Use of diclofenac in the preparation of a medicament for the treatment of diabetic retinopathy

Abstract

This invention provides the application of exifone in the preparation of drugs for treating diabetic retinopathy. Exifone binds near the lactation modification site of HDAC1 K412, thereby specifically inhibiting the lactation modification of HDAC1 at the K412 site. This leads to enhanced deacetylation activity of HDAC1 on H3K56, thereby inhibiting H3K56ac-mediated VEGFA transcriptional activation and pathological angiogenesis, and thus improving the vascular pathological damage of diabetic retinopathy. This application provides a new target and candidate drug for the clinical treatment of diabetic retinopathy. The research results of this application provide experimental evidence that exifone can be used as a clinically specific treatment for diabetic retinopathy.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the use of ethoxyphenone in the preparation of medicaments for the treatment of diabetic retinopathy. Background Technology

[0002] Diabetic retinopathy (DR) is a hallmark microvascular complication of diabetes and a leading cause of vision impairment. Its driving factors are hyperglycemic metabolic reprogramming and aberrant angiogenesis transcription. The core pathological feature of DR progression is aberrant angiogenesis, where glucose homeostasis disturbances exacerbate microvascular dysfunction through complex metabolic-epigenetic interactions. Although the pathogenesis of DR is not fully understood, recent evidence suggests that hyperglycemia partially promotes retinal vascular dysfunction by aberrantly activating the transcriptional processes of angiogenesis genes. Aberrant transcriptional activation of VEGFA is a key step driving pathological angiogenesis.

[0003] Currently, inhibiting the VEGF signaling pathway is the main strategy for the clinical treatment of DR. However, existing drugs have limitations in efficacy, differences in patient tolerance, and safety concerns regarding long-term use. For example, repeated intraocular injections can lead to poor compliance, drug resistance in some patients, and the risk of ocular inflammation. Therefore, there is an urgent need to develop new therapeutic targets and specific drugs. Summary of the Invention

[0004] Based on this, the purpose of this invention is to propose the application of exifenone in the preparation of drugs for treating diabetic retinopathy, so as to develop new therapeutic targets and specific drugs for the treatment of diabetic retinopathy.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] The use of exifone in the preparation of drugs for the treatment of diabetic retinopathy.

[0007] Exicophenone can bind near the lactation modification site of HDAC1 K412, thereby specifically inhibiting the lactation modification of HDAC1 at the K412 site. This leads to HDAC1 enhancing its deacetylation activity on H3K56, thereby inhibiting H3K56ac-mediated VEGFA transcriptional activation and pathological angiogenesis, and thus improving the vascular pathological damage of diabetic retinopathy.

[0008] Beneficial effects:

[0009] 1. This application demonstrates through in vitro cell experiments that treatment with ethoxybenzone significantly reduces the lactation level at the HDAC1 K412 site induced by high glucose or lactate stimulation in a dose-dependent manner. Simultaneously, the acetylation level of its downstream signaling molecule H3K56ac and the protein expression of VEGFA are also inhibited accordingly.

[0010] 2. This application verified the effect of ethoxybenzone on inhibiting retinal angiogenesis and reducing vascular VEGFA expression by constructing an HDAC1-K412A gene knock-in STZ-induced diabetic mouse model;

[0011] 3. This application found through chicken chorioallantoic membrane (CAM) experiments that injection of ethoxybenzone can significantly inhibit the formation of new blood vessels in developing embryos.

[0012] 4. This application discovers that the small molecule drug exifone can inhibit abnormal angiogenesis in diabetic mice. Further investigation reveals that exifone specifically inhibits the lactation modification of HDAC1 K412, which leads to enhanced deacetylation activity of HDAC1 on H3K56, thereby downregulating VEGFA transcription and ultimately inhibiting pathological retinal angiogenesis. Animal experiments show that exifone effectively alleviates STZ-induced retinal neovascularization in a diabetic mouse model. Therefore, this application provides a new target and candidate drug for the clinical treatment of diabetic retinopathy. The results of this application provide experimental evidence that exifone can be used as a specific clinical treatment for diabetic retinopathy. Attached Figure Description

[0013] Figure 1 This is a schematic diagram illustrating how ethoxybenzone inhibits retinal angiogenesis and VEGFA expression in a diabetic mouse model.

