Application of BPS in treatment of retinal neovascularization related diseases

Abstract

The invention provides application of BPS in treatment of retinal neovascularization related diseases, and relates to the field of biological medicine. The BPS has a remarkable inhibition effect on pathological endothelial cell migration and angiogenesis in a macrophage and human retina microvascular endothelial cell co-culture model, has a good application prospect in preparation of drugs for treating diseases related to retinal neovascularization, and provides a research tool for research on the diseases related to retinal neovascularization.

Description

TECHNICAL FIELD

[0001] The present application relates to the application of BPS in treating retinal neovascularization related diseases, and belongs to the field of biological medicine. BACKGROUND

[0002] Pathological neovascularization is the core mechanism of irreversible visual function damage caused by ischemic retinopathy (such as diabetic retinopathy, retinal vein occlusion, etc.). Its occurrence is closely related to the cascade release of pro-angiogenic factors (such as VEGF) and inflammatory factors induced by retinal ischemia and hypoxia.

[0003] Recent studies have shown that retinal pathological angiogenesis is a complex process of interaction between angiogenesis and inflammatory response. Myeloid cells (especially pro-inflammatory M1 macrophages) can activate NF-κB signaling pathway by secreting pro-inflammatory factors such as TNF-α and IL-1β and directly interacting with endothelial cells, and drive vascular endothelial cell migration and abnormal angiogenesis. The existing anti-angiogenic therapy combined with immunomodulation strategy is not clear, and there is no anti-retinal neovascularization drug with macrophage function as the core target in clinic. SUMMARY

[0004] In view of the above problems, the present application based on the co-culture model of macrophages and human retinal microvascular endothelial cells, found that specific intervention methods can significantly inhibit pathological endothelial cell migration and angiogenesis activity. Specifically, the application of BPS in treating retinal neovascularization related diseases is provided.

[0005] To achieve the above purpose, the specific technical solutions of the present application are as follows: The first aspect of the present application provides the application of BPS in preparing a drug for treating retinal neovascularization related diseases.

[0006] In the present application, BPS refers to bisphenol S (Bisphenol S), the chemical name is 4,4'-sulfonyl bisphenol (4,4'-sulfonyldiphenol), the molecular formula is C 12 H 10 O4S. Its structural characteristics are that two benzene rings are connected by sulfonyl (-SO2-), each benzene ring contains one hydroxyl group (-OH), forming a symmetrical structure. Its physical property is white crystalline powder, soluble in organic solvents (such as acetone, ethanol), slightly soluble in water, resistant to high temperature and high chemical stability.

[0007] Further, the BPS treats retinal neovascularization related diseases by at least one of the following ways: 1) inhibiting the formation of pathological neovascularization; 2) inhibiting the migration of pathological neovascularization.

[0008] In some embodiments, the dosage form of the drug is any one of injection, tablet, pill, powder, suspension, gel, emulsion, granule, capsule, and spray.

[0009] In some embodiments, the medicament further includes a pharmaceutically acceptable carrier.

[0010] In this invention, the pharmaceutically acceptable carrier includes, but is not limited to, any adjuvant, carrier, excipient, gliding agent, sweetener, diluent, preservative, dye / coloring agent, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, surfactant, or emulsifier that has been approved by the U.S. Food and Drug Administration or the China National Medical Products Administration for use in humans or livestock. The carrier should be biologically acceptable, meaning it is compatible with other components in the formulation and harmless to the patient, and will not elicit an adverse reaction (e.g., immune response) when administered to the host.

[0011] In some embodiments, the pharmaceutically acceptable carrier is selected from one or more of diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH adjusters, antioxidants, or antibacterial agents.

[0012] In some embodiments, the retinal neovascularization-related diseases are selected from at least one of neovascular glaucoma, ischemic retinopathy, ischemic choroidal disease and secondary retinopathy, proliferative diabetic retinopathy, retinopathy of prematurity, and age-related macular degeneration.

[0013] In some embodiments, the ischemic retinopathy is selected from at least one of retinal vein occlusion, central retinal artery occlusion, branch retinal artery occlusion, ciliary retinal artery occlusion, distal retinopathy, Purtscher-like retinopathy, retinal periphlebitis, ocular ischemia syndrome, aortitis fundus lesions, radiation retinopathy, and familial exudative vitreoretinopathy.

[0014] In some embodiments, the ischemic choroidal lesion is selected from at least one of polypoid choroidal vascular lesions, high myopia choroidal neovascularization, and idiopathic choroidal neovascularization.

[0015] In some embodiments, the secondary retinal lesion is selected from proliferative vitreoretinopathy, vitreous hemorrhage, fibrosis, and tractional retinal detachment.

[0016] A second aspect of the present invention provides a pharmaceutical composition for treating retinal neovascularization-related diseases, the pharmaceutical composition comprising BPS and a pharmaceutically acceptable carrier.

[0017] In some embodiments, the pharmaceutically acceptable carrier is selected from one or more of diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH adjusters, antioxidants, or antibacterial agents.

[0018] The pharmaceutical compositions of the present invention can be prepared into various dosage forms as needed. These dosage forms include, but are not limited to, tablets, solutions, granules, patches, ointments, capsules, aerosols, or suppositories for use via the skin, mucous membranes, nose, cheek, sublingual, or oral routes.

[0019] In some embodiments, the pharmaceutical composition further includes at least one additional active ingredient.

[0020] In some embodiments, the additional active ingredient includes anti-VEGF drugs and hormone drugs.

[0021] The mechanism of action of the anti-VEGF drugs is to inhibit vascular endothelial growth factor (VEGF), thereby blocking angiogenesis and leakage. In some embodiments, the anti-VEGF drugs include ranibizumab, aflibercept, bevacizumab, busizumab, etc.

[0022] The mechanism of action of the hormonal drugs is to inhibit inflammatory factors (such as IL-6 and TNF-α), reduce vascular leakage, and indirectly inhibit angiogenesis. In some embodiments, the hormonal drugs include dexamethasone implants, fluocinolone acetonide implants, etc.

[0023] In some embodiments, the additional active ingredient may also include other adjuvant drugs, such as PKC inhibitors, integrin inhibitors, etc.

[0024] Advantages and beneficial effects of the present invention: The present invention provides bisphenol S (BPS), which has a significant inhibitory effect on the migration and angiogenesis of pathological endothelial cells in a co-culture model of macrophages and human retinal microvascular endothelial cells. It has good application prospects in the preparation of drugs for the treatment of retinal neovascularization-related diseases and provides a research tool for the study of retinal neovascularization-related diseases. Attached Figure Description

[0025] Figure 1 The graph shows the results of macrophage viability after PMA induction by BPS and NAC as determined by the CCK-8 assay. In the graph, A is the OD450 statistical graph and B is the survival rate statistical graph.

