Application of ferritin light chain as pulmonary fibrosis disease prevention and / or treatment target

Abstract

The invention relates to the technical field of biological medicines, and mainly relates to an application of a ferritin light chain (FTL) as a prevention and / or treatment target in pulmonary fibrosis. The technical problem to be solved by the invention is to provide a new target selection for prevention and / or treatment of pulmonary fibrosis. According to the technical scheme for solving the technical problem, the application of the FTL (ferritin light chain) as a pulmonary fibrosis prevention and / or treatment target is provided. It is found that FTL is highly expressed in pulmonary fibrosis lung tissue, and expression of extracellular matrix genes and protein of human lung fibroblasts can be increased. It is found that by means of FTL knockout in macrophages, expression of Collagen I and Collagen III in co-cultured lung fibroblasts can be remarkably reduced, and therefore the progress of pulmonary fibrosis is inhibited. The invention provides a new target selection for prevention and / or treatment of pulmonary fibrosis, and has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of ferritin light chain (FTL) as a target for the prevention and / or treatment of pulmonary fibrosis. Background Art

[0002] Pulmonary fibrosis is an irreversible disease induced by multiple factors such as environment, genetics, and infection. It is characterized by the massive proliferation and differentiation of lung fibroblasts and the secretion of excessive extracellular matrix such as Collagen I, Collagen III, Fibronectin, etc., which causes the original loose alveolar structure to be replaced by scar tissue and the extracellular matrix it produces, affecting the gas exchange in the lungs, and the patient eventually dies of respiratory failure. Pulmonary fibrosis includes pulmonary fibrosis induced by inhaled particles, pulmonary fibrosis induced by infection, pulmonary fibrosis induced by autoimmune diseases, pulmonary fibrosis induced by radiation exposure, secondary pulmonary fibrosis induced by chronic inflammation, and idiopathic pulmonary fibrosis caused by unknown reasons. Dust-induced silicosis fibrosis, especially silica dust-induced silicosis fibrosis, is more common in clinical practice. However, as a terminal disease, there is currently no cure for pulmonary fibrosis.

[0003] Ferritin light chain (FTL) is one of the key proteins to maintain iron homeostasis in organisms. It forms ferritin together with ferritin heavy chain (FTH1). When the body has excess iron, it stores excess iron ions inside ferritin to avoid damage to cells and tissues caused by excess iron. In recent years, studies have found that ferritin is highly expressed in a variety of disease states, such as liver disease and infectious diseases, and high levels of ferritin are associated with poor prognosis of the disease, suggesting that ferritin or its components may be involved in the progression of the disease. However, whether ferritin and its components are related to the progression of pulmonary fibrosis has not been reported. Summary of the invention

[0004] The technical problem to be solved by the present invention is that there is still a lack of simple and effective means for the diagnosis and treatment of fibrosis, and a new target selection is provided for the prevention and / or treatment of pulmonary fibrosis. The technical solution of the present invention to solve the above technical problem is to provide the application of FTL (ferritin light chain) as a target for the prevention and / or treatment of pulmonary fibrosis.

[0005] The pulmonary fibrosis described in the above application includes at least one of pulmonary fibrosis induced by inhaled particulate matter, pulmonary fibrosis caused by infection, pulmonary fibrosis caused by autoimmune diseases, radiation-induced pulmonary fibrosis and idiopathic pulmonary fibrosis of unknown cause.

[0006] Among them, the pulmonary fibrosis induced by the above-mentioned inhaled particulate matter is dust-induced silicosis fibrosis.

[0007] At the same time, the present invention provides the use of a reagent or a diagnostic device for detecting the level of FTL in preparing or screening a reagent or a kit for detecting, diagnosing, monitoring and / or predicting pulmonary fibrosis.

[0008] Among them, the above-mentioned reagents for detecting FTL levels are used for in vitro diagnosis and risk stratification of pulmonary fibrosis.

[0009] Wherein, the above reagent or kit is used to measure the expression level and / or concentration of FTL in a sample.

[0010] Furthermore, the above reagent or kit detects the expression level and / or concentration of the FTL polypeptide in the sample by ELISA.

[0011] Wherein, the above reagent or kit comprises an antibody against the FTL polypeptide.

[0012] The anti-FTL polypeptide antibody is at least one of a monoclonal antibody, a polyclonal antibody, a single-chain antibody or a nanobody. For example, the anti-FTL polypeptide antibody can be antibody JM10-37.

[0013] The reagent or kit described in the above application detects the expression level and / or concentration of FTL in the sample by at least one of quantitative WB method, PCR method, fluorescence in situ hybridization (FISH) method, real-time fluorescence quantitative PCR method (qPCR) method, and immunohistochemistry staining method (IHC).

[0014] The above reagents or kits contain primers, primer pairs or primer sets for amplifying the FTL polypeptide encoding gene or CDS sequence or amplifying the FTL polypeptide encoding gene fragment or the fragment of its CDS sequence.

