Application of Furin inhibitor in preparation of medicine for preventing, delaying or treating idiopathic pulmonary fibrosis

Abstract

The invention belongs to the technical field of biological medicine, and discloses application of a Furin inhibitor in preparation of a medicine for preventing, delaying or treating idiopathic pulmonary fibrosis, the effect of the Furin inhibitor in inhibiting generation of P61-Sema3E so as to delay the idiopathic pulmonary fibrosis is found for the first time, the key effect of Furin and P61-Sema3E in generation and development of IPF is further determined, and the application of the Furin inhibitor in preparation of the medicine for preventing, delaying or treating the idiopathic pulmonary fibrosis is provided for the first time. And a new target can be provided for prevention and treatment of IPF. The invention determines that the Furin inhibitor inhibits activation, proliferation and migration of human primary lung fibroblasts by inhibiting the enzyme activity of Furin, reducing the conversion from P87-Sema3E to P61-Sema3E and reducing the generation of P61-Sema3E, so that a new treatment strategy is provided for IPF.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to the use of furin inhibitors in the preparation of drugs for preventing, delaying or treating idiopathic pulmonary fibrosis. Background Art

[0002] Pulmonary fibrosis (PF) is a fatal interstitial lung disease characterized by progressive lung parenchymal damage, abnormal fibroblast proliferation, and excessive deposition of extracellular matrix. Idiopathic pulmonary fibrosis (IPF) is the most common form. Patients have a median survival of only 2-3 years after diagnosis, with a 5-year survival rate below 30%. The mortality rate is higher than that of most malignancies (such as breast cancer and colorectal cancer). The global prevalence of IPF is increasing at an annual rate of 11%. In my country, the number of patients exceeds one million, and the incidence continues to rise due to environmental exposures (smoking, air pollution) and an aging population. Patients experience progressive loss of lung function, requiring oxygen support in the late stages of respiratory failure. This significantly reduces their quality of life and places a significant burden on their families and healthcare systems. Existing treatments (pirfenidone and nintedanib) can only slow the decline in lung function but cannot reverse the progression of fibrosis. Lung transplantation is the only curative option for end-stage patients, but donors are scarce, the surgery is risky, and postoperative survival is limited. Faced with the irreversible and fatal progression of pulmonary fibrosis and extremely limited treatment options, the development of drugs with novel mechanisms of action that can effectively block the fibrosis process and even repair lung tissue has become an urgent need in the global respiratory disease field. To overcome the bottlenecks of existing therapies, it is urgent to address the source of the disease and provide patients with more efficient and safe treatment strategies.

[0003] The semaphorin family is a class of secreted or membrane-bound signaling proteins that regulate cell migration, proliferation, and differentiation. Recent studies have revealed that some semaphorin members may participate in organ fibrosis by regulating immune cell infiltration or fibroblast transformation. Sema3E, a member of the semaphorin family, exists in two isoforms in vivo: P87-Sema3E and P61-Sema3E. P87-Sema3E is cleaved by furin, a proprotein convertase localized to the Golgi apparatus, to produce P61-Sema3E. Our study found that compared with healthy controls, the expression of Sema3E and its receptor, Plexin D1, was significantly increased in the lung tissue and plasma of patients with IPF, with Sema3E primarily present as P61-Sema3E. Further correlation analysis revealed a significant negative correlation between plasma Sema3E levels and lung function in IPF patients. In vitro, addition of P61-Sema3E to primary human lung fibroblasts significantly enhanced fibroblast activation, proliferation, and migration. Finally, we found that furin inhibition significantly reduced fibroblast activity by reducing the production of p61-Sema3E. These findings highlight the key role of the p61-Sema3E-Plexin D1 pathway in the pathogenesis of IPF and suggest that targeting this pathway by inhibiting furin may offer hope for the development of new strategies for the treatment of IPF. Summary of the Invention

[0004] In response to the problems existing in the prior art, the purpose of the present invention is to provide the use of furin inhibitors in the preparation of drugs for preventing, delaying or treating idiopathic pulmonary fibrosis. Furin inhibitors inhibit furin enzyme activity → inhibit the conversion of P87-Sema3E to P61-Sema3E → reduce P61-Sema3E production → inhibit lung fibroblast activation / proliferation / migration, providing new intervention targets for the development of new and effective therapeutic drugs for idiopathic pulmonary fibrosis.

