Use of dihydroxyqueoside in the preparation of a drug for preventing or treating diabetic retinopathy

Abstract

The application discloses application of dihydrogen baicalin in preparation of a medicine for preventing or treating diabetic retinopathy, belongs to the field of medicine, and relates to a new use of a known compound. The application requests protection of application of dihydrogen baicalin or a pharmaceutically acceptable salt thereof in preparation of a medicine for preventing or treating diabetic retinopathy. The application finds that dihydrogen baicalin can effectively improve the proliferation activity of retinal pigment epithelial cells in an in-vitro model of diabetic retinopathy, reduce the expression level of VEGF, antagonize angiogenesis, and then improve diabetic retinopathy; in an in-vivo model of diabetic retinopathy, dihydrogen baicalin can effectively improve the retinal thickness and pathological damage. Therefore, dihydrogen baicalin or the pharmaceutically acceptable salt thereof has a prospect of being developed into a medicine for preventing or treating diabetic retinopathy.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field and relates to new uses of known compounds, specifically the use of dihydrobaicalin in the preparation of drugs for the prevention or treatment of diabetic retinopathy. Background Technology

[0002] Diabetic retinopathy (DR) is an eye disease caused by damage to the microvessels of the retina due to diabetes, and it is one of the leading causes of blindness in working adults. Long-term hyperglycemia continuously damages the retinal microvessels, leading to a series of complications including microaneurysms, retinal hemorrhages, hard exudates, cotton wool spots, and vascular occlusion. As the disease progresses, it can induce abnormal neovascularization in the retina, further causing vitreous hemorrhage and tractional retinal detachment, resulting in irreversible visual impairment. In the International Classification of Diseases, 10th Revision (ICD-10), diabetic retinopathy is primarily classified as a complication of diabetes, with its code attached to the main category of diabetes (E10.3, E11.3, etc.), reflecting its status as a secondary damage to systemic diabetes. However, in the International Classification of Diseases, 11th Revision (ICD-11) published by the World Health Organization, the classification system for this disease has been significantly adjusted, classifying diabetic retinopathy as an independent retinal disease entity under Chapter 9, Diseases of the Visual System – Posterior Segment Disorders – Retinal Disorders.

[0003] Drug treatment for diabetic retinopathy primarily involves intraocular local administration. Systemic hypoglycemic agents can only slow disease progression and cannot reverse local retinal damage. Anti-VEGF biologics are first-line clinical treatments, administered intravitreally to inhibit the vascular endothelial growth factor pathway, improve diabetic macular edema, and inhibit pathological neovascularization. However, this therapy requires repeated intraocular injections, posing a heavy treatment burden, a risk of intraocular infection, and poor response in some patients. Glucocorticoids can be used as second-line anti-inflammatory therapy to relieve macular edema, but they have side effects such as increased intraocular pressure and cataract induction. Currently, there is a lack of effective local treatments for early-stage lesions, and existing treatment methods still have significant limitations. There is an urgent need to develop safer, more convenient, and more effective drugs or formulations.

[0004] Dihydrobaicalin is a naturally occurring dihydroflavonoid glycoside (flavonoid glycoside), CAS number 56226-98-3, molecular formula C2. 21 H 20 O 11It has a molecular weight of 448.38. Structurally, it is a reduction product of the C-2,3 double bond of baicalin—the parent nucleus changes from a flavonoid to a dihydroflavonoid (flavanone) form, while the β-D-glucuronic acid group remains attached at position 7. Currently, there are no reports of dihydrobaicalin exhibiting preventative or therapeutic activity against diabetic retinopathy. Summary of the Invention

[0005] The present invention aims to provide the use of dihydrobaicalin in the preparation of medicaments for the prevention or treatment of diabetic retinopathy.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution:

[0007] This invention provides the use of dihydrobaicalin or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention or treatment of diabetic retinopathy.

[0008] In one specific embodiment, the medicament comprises dihydrobaicalin or a pharmaceutically acceptable salt thereof for the prevention or treatment of diabetic retinopathy, and pharmaceutically acceptable excipients, formulated into a pharmaceutically acceptable dosage form.

[0009] In one specific embodiment, the medicament further comprises one or more other ingredients for the prevention, treatment, or adjunctive prevention or treatment of diabetic retinopathy.