[0015] Furthermore, the above-mentioned primers, primer pairs or primer sets include:

[0016] Forward(SEQ ID No.1):5′-ATGAGCTCCCAGATTCGTCAG-3′,

[0017] Reverse (SEQ ID No. 2): 5′-GCCCAGAGAGAGGTAGGTGT-3′;

[0018] Or include:

[0019] Forward(SEQ ID No.3):5′-TACACCTACCTCTCTCTGGGC-3′,

[0020] Reverse (SEQ ID No. 4): 5′-CCCGCGATCGTTCTGAAACT-3′.

[0021] The sample described in the above application includes plasma, serum or blood extract, brushing, biopsy or surgically removed tissue or liquid sample from the subject.

[0022] Furthermore, the above-mentioned samples are in vitro samples.

[0023] Wherein, the reagent described in the above application is used to diagnose or monitor the existence and / or process and / or severity and / or prognosis of pulmonary fibrosis.

[0024] The present invention also provides the use of the FTL inhibitor in preparing a medicine for preventing and / or treating pulmonary fibrosis.

[0025] The FTL inhibitor is a gene inhibitor of FTL. Furthermore, the gene inhibitor is an inhibitor that inhibits the expression of the FTL gene or its transcribed mRNA.

[0026] The gene inhibitor is at least one of antisense oligonucleotides, siRNA, esiRNA, dsRNA ribozyme, shRNA, microRNA or FTL gene promoter inhibitors that inhibit the expression of the FTL gene or its transcribed mRNA.

[0027] Among them, the above-mentioned gene inhibitor includes sgRNA or crRNA that inhibits the expression level of the FTL gene or its transcribed mRNA as the target of action.

[0028] Wherein, the above-mentioned gene inhibitor is siRNA, and the siRNA is at least one of the following:

[0029] 1: 5′-CCAGAUUCGUCAGAAUUAU-3′ (SEQ ID No. 5),

[0030] 5′-AUAAUUCUGACGAAUCUGG-3′ (SEQ ID No. 6);

[0031] or:

[0032] 2: 5′-GAAGUGAAGCUUAUCAAGA-3′ (SEQ ID No. 7)

[0033] 5′-UCUUGAUAAGCUUCACUUC-3′ (SEQ ID No. 8);

[0034] or:

[0035] 3: 5′-GGCGAGUAUCUCUUCGAAA-3′ (SEQ ID No. 9)

[0036] At least one of 5′-UUUCGAAGAGAUACUCGCC-3′ (SEQ ID No. 10).

[0037] Wherein, the above-mentioned FTL inhibitors include FTL polypeptide inhibitors.

[0038] Wherein, the inhibitor of the FTL polypeptide is an anti-FTL antibody.

[0039] Wherein, the anti-FTL antibody is at least one of a monoclonal antibody, a polyclonal antibody, a single-chain antibody or a nanobody.

[0040] The present invention also provides a method for establishing a pulmonary fibrosis cell model, which comprises the following steps: applying FTL to cells to be treated, or transfecting them with an expression vector capable of expressing FTL.

[0041] Wherein, the cells to be treated in the above method are lung fibroblasts.

[0042] Furthermore, the above method includes inoculating lung fibroblasts in a cell culture plate, applying FTL to the lung fibroblasts, and increasing the expression of fibrosis-related genes and proteins in the lung fibroblasts.

[0043] At the same time, the present invention also provides the application of the pulmonary fibrosis cell model established by the above method in any of the following: a. application in the study of the mechanism of pulmonary fibrosis; b. application in the screening of anti-pulmonary fibrosis drugs; c. application in the observation and / or evaluation of the efficacy of anti-pulmonary fibrosis drugs.

[0044] In addition, the present invention also provides a method for screening drugs for preventing or treating pulmonary fibrosis. The method comprises the following steps: a. establishing a model group of a pulmonary fibrosis cell model or an animal model, and setting up a normal control group; b. applying the drug to be screened to the model group according to a dose gradient, and then detecting the level of FTL polypeptide or its mRNA content therein; c. comparing the detection results with the normal control group, and the drug to be screened that can make the level of FTL protein or its mRNA content in the model group significantly approach that of the normal control group is selected.

[0045] Wherein, the pulmonary fibrosis cell model described in the above method is a cell model of co-culture of pulmonary fibroblasts and macrophages.

[0046] The present invention has the following beneficial effects:

[0047] The present invention found that FTL is highly expressed in the lung tissue of patients with pulmonary fibrosis, and further found that FTL in the lung tissue is derived from macrophages. Knocking down the expression of FTL in macrophages can inhibit the differentiation of fibroblasts to reduce the production of extracellular matrix, thereby slowing down the progression of pulmonary fibrosis. Further, the application of FTL (ferritin light chain) as a target for the prevention and / or treatment of pulmonary fibrosis is provided. The present invention found that FTL is highly expressed in pulmonary fibrosis and can be further used as a biomarker for diagnosing pulmonary fibrosis. Reagents or diagnostic devices for detecting the level of FTL in a sample can be applied to the detection, diagnosis, treatment monitoring and / or prediction of pulmonary fibrosis to expand the scope of the applicable population for pulmonary fibrosis screening and reduce the risks of puncture in pathological histological diagnosis.