[0005] In order to achieve the above objectives, this application adopts the following technical solutions: The present invention provides the use of a furin inhibitor in preparing a medicament for preventing, delaying or treating idiopathic pulmonary fibrosis.

[0006] In the above technical solution, the Furin inhibitor achieves the prevention, delay or treatment of idiopathic pulmonary fibrosis by reducing the production of p61-Sema3E.

[0007] In the above technical solution, the furin inhibitor is a D-arginine hexamer.

[0008] In the above technical solution, the Furin inhibitor inhibits the conversion of P87-Sema3E to P61-Sema3E, thereby reducing the production of P61-Sema3E.

[0009] In the above technical solution, the Furin inhibitor achieves the prevention, delay or treatment of idiopathic pulmonary fibrosis by inhibiting the activation, proliferation and migration of human primary lung fibroblasts.

[0010] In the above technical solution, the furin inhibitor inhibits the enzymatic activity of furin, reduces the conversion of P87-Sema3E to P61-Sema3E, and reduces the production of P61-Sema3E, thereby inhibiting the activation, proliferation and migration of human primary lung fibroblasts.

[0011] In the above technical solution, the drug also includes pharmaceutically acceptable excipients.

[0012] In the above technical solution, the auxiliary materials include fillers, diluents, adhesives, disintegrants, and emulsifiers.

[0013] The beneficial effects of the present invention are as follows: the present invention has discovered for the first time the role of furin inhibitors in inhibiting the production of P61-Sema3E and thus delaying idiopathic pulmonary fibrosis, further confirming the key role of furin and P61-Sema3E in the development and progression of IPF, and providing a new target for the prevention and treatment of IPF. The present invention has determined that furin inhibitors inhibit the activation, proliferation, and migration of human primary lung fibroblasts by inhibiting the enzymatic activity of furin, reducing the conversion of P87-Sema3E to P61-Sema3E, and reducing the production of P61-Sema3E, thereby providing a new treatment strategy for IPF. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The following is the basic expression of Sema3E in plasma and lung tissue of normal and IPF subjects; Figure 1 A is the plasma Sema3E content (ng / ml) in normal subjects (n=24) and IPF subjects (n=63); Figure 1 B is the protein level of Sema3E in plasma proteins of normal subjects (n=24) and IPF subjects (n=63); Figure 1 C is a statistical analysis of the protein levels and related grayscale values ​​of Sema3E and its receptor PlexinD1 in lung tissues of normal subjects (n=10) and IPF subjects (n=12); Figure 1 D is the correlation analysis between the plasma Sema3E content in IPF patients and their lung function index forced vital capacity percentage predicted value (FVC%pred); Figure 1 E is the correlation analysis between the plasma Sema3E content in IPF patients and their lung function index, the percentage of predicted value of the diffusion capacity for carbon monoxide (DLCO%pred); Figure 1 F is the correlation analysis between the Sema3E content in the plasma of IPF patients and their lung function index, the percentage of predicted total lung capacity (TLC%pred); Figure 1 G is the correlation analysis between the plasma Sema3E content in IPF patients and their lung function index, the percentage of predicted forced expiratory volume in one second (FEV1%pred); among them, *P<0.05, ***P<0.001.

