Use of an xor inhibitor for the preparation of a medicament for the prevention or treatment of diabetic retinopathy

Abstract

This invention relates to the application of XOR inhibitors in the preparation of drugs for the prevention and treatment of diabetic retinopathy, belonging to the field of biomedical technology. To address the lack of effective early intervention methods for diabetic retinopathy, this invention provides the application of XOR inhibitors in the preparation of drugs for the prevention and treatment of diabetic retinopathy. The XOR inhibitor is febuxostat or its pharmaceutically acceptable salts, esters, or solvates. Through cell and animal experiments, this invention demonstrates that febuxostat can significantly improve retinal vascular permeability, reduce leakage, repair retinal tissue structure and endothelial cell ultrastructural damage, correct high glucose-induced endothelial cell aging and DNA damage, maintain normal retinal structure and function, delay disease progression, and achieve early and precise intervention for diabetic retinopathy. This provides a new pathway for early clinical prevention and control and has significant application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of XOR inhibitors in the preparation of drugs for the prevention and treatment of diabetic retinopathy. Background Technology

[0002] Diabetic retinopathy (DR) is the most common microvascular complication of diabetes, seriously threatening the visual health and quality of life of diabetic patients. The continuous stimulation of chronic hyperglycemia gradually leads to abnormalities in the structure and function of retinal microvessels. Damage to the blood-retinal barrier (BRB) and abnormally increased microvascular permeability are core pathological features of the early progression of DR. Retinal endothelial cells (RECs), as a core component of the BRB, directly determine the rate of DR occurrence and progression through their functional homeostasis. Early protective interventions targeting retinal endothelial cells are of significant clinical importance in delaying the progression of DR and preventing blindness.

[0003] Currently, existing clinical treatments for diabetic retinopathy (DR) have significant limitations. Mainstream approaches are primarily salvage interventions for advanced stages, including retinal laser photocoagulation and intravitreal injections of anti-vascular endothelial growth factor (VEGF) drugs. These are only suitable for patients with severe non-proliferative or proliferative DR, and are invasive, failing to effectively control DR in its early stages. For early-stage DR patients, there are no specific antiviral drugs; treatment focuses on slowing disease progression through basal metabolic interventions such as blood sugar control, lipid regulation, blood pressure management, and smoking cessation. However, these interventions have limited effectiveness and cannot fundamentally prevent early retinal microvascular damage. Therefore, developing novel intervention programs that target early pathological stages of DR and specifically address retinal microvascular damage is a critical clinical challenge.

[0004] Xanthine oxidoreductase (XOR) belongs to the highly conserved molybdate xanthine family and is widely distributed in various prokaryotes and eukaryotes. Its core physiological function is to participate in the catabolism of purines, acting as a key rate-limiting enzyme to catalyze the oxidation of hypoxanthine and xanthine, ultimately producing uric acid. It is an important functional enzyme in the body's purine metabolic pathway. Research on xanthine oxidoreductase has largely focused on hyperuricemia and gout-related diseases; no studies have yet publicly disclosed its association with the occurrence and development of diabetic retinopathy. Summary of the Invention

[0005] To address the lack of effective early intervention methods for diabetic retinopathy, this invention provides the application of XOR inhibitors in the preparation of drugs for the prevention and treatment of diabetic retinopathy.

[0006] The technical solution of the present invention:

[0007] The application of XOR inhibitors in the preparation of drugs for the prevention and treatment of diabetic retinopathy, wherein the XOR inhibitor is febuxostat or a pharmaceutically acceptable salt, ester, or solvate thereof.

[0008] Furthermore, the XOR inhibitor is febuxostat.

[0009] Furthermore, the drug for preventing and treating diabetic retinopathy is administered orally.

[0010] Furthermore, the dosage of the drug for preventing and treating diabetic retinopathy is 5 mg febuxostat per kg body weight per day.

[0011] Furthermore, the drug for preventing and treating diabetic retinopathy also includes pharmaceutically acceptable carriers, diluents, or excipients.

[0012] Furthermore, the dosage form of the drug for preventing and treating diabetic retinopathy is tablets, capsules, suspensions, or oral liquids.

