treatment of pulmonary fibrosis.

[0051] The applicant declares that the above description is only a specific embodiment 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 conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. Use of a reagent for detecting the expression level of interferon regulatory factor 8 in bone marrow vascular endothelial cells in the manufacture of a product for diagnosing idiopathic pulmonary fibrosis, characterized in that, The expression level of interferon regulatory factor 8 in the bone marrow vascular endothelial cells is decreased in an idiopathic pulmonary fibrosis mouse model compared with healthy controls.

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