Application of Sema3E inhibitor in preparation of medicine for preventing, delaying or treating COPD small airway fibrosis

Abstract

The invention belongs to the technical field of biological medicines, discloses an application of a Sema3E inhibitor in preparation of a medicine for preventing, delaying or treating COPD small airway fibrosis, and proposes that the Sema3E inhibitor can be used for treating, preventing or delaying the COPD small airway fibrosis for the first time. The Sema3E inhibitor plays a role by inhibiting the expression of Sema3E. The specific molecular mechanism is as follows: by inhibiting the expression of Sema3E, inhibiting the expression of Wnt5a / b, beta-Catenin, increasing the expression of p-beta-Catenin, inhibiting the activation of a Wnt / beta-Catenin pathway, and inhibiting the activation / proliferation / migration of small airway fibroblasts. Through the mechanism, the effect of preventing, delaying or treating the COPD is achieved, and a new drug intervention target, namely Sema3E, is provided for treatment of the COPD.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of Sema3E inhibitors in the preparation of drugs for the prevention, delay or treatment of small airway fibrosis in COPD. Background Technology

[0002] Chronic obstructive pulmonary disease (COPD) is a common, preventable, and treatable disease characterized by persistent respiratory symptoms and airflow limitation. Its pathogenesis is associated with an enhanced chronic inflammatory response of the airways and lungs to toxic particles or gases. According to the World Health Organization (WHO), COPD is the third leading cause of death globally, affecting hundreds of millions of people worldwide and imposing a heavy socioeconomic burden.

[0003] The pathological changes in COPD are mainly concentrated in the small airways (membranous bronchioles with a diameter <2 mm) and lung parenchyma. In recent years, numerous studies have confirmed that small airway fibrosis is one of the core pathological mechanisms leading to airflow limitation and progressive disease progression in COPD patients. In the early stages of the disease, long-term exposure to smoke and inflammation activates epithelial cells, fibroblasts, and immune cells, leading to the massive release of various pro-fibrotic mediators (such as TGF-β, PDGF, CTGF, etc.). These mediators drive the transformation of small airway fibroblasts into myofibroblasts, and their abnormal proliferation and activation, thereby synthesizing and depositing excessive amounts of extracellular matrix (ECM), such as collagen and fibronectin, resulting in thickening of the small airway walls, narrowing of the lumen, loss of elasticity, and ultimately irreversible airflow limitation.

[0004] Currently, the standard clinical treatment for COPD mainly includes bronchodilators (such as β2 receptor agonists and anticholinergic drugs) and inhaled corticosteroids (ICS), aiming to relieve symptoms, reduce acute exacerbations, and improve quality of life. However, existing therapies primarily focus on anti-inflammation and relieving bronchospasm, and are almost ineffective in reversing or halting small airway fibrosis, the core structural lesion. Once the fibrotic process begins, it is characterized by self-sustaining and progressive development, leading to an irreversible decline in lung function.

[0005] Therefore, developing novel targeted therapies that can specifically intervene in fibroblast activation, inhibit excessive ECM deposition, or promote the degradation of fibrotic tissue, particularly in the critical aspect of small airway fibrosis in COPD, has become an urgent and unmet clinical need in the current COPD treatment field. This invention is proposed against this backdrop, aiming to provide a novel technical solution to address the aforementioned problems.

[0006] The semaphorin family consists of secreted or membrane-bound signaling proteins that regulate cell migration, proliferation, and differentiation. Recent studies have found that some semaphorin members participate in organ fibrosis by regulating immune cell infiltration or fibroblast transformation; Sema3E is a member of this family. Our study found that COPD patients, compared to healthy individuals, showed significantly increased expression of Sema3E and its receptor Plexin D1 in lung tissue and plasma, with Sema3E primarily existing in the P61-Sema3E form. In vitro, Sema3E significantly enhanced cell activation, proliferation, and migration by activating the Wnt / β-Catenin pathway in small airway fibroblasts. Finally, in vivo experiments revealed that Sema3E fibroblast-specific knockout mice, after being exposed to smoke, showed significantly improved small airway fibrosis compared to control smoke-exposed mice. These findings highlight the crucial role of Sema3E in the pathogenesis of COPD and suggest that inhibiting Sema3E may offer hope for developing new strategies for COPD treatment. Summary of the Invention