[0013] Furthermore, the drug for preventing and treating diabetic retinopathy has at least one of the following uses:

[0014] (1) Improves retinal vascular leakage caused by diabetic retinopathy;

[0015] (2) Improves the disordered retinal tissue structure caused by diabetic retinopathy;

[0016] (3) Improves the ultrastructural damage of retinal endothelial cells caused by diabetic retinopathy;

[0017] (4) Downregulate the abnormally high expression of XOR protein in diabetic retinopathy.

[0018] The beneficial effects of this invention are:

[0019] This invention demonstrates through cell and animal experiments that febuxostat can significantly improve retinal vascular permeability, reduce vascular leakage, repair retinal tissue structural disorders and endothelial cell ultrastructural damage, correct endothelial cell aging and DNA damage induced by high glucose, thereby maintaining normal retinal structure and function, avoiding disease progression caused by vascular damage and tissue disorders, reducing the risk of blindness, and achieving early and precise intervention for diabetic retinopathy.

[0020] This invention applies the XOR inhibitor febuxostat to the early intervention of diabetic retinopathy, effectively overcoming the limitation of traditional treatments that only target advanced lesions. This invention uses an oral administration method, which is simple to operate, highly safe, and has good patient compliance. It requires no invasive intervention, filling the technological gap in early intervention for DR and providing a novel technical approach for the early prevention and control of diabetic retinopathy, possessing extremely high clinical application value. Attached Figure Description

[0021] Figure 1 This is an immunofluorescence colocalization map of retinal XOR and endothelial marker CD34 in Example 1;

[0022] Figure 2 The images shown are XOR immunohistochemical staining images of the retina in Example 1. A is a low-power image of the entire retina of the CON group mice, B is a high-power magnified image of the GCL / INL region of the retina of the CON group mice, C is a low-power image of the entire retina of the DR group mice, and D is a high-power magnified image of the GCL / INL region of the retina of the DR group mice.

[0023] Figure 3 This is a Western blot result of XOR protein expression in HRMECs induced by high glucose in Example 1;

[0024] Figure 4 The image shows the immunofluorescence staining results of XOR protein in high glucose-induced HRMECs in Example 1. A is the XOR immunofluorescence staining image in high glucose-induced HRMECs, and B is the quantitative analysis of the average optical density of XOR protein immunofluorescence.

[0025] Figure 5 This is a growth curve diagram of the three groups of cells in Example 2;

[0026] Figure 6 This is a comparison of intracellular ROS levels in three groups of cells in Example 2. A is a histogram of intracellular ROS levels, and B is a comparison of average ROS fluorescence intensity.

[0027] Figure 7 This is a comparison diagram of cell permeability in the three groups of cells in Example 2;

[0028] Figure 8 The images show SA-β-GAL staining images of three groups of cells in Example 2, where A is a microscopic image of SA-β-Gal staining and B is a comparison of relative β-galactosidase activity.

[0029] Figure 9 These are images from the three groups of cell DNA damage comet experiments in Example 2. A is a fluorescence imaging image of the comet experiment, and B is a comparison image of comet tail length.

[0030] Figure 10The images show the morphological verification of the retinal blood vessels and tissue structures of the three groups of mice in Example 3. A is a fluorescence image of FITC-dextran retinal blood vessel permeability detection, B is a light micrograph of HE-stained pathological sections of retinal tissue, and C is an ultrastructural image of retinal endothelial cells under a transmission electron microscope. Detailed Implementation

[0031] The technical solution of the present invention will be further described below with reference to embodiments, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be covered within the protection scope of the present invention. In the following embodiments, the process equipment or apparatus not specifically specified are all conventional equipment or apparatus in the art. Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commercially available; unless otherwise specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0032] All experimental data in this invention were statistically analyzed using GraphPad Prism 8.0 software. Statistical analysis was performed using methods such as one-way ANOVA among three groups and t-test among two groups. A p-value < 0.05 was considered statistically significant.

[0033] Example 1

[0034] This embodiment clarifies the expression characteristics and cellular localization of XOR in diabetic retinopathy.