[0026] Figure 2 The graph shows the malondialdehyde (MDA) detection results, where A is the protein standard curve and B is the statistical graph of MDA detection.

[0027] Figure 3The image shows the ROS detection results of DCFH-DA staining, where A is a statistical graph of DCFH-DA staining fluorescence intensity and B is a representative fluorescence graph.

[0028] Figure 4 The graphs show the ROS detection results by flow cytometry, where A is the flow cytometry detection result graph and B is the flow cytometry detection statistical graph.

[0029] Figure 5 The results of Western blot detection of autophagy proteins in macrophages are shown in Figure A, where WB shows the protein bands of autophagy proteins LC3α, LC3β, p62, p-mTOR and mTOR, and BE shows the expression levels of LC3α (B), LC3β (C), p62 (D) and p-mTOR / mTOR (E).

[0030] Figure 6 The figures are statistical graphs from transmission electron microscopy analysis. Figure A shows the number of autolysosomes (ASS), and Figure B shows the number of autolysosomes (ASS) + autophagosomes (AP).

[0031] Figure 7 These are representative images from transmission electron microscopy (TEM) analysis, where A represents the Control group, B represents the BPS group, and C represents the BPS+NAC group. The scale bar for the left image is 5.0 μm, and the scale bar for the right image is 1.0 μm.

[0032] Figure 8 This is a graph showing the immunofluorescence results of autophagy-related genes.

[0033] Figure 9 The images show immunofluorescence statistics of autophagy-related genes, where A is the fluorescence intensity statistics of LAMP1 and B is the fluorescence intensity statistics of LC3β.

[0034] Figure 10 The image shows the results of Western blot detection of autophagy proteins in co-cultured endothelial cells and macrophages. In the image, A is the protein band diagram of autophagy proteins LC3α, LC3β, p62, p-mTOR and mTOR detected by WB, and BE is the statistical diagram of the expression levels of LC3α (B), LC3β (C), p62 (D) and p-mTOR / mTOR (E).

[0035] Figure 11 The figures show the results of the scratch test, where A is the result of the scratch test, B is the 12-hour migration ability statistics, and C is the 24-hour migration ability statistics.

[0036] Figure 12 The diagram shows the results of the pipe-forming experiment, where A is the result of the pipe-forming experiment, and BD is a statistical diagram of the pipe-forming intersection node (B), branch length (C), and trunk length (D).

[0037] Figure 13 The images show the immunofluorescence results of autophagy-related genes. In the images, A represents the representative results of CD63 and LC3β immunofluorescence, and BC represents the statistical graphs of the immunofluorescence intensity of CD63 (B) and LC3β (C).

[0038] Figure 14 The image shows the NTA detection results of macrophage exosomes, where A represents the Control group, B represents the BPS group, and C represents the 3-MA+BPS group.

[0039] Figure 15 Scanning electron micrograph of macrophage exosomes.

[0040] Figure 16 The images show the results of the Confocal experiments in the BPS group. Merge1 shows the synthesis results of green and red fluorescence, and Merge2 shows the synthesis results of green, red, and blue fluorescence. Magnification: 600x, Scale bar: 50 μm. Detailed Implementation

[0041] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] Example 1 BPS induces oxidative stress damage in macrophages and affects autophagy I. Experimental Methods 1. Cell culture and passage 1.1 Cell Culture THP-1 cells were prepared using RPMI-1640 medium (containing β-mercaptoethanol): RPMI-1640 medium (Gibco™, 11875119) + 10% fetal bovine serum (Gibco™, 10099141) + 0.05 mM β-mercaptoethanol + 1% penicillin and streptomycin (100 U / ml penicillin, 100 U / ml streptomycin, Gibco™, 15140163). The cells were incubated at 4 ℃ and then incubated in a 37 ℃ water bath before use.

[0043] 1.2 Cell resuscitation 1) Preheat the complete culture medium to 37°C in a water bath.

[0044] 2) Remove the cryovial and quickly place it into a preheated water bath to thaw rapidly. Shake it constantly to allow the liquid inside to melt quickly. Remove the cryovial before the liquid inside has completely melted.

[0045] 3) Prepare a cell suspension by transferring the cells to a 15 ml centrifuge tube and adding preheated culture medium dropwise while shaking the centrifuge tube; the amount of culture medium added should be at least 10 ml. 4) Centrifuge at 800 rpm for 5 minutes on a low-speed centrifuge; remove the supernatant, then resuspend the cells in 1 ml of culture medium, add the cell suspension to a cell culture flask (T25), and place the culture flask in an incubator containing 5% CO2 at 37°C for culture.

[0046] 1.3 Cell passage culture 1) Preheat the complete culture medium to 37°C in a water bath; 2) Transfer the cell suspension to a centrifuge tube, centrifuge at 800 rpm for 5 min, and discard the supernatant.

[0047] 3) Resuspend the cells using fresh culture medium. Use a pipette to draw an appropriate amount of cell suspension (1:2 passage), transfer it to a new culture flask, add an appropriate amount of fresh culture medium, and continue culturing.

[0048] 2. CCK-8 assay for cell viability 1) Remove cells that have grown to 80%, transfer the cell suspension to a centrifuge tube, centrifuge at 800 g for 5 min, discard the supernatant, wash the cells once or twice with PBS, add fresh complete culture medium, and use a pipette to mix the cells to prepare a single-cell suspension.

[0049] 2) Cells were counted using a cell counter and the cell density was adjusted to 1×10⁶ cells / mL using complete culture medium containing PMA (BBI, Order NO. A606759, 100 ng / mL). 5 / mL.

[0050] 3) Take out the 96-well plate and seed 100 μl at a density of 1×10⁶. 5 Cell suspension of / mL was cultured at 37℃ and 5% CO2 for 48 h.

[0051] 4) Bisphenol S (4,4-dihydroxydiphenyl sulfone, Shanghai Yuanye, S30430) was dissolved in DMSO and prepared into a complete culture medium containing 10 -8 Culture medium for M's BPS.

[0052] 5) Dissolve NAC (N-acetyl-L-cysteine, Shanghai Yuanye, S20137) in ddH2O and prepare a medium containing 10 μM NAC using complete culture medium.

[0053] 6) Control group: After 48 h of PMA induction, the culture medium was removed and replaced with fresh complete culture medium for 6 h of culture.

[0054] 7) BPS group: After PMA induction for 48 h, the culture medium was removed and replaced with a culture medium containing 10-8M BPS for 6 h.

[0055] 8) NAC+BPS group: After PMA induction for 48 h, the culture medium was removed, and the medium was pretreated with 10 μM NAC for 30 min. Then the culture medium was removed and replaced with 10-8 M BPS for 6 h.

[0056] 9) After processing, add 10 μl of CCK-8 detection working solution to each well, avoiding the generation of air bubbles. Place the culture plate in an incubator and incubate for 4 hours. After 4 hours, transfer the liquid in the 96-well plate to the microplate.