[0010] In one specific embodiment, the components for preventing, treating, or adjunctive preventing or treating diabetic retinopathy are selected from anti-vascular endothelial growth factor drugs, glucocorticoid drugs, antioxidants, and neurotrophic factors or neuroprotective agents. Specifically, anti-vascular endothelial growth factor drugs primarily target the core pathological feature of diabetic retinopathy—abnormal neovascularization of the retina—and can effectively inhibit excessive vascular proliferation and reduce retinal hemorrhage and exudation; glucocorticoid drugs can strongly inhibit local inflammatory responses in the fundus, alleviate retinal edema, and reduce inflammation-mediated microvascular damage; antioxidants can scavenge excess free radicals in the fundus, alleviate oxidative stress damage to retinal tissue, and block the core inducing factor of disease progression; neurotrophic factors or neuroprotective agents can target and repair damaged retinal nerve cells, improving retinal nerve function deficits.

[0011] In one specific embodiment, the anti-vascular endothelial growth factor drug is bevacizumab, ranibizumab, aflibercept, conbercept, or brogluzumab. All of these drugs are first-line targeted therapies used in clinical practice for the treatment of retinal vascular diseases, with clear pharmacological mechanisms and clinical safety and efficacy verified through numerous clinical trials. Among them, ranibizumab and conbercept are targeted drugs specifically for local retinal administration, possessing high affinity and sufficient local drug concentrations, precisely blocking vascular endothelial growth factor-mediated abnormal angiogenesis and rapidly improving symptoms of retinal exudation, hemorrhage, and neovascularization in patients with diabetic retinopathy. Bevacizumab, aflibercept, and brogluzumab have broad-spectrum targets and long-lasting effects, effectively inhibiting abnormal microvascular proliferation and delaying the progression of severe diabetic retinopathy and proliferative retinopathy. Combining them with dihydrobaicalin or its pharmaceutically acceptable salts can further enhance preventative or therapeutic effects.

[0012] In one specific embodiment, the glucocorticoid drug is dexamethasone, triamcinolone, fluocinolone, or prednisolone. These drugs possess potent and broad-spectrum anti-inflammatory and anti-edema effects. By inhibiting the release of inflammatory factors in retinal tissues and blocking the inflammatory cascade, they can rapidly alleviate typical symptoms of diabetic retinopathy, such as macular edema, retinal tissue swelling, and inflammatory exudation, effectively improving clinical manifestations such as blurred vision and decreased visual acuity. Dexamethasone has a strong anti-inflammatory effect and rapid onset of action, making it suitable for acute intervention in acute inflammatory attacks and severe edema. Triamcinolone has a long-lasting effect and good sustained-release properties, making it suitable for long-term control of chronic inflammation. Fluocinolone and prednisolone have low irritation and high safety, making them suitable for long-term local administration to the retina. Combined with dihydrobaicalin or its pharmaceutically acceptable salts, they can effectively avoid the problems of lesion aggravation and irreversible visual impairment caused by continuous inflammatory damage.

[0013] In one specific embodiment, the antioxidant is vitamin C, vitamin E, taurine, or lipoic acid. The development of diabetic retinopathy is closely related to an imbalance of oxidative stress in the fundus. A high-sugar environment induces the production of a large number of oxygen free radicals in retinal tissue, continuously damaging microvascular endothelial cells and nerve cells, which is a key factor in the progression of the disease. Vitamin C and vitamin E are classic water-soluble and fat-soluble antioxidant vitamins that can synergistically scavenge free radicals in the fundus, enhance the antioxidant capacity of retinal tissue, and protect the integrity of microvascular endothelium. Taurine can stabilize the retinal cell membrane structure, reduce oxidative damage, and improve fundus microcirculation. Lipoic acid, as a potent broad-spectrum antioxidant, can simultaneously scavenge water-soluble and fat-soluble free radicals, reverse high-sugar-induced oxidative stress damage, and block the pathological progression of retinopathy. When combined with dihydrobaicalin or its pharmaceutically acceptable salts, it can further enhance the antioxidant efficacy of the drug and improve the preventive or therapeutic effects.

[0014] In one specific embodiment, the neurotrophic factor or neuroprotective agent is brain-derived neurotrophic factor, ciliary neurotrophic factor, or retinal pigment epithelial-derived factor. Diabetic retinopathy not only involves microvascular complications but also progressive retinal nerve cell damage, apoptosis, and neurological degeneration, which is a major cause of irreversible visual impairment in patients. Brain-derived neurotrophic factor can promote the survival of retinal ganglion cells and repair damaged synaptic function; ciliary neurotrophic factor can inhibit retinal nerve cell apoptosis and delay retinal neurodegenerative diseases; retinal pigment epithelial-derived factor can simultaneously exert multiple effects such as neuroprotection, inhibition of abnormal angiogenesis, and antioxidation, effectively repairing retinal pigment epithelial cell damage. These three components can precisely compensate for the shortcomings of ordinary drugs that only target vascular lesions and neglect nerve damage. Combined with dihydrobaicalin or its pharmaceutically acceptable salts, vascular protection and nerve repair can be achieved simultaneously.