[0048] The present invention found that inhibiting the increase of FTL may be beneficial for the treatment of pulmonary fibrosis, and also proved in cell co-culture experiments and in vivo animal experiments that knocking down the expression of FTL in macrophages can reduce pulmonary fibrosis induced by silica particles. Therefore, FTL has the role of being a target for the treatment of pulmonary fibrosis, and agents that inhibit the FTL activity of lung tissue or reduce the expression of FTL therein have the potential to be developed as therapeutic drugs for pulmonary fibrosis, providing a new direction for the prevention and treatment of pulmonary fibrosis.

[0049] The present invention also uses FTL or FTL expression vector as an inducing factor to establish a pulmonary fibrosis cell model, which provides a new and effective option for applications in pulmonary fibrosis mechanism research, screening of anti-pulmonary fibrosis drugs, and drug evaluation, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Immunohistochemical staining of FTL in lung tissue of patients with silicosis and pulmonary fibrosis.

[0051] Figure 2 is the expression level of FTL in the serum of patients with silicosis and pulmonary fibrosis.

[0052] Figure 3 H&E staining of lung tissue of mice exposed to silica dust.

[0053] Figure 4 is the expression level of FTL gene in the lung tissue of mice exposed to silica dust.

[0054] Figure 5 is the expression level of FTL protein in the lung tissue of mice exposed to silica dust.

[0055] Figure 6 H&E staining of mouse lung tissue after bleomycin induction.

[0056] Figure 7is the expression level of Ftl1 protein in bleomycin-induced mouse lung tissue.

[0057] Figure 8 The expression levels of Collagen I and Collagen III genes in human lung fibroblasts after FTL intervention.

[0058] Fig. 9 The expression levels of Collagen I and Collagen III proteins in human lung fibroblasts after FTL intervention.

[0059] Fig.10 is the expression level of FTL mRNA in macrophages after dust exposure.

[0060] Fig.11 is the expression level of FTL protein in macrophages after dust exposure.

[0061] Fig.12 is the expression level of FTL gene in macrophages after intervention with FTL gene inhibitor.

[0062] Fig.13 is the expression level of FTL protein in macrophages after intervention with FTL gene inhibitor.

[0063] Fig.14 This is the expression level of Collagen I and Collagen III proteins in human lung fibroblasts after macrophage-fibroblast co-culture.

[0064] Fig.15 This is the expression level of Ftl1 protein in mouse lung tissue after AAV6-F4 / 80-Ftl1 intervention.

[0065] Fig.16 H&E staining of mouse lung tissue after AAV6-F4 / 80-Ftl1 intervention. DETAILED DESCRIPTION

[0066] In view of the deficiencies of existing research, the present invention provides the application of FTL as a target for the prevention and / or treatment of pulmonary fibrosis. The inventors have found that FTL is highly expressed in the lung tissue of patients with pulmonary fibrosis and mouse models, and is mainly derived from macrophages. Exogenous FTL intervention has been found to significantly increase the expression of Collagen I and Collagen III proteins in human lung fibroblasts, while inhibiting the expression of FTL can alleviate mouse pulmonary fibrosis.

[0067] Based on the above unexpected discovery, in-depth research was conducted, which provided the application of FTL (ferritin light chain) as a target for the prevention and / or treatment of pulmonary fibrosis and a series of related technical solutions.

[0068] At present, the main diagnostic gold standard for pulmonary fibrosis is imaging detection and pathological diagnosis, and both diagnostic methods have high requirements for hospital equipment and environment, as well as the professional skills of operators, and imaging and pathological diagnosis methods cannot be used in some special patients, such as pregnant women, people with a greater risk of lung puncture, etc., and there is still a lack of biomarkers that can be quickly used for diagnosis. The present invention found that FTL is highly expressed in pulmonary fibrosis and can further be used as a biomarker for diagnosing pulmonary fibrosis. FTL can be detected in serum, so the hematological detection method for FTL can be applied to the diagnosis and treatment monitoring of pulmonary fibrosis, so as to expand the scope of the applicable population for pulmonary fibrosis screening and reduce the risks of puncture in pathological histological diagnosis.

[0069] The present invention provides the use of a reagent or diagnostic device for detecting the level of FTL in a sample in the preparation or screening of a reagent or kit for detecting, diagnosing, monitoring and / or predicting pulmonary fibrosis. The reagent for detecting the level of FTL is used for in vitro diagnosis and risk stratification of pulmonary fibrosis. The sample to be detected may include plasma, serum or blood extracts, brushings, biopsies or surgically removed tissue or liquid samples from a subject.

