Use of alpha-ketobutyric acid for the preparation of a medicament for the prevention and / or treatment of vascular calcification

Abstract

The application belongs to the technical field of medicine, and particularly relates to application of alpha-ketobutyric acid in preparation of a medicine for preventing and / or treating vascular calcification. Through a high-phosphorus-induced vascular smooth muscle cell calcification model and a vitamin D3-induced mouse aorta calcification model, the application first proves that alpha-ketobutyric acid can significantly inhibit vascular calcium salt deposition, reduce osteogenic-like transdifferentiation of vascular smooth muscle cells, and down-regulate expression of osteogenic-related markers such as RUNX2 and BMP2. In addition, alpha-ketobutyric acid and sodium hydrosulfide in combination show a synergistic effect. The application provides a new medicine selection and strategy for prevention and treatment of vascular calcification and related cardiovascular diseases.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the use of α-ketobutyric acid in the preparation of drugs for the prevention and / or treatment of vascular calcification. Background Technology

[0002] Vascular calcification is a common pathological mineralization process in the cardiovascular system, characterized by the abnormal deposition of hydroxyapatite crystals in the blood vessel walls, accompanied by the transdifferentiation of vascular smooth muscle cells into osteoblast-like cells. This process is a core pathological link in diseases such as atherosclerosis, diabetic vascular complications, chronic kidney disease, and aging, leading to increased vascular stiffness and decreased compliance, and can significantly increase the risk of cardiovascular events such as myocardial infarction, heart failure, and stroke. Currently, there are no specific drugs in clinical practice that can effectively reverse or significantly inhibit the progression of vascular calcification. Existing treatments mainly target underlying diseases (such as lipid-lowering, blood pressure-lowering, and blood sugar-lowering drugs) or use bisphosphonates, but the latter have limited efficacy and potential side effects (such as osteonecrosis of the mandible and atypical femoral fractures). Therefore, the development of novel drugs that can target and intervene in the pathological process of vascular calcification has urgent clinical needs and significant social value.

[0003] α-Ketobutyrate is an important intermediate metabolite in amino acid metabolism and the tricarboxylic acid cycle in mammals. Existing studies have shown that α-ketobutyrate plays a regulatory role in energy metabolism and signal transduction, but its specific role in vascular calcification diseases has not yet been reported. Summary of the Invention

[0004] The present invention aims to overcome the shortcomings of the prior art and provide new uses for α-ketobutyric acid and its derivatives in the prevention and treatment of vascular calcification.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides the use of α-ketobutyric acid or a pharmaceutically acceptable salt, ester, solvate or prodrug thereof in the preparation of medicaments for the prevention and / or treatment of vascular calcification.

[0007] α-Ketobutyric acid (α-KB), also known as 2-oxobutyric acid or 2-oxo-n-butyric acid, is a four-carbon short-chain keto acid. Its molecular structure is CH3-CH2-CO-COOH, meaning it contains a ketone (carbonyl) group at the α-carbon position (C2 position) of the butyric acid chain, and terminates with a carboxyl group. Its molecular formula is C4H6O3. This compound has the CAS number 600-18-0 and is an important intermediate in the amino acid metabolic pathway.

[0008] Preferably, the drug further includes sodium hydrosulfide. When α-ketobutyric acid is used in combination with sodium hydrosulfide (NaHS), a donor of the endogenous gaseous signaling molecule hydrogen sulfide (H2S), synergistic or additive anti-calcification effects are observed in cell and animal models, indicating that the combination is a more preferred implementation.

[0009] Preferably, the vascular calcification manifests as osteoblastic transdifferentiation of vascular smooth muscle cells and / or abnormal deposition of calcium salts in the vascular wall.

[0010] The α-ketobutyric acid of this invention (alone or in combination with NaHS) exerts its role in preventing and treating vascular calcification through multiple pathways:

[0011] Inhibits calcium salt deposition: Significantly reduces abnormal calcium ion deposition in vascular tissues (such as the aorta).

