Application of amantadine in inhibiting ITGAV in preparing medicine for treating dry age-related macular degeneration

Abstract

The invention discloses application of amantadine in inhibition of ITGAV in preparation of medicines for treating dry age-related macular degeneration, and belongs to the technical field of biological medicines. At present, no report for researching the ITGAV gene in the dry AMD exists, the specific action mechanism is not clear, and amantadine adaptation diseases do not include the dry AMD; according to the application disclosed by the invention, the gene target ITGAV is screened from mutual hair generation of microglial cells and RPE cells in a retina microenvironment, and amantadine is used for inhibiting ITGAV protein to relieve the progress of AMD, so that a new thought is provided for treating dry AMD. Experiments show that amantadine can inhibit ITGAV and delay EMT transformation of RPE cells. In a dry AMD mouse model induced by sodium iodate, RPE cell damage can be remarkably relieved by intraocular injection of amantadine, and the structure and function of a retina layer are improved. These results indicate that it may function in early intervention of dry AMD.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of amantadine in inhibiting ITGAV in the preparation of drugs for treating dry age-related macular degeneration. Background Technology

[0002] AMD, or age-related macular degeneration, is a common eye disease. It primarily affects people over 50 years of age and continuously impairs vision, severely impacting their quality of life. With the accelerating aging of my country's population, the number of people suffering from visual impairment due to AMD is expected to increase dramatically, placing a heavy economic burden on society.

[0003] AMD is mainly classified into two types: dry and wet. Dry AMD, also known as non-exudative macular degeneration, accounts for 85% to 90% of all AMD cases. In recent years, the treatment of wet AMD has made significant breakthroughs due to the application of anti-vascular endothelial growth factor drugs. However, there are currently no specific treatments or drugs for dry AMD. Therefore, exploring the pathogenesis of AMD and finding therapeutic targets has become particularly important and urgent. Summary of the Invention

[0004] The purpose of this invention is to provide the application of amantadine in inhibiting ITGAV in the preparation of drugs for treating dry age-related macular degeneration. By targeting the ITGAV protein, amantadine inhibits the function of the ITGAV protein, thereby protecting the vision and retinal structural integrity of animal models of dry AMD.

[0005] The objective of this invention can be achieved through the following technical solutions: This invention provides the use of amantadine to inhibit ITGAV in the preparation of medicaments for the treatment and / or prevention of age-related macular degeneration.

[0006] Furthermore, the amantadine exerts its therapeutic effect by inhibiting ITGAV, which is a therapeutic target for age-related macular degeneration.

[0007] Furthermore, the age-related macular degeneration is dry age-related macular degeneration.

[0008] Furthermore, the treatment and / or prevention of age-related macular degeneration includes maintaining the structural integrity of the retina and / or choroid.

[0009] The present invention also provides a drug for treating and / or preventing age-related macular degeneration, characterized in that the drug has amantadine as the main active ingredient.

[0010] Furthermore, the age-related macular degeneration is dry age-related macular degeneration.

[0011] Furthermore, the drug also includes pharmaceutically acceptable carriers and / or excipients; Furthermore, the dosage form of the drug is an oral preparation, an injectable preparation, a spray, or an ophthalmic preparation.

[0012] Amantadine is a small-molecule synthetic adamantane tricyclic amine, commercially available in hydrochloride or sulfate forms, administered orally in immediate-release or sustained-release formulations and intravenously. Although amantadine has been used for over 50 years, its mechanism of action is not fully understood.

[0013] Disease-associated microglia (DAMs) play a crucial role in the progression of retinal neurodegenerative diseases. Studies have suggested that inhibiting DAMs may be a potential strategy for treating these diseases. However, current research on the role of DAMs in retinal degenerative diseases is still in its early stages, particularly regarding their regulation of retinal pigment epithelium (RPE) cell function. Under specific pathological conditions, RPE cells may undergo epithelial-mesenchymal transition (EMT) and migrate to the neuroretina. These migrating RPE cells typically exhibit low differentiation but retain some pigment deposition capacity. Increased pigmentation in the macular region may originate from a group of RPE cells migrating to this area to compensate for local pigment loss. Furthermore, these migrating RPE cells may undergo mesenchymal-epithelial transition (MET) in the neuroretina, thereby accelerating the pigment deposition process. The specific interactions between retinocyte proliferator-derived retinal cells (RPE cells) and microglia under aging and pathological conditions are crucial for elucidating the pathological mechanisms of age-related macular degeneration (AMD). Their interaction may induce AMD-related inflammatory responses, thereby exacerbating disease progression. With age, microglia undergo functional changes, including decreased phagocytic capacity and increased expression of pro-inflammatory factors, which may be closely related to the occurrence and progression of AMD. Furthermore, RPE cells may migrate and transform within the neuroretina through endogenous metastasis (EMT) and subsequent mesenchymal transition (MET), further influencing the pathological progression of AMD. Elucidating these mechanisms will contribute to a deeper understanding of the development of AMD and provide a theoretical basis for the formulation of relevant treatment strategies.

