Use of a small molecule antioxidant to slow blood vessel damage in type 2 diabetes

Abstract

This invention belongs to the field of pharmaceutical technology, specifically relating to the application of a small-molecule antioxidant in mitigating vascular damage in type 2 diabetes. To develop an effective solution for addressing vascular damage in diabetic patients, this invention, through research, discovered that Lithospermoside can specifically inhibit XO in the cell membrane anchoring state, effectively reducing ROS production in the thoracic aorta, thereby significantly reducing BUN, E-selectin, MDA, and UACR. Simultaneously, it inhibits the fluorescence intensity of nitrotyrosine staining, thus alleviating vascular endothelial damage in type 2 diabetes with persistent hyperglycemia. It exerts an antioxidant effect to slow down diabetic vascular damage and protect blood vessels, providing an effective material basis and new ideas for the prevention and treatment of vascular damage in type 2 diabetes.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of a small molecule antioxidant in slowing down vascular damage in type 2 diabetes. Background Technology

[0002] Patients with type 2 diabetes have an increased risk of cardiovascular disease (CVD) and a higher likelihood of dying from it. Oxidative stress is considered one of the causes of vascular damage in diabetic patients. Factors triggering oxidative stress mainly include increased production of advanced glycation end products (AGEs), activation of the polyol pathway, enhanced activity of protein kinase C and xanthine oxidase (XO), and uncoupling of endothelial nitric oxide synthase. Therefore, inhibiting oxidative stress is crucial for preventing the progression of vascular disease. However, effective strategies for managing CVD are currently lacking.

[0003] Xanthine oxidoreductase (XOR) catalyzes two key reaction steps: the synthesis of xanthine from hypoxanthine and the further synthesis of uric acid from xanthine. Under physiological conditions, XOR mainly exists as xanthine dehydrogenase (XDH), which uses nicotinamide adenine dinucleotide as an electron acceptor. However, under specific conditions, XDH can be converted to XO through limited proteolytic activity. XO uses molecular oxygen as an electron acceptor and generates superoxide anions during the reaction. XO is also considered to be associated with endothelial dysfunction, hypertension, and heart failure, mainly due to the presence of reactive oxygen species (ROS) both intracellularly and extracellularly. Extracellular ROS exist in two forms: a free state, which can exist in circulating blood; and an anchored state, which is bound to proteoglycans on the surface of endothelial cells.

[0004] Studies have shown that injection of allopurinol into cholesterol-loaded rabbits improved endothelial cell-dependent vasodilatory responses, and this effect was independent of serum uric acid concentration. Other reports indicate that XOR activity in the diseased coronary arteries of patients with coronary artery disease was significantly higher than in healthy controls. These findings suggest that excessive XOR activation may be involved in the abnormal functional processes of large artery endothelial cells.

[0005] Lithospermoside is extracted from the roots of Lithospermum purpurocaeruleum and Lithospermum officinale (both belonging to the Boraginaceae family), as well as Thalictrum rugosum and Thalictrum dasycarpum (both belonging to the Ranunculaceae family). It has antioxidant and antitumor effects.

[0006] Although lithospermoside (a small-molecule antioxidant) is currently used to treat hyperuricemia and gout, studies have found that administration of febuxostat or lithospermoside to db / db mice can inhibit albuminuria. However, only lithospermoside exhibits a dose-dependent effect, and this effect is related to XOR activity in plasma. Plasma XOR can anchor to proteoglycans on the surface of endothelial cells, leading to the local production of superoxide anions, which react with nitrite (NO) in blood vessels to generate cytotoxic peroxynitroso groups.

[0007] It is hypothesized that lithospermoside can improve vascular endothelial function by inhibiting circulating XOR, anchored XOR, and intracellular XOR; however, the extent of its association with these three XOR types is currently unclear. Furthermore, no studies have yet demonstrated an association between lithospermoside and type 2 diabetes. Summary of the Invention

[0008] To overcome the shortcomings of the prior art, this invention investigated the effect of the small-molecule antioxidant Lithospermoside on vascular ROS in type 2 diabetic rats. In a streptozotocin-induced type 2 diabetic rat model, XO is increased or activated, and ROS generation in large blood vessels (thoracic aorta) is increased; Lithospermoside can effectively alleviate this response, thereby protecting blood vessels in a diabetic state from damage.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] This invention provides the application of lithospermoside in the preparation of drugs that alleviate vascular damage in type 2 diabetes.