[0012] Inhibits cell transdifferentiation: Effectively inhibits the unfavorable transdifferentiation of vascular smooth muscle cells into osteoblast-like cells.

[0013] Downregulation of osteogenic markers: At both the protein and mRNA levels, the expression of key osteogenic-related markers in vascular tissues was reduced, including Runt-associated transcription factor 2 (RUNX2), bone morphogenetic protein 2 (BMP2), and muscle homeobox transcription factor 2 (MSX2).

[0014] Reduced biochemical indicators: Significantly reduced calcium content and alkaline phosphatase (ALP) activity in vascular tissue, both of which are classic biochemical indicators for measuring the degree of calcification.

[0015] Preferably, the drug further includes pharmaceutically acceptable excipients. Dosage forms include, but are not limited to, granules, tablets, capsules, oral liquid preparations, or injections.

[0016] Secondly, the present invention provides the use of α-ketobutyric acid in combination with sodium hydrosulfide in the preparation of medicaments for the prevention and / or treatment of vascular calcification.

[0017] Preferably, the concentration ratio of α-ketobutyric acid to sodium hydrosulfide is 1:1.

[0018] Thirdly, the present invention provides a pharmaceutical composition for the prevention and / or treatment of vascular calcification, comprising a therapeutically effective amount of an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient is selected from:

[0019] (a) α-Ketobutyric acid or its pharmaceutically acceptable salt, ester, solvate or prodrug; or

[0020] (b) α-Ketobutyric acid or its pharmaceutically acceptable salt, ester, solvate or prodrug, and sodium hydrosulfide.

[0021] Preferably, the dosage form of the pharmaceutical composition is an oral dosage form or an injectable dosage form. The oral dosage form includes, but is not limited to, granules, tablets, capsules, or oral liquid preparations.

[0022] The pharmaceutically acceptable excipients mentioned above include any one or a combination of at least two of the following: sustained-release agents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, and lubricants. The combination of at least two is, for example, a combination of binders and excipients, a combination of binders and flavoring agents, a combination of binders and fillers, etc. Any other combination is also acceptable, and will not be elaborated here.

[0023] The beneficial effects of this invention are as follows:

[0024] This invention, through establishing a high-phosphorus-induced vascular smooth muscle cell calcification model and a VD3-induced mouse aortic calcification model, demonstrates for the first time that α-ketobutyric acid has a significant anti-vascular calcification effect. Its mechanism of action includes at least:

[0025] Inhibits calcium salt deposition: Significantly reduces calcium content in vascular tissue.

[0026] Antagonistic cell transdifferentiation: Effectively reduces the transdifferentiation of vascular smooth muscle cells into osteoblast-like cells.

[0027] Downregulation of osteogenic factors: At the transcriptional and translational levels, the expression of key osteogenic transcription factors and proteins such as RUNX2, BMP2, and MSX2 is inhibited.

[0028] Reduced biochemical activity: Significantly inhibits the activity of alkaline phosphatase (ALP), which is closely related to the calcification process.

[0029] Furthermore, when α-ketobutyric acid is used in combination with sodium hydrosulfide, a donor of endogenous gaseous hydrogen sulfide, it shows a superior synergistic anti-calcification effect in cell and animal models.

[0030] This invention pioneers the use of the known compound α-ketobutyric acid for the novel indication of preventing and treating vascular calcification, providing a new drug candidate for clinical application. As an endogenous substance, α-ketobutyric acid has a potentially high safety profile. This discovery is of great significance for the development of new drugs to treat atherosclerosis, diabetic vascular complications, and cardiovascular diseases associated with chronic kidney disease. Attached Figure Description

[0031] Figure 1 Alizarin Red staining results (A) and Alkaline Phosphatase staining results (B) in Example 1.

[0032] Figure 2 Intracellular Ca in Example 1 2+ Content determination results.

[0033] Figure 3Immunofluorescence results of calcified vascular smooth muscle cells in Example 1.

[0034] Figure 4 In Example 1, Western blot was used to detect the expression of osteogenic markers Runx2, MSX2, and BMP2 proteins.

