Use of a drug taking acly gene as a target in preparation of a drug for preventing and treating vascular calcification

Abstract

The application relates to application of a medicine taking ACLY gene as an action target in preparation of a medicine for preventing and treating vascular calcification, and belongs to the technical field of biological medicine. In order to solve the problem that effective clinical means for treating vascular calcification is lacked at present, the application provides application of a medicine taking ACLY gene as an action target in preparation of a medicine for preventing and treating vascular calcification, the transcription or translation of the ACLY gene of vascular smooth muscle cells is efficiently and specifically inhibited, or the expression or activity of the ACLY protein of the vascular smooth muscle cells is efficiently and specifically reduced, the osteogenic differentiation of smooth muscle is reduced, the deposition of hydroxyapatite in the vascular media is reduced, and the progress of vascular calcification is alleviated. The medicine for preventing and treating vascular calcification is prepared on the basis of inhibition of the ACLY of vascular smooth muscle, has important clinical application potential for vascular calcification treatment, and can provide a new treatment option for patients with vascular calcification.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of drugs that target the ACLY gene in the preparation of drugs for the prevention and treatment of vascular calcification. Background Technology

[0002] Vascular calcification is a systemic vascular disease and an independent risk factor for mortality and poor prognosis in patients with cardiovascular disease. It promotes plaque instability in atherosclerosis and significantly increases the risk of myocardial infarction and stroke. Especially in patients with chronic kidney disease, diabetes, and the elderly, vascular calcification is significantly positively correlated with all-cause mortality. The pathological feature of this disease is mainly the ectopic deposition of hydroxyapatite in the vascular wall, a dynamic process that can affect all structural components, including the intima and media. From a pathogenesis perspective, media calcification is an active, cell-regulated osteoblast-like differentiation process. When vascular smooth muscle cells are exposed to a pathological microenvironment high in mineral ions for a long period, they undergo a phenotypic transformation towards osteo / chondrocyte-like cells: downregulation of calcification inhibitory factors, abnormal upregulation of osteoblast / osteoclast marker molecules, accompanied by changes in extracellular matrix composition and disturbances in matrix hydrolytic enzyme activity. Currently, clinical interventions for vascular calcification have limited efficacy, and specific targeted drugs are lacking.

[0003] ATP citrate lyase (ACLY) is a key enzyme linking carbohydrate metabolism and lipid synthesis, catalyzing the conversion of citrate to acetyl-CoA. Acetyl-CoA is not only an important component in fatty acid and cholesterol synthesis, but it also provides acetyl groups essential for histone acetylation and gene expression regulation. ACLY plays a crucial role in connecting metabolism and epigenetic modifications. Inhibition of ACLY has shown significant anti-tumor effects in preclinical models, and its pharmacological inhibitors have been approved by the FDA for lipid-lowering therapy. However, the role of ACLY in vascular calcification diseases is currently lacking in research, and no related drugs or treatment strategies have entered clinical trials. Summary of the Invention

[0004] To address the current lack of effective clinical treatments for vascular calcification, this invention provides the application of drugs targeting the ACLY gene in the preparation of drugs for the prevention and treatment of vascular calcification.

[0005] The technical solution of the present invention:

[0006] Application of drugs targeting the ACLY gene in the preparation of drugs for the prevention and treatment of vascular calcification, wherein the ACLY gene is located in the c41866916-41930542 region of the GRCh38 genome version of human chromosome 17.

[0007] Furthermore, the drug that targets the ACLY gene is prepared based on interfering with the ACLY gene to prevent and treat vascular calcification. The prepared drug can efficiently and specifically inhibit the transcription or translation of the ACLY gene in smooth muscle cells, or can efficiently and specifically reduce the expression or activity of ACLY protein in smooth muscle cells, thereby reducing smooth muscle osteogenic differentiation, reducing the deposition of hydroxyapatite in the vascular media, and alleviating the progression of vascular calcification.

[0008] Furthermore, the drug that targets the ACLY gene reduces the level of acetyl-CoA in vascular smooth muscle cells by inhibiting ACLY expression or activity, thereby alleviating the progression of vascular calcification.

[0009] Furthermore, the drug that targets the ACLY gene reduces the levels of H3K27ac and Runx2 in vascular smooth muscle cells by inhibiting the expression or activity of ACLY in these cells, thereby alleviating the progression of vascular calcification.