[0015] Figure 2 The expression of Sema3E and its receptor PlexinD1 in lung tissues of normal mice and bleomycin-induced pulmonary fibrosis model mice; Figure 2 A is the immunofluorescence co-staining image of Sema3E and α-SMA in lung tissue sections of normal mice (n=3) and bleomycin-induced pulmonary fibrosis model mice (n=3) and the corresponding fluorescence intensity statistical graph; Figure 2 B is the immunofluorescence co-staining image of PlexinD1 and α-SMA in lung tissue sections of normal mice (n=3) and bleomycin-induced pulmonary fibrosis model mice (n=3) and the corresponding fluorescence intensity statistical graph; Figure 2 C is the protein expression and corresponding grayscale value analysis of Sema3E and its receptor PlexinD1 in lung tissues of normal mice (n=5) and bleomycin-induced pulmonary fibrosis model mice (n=5); Figure 2 D is the content of Sema3E in the plasma of normal mice (n=5) and bleomycin-induced pulmonary fibrosis model mice (n=5); among them, *P<0.05, **P<0.01, ***P<0.001.

[0016] Figure 3 The expression of cell fibrosis indicators, proliferation ability and migration ability of human primary lung fibroblasts after adding different concentrations of P61-Sema3E recombinant protein for 48 hours; Figure 3 A is a validation diagram of P61-Sema3E and P87-Sema3E recombinant proteins; Figure 3 B is a statistical graph showing the protein expression and corresponding grayscale value analysis of fibrosis indicators Fibronectin, Col1a1, and -SMA in human primary lung fibroblasts after addition of different concentrations (0, 1ng / ml, 10ng / ml, 100ng / ml) of P61-Sema3E recombinant protein for 48 hours; Figure 3C is the detection graph and corresponding statistical graph of cell proliferation activity of human primary lung fibroblasts after adding different concentrations (0, 1ng / ml, 10ng / ml, 100ng / ml) of P61-Sema3E recombinant protein for 48 hours; Figure 3 D shows the detection graph and corresponding statistical graph of cell migration ability of human primary lung fibroblasts after adding different concentrations (0, 1ng / ml, 10ng / ml, 100ng / ml) of P61-Sema3E recombinant protein for 48 hours; among them, *P<0.05, **P<0.01, ***P<0.001.

[0017] Figure 4 Statistical analysis of protein expression and corresponding grayscale values ​​of fibrosis markers Fibronectin, Col1a1, and -SMA in human primary lung fibroblasts after addition of different concentrations of P87-Sema3E recombinant protein for 48 hours; Figure 4 A: Human primary lung fibroblasts were treated with different concentrations of P87-Sema3E recombinant protein for 48 hours and the protein expression levels of Fibronectin, Col1a1, and -SMA were detected by Western blotting. Figure 4 B is Figure 4 A. Statistical graph of gray value analysis of the corresponding bands.

[0018] Figure 5 Figure 2 shows the effect of P61-Sema3E recombinant protein addition on the proliferation, migration and expression of fibrosis markers in human primary lung fibroblasts after PlexinD1 knockdown. Figure 5 A is a graph showing the effect of adding P61-Sema3E recombinant protein to human primary lung fibroblasts and knocking down PlexinD1 on cell proliferation activity and the corresponding statistical graph; Figure 5 B is the detection graph and corresponding statistical graph of the effect of P61-Sema3E recombinant protein on the migration ability of human primary lung fibroblasts after knocking down PlexinD1; Figure 5 C is a statistical analysis of the protein expression and corresponding grayscale values ​​of fibrosis indicators Fibronectin, Col1a1, and -SMA in human primary lung fibroblasts after adding P61-Sema3E recombinant protein and knocking down PlexinD1; among them, *P<0.05, **P<0.01.

[0019] Figure 6 This is a graph showing the effects of adding Furin inhibitors to human primary lung fibroblasts on fibrosis indicators, cell proliferation activity, and cell migration ability; Figure 6A is the protein expression of Furin and the corresponding gray value analysis statistical graph after adding different concentrations (0, 2.5ng / ml, 5ng / ml, 10ng / ml) of TGF-β1 to human primary lung fibroblasts; Figure 6 B is a statistical analysis of the protein expression and corresponding grayscale values ​​of fibrosis indicators Fibronectin, Col1a1, and -SMA after adding different concentrations (0, 2.5uM, 5uM, 10uM) of the furin inhibitor D-arginine hexamer (Hexa-D-arginine) to human primary lung fibroblasts; Figure 6 C is a graph showing the effect of adding furin inhibitor Hexa-D-arginine on the proliferation of primary human lung fibroblasts and the corresponding statistical graph; Figure 6 D is the detection graph and corresponding statistical graph of the effect of furin inhibitor Hexa-D-arginine on the migration ability of human primary lung fibroblasts; among them, *P<0.05, **P<0.01, ***P<0.001. DETAILED DESCRIPTION

[0020] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. The present invention can be implemented in many different forms and should not be construed as limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that this disclosure will be thorough and complete and will fully convey the concept of the present invention to those skilled in the art. The present invention will be limited only by the claims.