[0035] Figure 5 Results of echocardiography of mice in Example 2.

[0036] Figure 6 Alizarin Red staining results of the entire aorta of mice in Example 2.

[0037] Figure 7 Results of masson staining, alizarin red staining and calcium salt staining of mouse aortic tissue in Example 2. Detailed Implementation

[0038] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0039] The processes, conditions, reagents, and experimental methods used in implementing this invention, except as specifically mentioned below, are all common knowledge and general knowledge in the field, and this invention does not have any particular limitations. Experimental methods in the embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0040] Unless otherwise stated, all technical terms and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. However, in the event of any conflict, the specification containing the definitions shall prevail.

[0041] The following is some information about the sources of the reagents used in the examples:

[0042] α-Ketobutyric acid (α-KB): Purchased from Shanghai Maclean Biochemical Technology Co., Ltd., product number C12040413.

[0043] Sodium hydrosulfide hydrate: purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number S106641.

[0044] Active vitamin D3 (cholecalciferol): purchased from Solarbio.

[0045] Mice: Male C57BL / 6J mice, SPF grade, purchased from the Department of Experimental Animals, Kunming Medical University (SCXK (Yunnan) K2020-0004).

[0046] High-sugar DMEM, purchased from Gibco.

[0047] Example 1: High-phosphorus-induced vascular smooth muscle cell calcification model experiment

[0048] 1. Preparation of main solutions

[0049] Complete culture medium: High-glucose DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin mixed antibiotics.

[0050] Calcification medium: Dissolve anhydrous sodium dihydrogen phosphate in ultrapure water to prepare a 300 mM stock solution, autoclave, and store at 4°C. Before use, dilute 1:100 into the complete medium to a final concentration of 3 mM.

[0051] Drug solution:

[0052] α-KB solution: Sodium α-ketobutyrate was dissolved in ultrapure water to prepare a 40 mM stock solution, which was then filtered through a 0.22 μm filter membrane for sterilization and stored at 4°C.

[0053] NaHS solution: Dissolve NaHS in ultrapure water to prepare a 40 mM stock solution, filter it through a 0.22 μm filter membrane for sterilization, and store it at 4℃.

[0054] 2. Experimental grouping and drug dosage

[0055] The experiment was divided into the following 5 groups:

[0056] Normal control group (Ctrl): cultured in complete culture medium

[0057] High-phosphorus calcified group (Pi): cultured in calcified medium

[0058] α-KB administration group (Pi+α-KB): calcified medium + 800 μM α-KB

[0059] NaHS administration group (Pi+NaHS): calcified medium + 800 μM NaHS

[0060] Combined drug administration group (Pi + α-KB + NaHS): calcified medium + 400 μM α-KB + 400 μM NaHS

[0061] 3. Model establishment and dosing regimen

[0062] Vascular smooth muscle cells were seeded into appropriate culture dishes (6-well plates, T25 flasks, or 24-well plates, depending on subsequent testing requirements) and incubated at 37°C in a 5% CO2 incubator. When cell confluence reached 80%, the original culture medium was discarded, and the cells were washed twice with PBS. The culture medium was then replaced according to the cell grouping. The medium was changed every 2 days, and the cells were cultured for 6-7 days. When brownish-yellow calcium nodules were observed on the cell surface under a microscope, the calcification model was considered successfully established, and subsequent testing could proceed.

[0063] 4. Detection Method

[0064] (1) Alizarin Red staining

[0065] After model establishment, discard the culture medium, gently wash three times with PBS, and fix with 4% paraformaldehyde for 15 min. Discard the fixative, rinse three times with deionized water, add 2% alizarin red staining solution (pH 4.2), and incubate overnight at room temperature.

[0066] Observe the cell staining. After staining, gently wash away any residual stain with deionized water until the washing solution is clear and colorless. Observe the cleaned six-well plate under an upright white light microscope. Positive results are indicated by red calcified nodules (Alizarin Red).