[0057] 10) Use the microplate reader (Baoyede K3 Plus) to read the OD value at 450 nm and analyze the data.

[0058] 3. Malondialdehyde (MDA) testing 1) Remove cells that have grown to 80%, transfer the cell suspension to a centrifuge tube, centrifuge at 800 g for 5 min, discard the supernatant, wash the cells once or twice with PBS, add fresh complete culture medium, and use a pipette to mix the cells to prepare a single-cell suspension.

[0059] 2) Cells were counted using a cell counter and the cell density was adjusted to 1×10⁶ cells / mL using complete culture medium containing PMA (BBI, Order NO. A606759, 100 ng / mL). 6 / mL; 3) Remove the 6-well plate and inoculate with 1×10⁻⁶ cells / well. 6 Cells were cultured at 37°C and 5% CO2 for 48 h.

[0060] 4) Bisphenol S (4,4-dihydroxydiphenyl sulfone, Shanghai Yuanye, S30430) was dissolved in DMSO and prepared into a complete culture medium containing 10 -8 Culture medium for M's BPS.

[0061] 5) Dissolve NAC (N-acetyl-L-cysteine, Shanghai Yuanye, S20137) in ddH2O and prepare a medium containing 10 μM NAC using complete culture medium.

[0062] 6) Control group: After 48 h of PMA induction, the culture medium was removed and replaced with fresh complete culture medium for 6 h of culture.

[0063] 7) BPS group: After PMA induction for 48 h, the culture medium was removed and replaced with a medium containing 10 -8 Cultured in medium with M concentration of BPS for 6 hours.

[0064] 8) NAC+BPS group: After PMA induction for 48 h, the culture medium was removed, and the medium was pretreated with 10 μM NAC for 30 min. Then, the culture medium was removed and replaced with a medium containing 10 μM NAC. -8 Cultured in medium with M concentration of BPS for 6 hours.

[0065] 9) After the intervention, total protein was extracted from the cells of each group using a full protein extraction kit (Solarbio, BC3710). The specific method is as follows: A: Quantity of lysis buffer: 10 6 Each cell requires 0.5 ml of lysis buffer; B: Adherent cells: Discard the culture medium, wash twice with cold PBS, discard the PBS, and then add the calculated cell lysis buffer; scrape the cells off on ice with a cell scraper, transfer the scraped cell lysis buffer into an EP tube, and invert for lysis for 20-30 minutes.

[0066] C: After lysis, centrifuge the cell lysis buffer at 12000 g for 30 min at 4°C. D: Transfer the supernatant into a new EP tube; perform protein quantification.

[0067] Plotting the standard curve: Take an ELISA plate and add reagents according to Table 1.

[0068] Table 1. Enzyme-labeled reagents

[0069] Dilute the sample 15 times (mix 2 μl of sample with 28 μl of deionized water thoroughly). Take 25 μl of the diluted sample into an ELISA plate. Based on the quantity of sample and standard, prepare an appropriate amount of BCA working solution by adding 1 volume of BCA reagent B (Prod # 1859078) to 50 volumes of BCA reagent A (Prod # 23228) (50:1). Mix thoroughly and add 200 μl of BCA working solution to each well. Place the microplate on a shaker and shake for 30 seconds, incubate at 37°C for 30 minutes, and then read the absorbance at 570 nm. Plot a standard curve with protein concentration (μg / μl) on the ordinate and absorbance on the abscissa. Based on the absorbance value of the measured sample and the regression equation of the standard curve, the corresponding sample concentration can be obtained. Finally, multiplying by the dilution factor, the sample concentration can be obtained, with the unit being μg / μl.

[0070] 10) MDA testing shall be performed in accordance with the instructions for the malondialdehyde (MDA) test kit (Nanjing Jiancheng, A003-1).

[0071] 4. DCFH-DA staining for ROS detection 1) Remove cells that have grown to 80%, transfer the cell suspension to a centrifuge tube, centrifuge at 800 g for 5 min, discard the supernatant, wash the cells once or twice with PBS, add fresh complete culture medium, and use a pipette to mix the cells to prepare a single-cell suspension.

[0072] 2) Cells were counted using a cell counter and the cell density was adjusted to 1×10⁶ cells / mL using complete culture medium containing PMA (BBI, Order NO. A606759, 100 ng / mL). 5 / mL.

[0073] 3) Remove the 12-well plate and inoculate with 2×10⁶ cells / well. 5 Cells were cultured at 37°C and 5% CO2 for 48 h.

[0074] 4) Bisphenol S (4,4-dihydroxydiphenyl sulfone, Shanghai Yuanye, S30430) was dissolved in DMSO and prepared into a complete culture medium containing 10 -8 Culture medium for M's BPS.

[0075] 5) Dissolve NAC (N-acetyl-L-cysteine, Shanghai Yuanye, S20137) in ddH2O and prepare a medium containing 10 μM NAC using complete culture medium.

[0076] 6) Control group: After 48 h of PMA induction, the culture medium was removed and replaced with fresh complete culture medium for 6 h of culture.

[0077] 7) BPS group: After PMA induction for 48 h, the culture medium was removed and replaced with a medium containing 10 -8 Cultured in medium with M concentration of BPS for 6 hours.

[0078] 8) NAC+BPS group: After PMA induction for 48 h, the culture medium was removed, and the medium was pretreated with 10 μM NAC for 30 min. Then, the culture medium was removed and replaced with a medium containing 10 μM NAC. -8 Cultured in medium with M concentration of BPS for 6 hours.

[0079] 9) After the treatment is complete, remove the cell culture medium and wash the cells once with serum-free cell culture medium.

[0080] 10) Add 100 μl of reagent working solution and incubate the cells at 37°C in the dark for 45 min.

[0081] 11) Remove reagent 1 working solution and wash 2-3 times with serum-free cell culture medium to fully remove DCFH-DA that has not entered the cells.

[0082] 12) Detection was performed directly under a fluorescence microscope (excitation wavelength 488 nm).

[0083] 5. Flow cytometry detection of ROS 1) Remove cells that have grown to 80%, transfer the cell suspension to a centrifuge tube, centrifuge at 800 g for 5 min, discard the supernatant, wash the cells once or twice with PBS, add fresh complete culture medium, and use a pipette to mix the cells to prepare a single-cell suspension.

[0084] 2) Cells were counted using a cell counter and the cell density was adjusted to 1×10⁶ cells / mL using complete culture medium containing PMA (BBI, Order NO. A606759, 100 ng / mL). 5 / mL.

[0085] 3) Remove the 12-well plate and inoculate with 2×10⁶ cells / well. 5 Cells were cultured at 37°C and 5% CO2 for 48 h.