[0007] The purpose of this invention is to provide the application of Sema3E inhibitors in the preparation of drugs for the prevention, delay, or treatment of small airway fibrosis in COPD. This invention is the first to propose that Sema3E inhibitors can be used for the treatment, prevention, or delay of small airway fibrosis in COPD. Sema3E inhibitors exert their effects by inhibiting the expression of Sema3E. The specific molecular mechanism is as follows: by inhibiting the expression of Sema3E → inhibiting the expression of Wnt5a / b, β-Catenin, and increasing the expression of p-β-Catenin → inhibiting the activation of the Wnt / β-Catenin pathway → inhibiting the activation / proliferation / migration of small airway fibroblasts. Through the above mechanism, the preventive, delaying, or therapeutic effects on COPD are achieved, providing a new drug intervention target for COPD treatment—Sema3E.

[0008] To achieve the above objectives, this application adopts the following technical solution: In a first aspect, the present invention provides the use of Sema3E inhibitors in the preparation of medicaments for the prevention, delay or treatment of small airway fibrosis in COPD.

[0009] In the above technical solution, the Sema3E inhibitor inhibits the activation, proliferation and migration of small airway fibroblasts by inhibiting the Wnt / β-Catenin pathway in small airway fibroblasts, thereby inhibiting the process of small airway fibrosis.

[0010] In the above technical solutions, the Sema3E inhibitor uses gene knockout, gene knockdown, or chemical drugs to reduce the expression of the Sema3E gene.

[0011] In the above technical solutions, the Sema3E inhibitor includes a regulator that reduces Sema3E expression.

[0012] In the above technical solutions, the modulator includes a reagent that knocks out or silences Sema3E.

[0013] In the above technical solutions, the reagents include siRNA, shRNA, or miRNA.

[0014] In the above technical solutions, the COPD is COPD induced by cigarette smoke or cigarette smoke extracts.

[0015] Secondly, this invention provides the application of Sema3E inhibitors in the preparation of products that inhibit the Wnt / β-Catenin pathway.

[0016] Thirdly, this invention provides the application of a reagent for detecting Sema3E expression levels in the preparation of products for diagnosing COPD small airway fibrosis. If the Sema3E expression level is detected to be higher than that of healthy individuals, the patient is diagnosed with COPD small airway fibrosis.

[0017] Fourthly, the present invention provides a kit for diagnosing small airway fibrosis in COPD, including reagents for detecting Sema3E expression levels.

[0018] The beneficial effects of this invention are as follows: This invention has determined that Sema3E can enhance the activation, proliferation and migration of small airway fibroblasts by activating the Wnt / β-Catenin pathway, thereby promoting the process of small airway fibrosis and thus providing a new treatment strategy for COPD.

[0019] This invention analyzes the content of Sema3E in different populations and mice, and determines that the expression of Sema3E is significantly increased in the lung tissue of COPD patients and COPD model mice. By adding Sema3E to human primary small airway fibroblasts, it was found that Sema3E promotes the expression of Wnt5a / b and β-Catenin, inhibits the expression of p-β-Catenin, and activates the Wnt / β-Catenin pathway, thereby enhancing the activation, proliferation and migration of small airway fibroblasts, and thus promoting the process of small airway fibrosis.

[0020] This invention, by specifically knocking out Smea3E in mouse fibroblasts, inhibits the expression of Sema3E in mouse small airway fibroblasts, reduces the activation of small airway fibroblasts in COPD, successfully improves the pathological state of small airway fibrosis in COPD, and further confirms the key role of Sema3E in the occurrence and development of COPD. Attached Figure Description

[0021] Figure 1 This is a map showing the basic expression of Sema3E in plasma and lung tissue of non-smokers (NS), healthy smokers (HS), and COPD patients; where: Figure 1 A represents the plasma Sema3E levels (ng / ml) of non-smokers (NS) (n=30), healthy smokers (HS) (n=30), and people with COPD (n=30). Figure 1 B represents the protein levels of Sema3E and its receptor PlexinD1 in lung tissue of non-smokers (NS) (n=10), healthy smokers (HS) (n=10), and people with COPD (n=12). Figure 1 C is Figure 1 Statistical chart of grayscale value analysis of corresponding band B; Figure 1 D represents the immunofluorescence staining of Sema3E on lung tissue sections from non-smokers (NS) and COPD patients. *P<0.05, **P<0.01, ***P<0.001.