[0067] (2) Alkaline phosphatase staining

[0068] After model establishment, discard the culture medium, gently wash three times with PBS, and fix with 4% paraformaldehyde for 15 min. Discard the fixative, rinse three times with deionized water, add the prepared BCIP / NBT staining working solution, incubate overnight at room temperature, and observe cell staining. After staining, gently wash away residual staining with deionized water until the washing solution is clear and colorless. Observe the cleaned six-well plate under an upright white light microscope. Positive results are indicated by deep purple calcified nodules.

[0069] (3) Intracellular Ca 2+ Content determination

[0070] Cells from each group were collected, washed three times with cold PBS, and incubated on ice for 30 min with protein lysis buffer. Cells were then collected with a cell scraper and sonicated (200 W, 5 s on / 5 s off, total 30 s). Cells were centrifuged at 14,000 rpm for 30 min at 4°C, and the supernatant was transferred to a new centrifuge tube. Simultaneously, Ca2+ was prepared. 2+ Developing solution. Take a clean 96-well plate and set up blank wells, standard wells, and measurement wells. Add the solution in sequence, taking care to avoid air bubbles. Mix well and let stand at room temperature for 10 min. Zero the plate using the blank well and measure the absorbance at 610 nm using a spectrophotometer. Read the absorbance of the standard and measurement tubes and calculate the Ca. 2+ content.

[0071] (4) Immunofluorescence detection

[0072] Collect cells from successfully calcified 24-well plates, discard the original culture medium, gently wash three times with PBS, and fix with 1 ml of 4% paraformaldehyde for 15 min. Discard the paraformaldehyde and wash three times with PBS. Permeabilize each well with 1 ml of 0.1% Triton X-100 for 10 min. Discard the Triton and wash three times with PBS. Block each well with 1 ml of 5% BSA for 2 h. Discard the blocking solution and add the primary antibody directly, incubating overnight at 4°C. Recover the primary antibody and wash three times with 0.1% PBST for 35 min each time. Add the corresponding fluorescent secondary antibody and incubate at room temperature in the dark for 1.5 h. Recover the secondary antibody and wash three times with 0.1% PBST for 25 min each time. Remove the cells from the slides, stain with DAPI, and air-dry the slides in the dark before photographing.

[0073] (5) Western Blot detection

[0074] Collect T25 cells from successfully calcified culture flasks, discard the culture medium, wash three times gently with PBS, add 200 µl of cell lysis buffer, gently mix, and incubate on ice for 30 min. Tap the flask every 5 min to ensure complete cell lysis, and scrape off cells with a cell scraper. Collect the cells in centrifuge tubes and centrifuge at 14,000 rpm at 4°C for 30 min. Remove the centrifuge tubes and aspirate the protein from the middle layer. Remove the 96-well plates, perform BCA quantification of the protein, and calculate the loading volume. Add 5x loading buffer to the remaining protein, heat in a 95°C metal bath for 15 min to denature, then remove and cool to room temperature before storing at -20°C. Perform Western blotting (WB) to detect osteogenic markers in the collected protein samples.

[0075] 4. Experimental Results

[0076] (1) Alizarin Red Results of Calcified Vascular Smooth Muscle Cells

[0077] See results Figure 1 A. The results showed that, compared with the normal group (Ctrl), the vascular smooth muscle cells in the calcified group (Pi) stained a deeper red with alizarin red, indicating that calcification of vascular smooth muscle cells had occurred. The alizarin red staining of vascular smooth muscle cells in the α-KB-treated group (Pi+α-KB) and the NaHS-treated group (Pi+NaHS) was lighter, indicating that calcification of vascular smooth muscle cells was reduced. The alizarin red staining of vascular smooth muscle cells in the combined treatment group (Pi+α-KB+NaHS) was even lighter, indicating that the combined treatment of the two drugs had a better effect on inhibiting vascular smooth muscle cell calcification.