[0014] ITGAV protein, short for Integrin Subunit AlphaV, also known as CD51, belongs to the integrin α-chain family. Integrins are heterodimeric transmembrane proteins composed of α and β subunits, involved in cell surface adhesion and signal transduction. The ITGAV precursor protein undergoes proteolytic processing to generate light and heavy chains, which together form the αV subunit. This subunit can bind to the β1, β3, β5, β6, and β8 subunits, exerting different functions.

[0015] The beneficial effects of this invention are: 1. There are currently no reports on the study of the ITGAV gene in dry AMD, and the specific mechanism of action is still unclear. Amantadine is not indicated for dry AMD. This invention starts from the interaction between microglia and RPE cells in the retinal microenvironment, screens for the gene target ITGAV, and uses amantadine to inhibit the ITGAV protein to alleviate the progression of AMD, providing a new approach for the treatment of dry AMD.

[0016] 2. This invention demonstrates through experiments that amantadine can inhibit ITGAV and delay EMT transformation in RPE cells. In a sodium iodide-induced dry AMD mouse model, intraocular injection of amantadine significantly reduced RPE cell damage and improved retinal layer structure and function. These results suggest that it may play a role in the early intervention of dry AMD. Attached Figure Description

[0017] The invention will now be further described with reference to the accompanying drawings.

[0018] Figure 1 This is a schematic diagram of the construction of a single-cell retinal atlas in the NaIO3-induced dry AMD model of the present invention; wherein, (A) is a schematic diagram of the experimental procedure; (B) is a cluster analysis of retinal and choroidal cells from three paired control groups and NaIO3-treated mice; (C) is a tSNE plot of immune cell clusters; (D) is a bar chart showing the cell numbers of different immune cell subsets; (E) is a volcano plot showing differentially expressed genes of four immune cell types; (F) is an immunofluorescence image showing the IBA-1 stained retinal region in eye sections of control and NaIO3-treated mice, with white arrows indicating microglia; INL: Inner nuclear layer, OPL: Outerplexiform layer, ONL: Outer nuclear layer, SRS: Subretinal space; (G) is a bar chart showing the number of microglia in the retina of control and NaIO3-treated mice; (H) is a bar chart showing the thickness of the outer nuclear layer in eye sections of control and NaIO3-treated mice. Figure 2 This is a schematic diagram illustrating the identification of DAM-like cells with high Spp1 expression in this invention. (A) is a tSNE diagram showing microglia subpopulations highly expressing Spp1, Ptgds, and Cxcl2; (B) is a tSNE diagram showing the expression of Spp1 in microglia; (C) is Ptgds; and (D) is Cxcl2. (E) is a bar chart showing the proportions of different microglia subpopulations in control and NaIO3-treated mice. (F) is a bubble chart showing the expression levels of genes related to digestion, phagocytosis, and recognition in the three microglia subpopulations; (G) is a bubble chart showing the expression levels of genes related to aged