[0014] Figure 2 Schematic diagram of immunoprecipitation of Flag-HDAC1 transfected cells after treatment with different glucose concentrations (0 mM, 12 mM, 25 mM, 50 mM) and Western blot analysis of HDAC1 lactation using lactation antibody.

[0015] Figure 3 This is a schematic diagram of the direct identification results of the lactation modification at the HDAC1 K412 site based on high-resolution mass spectrometry.

[0016] Figure 4 This is a schematic diagram illustrating the changes in H3K56ac levels under different concentrations of L-lactic acid (0 mM, 0.5 mM, 1 mM, 2 mM, 4 mM, 8 mM) detected by Western blot. β-actin was used as an internal control.

[0017] Figure 5 This is a schematic diagram of Western blot analysis for detecting different glucose concentrations (5 mM, 25 mM) and H3K56ac levels under HDAC1 knockdown, with β-actin as an internal control.

[0018] Figure 6 This diagram illustrates the Western blot analysis of H3K56ac levels in L-lactate (L-lac)-treated and untreated HDAC1 wild-type (WT) transfected or HDAC1 mutant (K412R) transfected cells. H3 is the internal control for H3K56ac, and β-actin is the internal control for the whole protein.

[0019] Figure 7 This diagram illustrates the Western blot analysis of H3K56ac levels in D-lactate (D-lac)-treated and untreated HDAC1 wild-type (WT) transfected or HDAC1 mutant (K412R) transfected cells. H3 is the internal control for H3K56ac, and β-actin is the internal control for the whole protein.

[0020] Figure 8 Venn plot analysis of the overlap between upregulated genes shown by RNA-seq analysis and H3K56ac binding genes shown by CUT&Tag analysis enriched a total of 207 genes.

[0021] Figure 9 Bubble graphs for KEGG pathway enrichment analysis show the enrichment of genes in lipids and atherosclerosis, the MAPK signaling pathway, and endoplasmic reticulum protein processing.

[0022] Figure 10 GO analysis showed that the enriched genes were enriched in the response to low oxygen levels, the response to hypoxia (biological function), and chromosomes / endoplasmic reticulum (cellular components).

[0023] Figure 11 This is a volcano plot of RNA-seq data.

[0024] Figure 12 This is a graph of CUT & Tag data.

[0025] Figure 13 This is a schematic diagram of the relative expression of VEGFA under different sugar concentrations analyzed by ChIP-qPCR, including the Highglucose and Low glucose groups (*** p<0.001).

[0026] Figure 14 This diagram illustrates the Western blot analysis of VEGFA, p-eNOS, p-iNOS, AKT, and HDAC1 levels in L-lactate (L-lac)-treated and untreated si-NC (negative control) or si-HDAC1 (HDAC1 knockdown) transfected cells. β-actin serves as the total protein internal control.

[0027] Figure 15 Figure 1 shows the results of scratch (wound healing) experiments and migration statistics for HDAC1 wild-type (WT) and K412R mutant cells.

[0028] Figure 16 This serves as a molecular docking model for exifone with the HDAC1 protein, demonstrating that exifone can bind near its lactation modification site.

[0029] Figure 17 This diagram illustrates the Western blot analysis of HDAC1 K412lac, H3K56ac, and VEGFA levels in cells treated with different concentrations of exifone (0 μM, 2 μM, 4 μM, 8 μM, 10 μM). HDAC is the internal control for HDAC1 K412lac, and β-actin is the internal control for total protein.

[0030] Figure 18 This is a schematic diagram of the structure of the wild-type allele and the target allele (HDAC1K412 site) in the mouse CRISPR-Cas9 system.