[0015] In one specific embodiment, the excipients are selected from one or more of solid excipients, liquid excipients, and semi-solid excipients. Solid excipients include starch, microcrystalline cellulose, lactose, dextrin, magnesium stearate, crospovidone, etc., mainly used for the shaping, disintegration, filling, and lubrication of oral solid dosage forms and solid ophthalmic dosage forms. Liquid excipients include purified water, water for injection, glycerin, propylene glycol, ethanol, etc., mainly used as solvents, co-solvents, and humectants in liquid dosage forms, injectable dosage forms, and ophthalmic liquid dosage forms. Semi-solid excipients include petrolatum, lanolin, polyethylene glycol, carbomer, etc., mainly used for the matrix shaping of semi-solid dosage forms such as ophthalmic gels, ointments, and transdermal preparations. All excipients meet pharmacopoeia standards, are highly safe, have no toxic side effects, and can be effectively adapted to different formulation manufacturing processes, ensuring the stability, homogeneity, and safety of the drug.

[0016] In one specific embodiment, the dosage form is selected from ophthalmic preparations, injectable preparations, oral preparations, inhaled preparations, and transdermal preparations. Ophthalmic preparations include eye drops, ophthalmic gels, ointments, and ophthalmic microspheres, which can directly act on lesions in the fundus, achieving high local drug concentrations, rapid onset of action, and minimal systemic side effects, making them suitable for local symptomatic treatment of mild to moderate diabetic retinopathy. Injectable preparations include ocular injections and intravenous injections, which are rapidly absorbed and precisely targeted, suitable for emergency intervention in moderate to severe proliferative diabetic retinopathy. Oral preparations include tablets, capsules, and granules, which are convenient to take and have high compliance, suitable for long-term preventative medication and maintenance treatment of chronic diseases. Inhaled and transdermal preparations offer gentle and minimally invasive administration methods, making them suitable for special patient groups who cannot tolerate ophthalmic or injectable drug administration.

[0017] Beneficial effects:

[0018] This invention reveals that dihydrobaicalin can effectively enhance the proliferative activity of retinal pigment epithelial cells, reduce VEGF expression levels, and antagonize angiogenesis in an in vitro model of diabetic retinopathy, thereby improving diabetic retinopathy. In an in vivo model of diabetic retinopathy, dihydrobaicalin can effectively improve retinal thickness and pathological damage. Therefore, dihydrobaicalin or its pharmaceutically acceptable salts hold promise for development into drugs for the prevention or treatment of diabetic retinopathy. Attached Figure Description

[0019] Figure 1 This is a comparison of the relative proliferative activity of retinal pigment epithelial cells in each group in Example 1;

[0020] Figure 2 The left figure shows a comparison of VEGF protein expression levels in retinal pigment epithelial cells of different groups in Example 1 (Western blot analysis, right figure shows quantitative bar graph).

[0021] Figure 3 Comparison of blood glucose levels in each group of mice in Example 2;

[0022] Figure 4 A compares the optical coherence tomography (OCT) results of mice in different groups in Example 2; B compares the ILM-OPL thickness of the inner retinal layer of mice in different groups in Example 2; C compares the ILM-RPE thickness of the full-thickness retina of mice in different groups in Example 2.

[0023] Figure 5 For comparison of HE staining results of the mouse retina in each group in Example 2;

[0024] Figure 6 This is a comparison of the relative proliferative activity of retinal pigment epithelial cells in each group in Example 3;

[0025] Figure 7 The comparison of VEGF protein expression levels in retinal pigment epithelial cells of different groups in Example 3 (left figure is Western blot, right figure is quantitative bar chart). Detailed Implementation

[0026] The following describes the substantive content of the present invention in detail with reference to embodiments, but this does not limit the scope of protection of the present invention.

[0027] Example 1: Cell model based on retinal pigment epithelial cells (ARPE-19)

[0028] I. Experimental Materials

[0029] Retinal pigment epithelial cells (ARPE-19, Cell Bank of Chinese Academy of Sciences).

[0030] Dihydrobaicalin (DBA, Chengdu Pusi Biotechnology Co., Ltd., 56226-98-3);

[0031] 10× PBS (Kage, KGB50011);

[0032] South American fetal bovine serum (FBS, Gibco, 10270-106).

[0033] Pancreatic enzyme (Gibco, 25200072);

[0034] DMEM / F12 medium (containing double antibiotics) (KGI, KGL1204-500);

[0035] Dimethyl sulfoxide (DMSO, Solarbio, 67-68-5);

[0036] Calcium dobesilate (CaD, Hangzhou Taoshu Biotechnology Co., Ltd., 20123-80-2).