[0022] Figure 2 This diagram illustrates the basal expression of Sema3E in the lung tissues of air-exposed mice, smoke-exposed mice, and human primary small airway fibroblasts stimulated with different concentrations of cigarette smoke extract (CSE); where: Figure 2 A represents the protein levels of Sema3E and its receptor PlexinD1 in the lung tissue of air-exposed mice (n=10) and smoke-exposed mice (n=10). Figure 2 B is Figure 2 A. Statistical chart of grayscale value analysis of the corresponding stripes; Figure 2 C represents the protein levels of Sema3E and its receptor PlexinD1 in human primary small airway fibroblasts stimulated by different concentrations of cigarette smoke extract (CSE). Figure 2 D is Figure 2 Statistical analysis of grayscale values ​​of the corresponding bands in C. Where *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0023] Figure 3The graph shows the effects of adding different concentrations of recombinant Sema3E protein to human primary small airway fibroblasts for 48 hours on the expression of fibrosis markers, proliferation capacity, and migration capacity; among which: Figure 3 A. The protein expression of fibrosis markers Fibronectin, Col1a1, and α-SMA was measured after adding different concentrations (0, 1 ng / ml, 10 ng / ml, 100 ng / ml) of recombinant Sema3E protein to primary human small airway fibroblasts for 48 h. Figure 3 B is Figure 3 Statistical chart of grayscale value analysis of the corresponding bands of A; Figure 3 C is a graph showing the migration ability of primary human small airway fibroblasts after adding different concentrations (0, 1 ng / ml, 10 ng / ml, 100 ng / ml) of Sema3E recombinant protein for 48 h. Figure 3 D is a graph showing the detection of cell proliferation activity of primary human small airway fibroblasts after adding different concentrations (0, 1 ng / ml, 10 ng / ml, 100 ng / ml) of Sema3E recombinant protein for 48 h; where *P<0.05, **P<0.01, ***P<0.001.

[0024] Figure 4 The graph shows the expression of fibrosis markers, proliferation, and migration ability of primary human small airway fibroblasts after Sema3E knockdown followed by CSE stimulation for 48 hours; among which: Figure 4 A. Knockdown of Sema3E in primary human small airway fibroblasts followed by stimulation with CSE for 48 h resulted in the expression of fibrosis markers Fibronectin, Col1a1, and α-SMA. Figure 4 B is Figure 4 Statistical chart of grayscale value analysis of the corresponding bands of A; Figure 4 C is a graph showing the cell migration ability of primary human small airway fibroblasts after Sema3E knockdown followed by CSE stimulation for 48 hours. Figure 4 D is a graph showing the cell proliferation activity of primary human small airway fibroblasts after Sema3E knockdown followed by CSE stimulation for 48 hours; where *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0025] Figure 5 The graph shows the detection of Wnt / β-Catenin pathway markers in primary human small airway fibroblasts after adding different concentrations of recombinant Sema3E protein for 48 hours; where: Figure 5A. The protein expression of Wnt / β-Catenin pathway markers Wnt5a / b, β-Catenin, and p-β-Catenin was evaluated after adding different concentrations (0, 1 ng / ml, 10 ng / ml, 100 ng / ml) of Sema3E recombinant protein to primary human small airway fibroblasts for 48 h. Figure 5 B is Figure 5 Statistical chart of grayscale value analysis of the corresponding bands of A.

[0026] Figure 6 The graph shows the detection of Wnt / β-Catenin pathway markers and cell fibrosis markers after 48 hours of stimulation of human primary small airway fibroblasts with the Wnt / β-Catenin pathway inhibitor LGK974; among which: Figure 6 A. After stimulating primary human small airway fibroblasts with the Wnt / β-Catenin pathway inhibitor LGK974 for 48 h, the protein expression of Wnt / β-Catenin pathway markers Wnt5a / b, β-Catenin, p-β-Catenin, and fibrosis markers Fibronectin, Col1a1, and α-SMA was observed. Figure 6 B is Figure 6 Statistical chart of grayscale value analysis of the corresponding bands of A.