[0011] Preferably, the shikonin exerts its antioxidant effect by inhibiting anchored xanthine oxidase, thereby slowing down vascular damage in type 2 diabetes and protecting blood vessels.

[0012] This invention has demonstrated that lithospermoside can improve endothelial damage in the aorta of a type 2 diabetic animal model by inhibiting anchored XO. The results suggest that lithospermoside may have a protective effect against microvascular disease induced by type 2 diabetes. The next step is to conduct prospective clinical trials to evaluate the protective effect of lithospermoside against macrovascular disease caused by type 2 diabetes.

[0013] Preferably, the effective dose of the shikonin is 1-5 mg / kg.

[0014] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0015] More preferably, the excipients include at least one of the following: excipients, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrators, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculators, antioxidants, adsorbents, filter aids, and release inhibitors.

[0016] Preferably, the dosage form of the drug includes tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, or suppositories.

[0017] Compared with the prior art, the beneficial effects of the present invention are:

[0018] This invention reveals that the small-molecule antioxidant Lithospermoside can specifically inhibit XO in the cell membrane anchored state, effectively reducing ROS production in the thoracic aorta, thereby significantly reducing blood urea nitrogen (BUN), E-selectin, malondialdehyde (MDA), and the urinary albumin / creatinine ratio (UACR). Simultaneously, it inhibits the fluorescence intensity of nitrotyrosine staining, thus alleviating vascular endothelial damage in type 2 diabetes mellitus with persistent hyperglycemia, exerting an antioxidant effect to slow down diabetic vascular damage and protect blood vessels.

[0019] This groundbreaking discovery can specifically improve diabetes-related vascular complications, providing a highly targeted solution for improving these complications and offering an effective material basis and new ideas for the prevention and treatment of vascular damage in type 2 diabetes. Simultaneously, the small-molecule antioxidant Lithospermoside holds promise for development into a novel drug or intervention formulation, which can be conveniently applied clinically via oral or injection routes, providing more precise and efficient treatment options for patients with type 2 diabetes. Attached Figure Description

[0020] Figure 1 A: Bar chart of purine body concentration in plasma; B: Bar chart of hypoxanthine concentration; C: Bar chart of xanthine concentration; D: Bar chart of xanthine oxidoreductase (XOR) activity.

[0021] Figure 2To investigate the effect of Lithospermoside treatment on anchored XOR, this study used chemiluminescence immunoassay to detect the effect of Lithospermoside on anchored XOR. The data in the figure are based on a standard (5 μU / mL xanthine oxidase), and after dry weight correction, show the difference in luminescence intensity between the sample group and the blank control group. Black bars: no heparin added; gray bars: 1000 U / mL heparin added. The figure shows the bar chart results of superoxide generation in different groups. The vertical axis represents superoxide generation (measured by the standard ratio of chemiluminescence immunoassay), and the horizontal axis represents different groups, including the control group, the Streptozotocin (STZ) group, and different doses (0.3 mg / kg, 1 mg / kg, 3 mg / kg) of Lithospermoside.

[0022] Figure 3 To investigate the effect of Lithospermoside on intracellular XOR, specifically, the effect of Lithospermoside on intracellular superoxide dismutase (XOR) was detected using ethidium dihydrofluorescence staining. A: Observation of ethidium dihydrofluorescence signal in aortic tissue sections under confocal microscopy; B: Quantitative analysis of the fluorescence intensity of ethidium dihydrofluorescence in the intima region; C: Quantitative analysis of the fluorescence intensity of ethidium dihydrofluorescence in the media region.