[0035] I. Immunofluorescence Co-localization Analysis

[0036] After fixing, permeabilizing, and blocking the retinal tissue of the DR mouse model, XOR primary antibody and endothelial marker CD34 primary antibody were incubated. Subsequently, fluorescence staining was performed with Alexa Fluor-labeled secondary antibody and DAPI. Images were acquired using a fluorescence microscope to verify the cellular localization of XOR.

[0037] The results are as follows Figure 1 As shown, XOR and CD34 are highly co-localized, indicating that XOR is mainly located in retinal endothelial cells.

[0038] II. Immunohistochemical Detection

[0039] Retinal sections from DR mice and control CON mice were dewaxed and then subjected to the following steps: citric acid (pH 6.0) antigen retrieval, 3% H2O2 endogenous peroxidase blockade, and serum blocking. XOR primary antibody was then added and incubated overnight at 4°C. After washing with PBS, the sections were incubated with secondary antibody, developed with DAB, and counterstained with hematoxylin. Finally, images were acquired at 400x magnification to detect XOR protein expression levels.

[0040] The results are as follows Figure 2 As shown in the figure, NFL (Nerve Fiber Layer) represents the nerve fiber layer, GCL (Ganglion Cell Layer) represents the ganglion cell layer, IPL (Inner Plexiform Layer) represents the inner plexiform layer, INL (Inner Nuclear Layer) represents the inner nuclear layer, OPL (Outer Plexiform Layer) represents the outer plexiform layer, and ONL (Outer Nuclear Layer) represents the outer nuclear layer. In the low-power image of the full-thickness retina of the CON group mice, brown represents DAB-positive staining for XOR. It can be seen that the CON group only has physiological basic staining in the lowest retinal pigment epithelium layer, and the other nerve layers have almost no obvious positive signal. In the high-power magnification image of the GCL / INL region, only a few cells have very faint XOR-positive staining, indicating the low basal expression level of XOR in the CON control group. In the low-power image of the full-thickness retina of DR group mice, in addition to the retinal pigment epithelium, the neural layers such as GCL and INL also showed obvious brown positive staining. The overall staining intensity was significantly higher than that of the CON group. In the high-power magnification image of the GCL / INL region, a large number of cells showed obvious brownish-yellow XOR positive staining. The staining intensity and the number of positive cells were much higher than those of the CON group, which proved that the expression of retinal XOR protein was significantly increased in the DR state.

[0041] III. In vitro cell experiments

[0042] HRMECs (human retinal microvascular endothelial cells) were seeded in DMEM cell culture medium containing 10% FBS (supplemented with 1% penicillin and streptomycin) and cultured in a 37°C, 5% CO2 cell culture incubator. After adhesion, the cells were divided into a normal glucose NC group and a high glucose-induced HTI (High Glucose + TNFα + IL-6) group. The NC group cells were replaced with DMEM complete medium containing 5.5 mmol / L glucose (10% FBS + 1% penicillin and streptomycin), while the HTI group cells were replaced with DMEM complete medium containing 30 mmol / L glucose (10% FBS + 1% penicillin and streptomycin) + TNFα 1 ng / ml + IL-6 1 ng / ml. After 48 h of culture in a 37°C, 5% CO2 cell culture incubator, XOR protein expression was detected by Western blotting and immunofluorescence staining.

[0043] (1) Western Blot

[0044] Cells from the NC and HTI groups were homogenized after being added to protein lysis buffer. Protein concentration was determined by BCA, followed by metal bath denaturation, and then electrophoresis, membrane transfer, blocking, primary antibody, secondary antibody, and imaging were performed sequentially. The bands were analyzed using ImageJ software.

[0045] The results are as follows Figure 3 As shown, the relative expression level of XOR protein in HRMECs treated with high glucose in vitro was significantly higher than that in the normal glucose group.

[0046] (2) Immunofluorescence staining

[0047] Cells from the NC group and HTI group were fixed, permeabilized, and blocked, respectively. They were then incubated with XOR primary antibody and fluorescent secondary antibody, stained with DAPI, mounted, and images were acquired using a fluorescence microscope. The differences were statistically significant (P<0.05).

[0048] The results are as follows Figure 4 As shown, the XOR fluorescence intensity in HRMECs was significantly enhanced after high glucose treatment, consistent with the WB results, further verifying the high expression characteristic of XOR protein.