[0027] Figure 7 The images show pathological findings of small airway fibrosis in Sema3E small airway fibroblast-specific knockout mice after smoke exposure, and the detection of fibrosis markers by lung tissue proteins; among which: Figure 7 A is an HE staining image of lung tissue sections from Sema3E small airway fibroblast-specific knockout mice after exposure to smoke. Figure 7 B is a Masson staining image of lung tissue sections from Sema3E small airway fibroblast-specific knockout mice after exposure to smoke. Figure 7 C is a Sirius red staining image of lung tissue sections from Sema3E small airway fibroblast-specific knockout mice after exposure to smoke. Figure 7 D is a graph showing the protein expression of fibrosis markers Fibronectin, Col1a1, and α-SMA in lung tissue of small airway fibroblast-specific knockout mice after exposure to smoke. Figure 7 E is Figure 7 Statistical chart of grayscale value analysis of the corresponding bands of D. Detailed Implementation

[0028] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below in conjunction with specific embodiments. This invention can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the inventive concept to those skilled in the art. This invention will be defined only by the claims.

[0029] Example 1: Determining the differential expression of Sema3E in plasma and lung tissue of non-smokers (NS), healthy smokers (HS), and COPD patients. Human trials: Volunteers were recruited from non-smokers (NS), healthy smokers (HS), and people with COPD. Plasma and lung tissue were collected from the subjects, and the expression of Sema3E was detected.

[0030] 1. ELISA Immobilize the specific Sema3E antibody in the wells of an ELISA plate. This is done 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. Incubation is typically performed at room temperature for 1–2 hours. Wash the plate with washing buffer to remove unbound blocking buffer. Add the plasma sample to be tested and a series of standards at known concentrations to the ELISA plate. Incubate at 37°C for 1–2 hours to allow the antigen in the sample to bind to the coating antibody. Wash the plate again with washing buffer, add the specific primary antibody, and incubate at 37°C for 1–2 hours. Wash the plate again with washing buffer, add the secondary antibody conjugated with HRP, incubate at 37°C for 1 hour, wash again, add a substrate that can be catalyzed by HRP to produce a color change, and after a certain time, add stop solution to stop the reaction and stabilize the color change. Finally, read the results using a microplate reader.

[0031] like Figure 1 As shown in Figure A, the average level of Sema3E in the plasma of non-smokers (NS) is around 2 ng / ml, the average level of Sema3E in the plasma of healthy smokers (HS) is around 2.5 ng / ml, while the average level of Sema3E in the plasma of COPD patients is around 3.5 ng / ml, indicating that the level of Sema3E in the plasma of COPD patients is significantly elevated.

[0032] 2. Lung tissue proteins were extracted and the expression of Sema3E and its receptor PlexinD1 was detected by Western blot.

[0033] Take an appropriate amount of tissue and place it in a 2 ml EP tube. Add 300 μl of RIPA protein lysis buffer to submerge the tissue, add 3 grinding beads, and grind the tissue using a homogenizer (speed 3) for 20-30 seconds until pulverized. Transfer to a 1.5 ml EP tube, then centrifuge at 12000 rpm for 15 minutes at 4°C. Collect the supernatant as total protein. Separate the proteins using a 10% SDS-PAGE gel, then transfer to a PVDF membrane and block in 5% milk for 1-2 hours. Subsequently, incubate overnight with Sema3E primary antibody at 4°C, wash three times with TBST, incubate with secondary antibody for 1 hour, and finally expose with chemiluminescent solution.

[0034] like Figure 1 As shown in BC, the protein levels of Sema3E and its receptor PlexinD1 in the lung tissue of non-smokers (NS) and healthy smokers (HS) were significantly lower than those in COPD patients.

[0035] 3. Immunofluorescence staining of lung tissue sections to detect Sema3E expression. Fresh human lung tissue was removed and fixed with 4% paraformaldehyde for 48 hours, 70% ethanol for 1.5 hours once, 80% ethanol for 30 minutes once, 95% ethanol for 15 minutes twice, anhydrous ethanol for 10 minutes three times, xylene clearing twice for 15 minutes each time, paraffin I for 30 minutes, paraffin II for 90 minutes, and paraffin III for 6 hours. After embedding, it was sectioned.