[0021] The present invention analyzes the content of P61-Sema3E in the lung tissue and plasma of different populations and determines that the expression of P61-Sema3E in the lung tissue and plasma of COPD population is significantly increased; by adding furin inhibitors to human primary lung fibroblasts for experimental analysis, it is determined that furin inhibitors inhibit the enzymatic activity of furin, reduce the conversion of P87-Sema3E to P61-Sema3E, reduce the production of P61-Sema3E, and thus inhibit the activation, proliferation and migration of human primary lung fibroblasts; further confirms the key role of P61-Sema3E in the occurrence and development of IPF.

[0022] Example 1: Determining the difference in Sema3E expression in plasma and lung tissue between normal subjects and IPF subjects Human experiments: IPF patients and healthy volunteers were recruited, their plasma and lung tissues were collected, and the expression of Sema3E was detected.

[0023] 1.ELISA Specific Sema3E antibodies are immobilized in the wells of an ELISA plate. This is accomplished by diluting the antigen in coating buffer and incubating overnight at 4°C. After coating, unbound sites are blocked with blocking buffer to reduce nonspecific binding. The plate is typically incubated for 1-2 hours at room temperature. The plate is then washed with wash buffer to remove any unbound blocking buffer. The plasma sample to be tested and a series of standards of known concentrations are added to the ELISA plate. The plate is typically incubated for 1-2 hours at 37°C to allow the antigen in the sample to bind to the coated antibody. The plate is washed again with wash buffer, and the specific primary antibody is added and incubated at 37°C for 1-2 hours. The plate is washed again with wash buffer, and an HRP-conjugated secondary antibody is added and incubated at 37°C for 1 hour. Finally, the plate is washed again, and a substrate that produces a color change catalyzed by HRP is added. After a specified period of time, the reaction is stopped with stop buffer to allow the color change to stabilize. The results are then read on a microplate reader.

[0024] like Figure 1 As shown in A, the average level of Sema3E in the plasma of normal people is about 2.5 ng / ml, while the average level of Sema3E in the plasma of IPF people is about 4 ng / ml, indicating that the plasma Sema3E level in IPF patients is significantly increased.

[0025] 2. Extract plasma proteins and detect the expression of Sema3E by Western blot.

[0026] Collect whole blood in an anticoagulant tube and centrifuge at 1500 × g for 10 minutes at 4°C. The supernatant is plasma. Add 2 μL of protease inhibitor cocktail (containing PMSF, EDTA, etc.) to every 200 μL of extraction solution, mix thoroughly, and store on ice until ready to use. Add 100–200 μL of extraction solution to every 100 μL of plasma, mix thoroughly, and incubate at 4°C for 5 minutes. Centrifuge at 14,000 × g for 10 minutes at 4°C. The supernatant is plasma total protein.

[0027] Proteins were separated by 10% SDS-PAGE gel, transferred to PVDF membranes, and blocked in 5% milk for 1–2 hours. Subsequently, the membranes were incubated with Sema3E primary antibody overnight at 4°C, washed three times with TBST, and incubated with secondary antibody for 1 hour before exposure to chemiluminescent solution.

[0028] like Figure 1 As shown in Figure B, the protein level of Sema3E in the plasma of normal people is significantly lower than that in the plasma of IPF people, and Sema3E mainly exists in the form of P61-Sema3E.

[0029] 3. Extract lung tissue protein and detect the expression of Sema3E by Western blot.