[0049] This embodiment uses immunofluorescence, immunohistochemistry, and Western blotting experiments to confirm that the XOR gene and protein are significantly highly expressed in the retina of diabetic retinopathy mice and in high glucose-induced human retinal endothelial cells, and are specifically localized in microvascular endothelial cells. This indicates that XOR is a key gene involved in DR endothelial damage and can serve as an important target for the early prevention and treatment of diabetic retinopathy.

[0050] Example 2

[0051] This study investigated the effect of the XOR inhibitor Febuxostat in improving high glucose-induced retinal endothelial cell dysfunction. Febuxostat was purchased from MCE (Med Chem Express) and the cells were treated with diluted and optimized concentrations according to the manufacturer's instructions.

[0052] I. In vitro cell culture experiment

[0053] HRMECs were seeded in DMEM cell culture medium containing 10% FBS (supplemented with 1% penicillin and streptomycin) and cultured in a 37°C, 5% CO2 cell culture incubator. After adhesion, they were divided into a normal glucose CON group, a high glucose treatment-induced HTI group, and a high glucose + febuxostat intervention group.

[0054] The CON group cells were replaced with DMEM complete medium containing 5.5 mmol / L glucose (10% FBS + 1% penicillin antibody); the HTI group cells were replaced with DMEM complete medium containing 30 mmol / L glucose (10% FBS + 1% penicillin antibody) + TNFα 1 ng / ml + IL-6 1 ng / ml; the high glucose + febuxostat intervention group cells were replaced with DMEM complete medium containing 30 mmol / L glucose + TNFα 1 ng / ml + IL-6 1 ng / ml and 10 μmol / L febuxostat (10% FBS + 1% penicillin antibody); all three groups of cells were continued to be cultured in a 37℃, 5% CO2 cell culture incubator, with the corresponding medium being changed every 2 days.

[0055] II. Cell Count

[0056] HRMECs from the normal glucose CON group, the high glucose-induced HTI group, and the high glucose + febuxostat intervention group were seeded into 96-well plates, and the cell concentration was adjusted to 1×10⁶ cells after digestion. 5 Cells / mL were seeded into 96-well plates, with 6 replicates per group. On days 0, 2, 4, 6, 8, and 10 of culture, 10 μL of LCK-8 reagent was added to each well. After incubation at 37°C in the dark for 2 hours, the absorbance at 450 nm was measured using a microplate reader, converted to cell count, and a growth curve was plotted.

[0057] The results are as follows Figure 5 As shown, cells in the CON group exhibited normal logarithmic proliferation; compared with the CON group, cell proliferation in the HTI group was significantly inhibited, with the cell number on day 10 being only 23% of that in the CON group; compared with the HTI group, cell proliferation in the HTI + febuxostat intervention group was significantly improved (P<0.01), with the cell number on day 10 recovering to 65% of that in the CON group, confirming that febuxostat can effectively reverse the high glucose-induced inhibition of HRMEC proliferation and improve retinal endothelial cell viability.

[0058] III. Detection of Intracellular ROS Levels

[0059] Intracellular reactive oxygen species (ROS) levels were detected using flow cytometry combined with a reactive oxygen species-specific fluorescent probe.

[0060] HRMECs from the normal glucose CON group, the high glucose-induced HTI group, and the high glucose + febuxostat intervention group were seeded into culture dishes, respectively. After culture, ROS probes were added for incubation. After washing, the intracellular fluorescence intensity was detected by flow cytometry to analyze the effect of febuxostat on ROS levels in high glucose-induced HRMECs.

[0061] The results are as follows Figure 6As shown, compared with the CON group, the ROS fluorescence intensity in HRMECs of the HTI group was significantly increased, showing a typical right-shifted distribution, indicating that high glucose stimulation can induce severe oxidative stress in retinal endothelial cells. Compared with the HTI group, the intracellular ROS fluorescence intensity in the HTI+febuxostat group was significantly reduced, the fluorescence peak position was significantly shifted to the left, and the ROS level was significantly decreased (P<0.01). This confirms that febuxostat can effectively reverse the excessive ROS production induced by high glucose by inhibiting XOR activity, reduce oxidative stress damage in endothelial cells, and functionally verify the protective effect of febuxostat against high glucose-mediated endothelial oxidative damage.