[0070] In the treatment of pulmonary fibrosis, current therapeutic drugs such as pirfenidone and nintedanib have significant side effects, such as liver damage, gastrointestinal damage, etc. In addition to high-risk and high-cost lung transplantation, there is no cure for pulmonary fibrosis in clinical practice. In the present invention, it is found that FTL increases in the lung tissue of patients and can induce the differentiation of fibroblasts to promote the progression of pulmonary fibrosis. Therefore, inhibiting the increase of FTL may be beneficial to the treatment of pulmonary fibrosis. The present invention also found in cell co-culture experiments and in vivo animal experiments that knocking down the expression of FTL in macrophages can reduce pulmonary fibrosis induced by silica particles, indicating that reducing the level of FTL in the microenvironment can indeed inhibit the progression of pulmonary fibrosis. Therefore, FTL has the effect of being a target for the treatment of pulmonary fibrosis, and inhibiting the FTL activity of lung tissue or reducing the FTL expression level therein can provide help for the treatment of pulmonary fibrosis. The reagent that inhibits the FTL activity of lung tissue or reduces the FTL expression level therein can be used to prepare a drug for the prevention and / or treatment of pulmonary fibrosis.

[0071] For example, antisense oligonucleosides, siRNA, esiRNA, dsRNA ribozymes, shRNA, microRNA or FTL gene promoter inhibitors that inhibit the expression of the FTL gene or its transcribed mRNA can be used to prepare drugs for preventing and / or treating pulmonary fibrosis.

[0072] The above-mentioned FTL inhibitors also include inhibitors of FTL polypeptides. Common inhibitors of FTL polypeptides are anti-FTL antibodies, such as monoclonal antibodies, polyclonal antibodies, single-chain antibodies or nano antibodies against FTL.

[0073] In terms of the establishment of pulmonary fibrosis models, the current modeling methods in this field mainly include: pneumoconiosis pulmonary fibrosis induced by inhaled particles such as silica, carbon black, etc., idiopathic pulmonary fibrosis induced by bleomycin, pulmonary fibrosis induced by radiation, chemical pulmonary fibrosis induced by paraquat, etc. However, these methods are only applicable to a certain type of pulmonary fibrosis and have defects to varying degrees. For example, the establishment of the pulmonary fibrosis model induced by inhaled particles is usually carried out by tracheal instillation, which cannot directly simulate the process of human inhalation through breathing, and idiopathic pulmonary fibrosis induced by bleomycin has a self-healing effect and cannot completely simulate the progression of clinical pulmonary fibrosis.

[0074] The present invention has found through research that FTL is highly expressed in silica-induced pulmonary fibrosis mice, bleomycin-induced fibrosis mice and clinical pneumoconiosis patients, and it is also found that FTL has the effect of promoting the occurrence and development of pulmonary fibrosis. Therefore, it can be inferred that FTL is stably highly expressed in pulmonary fibrosis induced by different factors. Based on the effect of FTL in promoting the proliferation and differentiation of fibroblasts, FTL or FTL expression vector can be used as an induction factor to establish a pulmonary fibrosis cell model.

[0075] The pulmonary fibrosis cell model established by the method provided by the present invention has application prospects in the following aspects: a. application in the study of the mechanism of pulmonary fibrosis; b. application in the screening of anti-pulmonary fibrosis drugs; c. application in the observation and / or evaluation of the efficacy of anti-pulmonary fibrosis drugs.

[0076] On this basis, the present invention also provides a method for screening drugs for preventing or treating pulmonary fibrosis. The method comprises the following steps: a. establishing a model group of a pulmonary fibrosis cell model or an animal model, and setting up a normal control group; b. using the drug to be screened to act on the model group according to a dose gradient, and then detecting the level of FTL polypeptide or its mRNA content therein; c. comparing the detection results with the normal control group, and the drug to be screened that can make the level of FTL protein or its mRNA content in the model group significantly approach that of the normal control group is screened. The method for screening drugs for preventing or treating pulmonary fibrosis is easy to operate and can be used in drug screening for pulmonary fibrosis caused by various factors.

[0077] The present invention is described in detail below in conjunction with specific embodiments. The following examples will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, some variations and improvements can also be made without departing from the inventive concept. These all belong to the protection scope of the present invention. The reagents or instruments used that do not indicate the manufacturer are all conventional products that can be obtained commercially.

[0078] Example 1 Detection of FTL expression in patients with pulmonary fibrosis

[0079] Materials: Lung puncture specimens and serum from patients with pulmonary fibrosis

[0080] Immunohistochemical staining of lung tissue of patients with pulmonary fibrosis: The hydrated sections were boiled in antigen repair solution under high pressure for 3 minutes, rinsed with cold water to cool down, circled the area of ​​the tissue section with an immunohistochemical pen, treated with endogenous peroxidase blocker for 10 minutes, and washed with PBS. The sections were placed in a dark box and blocked at room temperature for 10-15 minutes, then the primary antibody was added and incubated overnight at 4°C, and washed with PBS. Biotin-labeled mouse / rabbit secondary antibody was added according to the species of the primary antibody, incubated for 10-15 minutes, washed and DAB colorimetric solution was added, incubated at room temperature in the dark for 5-10 minutes, rinsed with tap water, and then hematoxylin was added for 2 minutes, rinsed with tap water until the washing solution was colorless, differentiated with 1% hydrochloric acid alcohol, and washed with tap water to reverse blue. Then the sections were dehydrated with ethanol solution, soaked in xylene and dried, and sealed with neutral gum, and observed under a microscope.