[0023] Figure 4 To investigate the effect of Lithospermoside on cytotoxicity, a fluorescence immunostaining method was used to detect the effect of Lithospermoside on cytotoxicity (frozen sections of the aorta were immunostained with anti-3-NT antibody and CD31 antibody, and co-stained with DAPI); A: Observation of fluorescence signals of 3-NT (green), CD31 (red) and DAPI (blue) in aortic tissue sections under a confocal microscope; B: Quantitative analysis of fluorescence intensity in the overlapping area of ​​CD31 and 3-NT fluorescence, scale bar is 50 μm. Detailed Implementation

[0024] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0025] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0026] In the following examples, the Krebs solution consisted of: 137.4 mM NaCl, 5.9 mM KCl, 1.2 mM MgSO4, 2.5 mM CaCl2, 15.5 mM NaHCO3, 1.2 mM KH2PO4, and 11.5 mM glucose. The solution was bubbled with 95% O2 and 5% CO2 to adjust the pH to 7.3–7.4. A modified Krebs-N-(2-hydroxyethyl)piperazine N′-2-ethanesulfonic acid (HEPES) buffer contained 99 mM NaCl, 4.7 mM KCl, 1.9 mM CaCl2, 1.2 mM MgSO4, 20 mM HEPES, 1.03 mM KH2PO4, 25 mM NaHCO3, and 11.1 mM glucose (pH 7.4). Krebs–Henseleit buffer contains 118.3 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3 and 11 mM glucose (pH 7.4).

[0027] XOR exists both intracellularly and extracellularly, inducing vascular damage by generating ROS. Based on this, this invention uses an animal model of type 2 diabetes with persistent hyperglycemia to investigate the effects of a small-molecule antioxidant, Lithospermoside, on ROS and its mechanism of action. Six-week-old male Sprague-Dawley rats were given 50 mg / kg Streptozotocin to induce diabetes; at eight weeks of age, the animals were administered Lithospermoside (0.3 mg / kg, 1 mg / kg, or 3 mg / kg) via mixed feeding for two weeks, after which aortic samples were collected. Compared with the Streptozotocin group, Lithospermoside at 3 mg / kg significantly reduced BUN, E-selectin, urinary MDA, and UACR. Compared with the Streptozotocin group, Lithospermoside at doses of 1 mg / kg and 3 mg / kg significantly reduced superoxide anion generation from cell membrane-anchored XO. 3 mg / kg Lithospermoside significantly inhibited the fluorescence intensity of nitrotyrosine staining. The above results indicate that Lithospermoside can reduce ROS production in the thoracic aorta by inhibiting anchored XO, thereby alleviating vascular endothelial damage in diabetic patients. Therefore, Lithospermoside exerts its antioxidant effect by inhibiting anchored XO, thus slowing down vascular damage in type 2 diabetes and protecting blood vessels.

[0028] To fully and clearly present the technical solution and significant advantages of the present invention, the present invention will be described in detail below with reference to specific embodiments.

[0029] 1. Experimental Methods

[0030] 1.1 Laboratory animals and their treatment

[0031] This study strictly followed the "Animal Experiment Operation Guidelines" of Shenzhen Second People's Hospital and was approved by the hospital's Animal Experiment Ethics Committee. Thirty-five Sprague-Dawley rats were purchased from the Guangdong Provincial Animal Center. All rats were fed standard laboratory feed and housed individually in a room with controlled temperature (23±2℃) and light (12-hour light / dark cycle), with free access to water. At 6 weeks of age, diabetes was induced by a single intravenous injection (iv) of 50 mg / kg streptozotocin in saline solution, while the control group received an equal volume of saline. Hyperglycemia was monitored for 7 days, and fasting blood glucose was measured from the tail vein at 7 weeks of age. Based on body weight and fasting blood glucose levels, the diabetic rats were divided into four groups: a control group, a streptozotocin group, and three Lithospermoside treatment groups (0.3 mg / kg / day, 1 mg / kg / day, and 3 mg / kg / day). The rats received intervention for 2 weeks starting at 8 weeks of age. Before tissue collection, the rats were fasted overnight, anesthetized with sevoflurane, and euthanized by exsanguination. After blood was drawn from the heart, plasma was separated by centrifugation at 3000×g for 15 minutes at 4℃ and stored at -80℃ for testing. The aorta was immediately removed and placed in Krebs solution to remove connective tissue, then stored in Krebs solution at 4℃ until use.

[0032] Preparation of 1mM Lithospermoside stock solution: Dissolve 1mg Lithospermoside in 3.0367mL of double-distilled water. After aliquoting the stock solution, store it in a freezer at -80℃ to avoid repeated freeze-thaw cycles. Dilute to the working solution concentration before use.