[0062] IV. Cell permeability detection

[0063] The barrier function and cell permeability of HRMECs in each group were detected using the Transwell assay.

[0064] HRMECs from the normal glucose CON group, the high glucose treatment-induced HTI group, and the high glucose + febuxostat intervention group were administered at 5 × 10⁻⁶ mg / L. 3 Cells were seeded at a density per well in the upper chamber of a Transwell chamber, and complete culture medium was added to the lower chamber. The chambers were incubated at 37°C in a 5% CO2 incubator until 100% cell confluence and a complete monolayer barrier structure was formed. The culture medium from both chambers was discarded. The chambers were gently washed twice with pre-cooled PBS to remove residual serum. Serum-free DMEM medium containing 1 mg / mL FITC-Dextran (40 kDa) was added to the upper chamber, and an equal volume of serum-free DMEM was added to the lower chamber. The chambers were incubated at 37°C in the dark for 2 hours. After incubation, the lower chamber culture medium was collected, and fluorescence intensity was detected using a multi-mode microplate reader at an excitation wavelength of 480 nm and an emission wavelength of 520 nm. The relative fluorescence intensity of each group was calculated using the fluorescence intensity of the CON group as a baseline.

[0065] The results are as follows Figure 7 As shown, compared with the CON group, the fluorescence intensity of FITC-glucan in the lower chamber of the HTI group was significantly increased, and the relative permeability was significantly upregulated (P<0.01), indicating that high glucose stimulation can significantly damage the barrier integrity of retinal endothelial cells, leading to abnormally increased cell permeability. Compared with the HTI group, the fluorescence intensity in the lower chamber of the HTI+febuxostat group was significantly decreased, and the relative permeability was significantly reduced (P<0.05), confirming that febuxostat can effectively reverse the high glucose-induced increase in endothelial cell permeability and restore the barrier function of retinal endothelial cells, providing direct functional evidence for febuxostat to improve blood-retinal barrier damage in diabetic retinopathy.

[0066] V. Cellular Senescence Detection

[0067] The effect of febuxostat on high glucose-induced senescence of HRMECs was detected by senescence-associated β-galactosidase (SA-β-Gal) staining assay.

[0068] HRMECs from the normal glucose CON group, the high glucose treatment-induced HTI group, and the high glucose + febuxostat intervention group were administered at 1×10⁻⁶ mg / L. 5 Cells were seeded at a density of [number] cells / mL in 6 cm culture dishes and cultured at 37°C in a 5% CO2 incubator until the logarithmic growth phase. The cell culture supernatant was discarded, cells were washed twice with PBS, and SA-β-Gal staining fixative was added and fixed at room temperature for 15 min. The fixative was discarded, cells were washed three times with PBS, and β-galactosidase staining working solution prepared according to the kit instructions was added to each dish. The dishes were incubated overnight at 37°C in a CO2-free incubator. The next day, the staining solution was discarded, cells were washed twice with PBS, and the staining results were observed and images were acquired under an optical microscope. The relative β-galactosidase activity was calculated.

[0069] The results are as follows Figure 8 As shown, compared with the CON group, the proportion of senescent cells with SA-β-Gal positive staining in HRMECs in the HTI group was significantly increased, and the relative β-galactosidase activity was significantly upregulated (P<0.01), indicating that high glucose stimulation can significantly induce senescence of retinal endothelial cells. Compared with the HTI group, the proportion of senescent cells in the HTI+febuxostat group was significantly decreased, and the relative β-galactosidase activity was significantly decreased (P<0.01), confirming that febuxostat can effectively inhibit high glucose-induced endothelial cell senescence and play a significant protective role against high glucose-mediated endothelial cell senescence damage.

[0070] VI. Detection of Cellular DNA Damage

[0071] The protective effect of febuxostat against high glucose-induced DNA damage in HRMECs was detected using a comet assay.