[0036] Immerse the slide in xylene three times, 5-10 minutes each time. Then immerse the slide sequentially in a gradient of alcohols: 100% ethanol I: 3-5 minutes, 100% ethanol II: 3-5 minutes, 95% ethanol: 3-5 minutes, 70% ethanol: 3-5 minutes, and distilled water. PBS or 5 minutes. Place the slide in a heat-resistant container containing antigen retrieval buffer. Place the container in a microwave oven and heat on high until boiling. Then reduce to medium-low heat and maintain a gentle boil for 10-20 minutes (be careful to prevent drying). Remove and cool to room temperature for 20-30 minutes. After antigen retrieval, rinse the slide three times with PBS (pH 7.4), 5 minutes each time. Immerse the slide in PBS containing 0.1-0.5% Triton X-100 or Tween 20 and incubate at room temperature for 5-15 minutes. Rinse three times with PBS, 5 minutes each time. Add sufficient blocking solution to the slide to ensure complete coverage. Incubate at room temperature for 30-60 minutes. Add sufficient primary antibody working solution to the slide and incubate overnight at 4°C. After warming up the next day, add sufficient fluorescently labeled secondary antibody working solution to the slide and incubate at room temperature in the dark for 1-2 hours. Under dark conditions, rinse the slide three times with PBS or TBST, 5 minutes each time. Finally, after reigniting the cell nuclei with DAPI dye for 10 minutes, add 1-2 drops of anti-fluorescence quenching mounting medium to the tissue, mount and photograph.

[0037] like Figure 1 As shown in Figure D, the red area represents FSP1 used to label small airway fibroblasts, the green area represents Sema3E, and the blue area represents nuclear DAPI. The red-green fluorescence intensity of lung tissue sections from COPD patients is significantly higher than that from non-smokers (NS), indicating a significant increase in Sema3E expression in the lung tissue of COPD patients. Furthermore, the red-green overlap (indicated by the white arrow) in the lung tissue sections from COPD patients is significantly greater than that in non-smokers (NS), indicating a significant increase in Sema3E expression in small airway fibroblasts in COPD patients.

[0038] Example 2: Determining the differential expression of Sema3E in lung tissues of normal mice and smoke-exposed COPD model mice 1. Constructing a mouse COPD model All mice (8-12 weeks old, male) were housed in SPF-grade animal isolation cages at Tongji Hospital, with a 12-hour light-dark cycle. They were exposed to indoor air or smoke for approximately 3 hours daily (12 cigarettes burned for 45 minutes each time, 4 times a day), 5 days a week, for 3 months, using Hongjinlong cigarettes.

[0039] 2. Proteins were extracted from mouse lung tissue, and the expression of Sema3E and its receptor PlexinD1 was detected by Western blot. The method is as described above.

[0040] like Figure 2As shown in AB, the expression of Sema3E and PlexinD1 in the lung tissue of COPD model mice was significantly higher than that in normal mice, indicating that the expression of Sema3E and its receptor PlexinD1 in the lung tissue of COPD model mice was significantly increased.

[0041] 3. Extraction and culture of primary human small airway fibroblasts Small airway fibroblasts were isolated from the same peripheral lung tissue using an explant culture method. The simplified procedure is as follows: After rinsing fresh human lung tissue with Hanks' Balanced Salt Solution (HBSS), visible small airways (<2 mm in diameter) and surrounding lung parenchyma were isolated from the same peripheral lung tissue using forceps and scissors. Three to five 0.5 cm² airway aliquots (taken from multiple lung tissue regions) were transferred to 10 cm culture dishes, and 3 mL of complete DMEM medium (containing DMEM from Sigma-Aldrich UK, 10% fetal bovine serum from BioSera UK, 1% glutamine from Sigma-Aldrich UK, 1% penicillin / streptomycin from Sigma-Aldrich UK, and 1% amphotericin B from Gibco UK) were added. Fibroblast culture was performed at 37°C and 5% CO2. The explant culture was continued for 2 weeks (medium changed every 4 days) to promote fibroblast growth, after which the tissue was removed and the cells were passaged. These cells exhibited typical fibroblast morphology, were positive for vimentin staining but negative for pancytokeratin staining, and all experiments used cells from passage 2 to 5.

[0042] 4. Total cellular protein was extracted and the expression of Sema3E and its receptor PlexinD1 was detected by Western blot. Cells were seeded in 12-well plates and exposed to different concentrations of CSE (cigarette smoke extract) for 48 h. Total cellular protein was extracted as described above and subjected to Western blot.