[0010] Furthermore, the drugs that target the ACLY gene are nucleic acid molecules, carbohydrates, lipids, small molecule chemicals, antibody drugs, peptides, proteins, or adeno-associated viruses.

[0011] Furthermore, the nucleic acid molecule is an antisense oligonucleotide, double-stranded RNA, small interfering RNA, or short hairpin RNA; the nucleic acid molecule silences the ACLY gene, inhibits the expression of ACLY protein in smooth muscle cells, weakens osteogenic differentiation of smooth muscle cells, and thus alleviates vascular calcification.

[0012] Furthermore, the adeno-associated virus contains a nucleotide sequence that interferes with the expression of the ACLY gene. This adeno-associated virus can silence the ACLY gene, reduce the expression of ACLY protein in smooth muscle cells, weaken the osteogenic differentiation of smooth muscle cells, and thus alleviate vascular calcification.

[0013] Furthermore, the drug for preventing vascular calcification also contains pharmaceutically acceptable excipients.

[0014] Furthermore, the excipient is one or more of glucose, sucrose, sorbitol, mannose, starch, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, or water.

[0015] Furthermore, the drug for preventing vascular calcification is in the form of tablets, pills, powders, or injections.

[0016] The beneficial effects of this invention are:

[0017] This invention demonstrates through animal and cell experiments that drugs targeting the ACLY gene can efficiently and specifically inhibit the transcription or translation of the ACLY gene in vascular smooth muscle cells, or reduce the expression or activity of ACLY protein in vascular smooth muscle cells, thereby reducing smooth muscle osteogenic differentiation, decreasing hydroxyapatite deposition in the vascular media, and alleviating the progression of vascular calcification. Based on the inhibition of ACLY in vascular smooth muscle, this invention has prepared a drug for the prevention and treatment of vascular calcification, which has significant clinical application potential and is expected to provide new treatment options for patients with vascular calcification. Attached Figure Description

[0018] Figure 1 From left to right, the graph shows the comparison of serum creatinine, blood urea nitrogen, and serum phosphorus levels in mice in the sham-operated group and the model group in Example 1.

[0019] Figure 2 This is a comparison of the results of alizarin red staining and silver nitrate staining of aortic sections from mice in the sham-operated group and the model group in Example 1;

[0020] Figure 3 This is a schematic diagram illustrating the treatment of vascular calcification induced by 5 / 6 nephrectomy in mice using AAV9-ACLYshRNA in Example 3.

[0021] Figure 4 This is a comparison of the ACLY level protein imprinting results in the aortic tissues of two groups of mice after treatment with AAV9-ACLYshRNA in Example 3;

[0022] Figure 5 This is a comparison of acetyl-CoA levels in the aortic tissue of two groups of mice after treatment with AAV9-ACLYshRNA in Example 3.

[0023] Figure 6 This is a comparison of the results of alizarin red staining and silver nitrate staining of aortic sections from two groups of mice after treatment with AAV9-ACLYshRNA in Example 3.

[0024] Figure 7 From left to right, the graph shows a comparison of aortic calcium content and alkaline phosphatase activity in two groups of mice after treatment with AAV9-ACLYshRNA in Example 3.

[0025] Figure 8 This is a schematic diagram illustrating the treatment of vascular calcification induced by 5 / 6 nephrectomy in mice using the ACLY inhibitor BMS303141 in Example 4.

[0026] Figure 9 This is a comparison of acetyl-CoA levels in the aorta of two groups of mice after using BMS303141 in Example 4.

[0027] Figure 10This is a comparison of the results of alizarin red staining and silver nitrate staining of aortic sections from two groups of mice after using BMS303141 in Example 4.

[0028] Figure 11 From left to right, the graph shows a comparison of aortic calcium content and alkaline phosphatase activity in two groups of mice after using BMS303141 in Example 4.

[0029] Figure 12 This is a comparison of the alizarin red staining results of the two groups of cells after treatment with ACLYsiRNA and NCsiRNA in Example 5.

[0030] Figure 13 This is a comparison of the expression levels of ACLY and Runx2 proteins in two groups of cells after treatment with ACLYsiRNA and NCsiRNA in Example 5.

[0031] Figure 14 From left to right, the graph shows the comparison of H3K27ac levels, Sox9 and Runx2 mRNA expression levels in the two groups of cells after treatment with ACLYsiRNA and NCsiRNA in Example 5.