[0037] D-(+)-glucose (D-Glu, Sigma, 50-99-7);

[0038] MTT Cell Proliferation Detection Kit (APExBIO, K2249).

[0039] PMSF (Shanghai Beyotime Biotechnology Co., Ltd., ST506);

[0040] Phosphatase inhibitor (Shanghai Beyotime Biotechnology Co., Ltd., P1087).

[0041] Anti-VEGFA antibody (Abcam, ab46154).

[0042] Red Loading Buffer Pack (CST, 7723).

[0043] II. Experimental Methods

[0044] 1. Preparation of DBA solution

[0045] The compound dihydrobaicalin was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution, which was then diluted with culture medium to the required concentration for the experiment.

[0046] 2. Cell Culture

[0047] ARPE-19 cell lines were cultured in DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin / streptomycin, and passaged in a conventional incubator at 37°C and 5% CO2. Experiments were conducted using F5-F15 generation cells.

[0048] 3. MTT assay for cell proliferation activity of retinal pigment epithelial cells

[0049] (1) Take ARPE-19 cells in good growth condition, resuspend them in DMEM / F12 medium containing 10% FBS, and seed them in 96-well plates with 100 μL of cell suspension per well (approximately 3000-4000 cells per well). Divide the cells into a normal group (Control), a model group (60 mM D-Glu), a calcium dobesilate group (10 μM CaD group, high glucose + 10 μM CaD), a low-dose dihydrobaicalin group (10 μM dihydrobaicalin group, high glucose + 10 μM DBA), a medium-dose dihydrobaicalin group (20 μM dihydrobaicalin group, high glucose + 20 μM DBA group), and a high-dose dihydrobaicalin group (30 μM dihydrobaicalin group, high glucose + 30 μM DBA). After culturing in a 37℃, 5% CO2 incubator for 24 h, the normal group was replaced with fresh medium, and the model group was replaced with DMEM / F12 medium containing 60% FBS. The fresh medium containing mM D-Glu was used for the calcium dobesilate group, while the fresh medium containing 60 mM D-Glu and 10 μM CaD was used for the calcium dobesilate group. The fresh medium containing 60 mM D-Glu and 10 μM, 20 μM, and 30 μM DBA was used for the different doses of dihydrobaicalin groups, respectively.

[0050] (2) After culturing in groups for 48 h, add 10 μL of MTT working solution to each well and then incubate in a cell culture incubator for 4 h.

[0051] (3) Add 100 μL of Formazan Solvent to each well, mix well, and incubate in a cell culture incubator for 3 hours.

[0052] (4) The absorbance (A) at 570 nm was measured using an ELISA reader. The relative cell proliferation activity was obtained by calculating the ratio of the absorbance of the drug-treated group to that of the control group.

[0053] 4. Western blot detection of VEGF expression in retinal pigment epithelial cells

[0054] (1) Take ARPE-19 cells in good growth condition, resuspend them in DMEM / F12 medium containing 10% FBS, and seed the cells in 6-well plates at 2×10⁶ cells per well. 5 Each cell / 2 mL, the modeling and drug administration procedures are the same as in "3. MTT assay of retinal pigment epithelial cell proliferation activity" in this example.

[0055] (2) Lyse cells and extract proteins using SDS loading buffer containing protease inhibitors and phosphatase inhibitors, boil in a metal bath at 100°C for 15 min, and cool for later use.

[0056] (3) Prepare a dodecyl sulfate-polyacrylamide gel electrophoresis to separate the protein and transfer it to a nitrocellulose membrane. Block with 5% skim milk at room temperature for 3 h, add primary antibody and incubate overnight at 4°C. On the second day, wash 3 times with TBST, incubate with secondary antibody at room temperature for 2 h, wash 3 times with TBST, incubate with ECL chemiluminescence buffer, and observe the blot using a fully automated chemiluminescence imaging analysis system.

[0057] 5. Statistical Analysis

[0058] The experimental results were statistically analyzed using GraphPad Prism 10.1 software, and the results are expressed as mean ± standard deviation (mean ± SD). One-way ANOVA was used to analyze the significant differences between each group. Compared with the normal group, ## This is represented as P<0.01. #### P<0.0001 is indicated by as follows: * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001. All these indicate that the difference is statistically significant.

[0059] III. Experimental Results

[0060] 1. Results of retinal pigment epithelial cell proliferation activity in each group are as follows: Figure 1 As shown in the figure, compared with the normal group, the proliferation activity of retinal pigment epithelial cells in the model group was significantly reduced, indicating that the high glucose injury model was successfully established. Compared with the model group, the calcium dobesilate group did not significantly increase cell proliferation activity, while the cell proliferation activity of retinal pigment epithelial cells in different doses of dihydrobaicalin groups was significantly increased in a dose-dependent manner, indicating that dihydrobaicalin has a significant effect on promoting the proliferation of retinal pigment epithelial cells.