microglia, DAMs, and IRMS in the three microglia subpopulations; (H) is Spp1. + Ptgds + and Cxcl2 + DAM score assessment of microglia; (I) Immunofluorescence images showing the staining of IBA-1 and SPP1 in eye sections of mice in the control and NaIO3-treated groups. (J) The corresponding bar chart, with white arrows indicating Spp1. + IBA-1 + cell; Figure 3 This invention is Spp1 + A schematic diagram of cell-RPE cell interaction via SPP1-ITGAV interaction; where (A) is a schematic diagram of the intercellular communication network of retinal cells in the control group mice; (B) is a schematic diagram of the intercellular communication network of retinal cells in the NaIO3-treated group mice; (C) is a bar chart showing the SPP1-ITGAV interaction in the control and NaIO3-treated groups mice. + Receptor interactions between microglia and other cells; (D) is a bar chart showing Spp1 in control and NaIO3-treated mice. + (E) shows the ligand interactions between microglia and other cells; (F) shows the expression levels of ligands interacting with Spp1 in RPE cells of mice in the control group and NaIO3 treatment group; (G) shows the experimental procedure for SPP1-ITGAV interaction between HMC3 cells and ARPE-19 cells; (H) shows the Western blot results of the immunoprecipitation experiment verifying the interaction between SPP1-ITGAV in ARPE-19 cells and HMC3 cells; (I) shows the Western blot results of the pull-down experiment verifying the interaction between SPP1-ITGAV in ARPE-19 cell lysate and recombinant SPP1 protein. Figure 4This diagram illustrates how antibody inhibition of ITGAV can alleviate visual function impairment caused by EMT in RPE cells. (A) shows HE staining of eye sections from mice in the control group, NaIO3-treated group, and NaIO3+ anti-ITGAV treatment group, with black arrows indicating RPE cells; (B) shows Arrestin immunofluorescence staining of eye sections from mice in the control group, NaIO3-treated group, and NaIO3+ anti-ITGAV treatment group; (C) shows retinal electrophysiological (ERG) waveforms from mice in the control group, NaIO3-treated group, and NaIO3+ anti-ITGAV treatment group; (D) shows a bar chart of statistical analysis of a-wave and b-wave. NS, P≥0.05; P<0.05; P<0.01; P < 0.001; Figure 5 This diagram illustrates the inhibition of dry AMD disease progression by targeting ITGAV with amantadine. (A) shows the molecular docking pattern between amantadine and ITGAV, revealing the potential binding between amantadine and SPP1. (B) is a schematic diagram of the experimental procedure for grouping mice to receive amantadine and NaIO3 (n=6). (C) shows the ERG waveforms of mice in the control group, NaIO3 treatment group, and NaIO3 + amantadine treatment group. (D) is a bar chart of statistical analysis of wave a and wave b. (E) shows HE staining of eye sections from mice in the control group, NaIO3 group, and NaIO3 + amantadine group (scale bar = 50 μm). Data are expressed as mean ± standard deviation (SD). One-way ANOVA was used, followed by Tukey's test to determine statistical significance. NS indicates P ≥ 0.05. This indicates that P < 0.05. This indicates that P < 0.01. This indicates that P < 0.001. Detailed Implementation