[0032] Figure 15 This is a comparison of the alizarin red staining results of cells in the control group and the BMS303141 treatment group in Example 6.

[0033] Figure 16 This is a comparison of Runx2 protein expression levels in cells of the control group and the BMS303141 treatment group in Example 6;

[0034] Figure 17 From left to right, the graph shows a comparison of the levels of H3K27ac, Sox9, and Runx2 mRNA expression in the control group and the BMS303141 treatment group in Example 6. Detailed Implementation

[0035] The technical solution of the present invention will be further described below with reference to embodiments, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be covered within the protection scope of the present invention. In the following embodiments, the process equipment or apparatus not specifically specified are all conventional equipment or apparatus in the art. Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commercially available; unless otherwise specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0036] The statistical analysis methods for Examples 1-6 are as follows:

[0037] All statistical tests were performed using SPSS or GraphPad software. Data are presented as mean ± SEM. The Mann-Whitney U test or Student's t-test was used to compare variables between the two groups. A p-value < 0.05 was considered statistically significant.

[0038] All animal studies were conducted in accordance with guidelines approved by the Animal Experiment Ethics Committee of the Second Affiliated Hospital of Harbin Medical University.

[0039] Example 1

[0040] This embodiment provides a method for constructing a mouse model of vascular calcification induced by 5 / 6 nephrectomy.

[0041] I. Laboratory Animals

[0042] Male C57Bl / 6J mice aged 6-7 weeks (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were selected and fasted for 24 hours before surgery, but were allowed to drink water normally.

[0043] II. Methods for establishing a mouse model of vascular calcification induced by 5 / 6 nephrectomy:

[0044] 5 / 6 nephrectomy: C57Bl / 6J mice were anesthetized with sevoflurane and the upper and lower poles of the left kidney, i.e., two-thirds of the left kidney, were removed. One week later, the right kidney was completely removed. One week after the 5 / 6 nephrectomy, the mice were given a 1.5% high-phosphate diet. Five weeks later, a mouse model of vascular calcification induced by 5 / 6 nephrectomy was successfully established.

[0045] Mice in the sham surgery group and the 5 / 6 nephrectomy group underwent the same surgery at the same time, but the renal capsule was removed only after the kidneys were exposed, and then the abdomen was closed.

[0046] III. Extraction of aortic tissue from each group of mice

[0047] Before sampling, each mouse was weighed. Mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital. Blood was collected from the apex of the heart, and the plasma was temporarily kept at 4°C. After sampling, the plasma was centrifuged to obtain serum. After opening the chest and abdomen, the blood in the systemic circulation was rinsed with physiological saline. The lungs, trachea, esophagus, liver, and other organs in front of the aorta were carefully cleaned to expose the aorta, which was then removed along with the heart. Subsequently, the connective tissue around the aorta was carefully dissected under a stereomicroscope. The cleaned aorta was preserved in a general tissue fixative and embedded in paraffin within one week for subsequent histopathological analysis, or the aorta was placed in a -80°C freezer for later use.

[0048] IV. Methods for detecting serum creatinine, blood urea nitrogen, and serum phosphorus levels in mice

[0049] (1) Serum creatinine test

[0050] Using the creatinine colorimetric assay kit (sarcosine oxidase method) (purchased from Elabscience), the required reagents were prepared according to the instructions. 12 μl of mouse serum and 180 μl of reagent one were added to each well. The mixture was incubated at 37°C for 5 minutes, and then 60 μl of reagent two was added. The OD value of each well was measured at 515 nm using an ELISA reader.

[0051] (2) Blood urea nitrogen test

[0052] Using the urea colorimetric assay kit (urease method) (purchased from Elabscience), the required reagents were prepared according to the instructions. 4 μl of mouse serum and 50 μl of enzyme working solution were added to each well. The mixture was incubated at 37°C for 10 minutes. Then, 125 μl of reagent one and reagent two were added. The OD value of each well was measured at 580 nm using an ELISA reader.

[0053] (3) Blood phosphorus test

[0054] Using the phosphorus colorimetric assay kit (phosphomolybdic acid method) (purchased from Elabscience), the required reagents were prepared according to the instructions. 35 μl of mouse serum and 200 μl of chromogenic working solution were added to each well. The mixture was incubated at 37°C for 30 minutes, and the OD value of each well was measured at 660 nm using a microplate reader.