[0061] 2. VEGF expression levels in retinal pigment epithelial cells of each group are as follows: Figure 2 As shown in the figure, the expression level of VEGF protein in the model group was significantly increased compared with the normal group; compared with the model group, the calcium dobesilate group and the different doses of dihydrobaicalin group significantly reduced VEGF expression, indicating that dihydrobaicalin can antagonize angiogenesis by reducing VEGF expression levels, thereby improving diabetic retinopathy.

[0062] It is well known in the art that in vitro cell models are important tools for studying the mechanisms of diabetic retinopathy and for initial screening of candidate drug activity. The human retinal pigment epithelial cell line ARPE-19 is a widely used in vitro cell model in the field of diabetic retinopathy research. These cells are derived from normal human retinal pigment epithelial tissue and can be stably passaged in vitro. Under high glucose stimulation, ARPE-19 cells can mimic the pathological phenotypes of retinal pigment epithelial cells in the diabetic pathological environment, including oxidative stress, inflammatory activation, secretion of pro-angiogenic factors such as VEGF, apoptosis, and barrier function impairment. They are often used to evaluate the protective effects of candidate drugs on retinal pigment epithelial cells and to explore the molecular damage mechanisms of retinal pigment epithelial cells in diabetic retinopathy. Calcium dobesilate is used as a microvascular protectant in the treatment of diabetic retinopathy. Its mechanism of action is to protect retinal capillary endothelial cells, reduce damage to the vascular basement membrane, decrease capillary permeability and fragility, inhibit platelet aggregation, improve blood rheology, and antagonize high glucose-induced oxidative stress and local inflammatory response. It also reduces retinal exudation, microbleeds and macular edema. It is mainly used for non-proliferative diabetic retinopathy to delay the progression of fundus microvascular disease.

[0063] The experimental results above show that, in terms of promoting cell proliferation, dihydrobaicalin is superior to calcium dobesilate at the same dosage; and in terms of anti-VEGF, dihydrobaicalin is comparable to calcium dobesilate at the same dosage.

[0064] Example 2: An animal model of STZ-induced diabetic retinopathy

[0065] I. Experimental Materials

[0066] Dihydrobaicalin (DBA, Chengdu Pusi Biotechnology Co., Ltd., 56226-98-3);

[0067] Metformin (Met, Shanghai Yuanye Biotechnology Co., Ltd., 1115-70-4).

[0068] SPF grade 6-8 week old male C57BL / 6J mice (Hangzhou Ziyuan Experimental Animal Technology Co., Ltd.);

[0069] Streptozotocin (STZ, MCE, HY-13753);

[0070] 0.1 mol / L sodium citrate buffer (Solepro, C1013);

[0071] Sodium hyaluronate eye drops (URSAPHARM Arzneimittel GmbH, National Drug Approval Number HJ20150150);

[0072] Ofloxacin eye ointment (Shenyang Xingqi Pharmaceutical Co., Ltd., National Drug Approval Number H10940177);

[0073] Compound Tropicamide Eye Drops (Changchun Dirui Pharmaceutical Co., Ltd., National Drug Approval Number H20103127);

[0074] Tribromoethanol (MCE, HY-B1372);

[0075] Sodium chloride injection (Shijiazhuang No. 4 Pharmaceutical Co., Ltd., National Drug Approval Number H13023200);

[0076] Blood glucose meter (Sannuo Premium);

[0077] Blood glucose test strips (Sannuo Premium);

[0078] Small animal ophthalmic multimodal imaging system (Nanjing Boshi Medical Technology Co., Ltd.).

[0079] II. Experimental Methods

[0080] 1. Grouping of experimental animals

[0081] SPF-grade male C57BL / 6J mice aged 6-8 weeks were randomly divided into a normal group (Control), a model group (Model), a metformin group (200 mg / kg Met), a low-dose dihydrobaicalin group (10 mg / kg DBA), and a high-dose dihydrobaicalin group (50 mg / kg DBA), with 6 mice in each group.

[0082] 2. Model Establishment

[0083] After one week of acclimatization feeding, mice were fasted for 6 hours. Except for the normal group, mice in the other groups were intraperitoneally injected with STZ (55 mg / kg) for 5 consecutive days to establish the model. Mice in the normal group were injected with an equal volume of sodium citrate buffer. Seven days after modeling, the glucose concentration in the tail vein of the mice was monitored. Mice with glucose levels > 16.7 mmol / L were considered to have successfully established the model.