[0019] The specific embodiments of the present invention will be described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments.

[0020] Example 1

[0021] The important role of ITGAV in dry AMD was discovered.

[0022] 1. Experimental Methods

[0023] 1.1 Construction of a dry AMD animal model

[0024] A NaIO3 (Sigma, S4007)-induced mouse model was used in this study. Six-week-old wild-type male mice were selected and divided into two groups: a normal control group and a NaIO3 treatment group. The experimental mice received a single intraperitoneal injection of NaIO3 at a dose of 25 mg / kg, while the control group received an equal volume of saline. For SPP1 antibody treatment, mice were first injected intravitreal with SPP1 antibody (1 μg) (R&D, AF808; Bioss, bs-0019R), followed by an intraperitoneal injection of NaIO3 30-60 minutes later. Retinal electrophysiological (ERG) changes were observed in the mice two weeks post-injection. Two weeks later, the mice were euthanized, and their eyeballs were extracted for HE staining.

[0025] 1.2 Single-cell transcriptome library construction and sequencing

[0026] Eyeballs were collected from mice with dry AMD, rinsed with saline, and retinal tissue was separated: (1) Retinal dissection was performed in 1×HBSS at 4°C, and the tissue was transferred to a 5mL centrifuge tube containing 1mL of retinal digestion solution. (2) The tube was then incubated at 8°C for 40 minutes, with gentle mixing every 10 minutes. Then, it was incubated again at 28°C for 10 minutes, with gentle mixing every 5 minutes. After the incubation was completed, the retina, which was still intact, settled to the bottom of the tube. (3) The digestion solution was removed. (4) 700µL of stop solution was added, and the retina was slowly mechanically ground 10 to 15 times. The grinding was stopped when the retina was significantly dissociated. (5) 700µL of pre-cooled retinal washing solution was added to the cell suspension, and the cell was centrifuged at 200g for 5 minutes at 4°C. (7) After removing the supernatant, resuspend the cells in 500 µL of DPBS containing 0.04% BSA and pass them through a 40 μm cell filter for cell counting. Once the viable cell rate reaches 80% or higher, the samples are processed according to the single-cell sequencing kit (10X single-cell sequencing library preparation kit, 10X Genomics, catalog number 1000120). Single-cell library preparation is performed according to the 10×Genomics Chromium Next GEM SingleCell kit. The Reagent Kits v3.1 User Guide is followed. The reagent formulations are as follows: Digestion solution: Add 5.5 mM glucose, 5.7 mM cysteine, and 40 U / ml papain to Hanks buffer, then titrate to pH 6.5 with potassium hydroxide, and incubate at 37°C for 30 minutes. After incubation, add 10 mM HEPES buffer to the solution, titrate to pH 7.4 with KOH, add approximately 120 U / ml DNase I, 10 U / ml superoxide dismutase, 10-25 U / ml catalase, and 0.02 mM D-α-tocopherol acetate, and then filter the solution using a 0.22 μm porous sterile filter. Stop solution: HBSS requires the addition of 11 mM glucose and 10 mM HEPES buffer, titrated to pH 7.4 with KOH. Subsequently, 120 U / ml DNase I, 0.07 mM protease inhibitor (antipain), 10 U / ml superoxide dismutase, 10-25 U / ml catalase, and 0.02 mM D-α-tocopherol acetate were added, and the solution was then filtered through a 0.22 µm porous sterile filter. Washing buffer: HBSS was prepared by adding 11 mM glucose, 0.04% BSA, and 10 mM HEPES buffer, titrating to pH 7.4, and then filtering through a 0.22 µm porous sterile filter.

[0027] 1.3 Obtaining the gene expression matrix and data quality control

[0028] After obtaining the single-cell transcriptome sequencing data, upstream analysis was first performed using the 10×Genomics Cell Ranger software package to generate a single-cell transcriptome expression matrix. Next, the expression matrix was read using the Seurat software package, and the following screening and filtering were performed: genes expressed in at least 3 cells were retained; genes with low expression levels in all cells were removed, retaining only genes expressed in at least 3 cells; cells expressing at least 200 genes were retained; and cells with a mitochondrial gene expression percentage below 0.4 were screened to reduce the impact of mitochondrial genes on subsequent analyses.

[0029] 1.4 Cell Clustering and Visualization

[0030] After upstream analysis and obtaining the gene expression matrix, the DESeq2 software package was used to perform differential gene analysis on different types in the NaIO3 and NC groups. Significantly upregulated / downregulated genes were screened using |Log2FC|≥0.25 and p<0.05 as filtering thresholds. GO and KEGG signaling pathway enrichment analyses were performed, and the top 10 enriched pathways were selected for visualization.

[0031] 1.5 Cell Type Annotation

[0032] The expression matrix was used, retaining at least 3 genes expressed in each cell type, and cells expressing at least 200 genes and those with mitochondrial gene expression percentages below 0.4 for subsequent analysis. Cell clustering and visualization: Principal component analysis was performed using the selected Top 2000 gene set with the highest coefficient of variation. Then, the FindClusters function was used for clustering analysis based on the Top 20 principal components showing significant differential expression across different cell types. The clustering results were visualized using t-SNE and UMAP algorithms. Cell type annotation: The FindAllMarkers function was used to select marker genes for each cell type. Combined with marker genes from databases such as CellMarker, cell type annotation was performed for different subclusters / sites in the whole retinal cell and microglia datasets from the single-cell transcriptome.

[0033] 2. Experimental Results

[0034] 2.1 Establishment of single-cell transcriptome sequencing maps in a dry AMD mouse model