[0030] Place an appropriate amount of tissue into a 2 ml EP tube. Add 300 μl of RIPA protein lysis buffer to submerge the tissue. Add three grinding beads and grind the tissue using a grinder (position 3) for 20–30 seconds until finely pulverized. Transfer the tissue to a 1.5 ml EP tube and centrifuge at 12,000 rpm at 4°C for 15 minutes. Collect the supernatant as the total protein. Perform Western blot analysis as described in 1.2.

[0031] like Figure 1 As shown in C, the protein level of Sema3E in the lung tissue of normal people is significantly lower than that in the lung tissue of IPF people, and Sema3E mainly exists in the form of P61-Sema3E.

[0032] 4. Correlation Analysis The plasma Sema3E levels and lung function indicators of IPF patients were collected, and the two were subjected to Pearson correlation analysis using Graphpadprism9.0 software.

[0033] like Figure 1 As shown in DG, the Sema3E content in the plasma of IPF people was significantly negatively correlated with their lung function indicators FVC%pred, DLCO%pred, TLC%pred, and FEV1%pred.

[0034] Example 2: Determination of the difference in Sema3E expression in lung tissue and plasma of normal mice and mice with bleomycin-induced pulmonary fibrosis 1. Establishment of a mouse pulmonary fibrosis model Male C57BL / 6J mice (6–8 weeks old) were purchased from Beijing Weitongli and housed at the SPF Animal Center of Tongji Hospital. To establish a mouse pulmonary fibrosis model, mice were anesthetized with 1% sodium pentobarbital (40 mg kg⁻¹) and then administered a single intratracheal injection of bleomycin (BLM, MedChemExpress, China) at 2 mg kg⁻¹ (dissolved in 50 μL of sterile saline) using a high-pressure airway atomizer. Mice were sacrificed 21 days after BLM model establishment for analysis of pulmonary fibrosis.

[0035] 2. Immunofluorescence staining of lung tissue sections to detect the expression of Sema3E and its receptor PlexinD1 Fresh mouse lung tissue was removed and fixed with 4% paraformaldehyde for 48 hours, 70% ethanol once for 1.5 hours, 80% ethanol once for 30 minutes, 95% ethanol twice for 15 minutes, anhydrous ethanol three times for 10 minutes, xylene clearing twice for 15 minutes each, paraffin I infiltration for 30 minutes, paraffin II infiltration for 90 minutes, paraffin III infiltration for 6 hours, and then embedded and sectioned.

[0036] Immerse slides in xylene three times for 5-10 minutes each. Then, immerse slides in a graded series of alcohols: 100% ethanol I for 3-5 minutes, 100% ethanol II for 3-5 minutes, 95% ethanol for 3-5 minutes, 70% ethanol for 3-5 minutes, and distilled water (dH2O) or PBS for 5 minutes. Place slides in a heat-resistant container filled with antigen retrieval buffer and microwave on high until boiling. Reduce to medium-low heat and maintain a gentle boil for 10-20 minutes (be careful not to dry out). Remove and cool at room temperature for 20-30 minutes. After antigen retrieval, rinse slides three times with PBS (pH 7.4) for 5 minutes each. Immerse slides in PBS containing 0.1-0.5% Triton X-100 or Tween 20 and incubate at room temperature for 5-15 minutes. Rinse slides three times with PBS for 5 minutes each. Apply enough blocking solution to the tissue slices to ensure complete coverage. Incubate at room temperature for 30-60 minutes. Apply an adequate amount of primary antibody working solution to the tissue slices and incubate overnight at 4°C. The next day, after warming, apply an adequate amount of fluorescently labeled secondary antibody working solution to the tissue slices and incubate at room temperature in the dark for 1-2 hours. Rinse the sections three times with PBS or TBST for 5 minutes each, protected from light. Finally, rinse the sections with DAPI dye for 10 minutes before applying 1-2 drops of anti-fluorescence quenching mounting medium to the tissue slices. Seal the sections and photograph.