[0043] like Figure 2 As shown in CD, small airway fibroblasts stimulated with 5% CSE and 10% CSE for 48 h showed significantly higher expression of Sema3E and PlexinD1 than those stimulated with 0% CSE, indicating that CSE stimulation of small airway fibroblasts increases the expression of Sema3E and its receptor PlexinD1.

[0044] Example 3: Determining the pro-fibrotic effect of Sema3E on human primary small airway fibroblasts 1. Western blot detection of fibrosis markers The method is as described above.

[0045] like Figure 3As shown in Figures AB, after adding different concentrations (0, 1 ng / ml, 10 ng / ml, 100 ng / ml) of Sema3E recombinant protein to human primary small airway fibroblasts for 48 h, the protein expression of fibrosis markers Fibronectin, Col1a1, and α-SMA was detected. It was found that with the increase of Sema3E recombinant protein concentration, the protein expression of fibrosis markers Fibronectin, Col1a1, and α-SMA also gradually increased, and the expression was most significant at 10 ng / ml, indicating the pro-fibrotic ability of Sema3E recombinant protein.

[0046] 2. Transwell method for detecting cell migration ability Digest the cells, resuspend them in serum-free medium, and adjust the density to 1-5×. Cells / mL: Place the Transwell chambers into 24-well plates. Add 100 μL of cell suspension to the upper chamber and 600 μL of culture medium containing 10% FBS to the lower chamber. After 2 hours, add different concentrations (0, 1 ng / ml, 10 ng / ml, 100 ng / ml) of Sema3E recombinant protein to the upper chamber. After 48 hours, remove the chambers, gently wash the surface of unmigrated cells with PBS, and gently wipe the surface of the upper chamber membrane with a moistened cotton swab (do not apply force). Fix with 4% PFA at room temperature for 15 minutes, stain with 0.1% crystal violet for 20 minutes, air dry, and observe and photograph under a microscope.

[0047] like Figure 3 As shown in Figure C, purple cells represent cells that migrate from the upper chamber to the lower chamber. As the concentration of Sema3E recombinant protein increases, the number of migrating cells also gradually increases, and the migration ability is most significant at 10 ng / ml, indicating that Sema3E recombinant protein promotes the migration ability of fibroblasts.

[0048] 3. Edu staining method for detecting cell proliferation activity Human primary small airway fibroblasts were seeded into 96-well plates. After adhesion, different concentrations (0, 1 ng / ml, 10 ng / ml, 100 ng / ml) of recombinant Sema3E protein were added for stimulation for 48 h. Culture medium containing EdU working solution was added, and the cells were incubated for 2 h. The culture medium was aspirated, and the cells were gently washed twice with pre-cooled PBS. The cells were fixed with 4% paraformaldehyde (PFA) at room temperature for 15 min, followed by washing with PBS 3 × 5 min. Finally, the cell nuclei were counterstained with DAPI.

[0049] like Figure 3As shown in Figure D, red light represents proliferating cells, indicating their proliferative activity, while blue light represents the cell nucleus. The number of cells emitting red fluorescence increases with increasing Sema3E recombinant protein concentration, reaching its peak at 10 ng / ml, indicating that Sema3E recombinant protein promotes fibroblast proliferation.

[0050] Example 4: Determining that Sema3E knockdown has a protective effect against CSE-induced fibrosis in human primary small airway fibroblasts. 1. Transfection of Sema3E small interfering RNA Sema3E small interfering RNA (PlexinD1 siRNA) was purchased from Sangon Biotech Ltd. Human primary small airway fibroblasts were seeded in 12-well plates. Once the cell density reached approximately 40%, transfection was performed. The solutions for each well were: Solution A: 2 μL siRNA + 50 μL Opti-MEM®; Solution B: Lipo3000 + 50 μL Opti-MEM®. Solutions A and B were prepared separately and incubated at room temperature for 5 minutes. Solutions A and B were then 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 to distribute it evenly. The medium was replaced with complete culture medium 6-8 hours after transfection.

[0051] 2. Western blot detection of fibrosis markers The method is as described above.

[0052] like Figure 4 As shown in AB, human primary small airway fibroblasts transfected with Sema3E siRNA showed significantly reduced protein expression of fibrosis markers Fibronectin, Col1a1, and α-SMA after 48 h of CSE stimulation compared to the CSE stimulation group alone, indicating that Sema3E knockdown has a protective effect against CSE-induced fibrosis in human primary small airway fibroblasts.