[0081] Serum FTL detection in patients with pulmonary fibrosis: ELISA method was used for detection. A standard working solution with a concentration gradient of 0-1500 ng / mL was prepared according to the instructions of the kit. 100 μL of the standard working solution and sample were added to the ELISA microplate and incubated for 90 minutes; the liquid in the well was discarded and 100 μL of biotinylated antibody working solution was added, incubated for 60 minutes, and after washing, the enzyme binding working solution was added and incubated for 30 minutes; after washing, the substrate solution was added to react for 15 minutes, and then 50 μL of stop solution was added to measure the OD value at 450 nm, and the level of FTL in the sample was calculated according to the standard curve.

[0082] Experimental results: Figure 1 As shown in the figure, compared with the control group, the expression of FTL in lung tissue of patients with pulmonary fibrosis was significantly increased. In terms of cell source, FTL in the control group was highly expressed in macrophages, while in patients with pulmonary fibrosis, FTL was highly expressed overall.

[0083] like Figure 2 As shown, compared with the control group, the expression level of FTL in the serum of patients with pulmonary fibrosis was increased, and the expression level increased with the severity of the disease.

[0084] Example 2 Establishment of dust-induced mouse pulmonary fibrosis model and detection of Ftl1 gene and protein expression.

[0085] Materials: C57BL / 6J, male mice, purchased from Beijing Weitonglihua Experimental Technology Co., Ltd.; silicon dioxide, purchased from Sigma, sterilized by dry heat at 200°C for 4 h and then dissolved in sterile saline for use.

[0086] Experimental methods:

[0087] After anesthesia, the mouse was fixed on the operating table, and the tongue of the mouse was gently clipped out with tweezers. The neck of the mouse was illuminated with a mercury lamp, and the tracheal opening was observed with the naked eye. The point that flickered with the mouse's breathing was the tracheal opening. The indwelling needle hose was inserted into the tracheal opening, and the silica suspension was quickly injected. After the indwelling needle was pulled out, the mouse was gently shaken to evenly distribute the silica in the lung tissue. The tongue of the mouse was then clipped out to avoid choking and coughing. After the mouse's breathing stabilized, the mouse was put back into the cage after the breathing stabilized, and the vital signs were observed until the mouse woke up. The experiment set up a control group and a silica model group.

[0088] After 12 weeks of silica exposure, the mice were sacrificed, and lung tissues were dissected and stained with H&E to evaluate the pathological conditions of the mouse lung tissues. RNA and protein were extracted from lung tissues, and q-PCR was used to detect the expression of Ftl1 mRNA.

[0089] Mouse FTL detection primers (mouse FTL gene is represented by Ftl1 in the examples):

[0090] forward:5′-TACACCTACCTCTCTCTGGGC-3′;

[0091] reverse:5′-CCCGCGATCGTTCTGAAACT-3′.

[0092] The expression of Ftl1 (mouse FTL protein is represented by Ftl1 in the examples) protein was detected by immunoblotting. The antibodies used were:

[0093] Anti-Ferritin Light Chain Recombinant Rabbit Monoclonal Antibody [JM10-37] (Cat. No.: ET1705-54, purchased from HUABIO (Hangzhou Huaan Biotechnology Co., Ltd.), Synthetic peptide within Human Ferritin Light Chain aa 126-175 / 175.).

[0094] Experimental results: After exposure to silica, the mice had obvious lesions in their lung tissues, such as Figure 3 Nodular infiltration appeared in the lung tissue, the alveolar wall was thickened, and inflammatory exudate was visible in the alveoli, indicating that the mouse pulmonary fibrosis model was successfully established.

[0095] like Figure 4 and Figure 5 As shown, compared with the control group, the expression of Ftl1 mRNA and protein in the lung tissue of mice in the silica dust exposure group was significantly increased, further confirming the results of Example 1, confirming that the expression of Ftl1 gene and protein increased during the occurrence of pulmonary fibrosis.

[0096] Example 3 Establishment of bleomycin-induced mouse pulmonary fibrosis model and detection of Ftl1 protein expression.

[0097] Materials: C57BL / 6J, male mice, purchased from Beijing Weitonglihua Experimental Technology Co., Ltd.; Bleomycin, purchased from MedChemExpress (MCE), was dissolved in sterile saline for use.

[0098] Experimental methods:

[0099] After anesthesia, the mouse was fixed on the operating table, and the tongue of the mouse was gently clipped out with tweezers. The neck of the mouse was illuminated with a mercury lamp, and the tracheal opening was observed with the naked eye. The point that flickered with the mouse's breathing was the tracheal opening. The indwelling needle hose was inserted into the tracheal opening, and the bleomycin solution was quickly injected. After the indwelling needle was pulled out, the mouse was gently shaken to evenly distribute bleomycin in the lung tissue. The tongue of the mouse was then clipped out to avoid choking. After the mouse's breathing stabilized, the mouse was put back into the cage, and vital signs were observed until the mouse woke up. The experiment set up a control group and a bleomycin model group.