[0072] Take a completely clean glass slide, and lay a 1% normal melting point agarose gel as the first layer. Allow it to solidify naturally at room temperature. Prepare single-cell suspensions of HRMECs from the normal glucose CON group, the high glucose-induced HTI group, and the high glucose + febuxostat intervention group, and adjust the cell concentration to 1×10⁻⁶. 5 cells / mL; Take the cell suspension and mix it thoroughly with 0.8% low melting point agarose gel preheated at 37℃ at a volume ratio of 1:10. Spread it on the first layer of gel as the second layer of cell gel and place it at 4℃ in the dark for 30 minutes to allow it to solidify completely.

[0073] The prepared slides were completely immersed in pre-chilled cell lysis buffer and incubated overnight (≥12h) at 4°C in the dark to allow cell lysis and release of DNA. The next day, the slides were removed and washed three times with pre-chilled PBS to remove residual lysis buffer. The slides were then placed horizontally in an electrophoresis tank, and pre-chilled alkaline electrophoresis buffer (pH>13) was added. Electrophoresis was performed at room temperature in the dark for 20 min. Subsequently, the electrophoresis parameters were adjusted to 25V and 300mA, and electrophoresis was performed for 20-30 min to allow the broken DNA fragments to migrate towards the anode and form a "comet tail". After electrophoresis, the slides were removed and placed in a petri dish. Neutral buffer and Propidium lodide Solution were added sequentially for neutralization and staining. The samples were observed and photographed under a fluorescence microscope. Comet analysis software was used to calculate parameters such as comet tail length and tail moment to quantify the degree of DNA damage.

[0074] The results are as follows Figure 9 As shown, compared with the CON group, the HTI group showed a significant increase in comet tail phenomenon in HRMECs, and the comet tail length was significantly increased (P<0.01), indicating that high glucose stimulation can induce severe DNA damage in retinal endothelial cells. Compared with the HTI group, the HTI+Febu group showed a significant reduction in comet tail phenomenon and a significantly shortened comet tail length (P<0.01), confirming that febuxostat can effectively alleviate high glucose-induced DNA damage in endothelial cells and protect the integrity of the cell genome, providing direct evidence at the DNA damage level for febuxostat to improve high glucose-mediated endothelial oxidative damage.

[0075] Example 3

[0076] This embodiment investigated the ability of the XOR inhibitor febuxostat to treat diabetic retinopathy mice, clarifying its ameliorative effect on DR retinopathy and its related mechanisms.

[0077] I. Laboratory Animals and Group Intervention

[0078] Healthy male C57BL / 6J mice aged 6-8 weeks, weighing 20-25g, were selected and acclimatized for 1 week. A type 1 diabetes model was induced using streptozotocin (STZ): After fasting for 12 hours, the mice were intraperitoneally injected with STZ sodium citrate buffer (pH 4.5) at a dose of 55mg / kg body weight for 5 consecutive days. Seven days after the last injection, mice with a fasting blood glucose level ≥16.7mmol / L were considered to have successfully established a diabetes model.

[0079] Diabetic model mice were randomly divided into two groups: the DR group and the DR + febuxostat group, with normal mice serving as the control group (CON). Ten mice were included in each group. The administration methods are as follows:

[0080] CON group: Administered an equal volume of normal saline by gavage once daily;

[0081] DR group: Administered an equal volume of normal saline by gavage once daily;

[0082] DR+ Febuxostat group: Febuxostat suspension was administered by gavage at a dose of 10 mg / kg body weight once daily for 12 weeks. Mouse body weight and blood glucose were monitored regularly during the intervention period to ensure model stability.

[0083] II. Retinal vascular permeability testing

[0084] Mice in three groups, after 12 weeks of intervention, were anesthetized with isoflurane. The thoracic cavity and right atrial appendage were incised and bloodletting was performed. FITC-dextran solution was slowly injected into the left ventricle. After 5 minutes of circulation, the end of circulation was confirmed by observing blood exudate and skin condition. The eyes were enucleated to prepare a flat retinal specimen. Fluorescent leakage of retinal vessels was observed under a fluorescence microscope, images were acquired, and vascular integrity was analyzed.