[0035] Single-cell RNA sequencing (scRNA-seq) was performed on ocular tissue from a dry AMD mouse model. Figure 1 A). After scRNA-seq and quality control, we obtained 5,958 retinal cells and 6,087 choroidal cells in the control group and 6,474 retinal cells and 4,593 choroidal cells in the NaIO3 treatment group. Transcriptome analysis identified 12 different cell populations based on specific cell type markers. Figure 1 B), including rod cells (Nr2e3) + ), cone cells (Opn1mw) + ), rod bipolar cells (Sebox) + ), cone bipolar cells (Lhx4) + ), horizontal cells (Calb2) + ), endothelial cells (Cdh5) + ), pericytes (Myh11) + Müller cells (Aqp4) + ), immune cells (Cd52) + ), non-long-processed neurons (Slc6a9) + ), retinal pigment epithelial (RPE) cells (Rpe65) + Schwann cells (Stx1b) + ), fibroblasts (Col1a1) + ), astrocytes (Slc1a3) + ) and ganglion cells (Scn7a +Notably, RPE cells were significantly reduced in the NaIO3-treated group, a key characteristic of the NaIO3-induced dry AMD model, validating its effectiveness. Furthermore, the immune cell population significantly increased in the NaIO3-treated group, suggesting that immune cells may play an important role in the pathogenesis of dry AMD. We performed statistical analysis on the number and distribution of the four immune cell types (…). Figure 1 D). Among these immune cell populations, microglia were the most numerous and significantly increased in the NaIO3 treatment group. This phenomenon suggests that microglia, as immune cells, may play an important role in the pathogenesis of AMD and warrants further investigation. Figure 1 E presents a volcano plot of four immune cell types, highlighting the three genes with the most significant differential expression. Notably, SPP1 showed the highest upregulation in microglia. To validate the findings of scRNA-seq, namely the increased number of microglia, we performed immunofluorescence staining experiments. Frozen sections were prepared from ocular tissues of control and NaIO3-treated mice, and microglia were labeled with IBA-1. The results confirmed that the number of microglia was significantly increased in the retina of NaIO3-treated mice. Figure 1 F and G). Furthermore, we observed structural disorder in the retina of the NaIO3-treated group, with a significant thinning of the outer nuclear layer (ONL). Figure 1 These results suggest that microglia may be closely related to the occurrence and development of retinal degeneration.

[0036] 2.2 Discovery of disease-associated microglia with high SPP1 expression

[0037] Given the heterogeneity of microglia, we re-extracted and clustered the microglia population, dividing it into three distinct subpopulations. Each subpopulation was named based on its most specifically expressed gene. The most expressed genes in these three subpopulations were Spp1, Ptgds, and Cxcl2, respectively; therefore, we named them Spp1-highly expressed microglia (Spp1...). high MG), Ptgds-overexpressing microglia (Ptgds) high MG) and Cxcl2-highly expressed microglia (Cxcl2) high MG) Figure 2 A). By analyzing the expression levels of Spp1, Ptgds, and Cxcl2, we observed significant differences in the expression of these genes among different cell populations. Figure 2BD). Further comparison of the proportions of these three microglia subsets in the control group and the NaIO3-treated group showed that Spp1 high Microglia showed the most significant increase after NaIO3 treatment. Figure 2 E). Because this subpopulation is significantly increased in the AMD model, we focused our study on it. Previous research has shown that microglia play an important phagocytic role in retinal degeneration; therefore, we analyzed three gene sets related to phagocytosis: "find-me," "eat-me," and "digest-me." The results showed that these genes are present in Spp1... high The upregulation trend was observed in all microglial subsets. Figure 2 F). Since disease-associated microglia (DAMs) exhibit enhanced phagocytic pathway activity in neurodegenerative diseases, we hypothesize that Spp1... high Microglia may belong to the category of retinopathy-associated microglia (DAMs). To test this hypothesis, we analyzed the expression of genes associated with DAMs. The results showed that DAM-related genes are expressed in Spp1. high Significantly upregulated in microglial subsets ( Figure 2 G), while genes associated with aging and type I interferon response in microglia (IRMs) did not show a significant increase in this subpopulation. To further validate this, we compared the expression scores of DAMs characteristic genes in different microglia subpopulations and found that Spp1 high Microglia showed significantly higher DAMs characteristic expression scores than other cell subsets. Figure 2 H). To further confirm this finding, we performed immunofluorescence staining experiments, labeling microglia with IBA-1 and SPP1 antibodies. In the control group, only a very small number of microglia expressed Spp1, while in the NaIO3-induced retinal injury model, Spp1 was abundant. high Microglia increased significantly ( Figure 2 (I and J). This result not only validated our sequencing data, but also suggests that Spp1 high Microglia may play an important role in retinal degeneration.

[0038] 2.3 Interaction between ITGAV receptor and SPP1 in microglia was discovered in RPE cells under AMD background.