[0031] Figure 19 The image shows the metabolic phenotype results of wild-type (WT) and HDAC1-K412A knock-in (KI) mice, including body weight (A), glucose tolerance (B), insulin sensitivity (C), and pyruvate tolerance (D). "ns" indicates no statistically significant difference, *P<0.05.

[0032] Figure 20 This is a schematic diagram of the experimental procedure for constructing a diabetes model and evaluating the retina in Hdac1-K412A knock-in (KI) mice. Day 1-Day 5: STZ injection to induce diabetes (50 mg / kg, intraperitoneal injection daily), Day 5-Day 12: blood glucose monitoring (blood glucose level ≥16.7 mM), Day 12: retinal evaluation.

[0033] Figure 21 The fluorescent images show schematic diagrams of retinal vessels in wild-type (WT) and Hdac1 K412A knock-in (KI) mice under different treatments (Vehicle / STZ). Collagen IV (red, marking the vascular basement membrane), Isolectin B4 (green, marking vascular endothelial cells), and Merge (red and green-yellow co-localized areas represent structurally normal vascular basement membranes, with white arrows indicating areas of typical pathological changes).

[0034] Figure 22To quantitatively analyze the difference in the number of cell-free capillaries between HDAC1 WT and KI mice in the STZ-induced diabetic retinopathy group, "ns" represents no statistically significant difference, *P<0.01.

[0035] Figure 23 This is a schematic diagram of the experimental procedure for establishing a diabetes model, drug intervention, and retinal assessment in C57BL / 6J mice. Day 1-Day 5: STZ injection to induce diabetes (50 mg / kg, intraperitoneal injection daily); Day 5-Day 12: Blood glucose monitoring (blood glucose level ≥16.7 mM); Day 12-Day 19: Exifone drug intervention (50 mg / kg, intraperitoneal injection, for 7 days); Day 19: Retinal assessment.

[0036] Figure 24 The fluorescent images show the retinal vessels of C57BL / 6J mice under different treatments (Vehicle / STZ) and in the untreated and treated exifone groups. Collagen IV (red, marking the vascular basement membrane), Isolectin B4 (green, marking vascular endothelial cells), and Merge (the yellow co-localized area of ​​red and green represents the structurally normal vascular basement membrane, and the white arrow is used to indicate the typical pathological changes).

[0037] Figure 25 To provide a quantitative statistical analysis of the effect of exifone on the STZ-induced diabetic retinopathy model in mice, "ns" indicates no statistically significant difference, ****P<0.0001.

[0038] Figure 26 To quantitatively analyze the difference in relative VEGFA expression between the untreated and treated exifone groups in STZ-induced mice, **P<0.01.

[0039] Figure 27 The retinal vessel morphology observation diagram shows the effect of exifone injection (8ng / kg or 40ng / kg) on ​​the formation of new blood vessels in early chicken embryonic development. The top row is an overall view of the retinal vessels, and the bottom row is a magnified view of the area within the box in the top row. White triangles are used to mark the small structural points around the vessels.

[0040] Figure 28 To provide a quantitative statistical analysis of the differences in Nb connectivity (number of vascular connectivity structures) under different concentrations of exifone (8 ng / kg, 40 ng / kg and DMSO control) (*P<0.05). Detailed Implementation