[0033] 1.2 Biochemical Analysis

[0034] Whole blood was used for glucose concentration determination. Fasting blood glucose was measured using the glucose oxidase method with a glucose meter (Sanwa Kagaku Kenkyusho Co., Ltd., Nagoya, Japan); plasma blood urea nitrogen (BUN) and urinary creatinine concentrations were measured using the L-type Wako UN kit (Catalog Nos. 416-55192, 412-55292, 419-41691, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and the L-type Wako Cre kit (Catalog Nos. 469-07594, 465-07694, 413-41591, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), respectively; urinary albumin concentration was measured using an enzyme-linked immunosorbent assay kit (Catalog No. E111-125, Bethyl Laboratory, AL, USA); and urinary malondialdehyde (MDA) concentration was measured using an MDA assay kit (Catalog No. E111-125, Bethyl Laboratory, AL, USA). Urinary MDA and albumin concentrations were measured using an ELISA kit (Catalog No. NWK-MDA01, Northwest Life Science Specialties LLC, OR, USA). Urinary MDA and albumin concentrations were expressed as the urinary creatinine ratio (UACR). Plasma E-selectin concentrations were measured using an ELISA kit (Catalog No. ELR-Eselectin, RayBiotech Life, GA, USA).

[0035] 1.3 Purine bodies and drug concentration

[0036] When determining purine bases, plasma was taken and added to Tris buffer (pH 8.5) containing sodium chloride, along with […]. 15 N2]-xanthine and [ 15 [N2]-uric acid was used as an internal standard, and the mixture was then heated at 95°C for 5 minutes. The resulting suspension was centrifuged at 4°C and 15000×g for 10 minutes. The supernatant was filtered through an ultrafiltration membrane and analyzed by liquid chromatography-mass spectrometry (LC / MS).

[0037] When determining the concentration of shikonin, plasma was taken and acetonitrile containing F10460 as an internal standard was added. After filtration through a membrane filter, the filtrate was evaporated, redissolved in 10% methanol, and used for liquid chromatography-tandem mass spectrometry (LC / MS / MS) analysis.

[0038] 1.4 XOR plasma activity assay

[0039] XOR activity determination: Plasma was added to a 20 mmol / L Tris buffer (pH 8.5), which contains [ 15 [N2]-xanthine (0.8 mmol / L), nicotinamide adenine dinucleotide (1 mmol / L), and oxaloacetic acid (0.013 mmol / L) were incubated at 37°C for 30 minutes. Then [ 13 C2, 15 [N2]-uric acid was used as an internal standard. The mixture was heated at 95°C for 5 minutes, and then centrifuged at 4°C and 15000×g for 10 minutes. The supernatant was filtered through an ultrafiltration membrane and the determination was performed by LC / MS. 15 N2]-uric acid concentration, activity as [ 15 [N2]-uric acid nmol / min / mg protein.

[0040] 1.5 Anchoring XOR activity

[0041] The effect of shikonin on anchored XOR activity was investigated by detecting the aortic segment (3 mm long) using the superoxide-sensitive chemiluminescent dye 8-amino-5-chloro-7-phenylpyrrole[3,4-d]pyrazine-1,4-(2H,3H)dione sodium salt and a chemiluminescence analyzer. The specific steps were as follows: The aortic segment was equilibrated at 37°C in a Krebs solution with 95% O2 / 5% CO2 for 30 minutes; a scintillation bottle containing modified Krebs–HEPES buffer (containing 100 μM L-012 and 50 μM xanthine) was placed in the chemiluminescence analyzer to determine the background signal; subsequently, the aortic segment was placed in the scintillation bottle, and the chemiluminescence signal was continuously tracked at 37°C for 30 minutes; after removal, the aortic segment was dried at 90°C for 24 hours and weighed.

[0042] During the detection process, the blank signal was subtracted from the chemiluminescence signal of the sample, and the difference was used to calculate the luminescence rate per minute. Chemiluminescence was expressed as a standard ratio (5 μU / mL xanthine oxidase) and corrected for dry weight. Furthermore, the effects of heparin (1000 U / mL) and superoxide dismutase (SOD) were evaluated to confirm whether these drugs could inhibit the effects of reactive oxygen species (ROS).