[0037] like Figure 2 As shown in A and B, the red color in the sections is α-SMA used to mark fibroblasts, the green color is Sema3E or PlexinD1, and the blue color is DAPI for the cell nucleus. The red and green fluorescence intensities of the lung sections of BLM-induced pulmonary fibrosis mice were significantly higher than those of the lung sections of normal mice, indicating that the expression of Sema3E and its receptor PlexinD1 in the lung tissues of BLM-induced pulmonary fibrosis mice was significantly increased.

[0038] 3. Extract mouse lung tissue protein and detect the expression of Sema3E and its receptor PlexinD1 by Western blot The method is as described above.

[0039] like Figure 2 As shown in Figure C, the expression of P61-Sema3E and PlexinD1 in the lung tissues of BLM-induced pulmonary fibrosis model mice was significantly higher than that in normal mice, while the expression of P87-Sema3E in the lung tissues of mice was very low. This indicates that the expression of Sema3E and its receptor PlexinD1 in the lung tissues of BLM-induced pulmonary fibrosis mice was significantly increased.

[0040] 4. Detection of Sema3E content in mouse plasma by ELISA The method is as described above.

[0041] like Figure 2 As shown in D, the average Sema3E content in the plasma of normal mice is about 2 ng / ml, while the average Sema3E content in the plasma of BLM-induced pulmonary fibrosis mice is about 3 ng / ml, indicating that the Sema3E content in the plasma of BLM-induced pulmonary fibrosis mice is significantly increased.

[0042] Example 3: Determination of the profibrotic effect of P61-Sema3E on primary human lung fibroblasts 1. Extraction of primary human lung fibroblasts In this experiment, fibroblasts were derived from three independent sources from three different donors. Lung tissue was obtained from human surgical specimens and perfused with PBS to remove residual blood. Tissue fragments were prepared in PBS and seeded onto 10 cm culture dishes in 2 mL of complete DMEM (10% fetal bovine serum and 1% penicillin / streptomycin). The fragments were then added to ensure adherence to the dish and did not float or aggregate. After 24 hours, 8 mL of fresh culture medium was added. On the third day, nonadherent cells were removed by washing or changing the medium. Spindle-shaped fibroblasts were observed migrating from the tissue fragments and, within six days, filled the dish bottom. These cells were then ready for passage. After serial passages, fibroblasts gradually became the dominant cell type, while epithelial cells and macrophages were gradually eliminated. Fibroblasts at passage 4 were used for this experiment. The cells were identified as lung fibroblasts by immunofluorescence staining for S100A4.

[0043] 2. Western blot detection of Sema3E recombinant protein The method is as described above.

[0044] like Figure 3 As shown in A, P87-Sema3E has a molecular weight of about 87 KDa and no band at about 61 KDa, while P61-Sema3E has a molecular weight of about 61 KDa and no band at about 87 KDa, indicating that the two recombinant proteins were well purified.

[0045] 3. Western blot detection of fibrosis index expression The method is as described above.

[0046] like Figure 3As shown in Figure B, after adding different concentrations (0, 1ng / ml, 10ng / ml, 100ng / ml) of P61-Sema3E recombinant protein to human primary lung fibroblasts for 48 hours, the protein expression of fibrosis indicators Fibronectin, Col1a1, and -SMA was detected. It was found that with the increase of the concentration of P61-Sema3E recombinant protein, the protein expression of fibrosis indicators Fibronectin, Col1a1, and -SMA also gradually increased, indicating the pro-fibrotic ability of P61-Sema3E recombinant protein.

[0047] 4. Edu staining method to detect cell proliferation activity Primary human lung fibroblasts were plated in 96-well plates and, after attachment, stimulated with P61-Sema3E recombinant protein at various concentrations (0, 1 ng / ml, 10 ng / ml, and 100 ng / ml) for 48 hours. Culture medium containing EdU working solution was then added and incubated for 2 hours. The culture medium was then aspirated and the cells were gently rinsed twice with pre-chilled PBS. The cells were fixed with 4% paraformaldehyde (PFA) for 15 minutes at room temperature and washed three times with PBS for 5 minutes. Finally, cell nuclei were counterstained with DAPI.