[0041] To facilitate understanding of the present invention, a more complete description will be given below with reference to various embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0043] Research has revealed that lactate-derived lysine lactation (Kla) plays a crucial role in cellular function regulation as a novel epigenetic regulatory mechanism. HDAC1, a key chromatin remodeling enzyme, is essential for embryonic development and angiogenesis. In the nucleus, HDAC1 influences chromatin structure and gene expression by regulating histone acetylation (e.g., H3K9ac, H3K27ac, and H3K56ac), thereby regulating the transcription of angiogenesis-related genes. Studies have confirmed that hyperglycemia can induce lactation modification of HDAC1 at the K412 site. HDAC1 K412 lactation leads to the loss of its deacetylase activity, ultimately resulting in the accumulation of H3K56 acetylation (H3K56ac), activating VEGFA transcription and promoting pathological angiogenesis. Notably, under physiological conditions, only L-lactate can serve as a donor for HDAC1 K412 lactation; D-lactate does not have this regulatory effect. This specificity provides an important basis for targeted therapy. These findings suggest that HDAC1 K412 lactation is a key molecular node linking hyperglycemic metabolic disorders with angiogenesis in diabetic diabetics, providing a potential therapeutic target for vascular complications of diabetes.

[0044] Exifone was initially used to improve cognitive impairment in elderly patients. Its mechanism of action includes activating neuronal glucose and oxygen metabolism, scavenging free radicals, and regulating the serotonin and dopamine pathways. Recently, it has been discovered to be a mixed, non-essential HDAC1 activator, capable of binding to both free and substrate-binding enzymes, thereby increasing the relative maximum rate of HDAC1-catalyzed deacetylation. However, whether exifone can treat diabetic retinopathy remains unclear.

[0045] This invention provides the use of ethoxyphenone in the preparation of a medicament for treating diabetic retinopathy. Please refer to [link to relevant documentation]. Figure 1Exicophenone can bind near the lactation modification site of HDAC1 K412, thereby specifically inhibiting the lactation modification of HDAC1 at the K412 site. This leads to HDAC1 enhancing its deacetylation activity on H3K56, thereby inhibiting H3K56ac-mediated VEGFA transcriptional activation and pathological angiogenesis, and thus improving the vascular pathological damage of diabetic retinopathy.

[0046] The present invention will now be described in detail.

[0047] 1. Methods and Materials

[0048] 1.1 Antibodies, chemicals, plasmids, and kits

[0049] Anti-HDAC1 antibody (Abcam, Cat# ab280198), anti-HDAC1 K412 lactation-specific antibody (PTMBIO, custom-made), anti-H3K56ac antibody (Cell Signaling, Cat# 4243), anti-VEGFA antibody (Abcam, Cat# ab32152), anti-β-actin antibody (Sigma-Aldrich, Cat# A5441), anti-H3 antibody (Abcam, Cat# ab176842)

[0050] 1.2 Cell Culture

[0051] Human umbilical vein endothelial cells (HUVECs) were cultured in Gibco endothelial cell growth medium containing 2% fetal bovine serum (FBS) and growth factors. HEK293T cells were cultured in DMEM (Gibco) supplemented with 10% FBS (Gibco) and 1% penicillin-streptomycin. Both cell lines were incubated at 37°C under humidified conditions of 5% carbon dioxide. Cells were passaged to 80%–90% confluence using 0.25% trypsin-EDTA, and all experiments used HUVECs from passages 3–7.

[0052] 1.3 Immunoblotting

[0053] Protein samples were separated by electrophoresis on a 10% SDS-PAGE gel under reducing conditions, transferred to a PVDF membrane, and blocked with 3% (w / v) bovine serum albumin (BSA, Sigma-Aldrich). After incubation with primary antibody, HRP-labeled anti-IgG antibody (Cell Signaling Technology) was added, and finally, detection was performed using an ECL kit (Thermo Fisher Scientific) on a chemiluminescence imaging system (Bio-Rad).

[0054] 1.4 Cell migration experiment

[0055] Wild-type and mutant HDAC1 cells were stored at 3 × 10⁶ cells per well. 4 Cells were seeded at a density of 100 μL in the upper chamber using 100 μL of serum-free DMEM medium. 600 μL of DMEM medium containing 10% fetal bovine serum (FBS) was added to the lower chamber. After incubation for 10 hours, unmigrated cells were carefully removed from the membrane. For single-cell populations, cells that had migrated in the lower chamber were washed with PBS, fixed, stained with crystal violet, and finally counted using an optical microscope.