[0100] Four weeks after bleomycin instillation, the mice were sacrificed, lung tissues were dissected, and H&E staining was performed to evaluate the pathological conditions of the mouse lung tissues; lung tissue proteins were extracted, and the expression of Ftl1 protein was detected by immunoblotting. The antibodies used were:

[0101] Anti-Ferritin Light Chain Recombinant Rabbit Monoclonal Antibody[JM10-37].

[0102] Experimental results: After bleomycin infusion, the lung tissue of mice developed obvious lesions, such as Figure 6 Nodular infiltration, thickening of alveolar walls, and proliferation of fibroblasts were observed in the lung tissue, indicating that the mouse pulmonary fibrosis model was successfully established.

[0103] like Figure 7As shown, compared with the control group, the expression of Ftl1 protein in the lung tissue of mice in the bleomycin group was significantly increased, further confirming the results of Example 1 and confirming that the expression of Ftl1 increases during the occurrence of pulmonary fibrosis.

[0104] Example 4 Detection of Collagen I and Collagen III expression after FTL intervention in human lung fibroblasts.

[0105] Materials: FTL, purchased from MedChemExpress (MCE); human pulmonary fibroblast (HPF), purchased from ATCC.

[0106] Experimental methods:

[0107] HPF cells in the logarithmic growth phase were digested with trypsin, centrifuged and resuspended, and seeded into 6-well plates at a density of 1×10^5 cells / mL. After adherence, human lung fibroblasts were intervened with 1000 ng / mL FTL for 48 hours, and RNA was extracted for detection.

[0108] Experimental results: Figure 8 and Fig. 9 As shown in the results, after FTL intervention, the mRNA expression of fibrosis-related markers COL1A1 and COL3A1 in HPF cells increased significantly; at the same time, the expression of Collagen I and Collagen III proteins also increased, suggesting that FTL can promote fibroblasts to produce extracellular matrix and thus participate in the progression of fibrosis.

[0109] Example 5 Detection of FTL expression in macrophages.

[0110] Materials: THP-1 macrophages were purchased from Wuhan Pronocell Life Science Co., Ltd.; silicon dioxide was purchased from Sigma, sterilized by dry heat at 200°C for 4 h and then dissolved in sterile saline for later use.

[0111] Experimental methods:

[0112] Studies have shown that FTLs are mainly derived from macrophages, and it can also be observed in Example 1 that FTLs are highly expressed in macrophages. In this study, dust will be used to intervene in macrophages to verify whether macrophages produce FTLs after dust exposure.

[0113] After centrifugation, macrophages THP-1 in the logarithmic growth phase were seeded into 6-well plates at a density of 1×10^6 cells / mL, and PMA was added to induce adherence to the wall for 24 hours to form macrophages. According to the results of previous studies, 0, 100, 200, and 400 μg / mL silica were used to intervene in macrophages, and the expression of FTL mRNA and protein in macrophages was detected after 48 hours.

[0114] Human FTL detection primers:

[0115] (forward:5′-ATGAGCTCCCAGATTCGTCAG-3′;

[0116] reverse:5′-GCCCAGAGAGAGGTAGGTGT-3′.

[0117] Immunoblotting was used to detect the expression of FTL protein. The antibodies used were:

[0118] Anti-Ferritin Light Chain Recombinant Rabbit Monoclonal Antibody[JM10-37].

[0119] Experimental results: Fig.10 and 11 As shown, silica intervention increased the expression of FTL mRNA and protein in macrophages in a dose-dependent manner, suggesting that macrophages are the source of FTL in lung tissue after dust exposure.

[0120] Example 6 Construction of FTL knockout macrophage model

[0121] Materials: FTL siRNA was synthesized by General Biotechnology (Anhui) Co., Ltd.

[0122] Experimental methods:

[0123] THP-1 cells in the logarithmic growth phase were seeded into 6-well plates at a density of 1×10^6 / mL, and PMA was added to induce adherence to macrophages for 24 hours. FTL-siRNA was transfected with LP3000 reagent at a final concentration of 100nM, and the culture medium was replaced with normal culture medium for 24 hours after transfection for 8-10 hours, and RNA and protein were extracted to detect the silencing effect of FTL.

[0124] There are three types of siRNA targeting FTL used in this example, namely:

[0125] FTL-siRNA1:

[0126] Sense strand: 5′-CCAGAUUCGUCAGAAUUAU-3′

[0127] Antisense strand: 5′-AUAAUUCUGACGAAUCUGG-3′;

[0128] FTL-siRNA2:

[0129] Sense strand: 5′-GAAGUGAAGCUUAUCAAGA-3′

[0130] Antisense strand: 5′-UCUUGAUAAGCUUCACUUC-3′;

[0131] FTL-siRNA3:

[0132] Sense strand: 5′-GGCGAGUAUCUCUUCGAAA-3′

[0133] Antisense strand: 5′-UUUCGAAGAGAUACUCGCC-3′.

[0134] Experimental results: Fig.12 and 13 As shown, the three FTL siRNA transfections provided by the present invention can significantly reduce the expression of FTL in THP-1 cells, indicating that the FTL knockdown macrophage model was successfully established. FTL-siRNA 3 was selected for the implementation of subsequent experiments.