[0055] The results are as follows Figure 1 As shown, compared with the sham surgery group, the serum creatinine, blood urea nitrogen, and serum phosphorus levels of mice in the 5 / 6 nephrectomy model group were significantly increased.

[0056] V. Evaluation of vascular histology using alizarin red and silver nitrate staining

[0057] (1) Alizarin Red staining: The aortic tissue sections of each group of mice were incubated in 2% Alizarin Red solution at room temperature for 10 minutes and rinsed with double distilled water. The positive staining parts were red / purple.

[0058] (2) Silver nitrate staining: The aortic tissue sections of each group of mice were incubated in silver nitrate solution and irradiated under ultraviolet light for 30 minutes. They were then rinsed with double distilled water. The positive staining parts were brown to black.

[0059] The results are as follows Figure 2 As shown, compared with the sham surgery group, the aorta of mice in the 5 / 6 nephrectomy model group showed significantly deeper Alizarin Red and Silver Nitrate staining, indicating that the calcium and phosphorus deposition in the blood vessels of the model group mice was significantly increased, and the vascular calcification mouse model was successfully established.

[0060] Example 2

[0061] This embodiment provides a method for constructing an adeno-associated virus—the AAV9 virus carrying the SM22 promoter.

[0062] Step 1: Using the short hairpin ribonucleic acid structure of the ACLY gene—the ACLY shRNA nucleic acid sequence—as the target gene, the following ACLY shRNA nucleic acid sequence was obtained through gene editing technology:

[0063] The nucleotide sequence of the positive strand of ACLY shRNA, as shown in SEQ ID NO:1, is as follows:

[0064] 5'-ATGGTGGAATGCTGGACAACATTAGTGAAGC-3';

[0065] The nucleotide sequence of the antisense strand of ACLY shRNA, as shown in SEQ ID NO:2, is as follows:

[0066] 5'-CACAGATGTAATGTTGTCCAGCATTCCACCAG-3'.

[0067] Step 2: Construct the AAV9 recombinant vector and verify it through sequencing:

[0068] The ACLY shRNA nucleic acid sequence obtained in step one was subcloned into the AAV9 expression vector (purchased from Jinan Boshan Company) by enzyme digestion-ligation-transformation. After obtaining the ACLY shRNA positive clone, the correctness of the inserted fragment was confirmed by sequencing, thus obtaining the ACLY shRNA plasmid.

[0069] Step 3: AAV9 virus packaging:

[0070] HEK293T cells with a polymerization degree of over 90% (purchased from ATCC) were transferred to discs at a ratio of 1:3, with approximately 2.5 × 10⁶ cells per disc. 6 One to two hours before plasmid transfection, the culture medium was replaced with serum-free medium, and transfection reagent (Lipofectamine 3000, purchased from Thermo) was used. The ACLY shRNA plasmid obtained in step two and the negative control NC shRNA plasmid were transfected into HEK293T cells, respectively. The medium was replaced with fresh serum-free medium 24 hours after transfection. After 72 hours, the virus was collected separately. The culture medium did not need to be discarded. The cells were centrifuged to obtain the supernatant and cell pellet. The virus in the supernatant was precipitated using PEG8000, and the pellets were collected after incubation overnight.

[0071] Step 4: Virus purification and concentration:

[0072] The viral mixture obtained in step three was purified by density gradient centrifugation with iodixanol, where the densities of iodixanol from top to bottom were 5%, 15%, 25%, 40%, and 54%. The centrifuged viral liquids were then concentrated in ultrafiltration tubes. The remaining liquid in the ultrafiltration tubes was repeatedly pipetted and transferred to viral storage tubes, and viral storage solution was added to obtain viral AAV9-ACLYshRNA carrying the SM22 promoter and negative control viral AAV9-NCshRNA, respectively.

[0073] Step 5, Virus titer detection: RT-PCR is used to determine the number of virus particles. Virus particle count (particles / ml) = relative value to standard.

[0074] Example 3

[0075] In this embodiment, the viral AAV9-ACLYshRNA carrying the SM22 promoter prepared in Example 2 was used as a therapeutic drug. Animal experiments were conducted to investigate the effect of the drug, which targets the ACLY gene, on alleviating the process of vascular calcification.