[0039] To further explore Spp1 highTo understand the role of microglia subpopulations in the retinal microenvironment, we performed cell-cell communication analysis. Using chord diagram visualization, we plotted the interaction network between three microglia subtypes and other retinal cell types. Figure 3 (A and B), and calculated and compared the control group with NaI. The strength of cell communication between treatment groups. Results showed that in NaI... After processing, Spp1 high Communication between microglia and other retinal cells was significantly enhanced, with both the number and intensity of interactions increasing markedly. We further explored Spp1... high Communication patterns of microglia in the retinal microenvironment, particularly their role as receptor providers ( Figure 3 C) and ligand providers ( Figure 3 The role of D). Analysis revealed that, compared with the control group, Spp1 high Microglia have a more significant impact on RPE cells, fibroblasts, and pericytes, highlighting their importance in regulating retinal pathological processes. Given that the protein encoded by Spp1 is a secretory protein capable of acting as an extracellular ligand, and given that Spp1... high SPP1 is expressed most highly in microglia, leading us to hypothesize that it may be a key mediator of microglia-retinal cell interactions. We focused on analyzing the interaction of SPP1 as a ligand with retinal cell receptors. Based on the ranking of interaction strength, we identified several integrin receptors (Itga8, Itga9, Itga4, Itgb6, Itgb5, Itgb1, Cd44, Itga5, Itgav), which are mainly distributed in retinal cells such as RPE cells. Figure 3 E). Considering NaI Treatment significantly reduced the number of RPE cells, and given the important role of RPE cells in the progression of AMD, we further investigated the integrin receptor that interacts with SPP1 in RPE cells. Analysis showed that Itgav expression was highest in RPE cells and significantly increased in the NaIO3 treatment group (…). Figure 3 F). This finding suggests that SPP1-ITGAV interaction may be a key mechanism in the progression of AMD retinal pathology. To verify this hypothesis, we first performed protein-protein interaction experiments. ARPE-19 cells (representing RPE cells) and HMC3 cells (representing microglia) were cultured separately, and their cell lysates were extracted and subjected to co-immunoprecipitation (co-IP) experiments. Figure 3 G). Experimental results confirmed that SPP1 and ITGAV physically interact in both cell types. Figure 3H). Since HMC3 cells co-express SPP1 and ITGAV, there is a possibility of intracellular interaction. To rule out this possibility, we further performed a pull-down experiment, co-incubating recombinant SPP1 protein with ARPE-19 cell lysate. The results further confirmed a strong interaction between SPP1 and ITGAV. Figure 3 I).

[0040] In summary, these findings suggest that Spp1 plays a crucial role in retinal pathological settings. high Disease-associated microglia (DAMs) can communicate with RPE cells through SPP1-ITGAV interactions, a process that may play a key role in the progression of AMD.

[0041] Example 2

[0042] Intraocular injection of amantadine inhibits the progression of AMD.

[0043] 1. Experimental Methods

[0044] 1.1 Intravitreal injection

[0045] Mice were deeply anesthetized via intraperitoneal injection, and topical anesthetic was applied to the eye to be injected. The mice were then transferred to a dissecting microscope, and mydriatic medication was instilled into the eye. Forceps were used to slide between the upper and lower eyelids to expose the sclera. A pre-incision was made behind the limbus using a 30G needle. A 33G injection needle filled with injection fluid was inserted into the pre-incision, with the tip penetrating approximately 1 mm, and a suitable amount of fluid was injected smoothly. After injection, the needle was held for approximately 5 seconds to prevent leakage, and then slowly withdrawn. Antibiotic eye ointment was applied to the cornea and injection area to prevent infection.

[0046] 1.2 Visual electrophysiological examination (ERG) detection

[0047] Prepare the stroboscopic stimulator, amplifier, and animal testing platform. The experiment should be conducted in a dark room, using dim red light for illumination to maintain the darkness. The experimental procedure is as follows: Administer an intraperitoneal injection to mice to achieve deep anesthesia. Secure the mice to the animal testing platform and dilate their pupils to ensure full pupil dilation. Fix the mice in front of the animal testing platform, ensuring both eyes are at the same height and fully exposing the eyes. Connect the electrodes to the skin of the mouse's tail and neck. Adjust the angle of the gold ring electrodes so that they gently contact the apex of the cornea. Connect the positive electrode of channel one to the right eye and the positive electrode of channel two to the left eye. Ensure that both gold ring electrodes contact the same apex of the cornea in both eyes at the same angle and manner. Push the animal testing platform with the mice into the stroboscopic stimulator. Ensure that the mice's eyes are fully exposed within the stroboscopic stimulator. Start the program to record data.