[0135] Example 7 Establishment of co-culture system of macrophages with low FTL expression and human lung fibroblasts and detection of collagen I and collagen III expression

[0136] Using a co-culture chamber, THP-1 cells were first inoculated and transfected in the upper layer of the chamber according to the method described in Example 6. After transfection, HPF cells were inoculated in the lower layer of the chamber at 1×10^5 cells / mL. After the HPF cells adhered to the wall, 200μg / mL of silica was added to the upper layer. After 48h, the expression of Collagen I and Collagen III in HPF cells was detected. The experiment set up a control group (THP-1 cells were transfected with nonsense sequence siRNA and not treated with silica), a silica group (THP-1 cells were transfected with nonsense sequence siRNA and treated with silica), and a silica + FTL knockdown group (THP-1 cells were transfected with FTL siRNA and treated with silica).

[0137] Experimental Results

[0138] like Fig.14As shown, after THP-1 cells were co-cultured with HPF cells, the expression of Collagen I and Collagen III proteins in HPF cells in the silica group was upregulated compared with the control group; compared with the silica group, the expression of Collagen I and Collagen III proteins in the silica + FTL knockdown group was significantly reduced, indicating that reducing the expression of FTL can inhibit the production of extracellular matrix in fibroblasts, thereby alleviating the progression of fibrosis, indicating that FTL can be used as a target for the prevention and / or treatment of pulmonary fibrosis. Example 8 Construction of mice with knockdown of macrophage Ftl1 and construction of mice with pulmonary fibrosis induced by silica and detection of Collagen I and Collagen III proteins

[0139] Materials: C57BL / 6J, male mice, purchased from Beijing Weitonglihua Experimental Technology Co., Ltd.; silicon dioxide, purchased from Sigma, sterilized by dry heat at 200°C for 4 h and then dissolved in sterile saline for use.

[0140] Experimental methods:

[0141] After anesthesia, the mouse was fixed on the operating table, and the tongue of the mouse was gently clipped out with tweezers. The neck of the mouse was illuminated with a mercury lamp. The tracheal opening was observed with the naked eye. The point that flickered with the mouse's breathing was the tracheal opening. The indwelling needle hose was inserted into the tracheal opening, and adenovirus containing Ftl1 siRNA was quickly injected. F4 / 80 was used as a promoter to target and inhibit the expression of Ftl1 in macrophages. After the indwelling needle was removed, the virus was mixed to evenly distribute in the mouse lungs. The treatment lasted for 2 weeks. After 2 weeks, one mouse was randomly selected to detect the expression of Ftl1 gene and protein in lung tissue to confirm whether Ftl1 was inhibited. Then, silica was instilled again to construct a mouse pulmonary fibrosis model. After 4 weeks of silica exposure, the mouse was sacrificed, lung tissue was dissected, and H&E staining was performed to evaluate the pathological condition of the mouse lung tissue.

[0142] The experiment set up a control group (tracheal instillation of normal saline + adenovirus-encapsulated nonsense sequence siRNA), a silica group (tracheal instillation of silica + adenovirus-encapsulated nonsense sequence siRNA), and a silica + Ftl1 knockdown group (tracheal instillation of silica + adenovirus-encapsulated Ftl1 siRNA).

[0143] Mouse FTL detection primers (mouse FTL gene is represented by Ftl1 in the examples):

[0144] forward:5′-TACACCTACCTCTCTCTGGGC-3′;

[0145] reverse:5′-CCCGCGATCGTTCTGAAACT-3′.

[0146] The expression of Ftl1 protein was detected by immunoblotting. The antibody used was Anti-Ferritin LightChain Recombinant Rabbit Monoclonal Antibody [JM10-37].

[0147] Experimental results: Fig.15 As shown in Figure 2, after Ftl1 siRNA treatment, the expression of Ftl1 mRNA and protein in lung tissue was significantly reduced, indicating that mice with low expression of Ftl1 in macrophages were successfully established; Fig.16 As shown in the figure, after silica exposure, mice had obvious lesions in their lung tissues, indicating that silica induced pulmonary fibrosis in mice. Compared with the silica model group, the degree of lesions in the lung tissues of mice with low Ftl1 expression was alleviated, and the expression of Collagen I and Collagen III proteins was also reduced, confirming that inhibiting the expression of Ftl1 can alleviate pulmonary fibrosis in mice.

Claims

1. Application of ferritin light chain (FTL) as a target for the prevention and / or treatment of pulmonary fibrosis.

2. The use according to claim 1, characterized in that: The pulmonary fibrosis is pulmonary fibrosis induced by inhaled particulate matter, pulmonary fibrosis caused by infection, pulmonary fibrosis caused by autoimmune disease, radiation-induced pulmonary fibrosis or idiopathic pulmonary fibrosis of unknown cause; further, the pulmonary fibrosis induced by inhaled particulate matter is dust-induced silicosis fibrosis.

3. Use of a reagent or diagnostic device for detecting the level of ferritin light chain in the preparation or screening of a reagent or kit for detecting, diagnosing, monitoring and / or predicting pulmonary fibrosis.