[0043] 1.6 Intracellular XOR activity

[0044] The effect of shikonin on intracellular XOR activity was investigated using ethidium dihydroacetate oxidation fluorescence staining. The experiment used aortic segments stored in the compound at 25°C (OCT). The specific procedures were as follows: Embedded frozen aortic segments were cut into 8 μm thick sections using a cryostat and fixed onto MAS-coated slides. The arterial sections were incubated in a CO2 incubator at 37°C for 20 minutes in Krebs-Henseleit buffer (pH 7.4) with or without 50 μM xanthine. Subsequently, they were incubated in Krebs-Henseleit buffer (pH 7.4) containing 1 μM ethidium dihydroacetate at 37°C for 30 minutes.

[0045] Images were acquired using a confocal laser scanning microscope system. Fluorescence intensity was measured in eight randomly selected regions from each slice, and the average value was calculated using digital image analysis software.

[0046] 1.7 Cytotoxicity test

[0047] The tissue section preparation procedure is as follows: The aorta was cut into 3 mm thick sections, fixed with 4% paraformaldehyde, and then incubated sequentially in 10% and 15% sucrose solutions for 4 hours each, followed by overnight incubation in 20% sucrose solution. After embedding with an optimal cutting temperature (OCT) compound, the specimen was rapidly frozen in liquid nitrogen. The frozen aorta was then cut into 6 μm thick sections using a cryostat and attached to MAS-coated glass slides (Matsunami Glass, Osaka, Japan).

[0048] 1.8. Fluorescent Immunostaining

[0049] Antigen activation was performed using HistoVT One solution. After rinsing with phosphate-buffered saline (PBS), sections were blocked with HistoOne blocking solution and then incubated overnight at 4°C with two primary antibodies: a mouse monoclonal antibody against 3-nitrotyrosine (3-NT) and a rabbit monoclonal antibody against the endothelial marker CD31.

[0050] The following day, the slides were washed with PBS and incubated at room temperature for 1 hour. The secondary antibody, goat anti-mouse immunoglobulin G (IgG) H&L, was used to label 3-NT (appearing green), and goat anti-rabbit IgG H&L was used to label CD31 (appearing red). A control experiment was also set up, in which PBS was used to treat the slides instead of the primary antibody as a secondary antibody control.

[0051] Finally, the sections were mounted and stained with a Vector TrueVIEW autofluorescence quenching kit containing 4,6-diamidinyl-2-phenylindole (DAPI). Images were acquired using a confocal laser scanning microscope system, and fluorescence intensity was measured in four randomly selected overlapping regions of CD31 and 3-NT fluorescence from each section. The average value was calculated using ImageJ software.

[0052] 1.9 Statistical Analysis

[0053] All results are reported as mean ± standard deviation, where n represents the number of rats used (each rat was used to provide only one segment for a specific experiment). Multiple group comparisons were performed using one-way ANOVA followed by Tukey's post-hoc test. Statistical significance was defined as p < 0.05.

[0054] 2. Experimental Results

[0055] 2.1 Biochemical characteristics and plasma purine body concentration

[0056] At 10 weeks of age, the body weight of rats in the control group was significantly higher than that in the Streptozotocin-treated group. Fasting blood glucose levels were significantly higher in the Streptozotocin-treated group compared to the control group. Fecal urea nitrogen (BUN) concentrations were significantly higher in the Streptozotocin group, while fecal BUN concentrations were significantly lower in the Lithospermoside 3 mg / kg group compared to the Streptozotocin group. Urinary MDA concentrations, corrected for creatinine, were significantly higher in the Streptozotocin group compared to the control group, but significantly lower in the Lithospermoside 1 mg / kg and 3 mg / kg groups. The urinary albumin-to-creatinine ratio (UACR) was significantly higher in the Streptozotocin group compared to the control group. Lithospermoside showed a dose-dependent significant decrease in UACR. Compared with the control group, the plasma E-selectin concentration in the Streptozotocin group was significantly increased; while compared with the Streptozotocin group, the plasma E-selectin concentration in the 1 mg / kg and 3 mg / kg Lithospermoside groups was significantly decreased (Table 1).

[0057] Meanwhile, no difference in uric acid concentration was observed between the Control group and the Streptozotocin group, but the concentration in the Lithospermoside 3 mg / kg group was significantly lower than that in the Streptozotocin group. Hypoxanthine and xanthine were detected only in the Lithospermoside 1 mg / kg and 3 mg / kg groups, as shown in the figure. Figure 1 As shown in AC, XOR activity in plasma was significantly higher in the Streptozotocin group than in the Control group, but significantly lower in the Lithospermoside 1 mg / kg and 3 mg / kg groups than in the Streptozotocin group. Figure 1 D).