[0053] 3. Transwell method for detecting cell migration ability The method is as described above.

[0054] like Figure 4 As shown in Figure C, the number of human primary small airway fibroblasts transfected with Sema3E siRNA that migrated after 48 hours of CSE stimulation was significantly lower than that of the CSE stimulation group alone, indicating that Sema3E knockdown has a protective effect on the migration ability of human primary small airway fibroblasts induced by CSE stimulation.

[0055] 4. Edu staining method for detecting cell proliferation activity The method is as described above.

[0056] like Figure 4 As shown in Figure D, the number of human primary small airway fibroblasts transfected with Sema3E siRNA emitting red fluorescence 48 h after CSE stimulation was significantly lower than that of the CSE stimulation group alone, indicating that Sema3E knockdown has a protective effect on the proliferation capacity of human primary small airway fibroblasts induced by CSE stimulation.

[0057] Additionally, it should be noted that, besides the Sema3E siRNA used in this example, other Sema3E inhibitors can achieve the same effect, including but not limited to gene knockout, gene knockdown, or chemical drugs to reduce Sema3E gene expression. Reagents for knocking out or silencing Sema3E include siRNA, shRNA, or miRNA.

[0058] Example 5: Determining the activation effect of Sema3E on the Wnt / β-Catenin pathway 1. Western blot detection of Wnt / β-Catenin pathway markers The method is as described above.

[0059] like Figure 5 As shown in Figures AB, after adding different concentrations (0, 1 ng / ml, 10 ng / ml, 100 ng / ml) of recombinant Sema3E protein to human primary small airway fibroblasts for 48 h, the protein expression of Wnt5a / b and β-Catenin, indicators of the Wnt / β-Catenin pathway, was significantly increased, while the protein expression of p-β-Catenin was significantly decreased, with the most significant increase observed at 10 ng / ml. This indicates that Sema3E activates the Wnt / β-Catenin pathway.

[0060] Example 6: Determining the pro-fibrotic effect of Sema3E on human primary small airway fibroblasts through the Wnt / β-Catenin pathway. 1. Western blot analysis of the expression of Wnt / β-Catenin pathway markers and fibrosis markers. The method is as described above.

[0061] like Figure 6As shown in Figures AB, stimulation of human primary small airway fibroblasts with 1 nM of the Wnt pathway inhibitor LGK974 for 48 h significantly reduced the protein expression of Wnt5a / b and β-Catenin compared to the group stimulated with Sema3E recombinant protein alone, and also significantly reduced the protein expression of p-β-Catenin, indicating that LGK974 successfully inhibited the activation of the Wnt / β-Catenin pathway. Subsequently, stimulation of human primary small airway fibroblasts with 1 nM of the Wnt pathway inhibitor LGK974 for 48 h significantly reduced the protein expression of fibrosis markers Fibronectin, Col1a1, and α-SMA compared to the group stimulated with Sema3E recombinant protein alone, indicating that Sema3E activates the pro-fibrotic effect of human primary small airway fibroblasts through the Wnt / β-Catenin pathway.

[0062] Example 7: In vivo experiments confirmed that Sema3E knockout has a protective effect against small airway fibrosis in COPD. 1. Construction of Sema3E fibroblast-specific knockout mice Background is C57BL / 6 The mice were constructed using CRISPR-Cas9 technology by Jicui Pharmaceutical (Jiangsu, China), with loxP sites inserted flanking exons 6-9. Col1a2-iCre transgenic mice were purchased from Cyagen Biosciences (Jiangsu, China). Mice obtained Col1a2-iCre+ after mating Mice (referred to as the Sema3E-CKO group, i.e. Sema3E fibroblast-specific knockout mice) were modeled by fumigation as described above.

[0063] 2. HE staining of lung tissue sections Fresh mouse lung tissue was extracted and fixed with 4% paraformaldehyde for 48 hours, followed by fixation with 70% ethanol for 1.5 hours, 80% ethanol for 30 minutes, 95% ethanol for 15 minutes twice, anhydrous ethanol for 10 minutes three times, clearing twice with xylene for 15 minutes each time, infiltration with paraffin I for 30 minutes, paraffin II for 90 minutes, and paraffin III for 6 hours. After embedding, the tissue was sectioned and baked at 55-60°C for 1 hour. Subsequently, it was dewaxed to water, stained with hematoxylin and eosin, differentiated with hydrochloric acid and alcohol, dehydrated, and mounted.