[0084] 3. Administration

[0085] After successful modeling, metformin group mice were administered the corresponding dose of metformin (200 mg / kg / d) by gavage, while dihydrobaicalin low-dose group mice and dihydrobaicalin high-dose group mice were administered the corresponding dose of dihydrobaicalin (10 mg / kg / d and 50 mg / kg / d) by intraperitoneal injection. The model group and normal group were given an equal volume of physiological saline. The administration was continued for 8 weeks.

[0086] 4. Observation Indicators

[0087] (1) Blood glucose detection: Blood was collected weekly to detect the random blood glucose levels of mice in each group and to observe the regulatory effect of dihydrobaicalin on blood glucose.

[0088] (2) Optical coherence tomography (OCT) to detect retinal thickness: After the last administration, compound tropicamide eye drops were instilled into both eyes of mice in each group to dilate the pupils. After anesthesia was induced by intraperitoneal injection of 0.01 mL / g tribromoethanol, ofloxacin eye ointment was applied to prevent dry eye. OCT was performed on both eyes using a small animal ophthalmology multimodal imaging system. The changes in retinal thickness of the inner retinal layer (ILM-OPL) and the full retinal layer (ILM-RPE) of mice in each group were statistically analyzed.

[0089] (3) Pathological examination of retinal tissue: After the experiment, the mouse retinal tissue was removed, stained with HE, and the morphological changes of the retinal tissue were observed.

[0090] III. Experimental Results

[0091] To verify the effect of dihydrobaicalin on diabetic retinopathy, we conducted a verification experiment using an STZ-induced diabetic retinopathy model in C57BL / 6J mice.

[0092] (1) Blood glucose level: Compared with the model group, the random blood glucose levels of mice in the metformin group and the high-dose dihydrobaicalin group were significantly lower (P<0.01 or P<0.05) (e.g. Figure 3 (As shown).

[0093] (2) Retinal thickness: OCT images showed that, compared with the normal group, the inner retinal layer (ILM-OPL) and total retinal thickness (ILM-RPE) of the model group mice were significantly reduced. Figure 4 In the middle A, B, and C groups, P < 0.01; compared with the model group, the retinal thickness of mice treated with metformin did not change significantly, while the inner retinal layer thickness and total retinal thickness of mice treated with high-dose dihydrobaicalin recovered. Figure 4 (Among A, B, and C, P < 0.01).

[0094] (3) Pathological changes in the retina: HE staining results showed that the retinal tissue of the model group mice exhibited obvious pathological changes, such as a decrease in the number of ganglion cells and disordered retinal structure. The pathological damage to the retinal tissue of mice in the low-dose dihydrobaicalin group and the high-dose dihydrobaicalin group was reduced (e.g., Figure 5 (As shown).

[0095] Example 3: Comparison of the efficacy of DBA and BA based on a retinal pigment epithelial cell (ARPE-19) cell model

[0096] I. Experimental Materials

[0097] Retinal pigment epithelial cells (ARPE-19, Cell Bank of Chinese Academy of Sciences).

[0098] Dihydrobaicalin (DBA, Chengdu Pusi Biotechnology Co., Ltd., 56226-98-3);

[0099] Baicalin (BA, Shanghai Yuanye Biotechnology Co., Ltd., 21967-41-9);

[0100] 10× PBS (Kage, KGB50011);

[0101] South American fetal bovine serum (FBS, Gibco, 10270-106).

[0102] Pancreatic enzyme (Gibco, 25200072);

[0103] DMEM / F12 medium (containing double antibiotics) (KGI, KGL1204-500);

[0104] Dimethyl sulfoxide (DMSO, Solarbio, 67-68-5);

[0105] D-(+)-glucose (D-Glu, Sigma, 50-99-7);

[0106] MTT Cell Proliferation Detection Kit (APExBIO, K2249).

[0107] PMSF (Shanghai Beyotime Biotechnology Co., Ltd., ST506);

[0108] Phosphatase inhibitor (Shanghai Beyotime Biotechnology Co., Ltd., P1087).

[0109] Anti-VEGFA antibody (Abcam, ab46154).

[0110] Red Loading Buffer Pack (CST, 7723).

[0111] II. Experimental Methods

[0112] 1. Prepare DBA and BA solutions

[0113] The compounds dihydrobaicalin and baicalin were dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution, which was then diluted with culture medium to the required concentration for the experiment.

[0114] 2. Cell Culture

[0115] ARPE-19 cell lines were cultured in DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin / streptomycin, and passaged in a conventional incubator at 37°C and 5% CO2. Experiments were conducted using F5-F15 generation cells.