[0056] 1.5 CUT & Tag Analysis of H3K56ac in HUVECs Endothelial Cells

[0057] The experiments were conducted at 37°C / 5% CO2 in either low-glucose medium (LG, 5 mM glucose) or high-glucose medium (HG, 25 mM glucose). In the CUT&Tag experiment, 2 × 10⁶ cells were collected when cell confluence reached 80%. 5 Cells were washed with PBS and then bound to concanavalin A-coated magnetic beads (Bangs Laboratories, BP531) in binding buffer (20 mM HEPES, pH 7.5, 10 mM potassium chloride, 1 mM calcium chloride, 1 mM manganese chloride). In situ antibody labeling and tagging were performed. Permeabilized cells were incubated overnight at 4°C with anti-H3K56ac primary antibody (Cell Signaling Technology, #4243; 1:100 dilution in digitalis saponin buffer). After washing, the samples were incubated with guinea pig anti-rabbit secondary antibody (Antibodies Online, ABIN101961; 1:100) at room temperature for 1 hour, followed by incubation with protein A-Tn5 fusion protein in digitalis saponin buffer (0.01% digitalis saponin, 20 mM HEPES, pH 7.5, 150 mM sodium chloride) at room temperature for 1 hour. Tagging was initiated by adding 10 mM MgCl2 and incubating at 37°C for 1 hour. All experimental steps, including library construction, Illumina sequencing, and downstream bioinformatics processing, were performed by Novogene Co., Ltd. (Beijing, China) under quality control conditions according to standardized operating procedures (SOPs). Data were uploaded to the GEO database (accession number: GSE305252).

[0058] 1.6 Real-time Polymerase Chain Reaction (PCR)

[0059] Total RNA was extracted from cells using TRIzol reagent (Invitrogen). Following the manufacturer's instructions, 2 µg of total RNA was reverse transcribed into complementary DNA (cDNA) using the PrimeScript™ FASTRT kit (Takara Inc.). Quantitative PCR (qPCR) reactions were performed in triplicate using SYBR Green Real-Time PCR Premix (Roche) and run on an ABI 7500 Real-Time PCR system. The following primers were used for RT-qPCR analysis:

[0060] Upstream primer: CCCAGTTTTGGGAACACCGA.

[0061] Downstream primer: CCCCAAAGCACAGCAATGTC.

[0062] 1.7 RNA Extraction and RNA-seq

[0063] Total RNA was isolated from cell / tissue samples using TRIzol reagent (Invitrogen, 15596026) according to the manufacturer's protocol. In short, samples were homogenized in TRIzol (per 1 × 10⁻⁶). 6 (1 mL of 50 mg tissue or 1 mL of chloroform) was added, and the mixture was incubated at room temperature for 5 minutes. After adding chloroform (0.2 mL / mL TRIzol), the phases were separated by centrifugation at 12,000 × g (15 minutes, 4 °C). The aqueous phase was mixed with isopropanol (1:1 v / v), incubated at room temperature for 10 minutes, and then centrifuged (12,000 × g, 10 minutes, 4 °C). The RNA precipitate was washed twice with 75% ethanol, air-dried, and dissolved in RNase-free water. RNA quality was verified by spectrophotometry, Qubit quantification, and Bioanalyzer analysis. For RNA-seq, HUVECs were cultured at 37 °C / 5% CO2 in low-glucose or high-glucose medium when they reached 80%–90% confluence. Total RNA was isolated according to the RNA extraction protocol. All experimental procedures, including library construction, Illumina sequencing, and downstream bioinformatics processing, were performed by Novogene Ltd. (Beijing, China) under quality control conditions according to standardized operating procedures (SOPs). The data was uploaded to the GEO database (ID: GSE305251).