[0086] 4) Bisphenol S (4,4-dihydroxydiphenyl sulfone, Shanghai Yuanye, S30430) was dissolved in DMSO and a medium containing 10-8 M BPS was prepared using complete culture medium.

[0087] 5) Dissolve NAC (N-acetyl-L-cysteine, Shanghai Yuanye, S20137) in ddH2O and prepare a medium containing 10 μM NAC using complete culture medium.

[0088] 6) Control group: After 48 h of PMA induction, the culture medium was removed and replaced with fresh complete culture medium for 6 h of culture.

[0089] 7) BPS group: After PMA induction for 48 h, the culture medium was removed and replaced with a medium containing 10 -8 Cultured in medium with M concentration of BPS for 6 hours.

[0090] 8) NAC+BPS group: After PMA induction for 48 h, the culture medium was removed, and the medium was pretreated with 10 μM NAC for 30 min. Then, the culture medium was removed and replaced with a medium containing 10 μM NAC. -8 Cultured in medium with M concentration of BPS for 6 hours.

[0091] 9) After the treatment is complete, remove the cell culture medium and wash the cells once with serum-free cell culture medium.

[0092] 10) Digest with trypsin at 37°C for 2 min, terminate digestion with 3 mL of complete culture medium, collect cells by centrifugation at 300 g for 5 min, wash once with PBS, and analyze by flow cytometry.

[0093] 6. Western blot detection of autophagy 6.1 Protein electrophoresis Protein electrophoresis was performed using a Precast Protein Plus Gel, 4-20%, 10 wells, Hepes-Tris 4-20% high-resolution precast gel (Hepes-Tris) (Yeasen, 36250ES10).

[0094] 1) Prepare denatured protein electrophoresis buffer. The concentrations of each component of the denatured protein electrophoresis buffer are: 50 mM Tris, 50 mM HEPES, 0.1% SDS, and 2 mM EDTA.

[0095] 2) Remove the Precast Protein Plus Gel from the packaging bag, tear off the tape at the bottom of the gel plate, slowly pull out the comb, and then fix the precast gel in the electrophoresis tank (Beijing Kaiyuan Bole Biotechnology Co., Ltd., MP-8001).

[0096] 3) Fill the inner tank of the electrophoresis tank with electrophoresis buffer, and the liquid level in the outer tank should be 1 / 3 higher than the height of the electrophoresis tank. Use a pipette or other tools to gently blow the electrophoresis buffer into the sample wells to remove any residual storage buffer and impurities.

[0097] 4) After drawing up the sample with a pipette, insert the pipette tip vertically into the sample well to load the sample. Be careful not to puncture the gel or insert the tip too far into the comb well, which could deform the gel plate and cause sample leakage.

[0098] 5) Electrophoresis conditions: 150 V, 30-40 min (Beijing Liuyi Instrument Factory, DYY-6C). Electrophoresis can be stopped when the bromophenol blue indicator band reaches the bottom of the gel or the predetermined position in the experiment.

[0099] 6) After electrophoresis, remove the gel and use a gel lifter or other suitable tool to insert into the gap between the two sides of the gel plate. Slowly pry up and down the three different positions (top, middle, and bottom). Then repeat the operation on the other side until both sides of the gel plate are completely opened.

[0100] 7) After opening the gel plate, the gel may stick to either side of the gel plate. Tilt the gel plate with the gel side into the water, gently stir the gel to let it fall freely into the container of water, shake to clean the gel, and then take it out for subsequent staining or transfer experiments.

[0101] 6.2 Transfer and antibody incubation 1) The membrane transfer was performed using the eBlot™ L1 rapid wet transfer instrument (Genscript, L00686C) and the eBlot L1 transfer and concentration kit (PVDF membrane base) (Genscript, L00726C).

[0102] 2) After the transfer is complete, remove the PVDF membrane (Millipore, IPVH00010) and gel. Rinse the PVDF membrane with 25 ml TBS for 5 min, place it in blocking solution with 5% skim milk powder-TBS, and place it on a shaker at 4°C and shake gently overnight (or block at room temperature for 1 h). Wash the PVDF membrane 3 times with 25 ml TBS, 5 min each time.

[0103] 3) Place the PVDF membrane into a small tank containing 3 ml of diluted LC3A antibody (Proteintech, Cat No: 18722-1-AP, 1:20000); LC3B (Proteintech, Cat No: 18725-1-AP, 1:1000); p62 antibody (Proteintech, Cat No: 18420-1-AP, 1:20000); mTOR (Proteintech, Cat No: 66888-1-Ig, 1:25000); Phospho-mTOR (Ser2448) (Proteintech, Cat No: 67778-1-Ig, 1:5000) or GAPDH (Abcam, ab8245, 1:10000) 5% skim milk powder-TBS solution, and place on a shaker at room temperature and shake gently for 1 h.

[0104] 4) Wash the PVDF membrane three times with 25 ml TBST, each time for 5 min.

[0105] 5) Place the PVDF membrane into a small tank containing 3 ml of diluted horseradish peroxidase-labeled secondary antibody solution, and gently shake it on a shaker at room temperature for 40 min (Secondary antibody: HRP Goat anti-Rabbit IgG (manufacturer: BOSTER, catalog number: BA1054, dilution ratio: 1:20000, incubation conditions: room temperature incubation for 40 min) or HRP Goat anti-Mouse IgG (manufacturer: BOSTER, catalog number: BA1051, dilution ratio: 1:20000, incubation conditions: room temperature incubation for 40 min).

[0106] 6) Wash the PVDF membrane three times with 25 ml TBST for 7 min each time, in preparation for subsequent experiments.

[0107] 6.3 Chemiluminescence, development, and fixing Chemiluminescence was performed using an ultrasensitive ECL chemiluminescence substrate kit (Biosharp, BL520B).

[0108] 1) Mix reagents A and B in equal volumes in a 1.5 ml EP tube; after 5 min, place the PVDF membrane protein side up on plastic wrap, add an appropriate amount of the mixture, wrap it up, and place it in a medical X-ray film holder.

[0109] 2) In a darkroom, pour 1× developer and fixer into separate plastic trays. Under a red light, remove the X-ray film, open the X-ray film holder, place the X-ray film on the film, close the X-ray film holder, press the film for 1 or 5 minutes, open the X-ray film holder, remove the X-ray film, and quickly immerse it in the developer. Once clear bands appear, immediately stop the development. Immerse the X-ray film in the fixer. Fixing time is generally 5 to 10 minutes, until the film is transparent. Rinse off any residual fixer with tap water and air dry at room temperature.

[0110] 6.4 Gel Image Data Analysis The film was scanned, and the net optical density values ​​of the strips were analyzed using a gel imaging system (Image-Pro Plus 6.0).