[0048] like Figure 3 As shown in Figure C, red light represents proliferating cells, indicating their proliferation activity, and blue light represents cell nuclei. As the concentration of P61-Sema3E recombinant protein increases, the number of cells emitting red fluorescence also increases, indicating that P61-Sema3E recombinant protein promotes the proliferation of fibroblasts.

[0049] 5. Transwell method to detect cell migration ability Digest the cells, resuspend them in serum-free medium, and adjust the density to 1-5×10 5 cells / mL, the Transwell chamber was placed in a 24-well plate, 100 μL of cell suspension was added to the upper chamber of the Transwell, and 600 μL of culture medium containing 10% FBS was added to the lower chamber. After 2 hours, different concentrations (0, 1ng / ml, 10ng / ml, 100ng / ml) of P61-Sema3E recombinant protein were added to the upper chamber. After 48 hours, the chamber was removed, the non-migrated cells on the surface were gently washed with PBS, and the cells on the surface of the upper chamber membrane were gently wiped with a wet cotton swab (do not use force), fixed with 4% PFA at room temperature for 15 minutes, stained with 0.1% crystal violet for 20 minutes, air-dried, and observed under a microscope.

[0050] like Figure 3As shown in D, the purple cells represent cells that migrated from the upper chamber to the lower chamber. As the concentration of P61-Sema3E recombinant protein increased, the number of migrated cells gradually increased, indicating that P61-Sema3E recombinant protein promoted the migration ability of fibroblasts.

[0051] Example 4: Determination of the lack of profibrotic effect of P87-Sema3E on primary human lung fibroblasts Western blot detection of fibrosis markers The method is as described above.

[0052] like Figure 4 As shown in A, after adding different concentrations (0, 1ng / ml, 10ng / ml, 100ng / ml) of P87-Sema3E recombinant protein to human primary lung fibroblasts for 48 hours, the protein expression of fibrosis indicators Fibronectin, Col1a1, and -SMA was detected. It was found that there was no significant difference in the protein expression of fibrosis indicators Fibronectin, Col1a1, and -SMA among the several groups of samples, indicating that P87-Sema3E recombinant protein has no pro-fibrotic effect on human primary lung fibroblasts. Figure 4 B is Figure 4 Gray value analysis statistical chart of A.

[0053] Example 5: Determining the Profibrotic Effects of the Sema3E-PlexinD1 Pathway on Primary Human Lung Fibroblasts Transfection of PlexinD1 small interfering RNA PlexinD1 small interfering RNA (PlexinD1 siRNA) was purchased from Bio-Tech Co., Ltd. Human primary fibroblasts were plated in 12-well plates and transfected when the cell density reached approximately 40%. Each well was prepared with the following system: Solution A: 2 μL siRNA + 50 μL Opti-MEM®; Solution B: Lipo3000 + 50 μL Opti-MEM®. Solution A / B were prepared separately and allowed to stand at room temperature for 5 minutes. Solution A and B were mixed gently and incubated at room temperature for 15-20 minutes. 100 μL of the complex was added dropwise to the cell culture medium, and the plate was gently shaken for even distribution. The culture medium was changed to complete medium 6-8 hours after transfection.

[0054] 2. Western blot detection of fibrosis markers The method is as described above.

[0055] like Figure 5As shown in A, after 48 hours of stimulation with P61-Sema3E recombinant protein, the protein expressions of fibrosis indicators Fibronectin, Col1a1, and -SMA in human primary lung fibroblasts transfected with PlexinD1 siRNA were significantly reduced compared with the group stimulated with P61-Sema3E recombinant protein alone, indicating that the pro-fibrotic effect of Sema3E on fibroblasts is exerted through its receptor PlexinD1.

[0056] 3. Edu staining method to detect cell proliferation activity The method is as described above.