[0064] 1.8 Molecular docking analysis

[0065] The HDAC1 protein structure was obtained using AlphaFold2, and the exifone small molecule structure was obtained using PubChem. Molecular docking was then performed using AutoDock software. Next, OpenBable was used in the Autodock suite to convert the small molecule structure into PDB format so that Autodock could dock the molecules. Binding energy, the number of hydrogen bonds formed, and root mean square deviation (RMSD) were used as indicators for screening results. Finally, the results were presented in different formats using the visualization software Pymol.

[0066] 1.9 Immunofluorescence

[0067] HUVECs were fixed with 4% paraformaldehyde (15 min, room temperature), permeabilized with 0.1% Triton X-100 (10 min), and blocked with 5% BSA / 0.3% glycine (1 h). Primary antibody (e.g., anti-HDAC1) was incubated overnight at 4°C. Secondary antibody labeled with Alexa Fluor 647 / 488 (1:1,000) and DAPI (1 μg / mL) were added sequentially. Z-axis stacked images were acquired at 0.5 μm intervals using a Zeiss confocal microscope (63x oil immersion), and signal quantification was performed using a blinded analysis protocol with ImageJ software.

[0068] 1.10 Immunoprecipitation

[0069] Cells were lysed in RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM sodium chloride, 1% NP-40) containing a protease / phosphatase inhibitor (Roche). Lysates (1 mg) were pre-cleaned with Protein G Dynabeads (Thermo Fisher Scientific) at 4°C for 1 h, followed by overnight incubation with 5 μg of anti-HA or anti-Flag antibody or IgG control. Magnetic beads were washed five times with ice-cold high-salt buffer (300 mM sodium chloride) and eluted in 2× Laemmli buffer (95°C, 5 min). Precipitated proteins were separated by 4%–20% SDS-PAGE (Bio-Rad Laboratories) and analyzed by Western blotting using antibodies.

[0070] 1.11 Chromatin grading cultured cells (HUVEC cells)

[0071] After washing with ice-cold PBS, cells were lysed on ice for 15 min in hypotonic buffer (10 mM HEPES pH 7.9, 10 mM potassium chloride, 1.5 mM MgCl2, 0.5% NP-40, 0.5 mM DTT, protease inhibitor). Nuclei were precipitated by centrifugation at 720 × g for 5 min at 4 °C, and the supernatant was retained as the cytoplasmic fraction. Nucleoproteins were extracted using high-salt buffer (20 mM HEPES pH 7.9, 400 mM sodium chloride, 25% glycerol, 0.2 mM EDTA), followed by sonication (3 × 10 sec pulses) and clarification at 16,000 × g. Fraction purity was verified by Western blotting using histone H3 (nuclear marker) and GAPDH (cytoplasmic marker). All procedures were performed at 4 °C using pre-cold buffer.

[0072] 1.12 Construction of Hdac1-K412A gene knock-in mice

[0073] Donor vectors carrying homologous arms were microinjected into the cytoplasm of C57BL / 6JGpt mouse zygotes along with a CRISPR / Cas9 ribonucleoprotein complex (containing sgRNA and high-fidelity Cas9 protein). Viable embryos were surgically transferred into the oviducts of pseudopregnant ICR surrogate females. Genomic DNA was extracted from tail and toe tissues of F0 generation pups collected on days 5–7 postnatally. Site-specific primers were used for target site amplification. The amplified products were analyzed using Sanger sequencing and T7 endonuclease I assay to verify insertion / deletion formation and homology-directed repair-mediated knock-in events. Genetically validated F0 generation founder individuals were mated with wild-type C57BL / 6JGpt mates. F1 generation offspring were biopsied on days 5–7 postnatally, and genotyped using the aforementioned PCR / sequencing methods. Strains demonstrating germline transmission and expected Mendelian proportions were amplified for subsequent analysis.