[0111] 7. Electron microscopy analysis 1) Sampling and fixation: Centrifuge to collect cell or bacterial pellets, ensuring the pellets are at least the size of a mung bean. Remove from culture medium, add electron microscopy fixative, resuspend at 4°C, mix well, and fix for 2-4 hours. Store and transport at 4°C.

[0112] 2) Agar Pre-embedding: Centrifuge cells or bacteria, discard the supernatant, add 0.1M phosphate buffer PB (pH 7.4), mix well, rinse for 3 min, centrifuge again, and repeat the washing 3 times. Prepare a 1% agarose solution by heating and dissolving in advance, and add it to an EP tube after slight cooling. Before the agarose solidifies, use tweezers to pick up the precipitate and suspend it in the agarose.

[0113] 3) Post-fixation: 1% osmium tetroxide was prepared in 0.1M phosphate buffer PB (pH 7.4) and fixed at room temperature in the dark for 2 hours. The solution was rinsed 3 times with 0.1M phosphate buffer PB (pH 7.4), 15 min each time.

[0114] 4) Room temperature dehydration: The tissue was sequentially immersed in 30%-50%-70%-80%-95%-100%-100% alcohol for upward dehydration, 20 minutes each time, and 100% acetone twice, 15 minutes each time.

[0115] 5) Infiltration embedding: Acetone: 812 embedding agent = 1:1, 37℃ for 2-4 hours; Acetone: 812 embedding agent = 1:2, 37℃ overnight; Pure 812 embedding agent, 37℃ for 5-8 hours. Pour pure 812 embedding agent into the embedding plate, insert the sample into the embedding plate, and bake in a 37℃ oven overnight.

[0116] 6) Polymerization: Place the embedding plate in a 60℃ oven for polymerization for 48 hours, then remove the resin block for later use.

[0117] 7) Ultrathin sectioning: The resin block is used to make ultrathin sections of 60-80nm using an ultrathin microtome, and the sections are retrieved using a 150-mesh copper screen.

[0118] 8) Staining: Stain copper mesh in 2% uranium acetate saturated alcohol solution for 8 min in the dark; wash 3 times with 70% alcohol; wash 3 times with ultrapure water; stain in 2.6% lead citrate solution for 8 min in the dark; wash 3 times with ultrapure water, and blot dry with filter paper. Place copper mesh sections in a copper mesh box and dry at room temperature overnight.

[0119] 9) Observe under a transmission electron microscope and collect and analyze images.

[0120] 8. Immunofluorescence 1) Cell permeabilization: After slightly drying the slide, use a histochemical pen to draw circles in the center of the coverslip where the cells are evenly distributed (to prevent antibody runoff). Add 50-100 μl of permeabilization working solution and incubate at room temperature for 20 min. Wash three times with PBS, 5 min each time. (If staining for membrane proteins, do not perform permeabilization.) 2) Serum blocking: Add 3% BSA to the tissue and evenly cover it, then block at room temperature for 30 minutes. (For primary antibodies derived from goats, use 10% donkey serum for blocking; for primary antibodies from other sources, use 3% BSA for blocking.) 3) Add the mixed reagent of the first and second primary antibodies: Mix the two primary antibodies from different sources, add the prepared primary antibody to the cell culture plate, and incubate the cell culture plate in a humidified chamber at 4°C overnight.

[0121] 4) Add secondary antibody: Place the cell plate on a decolorizing shaker and wash three times, 5 min each time. Add the corresponding secondary antibody and incubate at room temperature for 50 min.

[0122] 5) DAPI counterstaining of cell nuclei: Place the slides in PBS (pH 7.4) and wash three times on a destaining shaker for 5 minutes each time. After slightly drying the slides, add DAPI staining solution to the circle and incubate at room temperature in the dark for 10 minutes.

[0123] 6) Mounting: Place the slide in PBS (pH 7.4) and wash three times on a decolorizing shaker for 5 minutes each time. After slightly drying the slide, mount it with an anti-fluorescence quenching mounting medium.

[0124] 7) Image acquisition: DAPI excitation wavelength 330-380nm, emission wavelength 420nm; 488 excitation wavelength 465-495nm, emission wavelength 515-555 nm; CY3 excitation wavelength 510-560 nm, emission wavelength 590nm; CY5 excitation wavelength 608-648 nm, emission wavelength 672-712 nm.

[0125] DAPI channel positive results are displayed in blue in cell nuclei, 488 channel positive results are displayed in green, and CY3 channel positive results are displayed in red. Colocalization analysis was performed using ImageJ software.

[0126] II. Experimental Results 1. CCK-8 assay for macrophage viability: Results are as follows Figure 1 As shown, Control, 10 -8 M BPS and NAC+10 -8 There was no significant difference in cell viability among the M BPS groups, and the results of CCK-8 assay showed that the addition of BPS and NAC had no effect on the viability of macrophages after PMA induction.

[0127] 2. Effects of oxidants and reactive oxygen species (ROS) inhibitors on oxidative stress in macrophages: Sample protein concentrations are shown in Table 2, and the protein standard curves are plotted as follows. Figure 2 As shown in A in the diagram.

[0128] Table 2. Sample protein concentration

[0129] Malondialdehyde (MDA) is a major product of lipid peroxidation damage. MDA detection is generally involved in oxidative stress studies, and its concentration increases to varying degrees with the progression of oxidative stress. Experimental results are as follows... Figure 2 As shown in Figure B, compared with the control group, the addition of the oxidant BPS induced oxidative damage in macrophages, with a highly significant increase in MDA levels (P < 0.001). The addition of the reactive oxygen species (ROS) inhibitor NAC significantly reduced the occurrence of oxidative damage, and the MDA level in the NAC group was significantly lower than that in the BPS group (P < 0.001).

[0130] 3. DCFH-DA staining for ROS detection: ROS levels are an important marker of cellular oxidative damage. The fluorescent probe DCFH-DA was used to detect reactive oxygen species, and the results are as follows: Figure 3 As shown, BPS-induced oxidative damage in macrophages led to a sharp increase in ROS levels, with fluorescence intensity significantly higher than that in the control group and NAC group (P < 0.001). There was no significant difference in ROS levels between the control group and the NAC group.

[0131] 4. Flow cytometry detection of ROS: Flow cytometry was used to determine the relative intensity of ROS in live cells, with the logarithm of fluorescence readings on the x-axis and cell number on the y-axis. Results showed that compared to the control group, the addition of 10... -8 After M concentration BPS, the ROS level in macrophages increased sharply, significantly higher than that in the control group and NAC group (P<0.001).

[0132] 5. Western blot detection of autophagy proteins: WB results are as follows Figure 5 As shown, the autophagy pathway mTOR-p62-LC3 is activated by oxidative stress induced by the oxidant BPS, while the autophagy pathway is significantly inhibited after the addition of the ROS inhibitor NAC (P < 0.05).