[0076] I. Grouping Methods in Animal Experiments

[0077] (1) Treatment group:

[0078] Male C57Bl / 6J mice aged 6-7 weeks were selected and injected via tail vein with viral AAV9-ACLYshRNA carrying the SM22 promoter. 12 vg / mouse, and after two weeks, 5 / 6 nephrectomy-induced vascular calcification mice were obtained by constructing the model construction method provided in Example 1;

[0079] (2) Control group:

[0080] Male C57Bl / 6J mice aged 6-7 weeks were selected and injected with the negative control virus AAV9-NCshRNA via the tail vein. 12 vg / mouse, and after two weeks, 5 / 6 nephrectomy-induced vascular calcification mice were obtained by constructing the model construction method provided in Example 1;

[0081] II. Protein Imprinting Experiment

[0082] Mouse aortic tissues lysed by sonication or grinding were placed in RIPA lysis buffer, and protein quantification was performed using the BCA method, followed by SDS-PAGE gel electrophoresis. After transfer and blocking, the corresponding primary antibody was added, and the membrane was incubated overnight at 4°C. After washing with TBST, the membrane was incubated at room temperature with fluorescently labeled secondary antibody, and the protein blotting results were quantitatively analyzed using ImageJ.

[0083] III. Acetyl-CoA Detection

[0084] The test was performed using the Acetyl-Coenzyme A assay kit (purchased from Sigma). A tissue sample (20 mg) or a cell sample (10 mg) was obtained. 5 (1 cell), after flash freezing in liquid nitrogen, the sample was deproteinized using PCA. The acetyl-CoA detection solution was prepared according to the kit instructions and added to the sample. The sample was incubated at 37°C for 10 minutes, and the fluorescence intensity was detected using a fluorescence detector at an emission spectrum of 535.

[0085] The results are as follows Figure 4 and Figure 5 As shown, compared with the control group mice, the expression of ACLY in the aortic tissue of the treatment group mice was successfully inhibited, and the level of acetyl-CoA, a product of the citrate metabolism pathway involving ACLY, was also reduced.

[0086] IV. Assess vascular histology using alizarin red staining and silver nitrate staining, using the same methods as in Example 1.

[0087] The results are as follows Figure 6 As shown, in the control group, 5 / 6 kidney resection was performed to create a model of vascular calcification, and the vascular tissue of the mice showed obvious hydroxyapatite crystal deposition. In the treatment group, administration of AAV9-ACLY shRNA significantly alleviated the deposition of hydroxyapatite crystals in the aorta and relieved vascular calcification.

[0088] V. The deposition of hydroxyapatite in the vascular media was assessed by measuring aortic calcium content and alkaline phosphatase activity.

[0089] (1) Determination of aortic calcium content

[0090] The aortas of mice in each group were washed three times with calcium-free PBS, dried, and weighed. After drying, the aortas were decalcified with 0.1 M hydrochloric acid for 24 hours. The calcium content in the supernatant was measured using a calcium assay kit (purchased from Elabscience). The required reagents were prepared according to the instructions; 10 μl of sample and 260 μl of working solution were added to each well, and the mixture was incubated at room temperature for 5 minutes. The OD value was measured at 610 nm using a microplate reader. The calcium content was standardized to dry weight.

[0091] (2) Aortic alkaline phosphatase activity assay

[0092] Total protein was extracted from aortic tissue using RIPA lysis buffer without inhibitors. Protein content was measured using the biuret (BCA) protein assay (Thermo Fisher Scientific). Alkaline phosphatase activity was determined by colorimetric method (Elabscience). Reagents were prepared according to the instructions. 5 μl of sample was added to each well, followed by 50 μl each of working solutions I and II. The mixture was incubated at 37°C for 15 minutes, and then 150 μl of chromogenic solution was added. The OD value was measured at 520 nm using a microplate reader. Alkaline phosphatase activity was standardized to the total protein content of the aorta.

[0093] The results are as follows Figure 7 As shown, after 5 / 6 nephrectomy modeling, the control group mice showed a significant increase in aortic calcium content and alkaline phosphatase activity, while the AAV9-ACLYshRNA injected into the treatment group mice significantly reduced the levels of intravascular membrane calcium and alkaline phosphatase, effectively alleviating the progression of vascular calcification.

[0094] Example 4

[0095] This embodiment uses the ACLY inhibitor BMS303141 as a therapeutic drug and examines the effect of drugs that target the ACLY gene in alleviating the process of vascular calcification through animal experiments.