[0074] 1.13 Hdac1-K412A Knock-in Mouse Experiment Procedure

[0075] Eight-week-old Hdac1-K412A gene knock-in mice (wild-type control included) were selected and acclimatized for one week. On day 1 of the experiment, mice were fasted for 12 hours and injected intravenously with 50 mg / kg STZ (pre-cooled citrate buffer, freshly prepared). Glucose solution was administered after injection to prevent hypoglycemia. From day 5 onwards, blood glucose was monitored (≥16.7 mM indicated a diabetic model), along with body weight, glucose tolerance, insulin sensitivity, and pyruvate tolerance. On day 12, mice were sacrificed, and retina was harvested for angiogenesis assessment using fluorescence imaging and CD31 staining. Detailed experimental procedures are provided below. Figure 20 .

[0076] 1.14 C57BL / 6J mouse exifone intervention experiment procedure

[0077] Eight-week-old C57BL / 6J mice were selected. On day 1, a diabetic model was established by intravenous injection of 50 mg / kg STZ. Blood glucose was monitored from day 5 (≥16.7 mM was considered successful). Starting from day 12, the model mice were intraperitoneally injected with 50 mg / kg Exifone for 7 consecutive days. On day 19, the mice were sacrificed, and the retina was harvested. The density of neovascularization and the expression level of VEGFA were assessed by vascular staining and VEGFA immunohistochemistry.

[0078] 1.15 CAM angiogenesis experiment

[0079] First, the fertilized eggs of Qingjiao Ma chickens were weighed and incubated in an incubator at 37.8℃ and 60% humidity. To prevent the embryos from attaching to the egg membrane, the eggs needed to be turned more than twice a day. The first day the embryos were placed in the incubator was designated as day 0 (ED0). Before the experiment, the location of the air cell of the embryo was determined by candle positioning. After sterilizing the area above the air cell, a hole was drilled and sealed. From ED7 to ED9, the embryos were treated with different doses of exifone (0, 8 ng / kg, 40 ng / kg; n≥3) according to the established protocol. Images were taken 48–72 hours after treatment, and the blood vessel nodes were counted and analyzed using ImageJ software. Significant errors were eliminated through manual verification.

[0080] 2. Results

[0081] 2.1 Exifone inhibits HDAC1 K412 lactation and regulates the expression of downstream molecules.

[0082] Please see Figure 2 This application demonstrates that HDAC1 undergoes significant lactation under high glucose conditions, with the level of emulsification increasing with increasing glucose concentration. Mass spectrometry analysis also confirms this result, showing lactation modification at the K412 site, such as... Figure 3 As shown, the emulsification level at the K412 site changed significantly. H3K56ac showed a specific and concentration-dependent increase after L-lactic acid treatment, while other acetylation sites were unaffected, such as... Figure 4 As shown. To verify whether H3K56ac is regulated through HDAC1 lactation modification, the applicant found that inhibiting HDAC1 could eliminate the high glucose-induced increase in H3K56ac, as shown. Figure 5 As shown. More importantly, L-lactic acid significantly increased H3K56ac levels in cells expressing wild-type HDAC1, but had no such effect in cells expressing the K412R lactylation-deficient mutant, such as... Figure 6As shown. It is noteworthy that D-lactic acid did not exhibit this K412-dependent regulatory role, as... Figure 7 As shown.

[0083] To further investigate whether the H3K56ac binding gene has a functional association with angiogenesis, the applicant performed integrated RNA sequencing and cut & tag sequencing under high-glucose and low-glucose conditions. Using integrated RNA sequencing and cut & tag data, 207 overlapping targets were identified, such as... Figure 8 As shown, these targets are significantly enriched in lipid metabolism, atherosclerosis, MAPK signaling, and endoplasmic reticulum protein processing pathways, such as... Figure 9 As shown. GO analysis revealed relevant biological processes, including hypoxia responses and cellular components such as chromosomes and endoplasmic reticulum, such as... Figure 10 As shown. Transcriptome mapping and ChIP qPCR confirmed that the promoter region of VEGFA showed upregulated expression and enhanced enrichment of H3K56ac modification, such as... Figures 11 to 13 As shown, where, Figure 11 Volcano plot analysis of RNA-seq data showed that VEGFA transcription was significantly upregulated under high glucose conditions. Figure 12 CUT & Tag data analysis showed that the binding reads of H3K56ac in the VEGFA promoter region were significantly increased under high glucose conditions. Figure 13 The schematic diagram of relative VEGFA expression under different sugar concentrations, obtained by ChIP-qPCR analysis, verifies the increased binding of the VEGFA promoter peak H3K56ac. Inhibition of HDAC1 can block L-lactic acid-induced upregulation of VEGFA protein, such as... Figure 14 As shown. Meanwhile, the K412R lactylation-deficient mutant of endothelial cells exhibited stronger cell migration ability, such as... Figure 15 As shown.