[0048] 1.3 Frozen Slicing Procedure

[0049] After removing the eyeball, fix it for 5 minutes. Dissect the eyeball, separate the cornea, lens, and vitreous body to form an eye cup, and fix it in 4% paraformaldehyde for 1 hour. Wash twice in 1ml 1XPBS for 10 minutes each time. Equilibrate in 15% sucrose PBS solution at 4°C until it settles. Transfer to 30% sucrose PBS solution and equilibrate for 2-3 hours until it settles. Add the mold with the optic nerve facing one side. Immerse the cryogenic mold in a metal beaker containing isopropane for at least five minutes, then place the beaker in liquid nitrogen to one-third of its height. If liquid nitrogen is unavailable, freeze at -20°C first, then at -80°C. Remove the frozen block, wrap it in aluminum foil, and store it at -80°C, recording the tissue orientation with a pen. After adjusting the cryostat to between -20°C and -25°C, place the mold containing the embedded eye cup inside and allow it to equilibrate with the cryogenic temperature for one hour. Remove the microtome and begin cutting 10-micrometer-thick serial sections. Carefully attach the retinal portion to the labeled slide and store it in a slide cassette at -20°C or -80°C until ready for immunofluorescence.

[0050] 1.4 Immunofluorescence procedure

[0051] ① Permeabilization: Incubate the sample with PBS (containing 0.1% Triton X-100) for 10 minutes. Wash the cells with PBS 3 times, 5 minutes each time.

[0052] ② Blocking: Block with PBS containing 5% BSA at room temperature for 1 hour. Wash cells three times with PBS for 5 minutes each time.

[0053] ③ Antibody incubation: Primary antibody incubation: IBA-1 antibody (Wako, 019-19741) was added to 1% BSA in PBS at a dilution of 1:500 and incubated overnight at 4°C. Cells were washed three times with PBS for 5 minutes each time. Secondary antibody incubation: Rabbit secondary antibody (abcam, ab6721) was added to 1% BSA in PBS at a dilution of 1:500 and incubated at room temperature for 2 hours. Cells were washed three times with PBS for 5 minutes each time.

[0054] ④ Staining: Tyramide labeled with Alexa Fluor™ 488 (B40953) was added to the incubation of specific proteins using the Tyramide SuperBoost Kit (Thermo, B40922) to enhance the immune signal.

[0055] Prepare the dye according to the following proportions.

[0056] After preparation, add 100 μL to the tablet and incubate for 5 minutes.

[0057] ⑤ Wash the cells three times with PBS, 5 minutes each time. Observe the staining under a fluorescence microscope.

[0058] ⑥ Antibody elution: Preheat antibody elution buffer to 37°C, add 100 μL of antibody elution buffer to cover the sample, and incubate at room temperature for 3-5 minutes; discard the elution buffer; add another 200 μL of elution buffer to cover the sample (the amount of elution buffer should be enough to slightly bulge the slide, generally 150-300 μL), and incubate at 37°C for 40 minutes; wash 3 times with PBS.

[0059] ⑦ Repeat the above steps: Incubation with the new primary antibody: Incubate overnight at 4°C using SPP1 antibody (Abclonal, A23658). 3. Secondary antibody incubation: Add rabbit secondary antibody (abcam, ab6721) at a dilution of 1:500 to 1% BSA in PBS and incubate at room temperature for 2 hours. Staining: Add Alexa Fluor™ 647 (Thermo, B40958): to induce red fluorescence in the target protein.

[0060] ⑧ Nuclear staining: Finally, Hoechst 33342 (Sigma, AATB-17535) nuclear staining is performed to stain the cell nucleus blue, which is used to locate the cell nucleus.

[0061] 2. Experimental Results

[0062] 2.1 Intraocular injection of ITGAV antibody inhibits visual impairment in dry AMD mice