[0133] 6. Transmission electron microscopy analysis: Statistics on the number of ASSes and the total number of ASSes and APs are as follows. Figure 6 As shown.

[0134] Figure 7 Image A in the Control group shows a representative image, where cells are slightly enlarged, cell membrane structures are dissolved and damaged, pseudopodia are visible around the membrane, cytoplasm density is uneven, and organelles are swollen; the nucleus (N) is off-center from one side of the cell, with local invagination, uniform chromatin distribution, and a clear nuclear membrane; the mitochondrial (M) membrane is intact, the intracellular matrix is ​​homogeneous, and the cristae are clear; the rough endoplasmic reticulum (RER) shows slight local expansion, and perimembrane ribosomes are detached; the Golgi apparatus (Go) shows slight hyperplasia and hypertrophy; a small number of lipid droplets (LD) are present; and autolysosomes (ASS) are visible.

[0135] Figure 7 Image B in the image shows a representative image of the BPS group, where the cells are slightly larger, the cell membrane structure is locally damaged, pseudopodia are visible around the membrane, the cytoplasm is uniformly distributed, and the organelles are swollen; the nucleus (N) is off to one side of the cell, locally invaginated, mainly composed of euchromatin, and the nuclear membrane is clear; the mitochondrial (M) membrane is intact, the intracellular matrix density is slightly higher, and the cristae are expanded; the rough endoplasmic reticulum (RER) is slightly expanded locally, and ribosomes are sparse on the endoplasmic reticulum; the Golgi apparatus (Go) sac wall is slightly expanded; lipid droplets (LD) are present in small numbers; and autolysosomes (ASS) and autophagosomes (AP) are more numerous.

[0136] Figure 7 Image C in the image shows a representative image of the BPS+NAC group. The cells are slightly smaller, the cell membrane structure is intact, pseudopodia are visible around the membrane, the cytoplasm is uniform, and the organelles are swollen. The nucleus (N) is irregular and locally concave, mainly composed of euchromatin, the nucleolus (Nu) is large, and the nuclear membrane is clear. The mitochondria (M) are slightly swollen, the membrane is intact, the matrix is ​​slightly shallow, and the cristae are reduced. The rough endoplasmic reticulum (RER) is slightly expanded locally, and the surface ribosomes are sparse. The Golgi apparatus (Go) is slightly proliferating and expanded. There are fewer autolysosomes (ASS) and autophagosomes (AP).

[0137] Transmission electron microscopy revealed differences in autophagy levels among the three groups. In the BPS group, oxidant-induced autophagy in macrophages was observed, along with severe mitochondrial damage and a higher number of autolysosomes and autophagosomes. In the NAC intervention group, autophagy was reduced, mitochondrial damage was decreased, and the number of autolysosomes and autophagosomes was significantly reduced (P < 0.01).

[0138] 7. Immunofluorescence detection of autophagy: Immunofluorescence results showed that the number of autophagic vesicles in macrophages in the BPS group was significantly increased, and autolysosomes co-labeled by LC3β and LAMP1 were visible, with fluorescence intensity significantly higher than that in the control group (P < 0.0001). After NAC intervention, the number of autolysosomes decreased and the degree of autophagy was significantly reduced (P < 0.0001).

[0139] III. Conclusion Exogenous oxidant oxidative stress (BPS) can induce oxidative stress damage in macrophages and affect their autophagy levels. Intervention with the ROS inhibitor NAC can reduce oxidative stress and autophagy in macrophages. This suggests that BPS may trigger changes in macrophage polarization by activating oxidative stress pathways (such as ROS / NF-κB), thereby inhibiting the secretion of pro-angiogenic factors (such as VEGF and IL-6). Although the effect of NAC intervention suggests that oxidative stress is a key mediator of BPS action, the regulation of autophagy levels may indirectly inhibit angiogenesis by clearing damaged organelles (such as mitochondria) and mitigating the inflammatory response. This bidirectional regulation of the "oxidative stress-autophagy" axis may provide BPS with a unique anti-angiogenic mechanism.

[0140] Example 2 Effect of macrophage autophagy level on the proliferation and migration of endothelial cells in co-culture system I. Experimental Methods 1. Resuscitation and passage culture of human retinal microvascular endothelial cells (HRMECs) 1.1 Human retinal microvascular endothelial cells (HRMECs) (CP-H130) were purchased from Wuhan Pronosai Life Science Technology Co., Ltd., and cultured in a 37°C incubator containing 5% CO2 using complete human retinal microvascular endothelial cell culture medium (CM-H130, Wuhan Pronosai Life Science Technology Co., Ltd.).

[0141] 1.2 Cell resuscitation: The experimental procedure is the same as 1.2 in Example 1.

[0142] 1.3 Cell passage culture: The experimental procedure is the same as 1.3 in Example 1.

[0143] 2. THP-1 cell culture: The experimental procedure is the same as 1.1-1.3 in Example 1.

[0144] 3. Western blot detection of related protein expression: The experimental procedure is the same as 6.1-6.4 in Example 1.

[0145] 4. Scratch detection 1) Use a marker pen to draw a horizontal line on the back of the 6-hole board beforehand, passing through the holes.

[0146] 2) Before co-culturing, take out the 6-well plate inoculated with HRMECs and use a 200 μl pipette tip to make a vertical cut on the back of the plate. The pipette tip must be vertical and not tilted.

[0147] 3) Wash the cells three times with PBS to remove the crossed cells, and add serum-free culture medium.

[0148] 4) Transwell chambers of the non-intervention group were placed on culture plates and incubated at 37°C and 5% CO2 for 12 and 24 h. The chambers were then observed and photographed using an inverted microscope and analyzed using ImageJ software.

[0149] 5. Angiogenesis 1) In a clean bench, use a pre-cooled pipette tip to take 50 μl of Matrigel (40187ES10, Yisheng Biotechnology (Shanghai) Co., Ltd.) and add it to a pre-cooled 6-well plate. Gently spread the gel using the pipette tip. After spreading the gel, place the plate in a 4°C refrigerator for 24 h to allow the gel to spread evenly. Then, place the plate in a 37°C, 5% CO2 incubator and incubate for 0.5 h to allow the gel to solidify.

[0150] 2) Take 2 mL of cell suspension (5 × 10⁻⁶) 5 Add 1 × 10⁶ mL of the solution to each well of a 6-well plate that has completed incubation. 6 Cells were placed in Transwell chambers in the non-intervention group on culture plates and incubated in an incubator. After 12 hours of incubation, the cells were observed and photographed under a microscope.

[0151] 3) Perform tube analysis using ImageJ software.

[0152] II. Experimental Results 1. Western blot detection of autophagy proteins: WB results are as follows Figure 10 As shown, after treatment with BPS, p62 expression was reduced and the autophagy pathway was significantly activated in the co-culture group of endothelial cells and macrophages (P<0.05), while the addition of 3-MA could reverse this effect.