[0096] I. Grouping Methods in Animal Experiments

[0097] (1) Treatment group:

[0098] Following the model construction method provided in Example 1, mice were given a 1.5% high-phosphate diet one week after 5 / 6 nephrectomy, and were simultaneously treated by gavage with the ACLY inhibitor BMS303141 (purchased from MedChemExpress) at a dose of 50 mg / kg / day for 5 weeks.

[0099] (2) Control group:

[0100] Following the model construction method provided in Example 1, mice were fed a 1.5% high-phosphate diet one week after 5 / 6 nephrectomy, while corn oil was used as a control by gavage. The feeding period was 5 weeks.

[0101] 2. Five weeks later, mice in each group were sacrificed to examine the therapeutic effect of BMS303141. The acetyl-CoA level in the aortic tissue of mice was detected. The histology of blood vessels was assessed using alizarin red staining and silver nitrate staining. The deposition of hydroxyapatite in the vascular media was assessed by aortic calcium content and alkaline phosphatase activity. The methods for measuring acetyl-CoA level, pathological staining, and detecting calcium content and alkaline phosphatase activity were the same as in Example 3.

[0102] The acetyl-CoA levels in the two groups of mice were as follows: Figure 9 As shown, oral administration of the ACLY inhibitor BMS303141 effectively inhibited the production of acetyl-CoA, a product of the citrate metabolism pathway; the results of alizarin red staining and silver nitrate staining of the aorta in both groups of mice, as well as the detection results of aortic calcium content and alkaline phosphatase activity, are as follows. Figure 10 and Figure 11As shown, after 5 / 6 nephrectomy modeling, the control group mice developed chronic vascular calcification, with obvious calcium nodule formation in the aorta, increased vascular calcium content and alkaline phosphatase activity, while the treatment group, orally administered BMS303141, significantly reduced the deposition of hydroxyapatite in the vascular membrane, calcium and alkaline phosphatase levels, and significantly alleviated the progression of calcification.

[0103] Example 5

[0104] This embodiment uses ACLY siRNA as a therapeutic agent and examines the effect of drugs that target the ACLY gene on alleviating the process of vascular calcification through cell experiments.

[0105] I. Preparation method of primary mouse vascular smooth muscle cells

[0106] Primary aortic smooth muscle cells (VSMCs) were isolated from the thoracic aorta of 6- to 10-week-old C57Bl / 6J mice. The aorta was first separated from the body, and an incision was made at the aortic arch to obtain only the thoracic segment. The isolated aorta was washed twice with ice-cold PBS and cultured in Ham's F12 medium with 1 mL of 0.2% collagenase I solution at 37°C for 30 min. Under microscopic guidance, the adventitia of the aorta was removed using forceps. The aorta was longitudinally incised, and the endothelial cells were gently scraped away. The aorta was then cut into small pieces and placed at the bottom of a culture dish, and cultured for several days in DMEM / F-12 medium containing 15% fetal bovine serum and 1% penicillin-streptomycin at 37°C in a humidified environment of 5% CO2. Cells migrating from the explants were collected and maintained in growth medium. VSMCs from passages 3 to 6 were used for further experiments. The purity of VSMCs was confirmed by positive staining for SM22α and α-SMA.

[0107] II. Methods for inducing cell calcification models:

[0108] Primary mouse vascular smooth muscle cells were cultured in DMEM medium containing 10% fetal bovine serum, 1% penicillin and streptomycin, and 3.0 mM phosphate. The medium was changed regularly and the cells were cultured continuously for 7 days.

[0109] III. Cell Experiment Grouping Methods

[0110] (1) Treatment group:

[0111] Primary mouse vascular smooth muscle cells were treated with ACLY siRNA and then induced to develop a high-phosphate calcification model.

[0112] The nucleotide sequence of ACLY siRNA in this embodiment is as follows:

[0113] The nucleotide sequence of the positive strand of ACLY siRNA, as shown in SEQ ID NO:3, is as follows:

[0114] 5'-GUGGAAUGCUAAGCAACAUTT-3';

[0115] The nucleotide sequence of the antisense strand of ACLY siRNA, as shown in SEQ ID NO:4, is as follows:

[0116] 5'-AUGUUGUCCAGCAUUCCACTT-3'.

[0117] To comply with WIPO ST.26 standards, uracil in RNA is represented by T replacing the U at positions 2, 7, 10, and 19 at the 5' end in the nucleotide sequence shown in SEQ ID NO:3, and uracil in RNA is represented by T replacing the U at positions 2, 4, 5, 7, 14, and 15 at the 5' end in the nucleotide sequence shown in SEQ ID NO:4.