[0078] (2) Results of alkaline phosphatase in calcified vascular smooth muscle cells

[0079] See results Figure 1B. The results showed that, compared with the normal group (Ctrl), the alkaline phosphatase in the calcified group (Pi) was darker purple, indicating that calcification of vascular smooth muscle cells had occurred. The alkaline phosphatase in the α-KB group (Pi+α-KB) and the NaHS group (Pi+NaHS) was lighter purple, indicating that calcification of vascular smooth muscle cells was reduced. The alkaline phosphatase in the combined drug group (Pi+α-KB+NaHS) was even lighter purple, indicating that the combined drug had a better effect on inhibiting vascular smooth muscle cell calcification.

[0080] (3) Ca in calcified vascular smooth muscle cells 2+ Content detection results

[0081] See results Figure 2 The results showed that, compared with the normal group (Ctrl), the vascular smooth muscle cells in the calcified group (Pi) had significantly higher calcium levels. 2+ The significantly increased content indicates calcification of vascular smooth muscle cells. The groups treated with α-KB (Pi+α-KB), NaHS (Pi+NaHS), and the combination of both drugs (Pi+α-KB+NaHS) showed significantly higher levels of Ca in vascular smooth muscle cells. 2+ The decrease in content indicates that both α-KB and NaHS can inhibit calcification of vascular smooth muscle cells, and the combined use of the two leads to a decrease in Ca content. 2+ The more significant decrease in content indicates a synergistic effect when used in combination.

[0082] (4) Immunofluorescence results of calcified vascular smooth muscle cells

[0083] The experimental results are shown in Figure 3 The results showed that, compared with the normal group (Ctrl), the calcified group (Pi) had increased BMP2 fluorescence intensity and decreased SM22α fluorescence intensity, indicating osteogenic changes in vascular smooth muscle cells. In the groups treated with α-KB (Pi+α-KB) and NaHS (Pi+NaHS), BMP2 fluorescence intensity was decreased and SM22α fluorescence intensity was increased, indicating reduced calcification of vascular smooth muscle cells. In the group treated with the two drugs in combination (Pi+α-KB+NaHS), BMP2 fluorescence intensity was decreased and SM22α fluorescence intensity was increased more significantly, indicating that the combination of the two drugs had a better effect on inhibiting vascular smooth muscle cell calcification.

[0084] (5) Results of Western blot analysis of proteins in calcified vascular smooth muscle cells

[0085] See results Figure 4The results showed that compared with the normal group (Ctrl), the expression of osteogenic markers MSX2, BMP2, and RUNX2 in vascular smooth muscle cells of the calcified group (Pi) was increased, indicating that vascular smooth muscle cells underwent calcification. The expression of the three osteogenic markers in vascular smooth muscle cells of the group treated with α-KB (Pi+α-KB) and the group treated with NaHS (Pi+NaHS) was decreased, indicating that calcification of vascular smooth muscle cells was reduced. The expression of MXS2, BMP2, and RUNX2 in vascular smooth muscle cells of the two-drug combination group (Pi+α-KB+NaHS) was decreased more significantly, indicating that the combination of the two drugs had a better inhibitory effect on vascular smooth muscle cell calcification.

[0086] Example 2: VD3-induced mouse aortic calcification model experiment

[0087] 1. Laboratory Animals and Ethical Statement

[0088] Eight-week-old male C57BL / 6J mice, SPF grade, weighing 20-25 g, were selected. Mice were housed in separate cages of six, with the ambient temperature controlled at 26±2℃ and relative humidity at 55%±5%, maintaining a 12-hour light-dark cycle. During the experiment, mice had free access to standard feed (purchased from the Laboratory Animal Research Institute of Sichuan Provincial People's Hospital, Sichuan Academy of Medical Sciences) and autoclaved tap water. All animals underwent a one-week acclimatization period before the experiment. The experimental protocol was reviewed and approved by the Animal Ethics Committee of Kunming Medical University and strictly followed international guidelines for laboratory animal care and use.

[0089] 2. Preparation of main reagents and solutions

[0090] Vitamin D3 (VD3) injection: Weigh 125 mg of vitamin D3 powder and add it to 50 mL of corn oil. Stir in the dark until completely dissolved to prepare a working solution with a concentration of 2.5 mg / mL (equivalent to 100,000 IU / mL based on 1 mg = 40,000 IU). Store at 4°C in the dark for later use. The normal control group uses an equal volume of pure corn oil.