[0063] To evaluate whether ITGAV could serve as a potential therapeutic target, we conducted in vivo experiments, blocking the interaction between SPP1 and ITGAV molecules by intravitreal injection of an ITGAV antibody. Subsequently, mice were treated with NaIO3 to induce retinal damage, and their retinal structure and visual function were assessed. The specific experimental procedures were as follows: Two days before NaIO3 injection, a specific antibody was injected intravitreally, followed by intraperitoneal injection of NaIO3. Fourteen days later, visual function was assessed using ERG, and mouse eye tissue was collected for histological analysis. HE staining was used to analyze retinal structure (…). Figure 4 A). Compared with the PBS-injected control group, the NaIO3-treated group showed impaired retinal structure, with irregular and fragmented RPE layers. However, in mice treated with ITGAV antibodies, the retinal structure was relatively normal, and the RPE layer showed greater continuity and integrity, significantly superior to the IgG control group. Since RPE layer abnormalities can lead to photoreceptor cell damage and ultimately vision loss, we used Arrestin staining to label photoreceptor cells and analyzed their morphology and function. The results showed ( Figure 4In group B, Arrestin expression was significantly reduced in the NaIO3 treatment group, while SPP1 antibody treatment effectively halted this reduction. Furthermore, NaIO3 treatment led to the loss of photoreceptor cells in the outer nuclear layer (ONL), particularly near the abnormal RPE cell region. SPP1 antibody treatment effectively protected ONL thickness, indicating that inhibiting SPP1 can delay retinal degeneration and maintain the integrity of retinal structure. The primary purpose of protecting retinal structure is to maintain visual function. Therefore, we performed ERG analysis on mice in different treatment groups (…). Figure 4 (C and D) The PBS-injected control group showed normal ERG signaling, while the NaIO3-treated group showed a significant decrease in ERG signaling. In mice treated with ITGAV antibodies, the decreasing trend in a-wave and b-wave amplitudes was effectively halted, suggesting improved scotopic visual function. Statistical analysis further confirmed that ITGAV antibody treatment restored retinal function, as evidenced by the increase in a-wave and b-wave amplitudes. These results indicate that in vivo inhibition of ITGAV can protect the morphology and function of RPE cells and photoreceptor cells, highlighting the potential of ITGAV as a therapeutic target for dry AMD.

[0064] 2.2 Screening revealed that the ITGAV inhibitor amantadine prevented visual impairment in dry AMD mice.

[0065] To screen small molecule compounds targeting ITGAV, we used the ConPLex method to screen the FDA-approved drug database. Eight small molecules predicted to potentially interact with ITGAV were identified and ranked according to their ConPLex scores. Further molecular docking analysis was performed using AutoDock to assess the binding energy and interaction modes between these compounds and ITGAV. Among the candidate compounds, ITGAV was found to bind to amantadine via hydrophobic interactions through PHE-51, PHE-189, TYR-254, PHE-308, and TYR-436. Figure 5 A). The binding free energy calculated by docking simulations was -4.3 kcal / mol, with a lower limit of RMSD of 0.809 Å and an upper limit of 1.373 Å (optimal mode), indicating a strong binding affinity between amantadine and ITGAV. Next, we evaluated the therapeutic effect of amantadine on a mouse model of dry AMD. After administration of NaI... Two days ago, amantadine (25 mg / kg) was injected intravitreally. Figure 5 B). ERG records showed that amantadine treatment in NaI Induced damage resulted in partial preservation of visual function. Figure 5C and D). Histological analysis stained with hematoxylin and eosin (HE) showed that amantadine alleviated NaIO3-induced retinal thinning and RPE layer disorder (C and D). Figure 5 E).

[0066] These results suggest that adamantane may interfere with the progression of AMD by modulating the downstream signaling pathway of ITGAV.

[0067] The above-disclosed embodiments are merely a few specific examples of the present invention. However, the embodiments of the present invention are not limited thereto, and any variations that can be conceived by those skilled in the art should fall within the protection scope of the present invention.

Claims

1. The use of amantadine to inhibit ITGAV in the preparation of drugs for the treatment and / or prevention of age-related macular degeneration.

2. The application according to claim 1, characterized in that, The amantadine exerts its therapeutic effect by inhibiting ITGAV, which is a therapeutic target for age-related macular degeneration.

3. The application according to claim 1, characterized in that, The age-related macular degeneration mentioned is dry age-related macular degeneration.

4. The application according to claim 1, characterized in that, The treatment and / or prevention of age-related macular degeneration includes maintaining the structural integrity of the retina and / or choroid.

5. A medicament for treating and / or preventing age-related macular degeneration, characterized in that, The drug has amantadine as its main active ingredient.

6. The drug according to claim 5, characterized in that, The age-related macular degeneration mentioned is dry age-related macular degeneration.

7. The drug according to claim 5, characterized in that, The drug also includes pharmaceutically acceptable carriers and / or excipients.

8. The medicament according to claim 7, characterized in that, The dosage form of the drug is an oral preparation, an injectable preparation, a spray, or an ophthalmic preparation.

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