[0057] like Figure 5 As shown in B, after 48 hours of stimulation with P61-Sema3E recombinant protein, the number of red fluorescent cells in human primary lung fibroblasts transfected with PlexinD1 siRNA was significantly reduced compared with the group stimulated with P61-Sema3E recombinant protein alone, indicating that the proliferative effect of Sema3E on fibroblasts is exerted through its receptor PlexinD1.

[0058] 4. Transwell method to detect cell migration ability The method is as described above.

[0059] like Figure 5 As shown in Figure C, after 48 hours of stimulation with P61-Sema3E recombinant protein, the number of migrated cells in human primary lung fibroblasts transfected with PlexinD1 siRNA was significantly reduced compared with the group stimulated with P61-Sema3E recombinant protein alone, indicating that Sema3E's ability to promote fibroblast migration is through its receptor PlexinD1.

[0060] Example 6: Determining the Effect of Furin Inhibitors on Inhibiting Fibrosis by Reducing the Production of P61-Sema3E 1. Western blot detection of Furin expression The method is as described above.

[0061] like Figure 6 As shown in A, after fibroblasts were stimulated with different concentrations of TGF-β1 for 48 hours, the expression of furin protein by fibroblasts gradually increased with the increase of TGF-β1 concentration, indicating that activated fibroblasts increased the expression of furin.

[0062] 2. Western blot detection of P61-Sema3E and fibrosis markers The method is as described above.

[0063] like Figure 6As shown in Figure 2, after fibroblasts were stimulated with different concentrations of the furin inhibitor D-arginine hexamer (Hexa-D-arginine) for 48 hours, the protein level of P61-Sema3E gradually decreased with the increase of Hexa-D-arginine concentration, and the protein levels of fibrosis indicators Fibronectin, Col1a1, and -SMA also gradually decreased, indicating that the furin inhibitor Hexa-D-arginine inhibits fibrosis by reducing the production of P61-Sema3E.

[0064] 3. Edu staining method to detect cell proliferation activity The method is as described above.

[0065] like Figure 6 As shown in C, the number of fibroblasts emitting red fluorescence after TGF-β1 stimulation with the addition of the furin inhibitor Hexa-D-arginine was significantly reduced compared with the fibroblasts stimulated with TGF-β1 alone, indicating that the number of cells in the proliferation phase was significantly reduced, suggesting that the furin inhibitor Hexa-D-arginine reduced the proliferation activity of fibroblasts by reducing the production of P61-Sema3E.

[0066] 4. Transwell method to detect cell migration ability The method is as described above.

[0067] like Figure 6 As shown in D, the number of purple cells in fibroblasts stimulated with TGF-β1 was significantly reduced compared with fibroblasts stimulated with TGF-β1 alone, indicating that the number of migrating cells was significantly reduced, suggesting that the furin inhibitor hexa-D-arginine reduced the migration ability of fibroblasts by reducing the production of P61-Sema3E.

[0068] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. Use of furin inhibitors in the preparation of drugs for preventing, delaying or treating idiopathic pulmonary fibrosis.

2. The application according to claim 1, characterized in that: The Furin inhibitor achieves the prevention, delay or treatment of idiopathic pulmonary fibrosis by reducing the production of p61-Sema3E.

3. The application according to claim 1, characterized in that: The furin inhibitor is a D-arginine hexamer.

4. The application according to claim 1, characterized in that: The furin inhibitor inhibits the conversion of P87-Sema3E to P61-Sema3E and reduces the production of P61-Sema3E.

5. The application according to claim 1, characterized in that: The furin inhibitor prevents, delays or treats idiopathic pulmonary fibrosis by inhibiting the activation, proliferation and migration of human primary lung fibroblasts.

6. The application according to claim 5, characterized in that: The furin inhibitor inhibits the enzymatic activity of furin, reduces the conversion of P87-Sema3E to P61-Sema3E, and reduces the production of P61-Sema3E, thereby inhibiting the activation, proliferation and migration of human primary lung fibroblasts.

7. The use according to claim 1, characterized in that: The drug also includes pharmaceutically acceptable excipients.

8. The application according to claim 1, characterized in that: The auxiliary materials include fillers, diluents, binders, disintegrants and emulsifiers.

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