[0091] α-Ketobutyric acid solution: Dissolve sodium α-ketobutyrate in high-pressure secondary water to prepare an 8 mM stock solution, store at 4°C and use within 3 days.

[0092] Sodium hydrosulfide solution: Dissolve NaHS in physiological saline to prepare an 8 mM stock solution, which should be prepared and used immediately.

[0093] 3. Experimental Design and Animal Grouping

[0094] This experiment employed a two-stage design (induction period + intervention period), randomly dividing mice into 5 experimental groups, with 9 mice in each group (3 mice in each group, with 3 independent biological replicates): normal group (Ctrl), VD3 model group (VD3), α-KB treatment group (VD3+α-KB), NaHS treatment group (VD3+NaHS), and combined treatment group (VD3+α-KB+NaHS).

[0095] In this group, the normal group received subcutaneous or intraperitoneal injections of corn oil as a solvent control throughout the entire experimental period (26 days); the VD3 model group received subcutaneous injections of VD3 to induce calcification from days 1 to 6, and then received saline as a model positive control from days 7 to 26; the α-KB treatment group and the NaHS treatment group received oral α-KB or intraperitoneal injections of NaHS on day 1 of VD3 induction, respectively; and the combined treatment group received both drugs simultaneously and continued to receive them until day 26.

[0096] 4. Model establishment and dosing regimen

[0097] Induction period (days 1-6): Except for the normal group, the other four groups of mice received high-dose subcutaneous injections of VD3 to establish an acute aortic calcification model. The specific procedure was as follows: the dosage volume was calculated daily at 500,000 IU / kg (equivalent to 5 ml / kg). The mouse's back skin was gently pulled back with the left hand to form a "triangular tent" shape. A 1 mL syringe was inserted subcutaneously at a 30-45° angle with the right hand, and the VD3 solution was slowly injected. After confirming no leakage, the needle was withdrawn. The normal group received an equal volume of corn oil (5 mL / kg) in the same manner. Injections were repeated for 6 consecutive days. During the VD3 induction period, all treatment groups received simultaneous drug administration starting from day 1. The α-KB treatment group received 4.5 mL of 8 mM α-KB solution orally daily; the NaHS treatment group received an intraperitoneal injection of NaHS daily at a dose of 60 μM / kg; the combined treatment group received 2.25 mL of 8 mM α-KB solution orally daily and simultaneously received an intraperitoneal injection of NaHS at a dose of 30 μM / kg. Normal group and VD3 model group: Approximate volumes of physiological saline were administered as solvent controls. By administering the drug concurrently during the calcification induction phase, the aim was to inhibit vascular calcification in its early stages, thereby reducing the subsequent degree of vascular calcification.

[0098] Intervention period (days 7-26): VD3 injections were discontinued from day 7. Each experimental group continued to receive α-KB, NaHS, or a combination thereof according to the above-mentioned grouping protocol. The normal control group and the VD3 model group continued to receive the corresponding volume of physiological saline. This phase lasted 20 days, during which all mice were fed a standard diet.

[0099] Endpoint of observation: Euthanasia was performed on day 26 after echocardiography, and aortic tissue was collected for subsequent analysis.

[0100] 5. Detection Methods

[0101] (1) Echocardiographic examination: On day 26 of the experiment, mice were anesthetized with isoflurane (induction concentration 3%, maintenance concentration 1.5-2%) and fixed in a supine position on a heating blanket. The aortic arch and branches were measured through the left sternal view using a 30-40 MHz high-frequency ultrasound probe to assess the structural changes caused by vascular calcification.

[0102] (2) Sample collection: After ultrasound examination, mice were perfused with PBS. After perfusion, the mice were euthanized, and the heart and aorta (from the root of the aortic artery to the level of the diaphragm) were quickly removed by thoracotomy. Surrounding adipose connective tissue was removed. Some tissues were immediately flash-frozen in liquid nitrogen and then transferred to a -80°C freezer for storage for Western blotting and biochemical index detection; other tissues were fixed in 4% paraformaldehyde for 24 h for histopathological staining.