4. The use according to claim 3, characterized in that: The reagent for detecting FTL level is used for in vitro diagnosis and risk stratification of pulmonary fibrosis; or; the reagent is used for diagnosing or monitoring the existence and / or process and / or severity and / or prognosis of pulmonary fibrosis.

5. The use according to claim 3, characterized in that: The reagent is used to determine the expression level and / or concentration of FTL in a sample; further, the reagent or kit detects the expression level and / or concentration of the FTL polypeptide in the sample by ELISA, colloidal gold method or chemiluminescence method; further, the reagent or kit contains an antibody against the FTL polypeptide; Preferably, the anti-FTL polypeptide antibody is at least one of anti-FTL monoclonal antibody, polyclonal antibody, single-chain antibody or nanobody; further preferably, the anti-FTL polypeptide antibody is antibody JM10-37.

6. The use according to claim 3, characterized in that: The reagent or kit detects the expression amount and / or concentration of FTL in a sample by at least one of WB method, quantitative PCR method, fluorescence in situ hybridization method (FISH), real-time fluorescence quantitative PCR method (qPCR) or immunohistochemistry staining method (IHC); Further, the reagent or kit comprises a primer, a primer pair or a primer set for amplifying a FTL polypeptide encoding gene or a CDS sequence or amplifying a fragment of a FTL polypeptide encoding gene or its CDS sequence; Preferably, the primers include: forward:5′-ATGAGCTCCCAGATTCGTCAG-3′; reverse:5′-GCCCAGAGAGAGGTAGGTGT-3′; Or include: forward:5′-TACACCTACCTCTCTCTGGGC-3′; reverse:5′-CCCGCGATCGTTCTGAAACT-3′.

7. The use according to claim 3, characterized in that: The sample comprises plasma, serum or blood extract, brushing, biopsy or surgically removed tissue or liquid sample from the subject; further, the sample is an ex vivo sample.

8. Use of a ferritin light chain (FTL) inhibitor in the preparation of a drug for preventing and / or treating pulmonary fibrosis.

9. The use according to claim 8, characterized in that: The inhibitor is a gene inhibitor of FTL; further, the gene inhibitor is an inhibitor that inhibits the expression of the FTL gene or its transcribed mRNA as the target of action; further, the gene inhibitor is at least one of an antisense oligonucleoside, siRNA, esiRNA, dsRNA ribozyme, shRNA, microRNA or FTL gene promoter inhibitor that inhibits the expression of the FTL gene or its transcribed mRNA as the target of action; Alternatively, the gene inhibitor is an sgRNA or crRNA that inhibits the expression of the FTL gene or its transcribed mRNA; Preferably, the gene inhibitor is siRNA, and its nucleotide sequence is: 1: 5′-CCAGAUUCGUCAGAAUUAU-3′ and 5′-AUAAUUCUGACGAAUCUGG-3′; or 2: 5′-GAAGUGAAGCUUAUCAAGA-3′ and 5′-UCUUGAUAAGCUUCACUUC-3′; or 3: 5′-GGCGAGUAUCUCUUCGAAA-3′ and 5′-UUUCGAAGAGAUACUCGCC-3′.

10. Use of ferritin light chain protein or a nucleic acid molecule encoding ferritin light chain in preparing a cell model or an animal model of pulmonary fibrosis; further, the cell is a lung fibroblast.

11. The use according to claim 10, characterized in that The nucleic acid molecule encoding FTL is a DNA molecule or RNA molecule encoding FTL; or, further, the expression of Collagen I and Collagen III proteins in the lung fibroblasts is significantly increased.

12. A method for establishing a pulmonary fibrosis cell model, comprising the following steps: Apply FTL protein to the cells to be treated, or transfect them with an expression vector capable of expressing FTL; further, the cells to be treated are lung fibroblasts.

13. The method for establishing a pulmonary fibrosis cell model according to claim 12, characterized in that: The method comprises the steps of applying FTL treatment to lung fibroblasts, and then detecting the expression levels of fibrogenesis-related genes in the fibroblasts.

14. Use of the pulmonary fibrosis cell model established by the method of claim 12 in any of the following: a. Use in the study of the mechanism of pulmonary fibrosis; b. Use in screening anti-pulmonary fibrosis drugs; c. Use in the observation and / or evaluation of the efficacy of anti-pulmonary fibrosis drugs.

15. A method for screening drugs for preventing or treating pulmonary fibrosis, characterized in that The following steps are involved: a. Establish a model group of pulmonary fibrosis cell model or animal model, and set up a normal control group; b. Use the drug to be screened to act on the model group according to the dose gradient, and then detect the level of FTL polypeptide or its mRNA content; c. Compare the test results with the normal control group. The drug to be screened that can make the level of FTL protein or its mRNA content in the model group significantly approach that of the normal control group is selected.

16. The method for screening drugs for preventing or treating pulmonary fibrosis according to claim 15, characterized in that: The pulmonary fibrosis cell model is a co-culture model of macrophages and pulmonary fibroblasts.

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