[0116] 3. MTT assay for cell proliferation activity of retinal pigment epithelial cells

[0117] (1) Take ARPE-19 cells in good growth condition, resuspend them in DMEM / F12 medium containing 10% FBS, and seed them in 96-well plates with 100 μL of cell suspension per well (about 3000~4000 cells per well). They are divided into normal group (Control), model group (60 mM D-Glu), baicalin group (baicalin 10 μM group, high glucose + 10 μM BA), and dihydrobaicalin group (dihydrobaicalin 10 μM group, high glucose + 10 μM DBA). After culturing in a 37℃, 5% CO2 incubator for 24 h, the normal group is replaced with fresh medium, the model group is replaced with fresh medium containing 60 mM D-Glu, the baicalin group is replaced with fresh medium containing 60 mM D-Glu and 10 μM BA, and the dihydrobaicalin group is replaced with fresh medium containing 60 mM D-Glu and 10 μM DBA.

[0118] (2) After culturing in groups for 48 h, add 10 μL of MTT working solution to each well and then incubate in a cell culture incubator for 4 h.

[0119] (3) Add 100 μL of Formazan Solvent to each well, mix well, and incubate in a cell culture incubator for 3 hours.

[0120] (4) The absorbance (A) at 570 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader. Cell proliferation activity was obtained by calculating the ratio of the absorbance of the drug-treated group to that of the control group.

[0121] 4. Western blot detection of VEGF expression in retinal pigment epithelial cells

[0122] (1) Take ARPE-19 cells in good growth condition, resuspend them in DMEM / F12 medium containing 10% FBS, and seed the cells in 6-well plates at 2×10⁶ cells per well. 5 Each cell / 2 mL, the modeling and drug administration procedures are the same as in "3. MTT assay of retinal pigment epithelial cell proliferation activity" in this example.

[0123] (2) Lyse cells and extract proteins using SDS loading buffer containing protease inhibitors and phosphatase inhibitors, boil in a metal bath at 100°C for 15 min, and cool for later use.

[0124] (3) Prepare a dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to separate proteins and transfer them to a nitrocellulose membrane. Block with 5% skim milk at room temperature for 3 h, add primary antibody and incubate overnight at 4°C. On the second day, wash 3 times with TBST, incubate with secondary antibody at room temperature for 2 h, wash 3 times with TBST, incubate with ECL chemiluminescence buffer, and observe the blot using a fully automated chemiluminescence imaging analysis system.

[0125] 5. Statistical Analysis

[0126] The experimental results were statistically analyzed using GraphPad Prism 10.1 software, and the results are expressed as mean ± standard deviation (mean ± SD). One-way ANOVA was used to analyze the significant differences between each group. Compared with the normal group, # This is expressed as P<0.05. #### P < 0.0001 is indicated by as follows: * indicates P < 0.05, *** indicates P < 0.001, and **** indicates P < 0.0001, all indicating statistically significant differences. Compared with the baicalin group, & This indicates that P < 0.05. &&& This indicates that P < 0.001.

[0127] III. Experimental Results

[0128] 1. Results of retinal pigment epithelial cell proliferation activity in each group are as follows: Figure 6 As shown in Table 1, compared with the normal group, the proliferative activity of retinal pigment epithelial cells in the model group was significantly reduced, indicating that the high glucose injury model was successfully established. Compared with the model group, both the baicalin group and the dihydrobaicalin group significantly increased the proliferative activity of retinal pigment epithelial cells. Compared with the baicalin group, dihydrobaicalin significantly increased cell viability, indicating that both dihydrobaicalin and baicalin can restore the high glucose-induced decrease in ARPE-19 cell proliferation activity, and dihydrobaicalin has a better therapeutic effect than baicalin.

[0129] Table 1 Results of retinal pigment epithelial cell proliferation activity in each group

[0130]

[0131] 2. VEGF expression levels in retinal pigment epithelial cells of each group are as follows: Figure 7As shown in Table 2, compared with the normal group, the expression level of VEGF protein in retinal pigment epithelial cells of the model group was significantly increased; compared with the model group, both the baicalin group and the dihydrobaicalin group significantly decreased VEGF expression; compared with the baicalin group, dihydrobaicalin significantly decreased VEGF expression, indicating that dihydrobaicalin and baicalin can improve the hyperglycemic VEGF elevation, and dihydrobaicalin has a better therapeutic effect than baicalin.

[0132] Table 2. Relative VEGF expression levels in retinal pigment epithelial cells of each group

[0133]

[0134] This example, as a supplementary experiment to Example 1, aims to compare the differences in the activity of dihydrobaicalin and baicalin in improving diabetic retinopathy. The two substances differ in structure by only one double bond, and theoretically their activities should be somewhat similar. However, the experimental results show that dihydrobaicalin has significantly better proliferative activity and anti-VEGF expression activity against retinal pigment epithelial cells than baicalin, with the proliferative activity being approximately twice that of baicalin and the anti-VEGF expression activity being more than twice that of baicalin. This significant difference in activity cannot be expected from minor structural differences between the two substances.