[0058] Table 1 Biochemical characteristics of rats treated with Lithospermoside

[0059]

[0060]

[0061] Note: STZ stands for Streptozotocin group, and Lto stands for Lithospermoside group.

[0062] 2.2 Anchoring XOR activity

[0063] Compared with the control group, superoxide production was significantly increased in the streptozotocin group; while compared with the streptozotocin group, superoxide production was significantly decreased in the lithospermoside 1 mg / kg and 3 mg / kg groups. Furthermore, superoxide production was significantly inhibited in the following groups: control group, streptozotocin group, lithospermoside 0.3 mg / kg group, and lithospermoside 1 mg / kg group. Figure 2 This indicates that Lithospermoside significantly inhibits the activity of xanthine oxidase (XOR) in the circulatory system.

[0064] 2.3 Effects of intracellular XOR

[0065] Compared to the control group, the Streptozotocin group showed a significant increase in intracellular XOR-active superoxide production, while the Lithospermoside group showed no change. In contrast, Lithospermoside did not inhibit intracellular reactive oxygen species (ROS) produced by xanthine oxidase. Figure 3 AC).

[0066] Besides xanthine oxidase, various other enzyme systems in the blood vessel wall can produce reactive oxygen species (ROS), including nicotinamide adenine dinucleotide phosphate oxidase, mitochondrial respiratory chain-related enzymes, and endothelial nitric oxide synthase uncoupling enzymes. Lithospermoside alone may not be sufficient to inhibit intracellular ROS generation induced by oxidative stress from these complex factors. Figure 2 Chemiluminescence results and Figure 3 Dihydroethidium oxidation fluorescence staining results showed that the level of ROS generated by intracellular XOR was not affected. These results suggest that Lithospermoside can inhibit the increase of ROS generated by anchored XOR, and Lithospermoside may improve vascular injury by inhibiting anchored XOR.

[0067] 2.4 Cytotoxic Effects

[0068] Cytotoxicity assay results showed that, compared with the control group, the fluorescence intensity of nitrotyrosine in the Streptozotocin group was significantly increased; compared with the Streptozotocin group, the fluorescence intensity of nitrotyrosine in the Lithospermoside 3 mg / kg group was significantly decreased. Figure 4 AB).

[0069] Oxidative stress is a major contributing factor to vascular damage in diabetes. XOR activation promotes the uptake of low-density lipoprotein by macrophages, indicating that XOR is directly involved in the progression of atherosclerosis. Figure 4 Nitrotyrosine staining results suggest that Lithospermoside can improve aortic endothelial cell damage. These findings indicate that Lithospermoside may reduce aortic vascular damage by inhibiting reactive oxygen species (ROS), a change that has potential benefits for managing cardiovascular disease in diabetic patients.

[0070] In summary, Lithospermoside significantly reduces blood urea nitrogen (BUN), E-selectin, urinary malondialdehyde (MDA), and the urinary albumin / creatinine ratio (UACR) by inhibiting anchored XO enzymes and reducing the resulting reactive oxygen species (ROS). It also inhibits the fluorescence intensity of nitrotyrosine staining, thereby effectively improving endothelial damage in the aorta of diabetic animal models. The results of this study suggest that this drug may have a vascular protective effect in patients with diabetic macrovascular disease. Further prospective clinical trials targeting diabetic macrovascular disease should be conducted to further validate the vascular protective effect of Lithospermoside.

[0071] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. Use of shikimic cyanoride for the preparation of a medicament for slowing down the vascular damage in diabetes mellitus type 2, characterized in that, The shikonin exerts its antioxidant effect by inhibiting anchored xanthine oxidase, thereby slowing down vascular damage in type 2 diabetes and protecting blood vessels.

2. The application according to claim 1, characterized in that, The effective dose of the shikonin is 1-5 mg / kg.

3. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

4. The application according to claim 3, characterized in that, The excipients include at least one of the following: excipients, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, penetration promoters, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, antioxidants, adsorbents, filter aids, and release inhibitors.

5. The application according to claim 3, characterized in that, The dosage forms of the drug include tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, or suppositories.

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