[0064] like Figure 7 As shown in Figure A, the number of spindle-shaped fibroblasts around the small airways in the control group mice was significantly increased compared to the control group mice, while the number of spindle-shaped fibroblasts around the small airways in the Sema3E-CKO group mice was significantly decreased compared to the control group mice. This indicates that Sema3E knockout in vivo has a protective effect against small airway fibrosis in COPD.

[0065] 3. Masson staining of lung tissue sections Mouse lung tissue sections were dewaxed to water, and the cell nuclei were stained with hematoxylin for 5-10 minutes. The sections were then rinsed with running water for 10-15 minutes until the cell nuclei turned blue. Subsequently, the sections were stained with Masson's Ponceau S Acid Fuchsin for 5-10 minutes. The staining solution was discarded, and the sections were directly differentiated with 1% phosphomolybdic acid aqueous solution for 3-5 minutes. Without rinsing with water, the sections were directly counterstained with aniline blue solution for 3-5 minutes. After differentiation with hydrochloric acid and alcohol, the sections were dehydrated and mounted.

[0066] like Figure 7 As shown in Figure B, the number of blue collagen fibers around the small airways in the control group mice was significantly increased compared to the control group mice, while the number of collagen fibers around the small airways in the Sema3E-CKO group mice was significantly decreased compared to the control group mice. This indicates that Sema3E knockout in vivo has a protective effect against small airway fibrosis in COPD.

[0067] 4. Staining lung tissue sections with Sirius red. Mouse lung tissue sections were dewaxed to water, and the cell nuclei were stained with hematoxylin for 5 minutes. The sections were then rinsed with running water for 10-15 minutes until the cell nuclei turned blue. The sections were then immersed in Sirius red staining solution and stained at room temperature for 60 minutes. After differentiation and dehydration, the sections were mounted.

[0068] like Figure 7 As shown in Figure C, the number of reddish collagen fibers around the small airways in the control group mice was significantly increased compared to the control group mice, while the number of collagen fibers around the small airways in the Sema3E-CKO group mice was significantly decreased compared to the control group mice. This indicates that Sema3E knockout in vivo has a protective effect against small airway fibrosis in COPD.

[0069] 5. Western blot detection of fibrosis markers The method is as described above.

[0070] like Figure 7 As shown in the DE, the expression of fibrosis markers Fibronectin, Col1a1, and α-SMA in the lung tissue of mice in the control group was significantly increased compared to the control group, while the expression of Fibronectin, Col1a1, and α-SMA in the lung tissue of mice in the Sema3E-CKO group was significantly decreased compared to the control group. This indicates that Sema3E knockout in vivo has a protective effect against lung fibrosis in COPD mice.

[0071] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. Application of Sema3E inhibitors in the preparation of drugs for the prevention, delay or treatment of small airway fibrosis in COPD.

2. The application according to claim 1, characterized in that: The Sema3E inhibitor inhibits the activation, proliferation, and migration of small airway fibroblasts by suppressing the Wnt / β-Catenin pathway within them, thereby inhibiting the process of small airway fibrosis.

3. The application according to claim 1, characterized in that: The Sema3E inhibitor uses gene knockout, gene knockdown, or chemical drugs to reduce the expression of the Sema3E gene.

4. The application according to claim 1, characterized in that: The Sema3E inhibitors include regulators that reduce Sema3E expression.

5. The application according to claim 4, characterized in that: The modulators include reagents that knock out or silence Sema3E.

6. The application according to claim 5, characterized in that: The reagents include siRNA, shRNA, or miRNA.

7. The application according to claim 1, characterized in that: The COPD mentioned is COPD induced by cigarette smoke or cigarette smoke extracts.

8. Application of Sema3E inhibitors in the preparation of products that inhibit the Wnt / β-Catenin pathway.

9. The application of a reagent for detecting Sema3E expression levels in the preparation of products for diagnosing small airway fibrosis in COPD, characterized in that: If the Sema3E expression level is detected to be higher than that of healthy individuals, the patient is diagnosed with COPD small airway fibrosis.

10. A kit for diagnosing small airway fibrosis in COPD, characterized in that: This includes reagents for detecting Sema3E expression levels.

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