[0135] In summary, dihydrobaicalin can effectively enhance the proliferative activity of retinal pigment epithelial cells, reduce VEGF expression levels, and antagonize angiogenesis in an in vitro model of diabetic retinopathy, thereby improving diabetic retinopathy. In an in vivo model of diabetic retinopathy, dihydrobaicalin can effectively improve retinal thickness and pathological damage. Therefore, dihydrobaicalin shows promise as a drug for the prevention or treatment of diabetic retinopathy.

[0136] Example 4: A medicine for the prevention or treatment of diabetic retinopathy

[0137] A medicine for the prevention or treatment of diabetic retinopathy, wherein the medicine is an active ingredient of dihydrobaicalin or a pharmaceutically acceptable salt thereof (dihydrobaicalin has two phenolic hydroxyl groups and one carboxyl group, and theoretically has the potential to form salts, such as alkali metal salts and organic amine salts, which can help improve water solubility), and is formulated into a pharmaceutically acceptable dosage form (one of ophthalmic preparations, injectable preparations, oral preparations, inhaled preparations, and transdermal preparations) using pharmaceutically acceptable excipients (one or more of solid excipients, liquid excipients, and semi-solid excipients).

[0138] Example 5: A medicine for the prevention or treatment of diabetic retinopathy

[0139] A medicine for the prevention or treatment of diabetic retinopathy, wherein the active ingredient comprises dihydrobaicalin or a pharmaceutically acceptable salt thereof (dihydrobaicalin has two phenolic hydroxyl groups and one carboxyl group, theoretically allowing for salt formation, such as alkali metal salts or organic amine salts, which can improve water solubility) and another ingredient for the prevention, treatment, or adjunctive prevention or treatment of diabetic retinopathy (anti-vascular endothelial growth factor drugs such as bevacizumab, ranibizumab, aflibercept, conbercept, or brogluzumab; glucocorticoid drugs such as dexamethasone, triamcinolone, fluocinolone, or prednisolone; antioxidants such as vitamin C, vitamin E, taurine, or lipoic acid; or neurotrophic factors or neuroprotective agents such as brain-derived neurotrophic factor, ciliary neurotrophic factor, or pigment epithelial-derived factor), formulated into a pharmaceutically acceptable dosage form (one of ophthalmic preparations, injectable preparations, oral preparations, inhaled preparations, and transdermal preparations) using pharmaceutically acceptable excipients (one or more of solid excipients, liquid excipients, and semi-solid excipients).

[0140] The purpose of the above embodiments is to specifically illustrate the substantive content of the present invention, but those skilled in the art should know that the scope of protection of the present invention should not be limited to the specific embodiments.

Claims

1. The use of a dihydrobaicalin or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention or treatment of diabetic retinopathy.

2. The application according to claim 1, characterized in that: The drug comprises dihydrobaicalin or a pharmaceutically acceptable salt thereof for the prevention or treatment of diabetic retinopathy, and pharmaceutically acceptable excipients, formulated into a pharmaceutically acceptable dosage form.

3. The application according to claim 2, characterized in that: The medicine also contains one or more other ingredients for the prevention, treatment or adjunctive prevention or treatment of diabetic retinopathy.

4. The application according to claim 3, characterized in that, The ingredients used for the prevention, treatment, or adjunctive prevention and treatment of diabetic retinopathy are selected from anti-vascular endothelial growth factor drugs, glucocorticoid drugs, antioxidants, and neurotrophic factors or neuroprotective agents.

5. The application according to claim 4, characterized in that: The anti-vascular endothelial growth factor drug is bevacizumab, ranibizumab, aflibercept, conbercept, or broluzumab.

6. The application according to claim 4, characterized in that: The glucocorticoid drugs mentioned are dexamethasone, triamcinolone, fluocinolone acetonide, or prednisolone.

7. The application according to claim 4, characterized in that: The antioxidant is vitamin C, vitamin E, taurine, or lipoic acid.

8. The application according to claim 4, characterized in that: The neurotrophic factor or neuroprotective agent is brain-derived neurotrophic factor, ciliary neurotrophic factor, or pigment epithelium-derived factor.

9. The application according to any one of claims 2 to 8, characterized in that: The excipients are selected from one or more of solid excipients, liquid excipients, and semi-solid excipients.

10. The application according to any one of claims 2 to 8, characterized in that: The dosage form is selected from one of ophthalmic preparations, injectable preparations, oral preparations, inhaled preparations, and transdermal preparations.

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