[0118] The specific transfection steps for ACLY siRNA are as follows (taking a six-well plate as an example): Dilute 5 μl of transfection reagent lipo3000 with 100 μl opti-MEM (purchased from Thermo) to obtain solution A. Dilute ACLY siRNA with 100 μl opti-MEM to a concentration of 1 μM to obtain solution B. Slowly add solution B to solution A, incubate at room temperature for 15-20 minutes, and then add to the cell well plate.

[0119] (2) Control group:

[0120] Primary mouse vascular smooth muscle cells were treated with NC siRNA and then induced to develop a calcified hyperphosphatemic calcification model. The specific transfection procedure was the same as that in the treatment group.

[0121] The nucleotide sequence of the NC siRNA in this embodiment is as follows:

[0122] The nucleotide sequence of the positive strand of NC siRNA, as shown in SEQ ID NO:5, is as follows:

[0123] 5'-UUCUCCGAACGUGUCACGUTT-3';

[0124] The nucleotide sequence of the antisense strand of NC siRNA, as shown in SEQ ID NO:6, is as follows:

[0125] 5'-ACGUGACACGUUCGGAGAATT-3'.

[0126] To comply with WIPO ST.26 standards, uracil in RNA is represented by T replacing the U at positions 1, 2, 4, 12, 14, and 19 at the 5' end in the nucleotide sequence shown in SEQ ID NO:5, and uracil in RNA is represented by T replacing the U at positions 4, 11, and 12 at the 5' end in the nucleotide sequence shown in SEQ ID NO:6.

[0127] IV. Alizarin Red Staining Method for Cells

[0128] Vascular smooth muscle cells were fixed with 4% formaldehyde at room temperature, incubated with 2% alizarin red staining solution at room temperature for 10 minutes, and rinsed with double-distilled water. Positive staining areas appeared red / purple.

[0129] The results are as follows Figure 12 As shown, the control group cells exhibited multiple calcium crystals in the high phosphorus-induced cellular calcification, and the cells were stained dark red by Alizarin Red. The cells treated with ACLY-siRNA showed a significant reduction in calcium crystals, thus delaying the progression of calcification.

[0130] V. The same protein imprinting experiment as in Example 3 was used to examine the ACLY and Runx2 levels in each group of cells. The results are as follows: Figure 13 As shown, compared with the control group, the levels of ACLY and Runx2 proteins in calcified primary mouse vascular smooth muscle cells were significantly reduced after ACLY-siRNA treatment.

[0131] VI. H3K27ac Detection Method

[0132] The Acetyl-Histone H3 (Lys27) ELISA Kit (purchased from CST) was used. Smooth muscle cells were pooled to 80-90% confluence, washed with PBS, and then cell extraction buffer was added. The cells were incubated on ice for 5 minutes, centrifuged at 14000 rpm for 5 minutes to obtain the supernatant, and 50 μl of antibody was added. The plates were incubated at 400 rpm for 1 hour at room temperature on a plate shaker. The plates were then washed, and 100 μl of TMB substrate was added. The plates were incubated in the dark at room temperature at 400 rpm for 15 minutes. 100 μl of stop solution was added to each well, and the absorbance was read at 450 nm using a microplate reader.

[0133] The results are as follows Figure 14 As shown, the H3K27ac level in the control group increased significantly after high phosphorus calcification induction, while the H3K27ac level decreased after ACLY expression was inhibited using ACLYsiRNA.

[0134] VII. Real-time quantitative PCR detection

[0135] Total RNA was extracted from cells using Trizol, and after reverse transcription, it was converted into cDNA. Using cDNA as a template, primers for the target gene were added, and the target gene was amplified and detected using a real-time quantitative PCR kit. The relative expression of each gene was normalized using the internal control β-actin, and 2-... ΔΔCT The method is used for analysis.

[0136] The results are as follows Figure 14 As shown, after high phosphorus calcification induction, the levels of Runx2 and Sox9 mRNA in the control group were significantly increased, while the expression of Runx2 and Sox9 was significantly reduced after inhibiting ACLY expression with ACLYsiRNA.

[0137] Example 6

[0138] In this embodiment, the ACLY inhibitor BMS303141 was used as a therapeutic drug. Cell experiments were conducted to investigate the effect of drugs that target the ACLY gene on alleviating the process of vascular calcification.