[0153] 2. Scratch Assay: To investigate the effects of macrophages on endothelial cell migration under different conditions, a co-culture system of macrophages and endothelial cells was established. The migration function of endothelial cells was observed through a scratch assay. Co-culture of macrophages and endothelial cells is an important in vitro research model for vascular biology. It can be used to reproduce the interaction mechanism between macrophages and endothelial cells under physiological conditions. Furthermore, by adding the oxidant BPS, oxidative stress damage can be utilized to recreate the formation of new blood vessels under pathological conditions through co-culture of macrophages and endothelial cells.

[0154] Compared with the macrophage co-culture group, the addition of the oxidant BPS significantly reduced the migration function of endothelial cells (P<0.05), while the addition of 3-MA had no significant effect on the migration of endothelial cells. Figure 11 ).

[0155] 3. Tube Formation Experiment: Statistical analysis was performed on the tube formation intersections (Nb Junctions), branch lengths (Tot. branchinglength), and trunk lengths (Tot. segments length). The results are as follows: Figure 12 The results showed that, compared with the co-culture group, the number of nodes and the length of tubes in the BPS-added group were significantly reduced, indicating that BPS significantly inhibited the tube-forming function of endothelial cells (P<0.05). The addition of 3-MA had no significant effect on the tube-forming function of endothelial cells.

[0156] III. Conclusion Experimental results showed that BPS could inhibit the tube formation and migration functions of endothelial cells co-cultured with macrophages, but the addition of 3-MA did not significantly reverse this inhibitory effect. This suggests that the inhibitory effect of BPS on endothelial cells may be independent of the autophagy pathway, and more dependent on directly interfering with endothelial cell proliferation signals (such as the PI3K / Akt and MAPK pathways) or disrupting cytoskeleton homeostasis. This autophagy-independent inhibitory mechanism may reduce interference with normal vascular homeostasis and improve therapeutic selectivity.

[0157] Example 3 Effect of exogenous oxidants on macrophage exosomes I. Experimental Methods 1. THP-1 cell culture 1.1 The experimental procedure is the same as 1.1-1.3 in Example 1.

[0158] 1.2 THP-1-induced M0 and creeping 1) Remove cells that have grown to 80%, transfer the cell suspension to a centrifuge tube, centrifuge at 800 g for 5 min, discard the supernatant, wash the cells once or twice with PBS, add fresh complete culture medium, and use a pipette to mix the cells to prepare a single-cell suspension.

[0159] 2) Cells were counted using a cell counter and the cell density was adjusted to 1×10⁶ cells / mL using complete culture medium containing PMA (BBI, Order NO. A606759, 100 ng / mL). 5 / mL.

[0160] 3) Remove the 6-well plate and seed 800 μl at a density of 1×10⁻⁶. 5 A 1 mL / mL cell suspension was added to the slide.

[0161] 4) After incubation at 37℃ and 5% CO2 for 24 h, remove the culture medium and use a medium containing 10 -8 Treatment with 1 M BPS and 5 mM 3-MA (MCE, HY-19312) for 6 h.

[0162] 2. Double immunofluorescence labeling 1) After washing the cells 2-3 times with PBS, add 1 mL of 4% paraformaldehyde and fix at room temperature for 10 min; 2) After washing with PBS 3 times, take out the slide, shake it dry slightly, and use a histochemical pen to draw circles in the middle of the coverslip where the cells are evenly distributed (to prevent the antibody from flowing away). Add 50-100 μl of membrane breaking working solution, incubate at room temperature for 20 min, and wash with PBS 3 times, 5 min each time. 3) Serum blocking: Add 10% donkey serum to the cell circle and evenly cover the cells. Block at room temperature for 30 min. 4) After blocking, rinse three times with PBS, add the mixed primary antibody (LC3β, Proteintech, Cat No: 18725-1-AP, 1:400; CD63, abcam, ab193349, 1:40), evenly cover the cells, and incubate the cell culture plate in a humidified chamber at 4°C overnight.

[0163] 5) Add secondary antibodies: Add TBST to the cell wells, place on a decolorizing shaker and wash 3 times, 5 min each time. Add the corresponding secondary antibodies (Alexa Fluor 488-labeled goat anti-rabbit IgG, GB25303, Servicebio, 1:400; CY5-labeled goat anti-mouse IgG, GB27301, Servicebio, 1:400), and incubate at room temperature for 50 min.

[0164] 6) DAPI counterstaining of cell nuclei: Place the slide in PBS and wash three times on a destaining shaker for 5 min each time. After slightly drying the slide, add DAPI staining solution to the circle and incubate at room temperature in the dark for 10 min.

[0165] 7) Mounting: Place the slide in PBS and wash it three times on a decolorizing shaker for 5 minutes each time. After slightly drying the slide, mount it with an anti-fluorescence quenching mounting medium.

[0166] 8) Image acquisition: DAPI excitation wavelength 330-380 nm, emission wavelength 420 nm; 488 excitation wavelength 465-495 nm, emission wavelength 515-555 nm; CY5 excitation wavelength 608-648 nm, emission wavelength 672-712 nm.

[0167] DAPI (blue light): cell nucleus; Alexa Fluor 488 (green light): CD63; CY5 (red light): LC3B.

[0168] 3. Transmission electron microscopy 3.1 Exosome Extraction 1) Using a product containing 10 -8 Cells were treated with exosome-free complete culture medium containing MBPS and 5 mM 3-MA (MCE, HY-19312) for 6 h. After the intervention, 50 ml of cell culture supernatant was collected for exosome extraction. 2) Add the sample to a 50 ml centrifuge tube, centrifuge at 2000 g for 15 min, and collect the supernatant; 3) Centrifuge the sample in a high-speed refrigerated centrifuge at 4°C and 10,000 g for 30 min, and collect the supernatant; 4) After wetting the 0.22 μm needle filter with PBS, filter the supernatant of the previously centrifuged sample; 5) Transfer the filtered sample to a Himac ultracentrifuge tube, centrifuge at 4°C for 90 min, remove the supernatant, and resuspend the precipitate with PBS; 6) Centrifuge again at 4°C for 90 min, and resuspend the exosome precipitate in 150 μl of PBS.

[0169] 7) Store in a refrigerator at -80℃ for later use.

[0170] 3.2 Electron Microscopy Observation 1) Take 20 μl of exosome suspension and drop it onto a carbon membrane copper grid (Zhongxing Bairui, ZB-C1004) and let it stand for 3-5 min, then use filter paper to absorb the excess liquid.

[0171] 2) Drop 2% phosphotungstic acid (Saiwell, G1102) onto the copper mesh of the carbon support membrane and let it stand for 1-2 minutes. Use filter paper to absorb the excess liquid and let it dry at room temperature.