[0085] Figure 10 As shown in Figure A, FITC-dextran angiography revealed that the retinal vessels of mice in the CON group were intact, with no obvious fluorescein leakage and normal vascular permeability; the DR group showed extensive perivascular fluorescein leakage in the retina, indicating severe damage to the blood-retinal barrier; after febuxostat intervention, retinal vascular leakage in mice was significantly reduced and vascular integrity was significantly restored.

[0086] III. Pathological examination of retinal tissue by HE staining

[0087] After 12 weeks of intervention, eyeballs were harvested from mice in each group. Eyeball samples were fixed with 4% paraformaldehyde, then sequentially dehydrated with graded ethanol, cleared with xylene, and embedded in paraffin. Tissue sections were cut into 4μm thick slices, dewaxed, hydrated with graded ethanol, and stained with hematoxylin and eosin. After staining, the sections were dehydrated and cleared again, mounted with neutral resin, and images were acquired at 400x magnification under a Nikon microscope to observe retinal tissue structure and cell arrangement.

[0088] Figure 10 The HE-stained pathological sections shown in Figure B in the Chinese version indicate that the morphology and structure of the retina in the CON group mice are normal, with cells arranged neatly and tightly, and the layers are clear; the retina structure of the DR group mice is disordered, with a significant reduction in the number of cells in each layer, loose arrangement, and significantly widened intercellular spaces; febuxostat intervention can significantly improve the structural disorder of the retina in DR mice and restore the tightness of cell arrangement and the integrity of the layers.

[0089] IV. Ultrastructural observation of retinal endothelial cells by transmission electron microscopy (TEM)

[0090] To further clarify the ultrastructural changes, transmission electron microscopy was used to observe mouse retinal endothelial cells.

[0091] After 12 weeks of intervention, eyeballs were harvested from each group of mice. After pre-fixation, the eyeball samples were immediately fixed in 2.5% glutaraldehyde for 24 hours, followed by secondary fixation in 1% osmium tetroxide. The samples were dehydrated using a gradient of ethanol, embedded in epoxy resin, sectioned using an ultramicrotome, and then double-stained with lead citrate and uranium acetate. Finally, the field of view was observed and images were captured under a transmission electron microscope.

[0092] Figure 10 The TEM ultrastructure shown in Figure C indicates that the retinal endothelial cells of the CON group mice were compact in morphology, with uniform nuclear chromatin and intact intercellular connections; the endothelial cells of the DR group mice had a loose matrix, disrupted intercellular connections, and damaged barrier structure; after febuxostat treatment, the endothelial cells regained their compact morphology, intact intercellular connections, and significantly improved ultrastructure.

[0093] The above results suggest that Febuxostat can improve retinal vascular leakage, tissue structure disorder, and ultrastructure in DR mice.

Claims

1. Use of an XOR inhibitor for the preparation of a medicament for the prevention and treatment of diabetic retinopathy, characterized in that, The XOR inhibitor is febuxostat.

2. The application of the XOR inhibitor according to claim 1 in the preparation of drugs for the prevention and treatment of diabetic retinopathy, characterized in that, The medication for preventing and treating diabetic retinopathy is administered orally.

3. The application of the XOR inhibitor according to claim 2 in the preparation of drugs for the prevention and treatment of diabetic retinopathy, characterized in that, The dosage of the drug for preventing and treating diabetic retinopathy is 5 mg febuxostat per kg body weight per day.

4. The application of the XOR inhibitor according to claim 3 in the preparation of drugs for the prevention and treatment of diabetic retinopathy, characterized in that, The drugs for the prevention and treatment of diabetic retinopathy also include pharmaceutically acceptable excipients.

5. The application of the XOR inhibitor according to claim 4 in the preparation of drugs for the prevention and treatment of diabetic retinopathy, characterized in that, The dosage form of the drug for preventing and treating diabetic retinopathy is tablets, capsules, suspensions, or oral liquids.

6. The application of the XOR inhibitor according to claim 5 in the preparation of drugs for the prevention and treatment of diabetic retinopathy, characterized in that, The drug for preventing and treating diabetic retinopathy has at least one of the following uses: (1) Improves retinal vascular leakage caused by diabetic retinopathy; (2) Improves the disordered retinal tissue structure caused by diabetic retinopathy; (3) Improves the ultrastructural damage of retinal endothelial cells caused by diabetic retinopathy.

Images