[0139] (1) Treatment group:

[0140] Primary mouse vascular smooth muscle cells were treated with BMS303141 (final concentration 25 μM), and a high-phosphate-induced cell calcification model was established 12 hours later.

[0141] (2) Control group:

[0142] Primary mouse smooth muscle cells were treated with DMSO as a negative control, and a high-phosphate-induced cell calcification model was established 12 hours later.

[0143] III. The two groups of cells were stained using the same Alizarin Red staining method as in Example 4, and the results are as follows: Figure 15 As shown, the model group cells exhibited high phosphorus-induced cellular calcification with multiple calcium crystals. The cells were stained dark red with Alizarin Red. The calcium crystals in the cells treated with BMS303141 were significantly reduced.

[0144] IV. The same protein imprinting experiment as in Example 3 was used to examine the Runx2 levels in each group of cells. The results are as follows: Figure 16 As shown, compared with the control group, the Runx2 protein level in calcified primary mouse vascular smooth muscle cells treated with BMS303141 was significantly reduced. The H3K27ac level, Sox9 and Runx2 mRNA levels were detected using the same H3K27ac detection method and real-time quantitative PCR method as in Example 5. The results are as follows: Figure 17As shown, compared with the control group, BMS303141 treatment significantly reduced the levels of high phosphorus-induced H3K27ac, Sox9, and Runx2 mRNA, explaining the mechanism by which targeted inhibition of ACLY expression in vascular smooth muscle cells alleviates vascular calcification at the molecular level.

[0145] In summary, inhibiting ACLY reduces H3K27ac levels in smooth muscle cells, thereby downregulating the expression of the calcification-related transcription factor Runx2 and ultimately alleviating the progression of vascular calcification. Therefore, targeting and inhibiting ACLY expression in vascular smooth muscle cells is an effective strategy for the treatment of vascular calcification.

Claims

1. The application of drugs targeting the ACLY gene in the preparation of drugs for preventing and treating vascular calcification, characterized in that, The ACLY gene is located in the c41866916-41930542 region of the GRCh38 genome version of human chromosome 17; the drug targeting the ACLY gene is the ACLY inhibitor BMS303141, ACLY shRNA, or ACLY siRNA; the sense strand nucleotide sequence of the ACLY shRNA is shown in SEQ ID NO:1, and the antisense strand nucleotide sequence is shown in SEQ ID NO:2; the sense strand nucleotide sequence of the ACLY siRNA is shown in SEQ ID NO:3, and the antisense strand nucleotide sequence is shown in SEQ ID NO:

4.

2. The application of the drug targeting the ACLY gene according to claim 1 in the preparation of drugs for preventing and treating vascular calcification, characterized in that, The drug that targets the ACLY gene is prepared based on interfering with the ACLY gene to prevent and treat vascular calcification. The prepared drug can efficiently and specifically inhibit the transcription or translation of the ACLY gene in smooth muscle cells, or can efficiently and specifically reduce the expression or activity of ACLY protein in smooth muscle cells.

3. The application of the drug targeting the ACLY gene according to claim 2 in the preparation of drugs for preventing and treating vascular calcification, characterized in that, The drug that targets the ACLY gene reduces the level of acetyl-CoA in vascular smooth muscle cells by inhibiting ACLY expression or activity.

4. The application of the drug targeting the ACLY gene according to claim 2 in the preparation of drugs for preventing and treating vascular calcification, characterized in that, The drug that targets the ACLY gene reduces the levels of H3K27ac and Runx2 in vascular smooth muscle cells by inhibiting the expression or activity of ACLY in these cells.

5. The application of the drug targeting the ACLY gene according to claim 1 in the preparation of drugs for preventing and treating vascular calcification, characterized in that, The drug for preventing vascular calcification also contains pharmaceutically acceptable excipients.

6. The application of the drug targeting the ACLY gene according to claim 5 in the preparation of drugs for preventing and treating vascular calcification, characterized in that, The excipient is one or more of glucose, sucrose, sorbitol, mannose, starch, polyvinylpyrrolidone, cellulose, or water.

7. The application of the drug targeting the ACLY gene according to claim 1 in the preparation of drugs for preventing and treating vascular calcification, characterized in that, The drugs for preventing and treating vascular calcification are in the form of tablets, pills, powders, or injections.

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