[0172] 3) Observe and analyze images under a transmission electron microscope (HITACHI, HT7800).

[0173] 4. NTA testing 4.1 Instrument Calibration Turn on the instrument, computer, and software in sequence. The software will prompt "please make sure that the fluorescence filter is removed". After confirming that the position is correct, click OK. The instrument will start automatic calibration: 1. Select ultrapure water to rinse the instrument. After the instrument stops rinsing, use ultrapure water to dilute the 100nm polystyrene microsphere standard solution 250,000 times. Take 1 ml of the diluted standard solution for automatic instrument calibration.

[0174] 4.2 On-machine testing 1) The instrument can only be used for detection after it has passed automatic calibration. Rinse the instrument to ensure the particle count is in the single digits. Dilute the exosome sample with clean PBS solution to a suitable concentration so that the number of particles displayed on the instrument's detection interface is between 50 and 400, preferably around 200.

[0175] 2) Enter the dilution factor of the sample in the software interface and observe whether the number of particles displayed at the detection location is between 50 and 400.

[0176] 3) After confirming that the number of particles displayed at each detection location is within the range, click Measurement and Run Video Acquisition on the software interface in sequence. In the pop-up interface, set the sample name and data save path, select the appropriate SOP, and finally click OK to start the test.

[0177] 4.3 Data Analysis The instrument automatically completes the testing process, automatically analyzes the data, and then automatically generates a test report, providing information such as the particle size and concentration of the sample.

[0178] II. Experimental Results 1. Immunofluorescence: Autophagosomes and exosomes are closely linked in their synthetic pathways. Autophagosome endosomes can be co-labeled by the autophagosome marker protein LC3β and the exosome marker protein CD63. Immunofluorescence detection of CD63 and LC3β expression... Figure 13 The results showed that some CD63 molecules could be co-expressed with LC3β, indicating that autophagosome and exosome synthesis are related. The fluorescence intensity of the exosome marker protein CD63 in the BPS group was significantly lower than that in the control group (P < 0.0001), indicating that macrophages released fewer exosomes after oxidative stress injury, but 3-MA showed a tendency to reverse this effect.

[0179] 2. Transmission electron microscopy and NTA To further identify and compare the characteristics of exosomes produced by macrophages in different groups, this study used Nanosight to detect the particle size and concentration of extracted extracellular vesicles.

[0180] Figure 14Table 3 shows that most microvesicles in the control group had a particle size range of 91.1-202 nm and a concentration of 1.7E+11 particles / mL. Exosomes secreted by macrophages after oxidative damage in the BPS group had a particle size range of 95.2-242.3 nm and a concentration of 2.0E+10 particles / mL. Exosomes extracted from macrophages after 3-MA treatment had a particle size range of 92.6-223.7 nm and a concentration of 2.6E+10 particles / mL. Exosomes derived from macrophages after oxidative damage had a larger size and lower concentration compared to those secreted by normal macrophages. Inhibition of autophagy within macrophages by 3-MA reduced the exosome particle size. These results correspond to the immunofluorescence results.

[0181] Table 3. NTA Detection Results

[0182] Scanning electron microscope images such as Figure 15 As shown, the cup-shaped or saucer-like structure of exosomes can be observed, indicating that the morphology and size of the microvesicles extracted in this study are highly consistent with the characteristics of exosomes.

[0183] 3. Confocal laser scanning microscope Further investigation was conducted to detect the co-expression of CD63 and LC3β, such as... Figure 16 As shown in the figure. Immunofluorescence co-localization results indicate that some CD63 molecules can be co-expressed with LC3β, suggesting that autophagosome and exosome synthesis are associated.

[0184] III. Conclusion Experimental results show that exosomes were successfully extracted from macrophages in this study. Macrophages damaged by oxidative stress secreted fewer exosomes but with increased particle size, while 3-MA intervention reduced the particle size of exosomes. As important carriers of intercellular communication, changes in the particle size of exosomes may affect the packaging and delivery efficiency of their contents (such as miRNAs and proteins). BPS-induced exosome size increase may reduce the transmission of pro-angiogenic signals, and the effect of 3-MA intervention further suggests that autophagy regulation may indirectly regulate angiogenesis by affecting exosome biogenesis (such as the ESCRT complex). This finding provides a novel mechanism for BPS to inhibit angiogenesis through the exosomal pathway.

[0185] In summary, this application provides preliminary evidence from three aspects—macrophage oxidative stress, endothelial cell function inhibition, and exosome regulation—to demonstrate the potential role of BPS in inhibiting retinal neovascularization. Future research should focus on optimizing BPS intervention strategies and conducting preclinical studies to validate its safety and efficacy, thus providing new treatment options for retinal neovascularization-related diseases.

[0186] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. Application of BPS in the preparation of drugs for treating retinal neovascularization-related diseases.

2. The application according to claim 1, characterized in that, The BPS treats retinal neovascularization-related diseases through at least one of the following methods: 1) Inhibits the formation of pathological angiogenesis; 2) Inhibits the migration of pathological angiogenesis.

3. The application according to claim 1, characterized in that, The dosage form of the drug is any one of injection, tablet, pill, powder, suspension, gel, emulsion, granule, capsule and spray.

4. The application according to claim 1, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

5. The application according to claim 4, characterized in that, The pharmaceutically acceptable carrier is selected from one or more of the following: diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH adjusters, antioxidants, or antibacterial agents.

6. The application according to claim 1, characterized in that, The retinal neovascularization-related diseases are selected from at least one of neovascular glaucoma, ischemic retinopathy, ischemic choroidal disease and secondary retinopathy, proliferative diabetic retinopathy, retinopathy of prematurity, and age-related macular degeneration.

7. The application according to claim 6, characterized in that, The ischemic retinopathy is selected from at least one of the following: retinal vein occlusion, central retinal artery occlusion, branch retinal artery occlusion, ciliary retinal artery occlusion, distal retinopathy, Purtscher-like retinopathy, retinal periphlebitis, ocular ischemia syndrome, aortitis fundus lesions, radiation retinopathy, and familial exudative vitreoretinopathy. Preferably, the ischemic choroidal lesion is selected from at least one of polypoid choroidal vascular lesions, high myopia choroidal neovascularization, and idiopathic choroidal neovascularization; Preferably, the secondary retinal lesion is selected from proliferative vitreoretinopathy, vitreous hemorrhage, fibrous proliferation, and tractional retinal detachment.

8. A pharmaceutical composition for treating retinal neovascularization-related diseases, characterized in that, The pharmaceutical composition includes BPS and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutically acceptable carrier is selected from one or more of the following: diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH adjusters, antioxidants, or antibacterial agents.

10. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition further includes at least one additional active ingredient; Preferably, the additional active ingredients include anti-VEGF drugs and hormone drugs.

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