[0084] HDAC1 inhibits VEGFA expression by deacetylation of H3K56. This inhibition is eliminated when HDAC1 is lactated at the K412 site, leading to H3K56ac accumulation, VEGFA transcriptional activation, and subsequent endothelial cell proliferation. Molecular docking results show that exifone can form a stable binding region near the HDAC1 K412 site, interacting with surrounding amino acid residues through hydrogen bonds, such as... Figure 16 As shown, the binding of exifone to HDAC1 is dependent on the domain containing the K412 site. Immunoblotting results showed that exifone treatment reduced intracellular HDAC1 lactation modification, H3K56ac, and VEGFA levels in a dose-dependent manner. Figure 17As shown, hyperglycemia induces the accumulation of H3K56ac and VEGF transcriptional activation, thereby increasing pathological retinal angiogenesis.

[0085] 2.2 Exifone can inhibit abnormal angiogenesis in the retina of diabetic mice.

[0086] The applicant constructed an Hdac1-K412A gene knock-in mouse model, such as Figure 18 As shown. In the STZ-induced diabetes model, these mutant mice did not show significant changes in body weight and pyruvate tolerance, but exhibited poorer glucose tolerance and stronger insulin sensitivity, such as... Figure 19 As shown. Meanwhile, compared to the control group, these mutant mice exhibited significantly reduced retinal neovascularization, such as... Figure 21 and Figure 22 As shown.

[0087] like Figure 23 As shown, after the successful establishment of the STZ-induced diabetic mouse model, the results showed that after 8 weeks of treatment with exifone, compared with the control group, the retinal vascular morphological disorders in the exifone-treated group were significantly improved. Figure 24 As shown; the number of retinal neovascularizations is significantly reduced, such as Figure 25 As shown; VEGFA expression levels decreased, as Figure 26 As shown.

[0088] 2.3 Exifone can significantly inhibit the formation of new blood vessels in developing embryos.

[0089] To investigate whether this mechanism applies to physiological angiogenesis, experiments using the chicken chorioallantoic membrane (CAM) revealed that injection of exifone significantly inhibited the formation of new blood vessels in developing embryos. Figure 27 and Figure 28 As shown.

[0090] In summary, this application demonstrates that exifone inhibits pathological angiogenesis and improves the pathological damage of diabetic retinopathy by specifically binding to the region near the HDAC1 K412 site, inhibiting its lactation modification, enhancing the deacetylation activity of H3K56, reversing hyperglycemia-induced H3K56ac accumulation and VEGF transcriptional activation. As a potential HDAC1 lactation inhibitor, exifone exhibits good in vitro and in vivo activity and safety, providing a new drug option and target for the clinical treatment of diabetic retinopathy.

[0091] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. Application of ethoxyphenone in the preparation of drugs for the treatment of diabetic retinopathy.

2. The application according to claim 1, characterized in that, Exicophenone can bind near the lactation modification site of HDAC1 K412, thereby specifically inhibiting the lactation modification of HDAC1 at the K412 site. This leads to HDAC1 enhancing its deacetylation activity on H3K56, thereby inhibiting H3K56ac-mediated VEGFA transcriptional activation and pathological angiogenesis, and thus improving the vascular pathological damage of diabetic retinopathy.

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