[0103] (3) Histopathological examination: After the fixed aortic samples were thoroughly washed with PBS, the whole aorta was stained with alizarin red (whole staining method): the aorta was immersed in 2% alizarin red-potassium hydroxide staining solution (pH 4.2) and stained overnight at room temperature in the dark. The aorta was gently rinsed with 2% potassium hydroxide until the washing solution was colorless. The percentage of calcification area was observed and calculated under a stereomicroscope. At the same time, the aortic root was paraffin-embedded and 5 μm thick serial sections were prepared. Calcium salt staining (calcium salts are black), Masson's trichrome staining (collagen fibers are blue, muscle fibers are red), and alizarin red staining were performed to observe the integrity of the vascular wall structure, the degree of fibrosis, and the distribution of calcium salt deposition.

[0104] 6. Experimental Results

[0105] (1) Ultrasonic test results

[0106] See results Figure 5 Compared with the normal group (Ctrl), the aortic arch of the calcified group (VD3) had a blurred edge contour and a bright echo, indicating that the aorta of the VD3 group mice had calcification and the modeling was successful. The bright part of the aortic arch was reduced in the α-KB group (VD3+α-KB) and the NaHS group, and the aortic arch calcification was alleviated. The bright part of the aortic arch of the mice in the combination of the two drugs (VD3+α-KB+NaHS) was reduced even more significantly, indicating that the combined drug treatment was more effective in reducing aortic calcification.

[0107] (2) Results of Alizarin Red staining of the entire aorta of mice

[0108] See results Figure 6 The results showed that, compared with the normal group (Ctrl), the aorta of the calcified group (VD3) had a deeper alizarin red staining, indicating that aortic calcification had occurred. The alizarin red staining of the aorta in the α-KB group (VD3+α-KB) and the NaHS group (VD3+NaHS) was lighter, indicating that aortic calcification was reduced. The alizarin red staining of the aorta in the combined treatment group (VD3+α-KB+NaHS) was even lighter, indicating that the combined treatment of the two drugs had a better effect on reducing aortic calcification.

[0109] (3) Results of Masson staining, Alizarin Red staining (ARS staining), and Von Kossa staining of calcified aortic tissue in mice.

[0110] See results Figure 7 The results showed that, compared with the normal group (Ctrl), the aorta of the calcified group (VD3) had increased blue collagen fibers stained with masson's stain, indicating aortic lesions. Alizarin red staining was deeper, and calcium salt staining showed dark purple or even black precipitates, indicating aortic calcification. In the groups treated with α-KB (VD3+α-KB) and NaHS (VD3+NaHS), the aortic tissue showed decreased blue collagen fibers stained with masson's stain, increased red muscle fibers, lighter red stained alizarin red, and reduced dark purple calcium salt precipitates, indicating reduced aortic calcification. In the combined treatment group (VD3+α-KB+NaHS), the reduction in blue collagen fibers stained with masson's stain was more pronounced, the red stained alizarin red was lighter, and the reduction in dark purple calcium salt precipitates was even greater, indicating that the combined treatment of the two drugs was more effective in reducing aortic calcification.

[0111] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of α-ketobutyrate in the preparation of drugs for the prevention and / or treatment of vascular calcification.

2. The application according to claim 1, characterized in that, The drug also includes sodium hydrosulfide.

3. The application according to claim 1 or 2, characterized in that, The vascular calcification is manifested as osteoblastic transdifferentiation of vascular smooth muscle cells and / or abnormal deposition of calcium salts in the vessel wall.

4. The application according to claim 1 or 2, characterized in that, The drug also includes pharmaceutically acceptable excipients.

5. A pharmaceutical composition for preparing a treatment for preventing and / or treating vascular calcification, characterized in that, The pharmaceutical composition comprises a therapeutically effective amount of an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient comprises α-ketobutyrate and sodium hydrosulfide.

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