Application of medicine taking ACLY gene as action target in preparation of medicine for preventing and treating vascular calcification

Abstract

The invention relates to application of a medicine taking an 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 medicines. In order to solve the problem that an effective clinical means for treating vascular calcification is lacked at the present stage, the invention provides application of a medicine taking an ACLY gene as an action target in preparation of a medicine for preventing and treating vascular calcification. Or expression or activity of vascular smooth muscle cell ACLY protein is efficiently and specifically reduced, smooth muscle osteogenic differentiation is reduced, deposition of hydroxyapatite in blood vessel media is reduced, and the progress of vascular calcification is relieved. The medicine for preventing and treating vascular calcification is prepared on the basis of inhibiting vascular smooth muscle ACLY, has important clinical application potential in treatment of vascular calcification, and is expected to provide a new treatment choice for patients with vascular calcification.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to the use of a drug using the ACLY gene as a target in the preparation of a drug for preventing and treating vascular calcification. Background Art

[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 atherosclerotic plaque instability and significantly increases the risk of myocardial infarction and stroke. Vascular calcification is particularly positively correlated with all-cause mortality in patients with chronic kidney disease, diabetes, and the elderly. The pathological hallmark of this disease is 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 pathological perspective, medial calcification is an active, cell-regulated, osteoblast-like differentiation process. When vascular smooth muscle cells are chronically exposed to a pathological microenvironment high in mineral ions, they undergo a phenotypic transformation toward osteo- / chondrocyte-like cells: expression of calcification inhibitors is downregulated, osteoblast / osteoclast markers are abnormally upregulated, and this is accompanied by alterations in extracellular matrix composition and disrupted matrix hydrolase activity. Current clinical therapeutic interventions for vascular calcification have limited efficacy, and specific targeted drugs are lacking.

[0003] ATP citrate lyase (ACLY) is a key enzyme linking glucose metabolism and lipid synthesis, catalyzing the conversion of citrate to acetyl-CoA. Acetyl-CoA is not only an important component in the synthesis of fatty acids and cholesterol, but also provides the acetyl group required for histone acetylation and gene expression regulation. ACLY plays a key role in linking metabolism and epigenetic modification. Inhibition of ACLY has demonstrated significant anti-tumor effects in preclinical models, and its pharmacological inhibitors are FDA-approved for lipid-lowering therapy. However, the role of ACLY in vascular calcification is understudied, and no related drugs or therapeutic strategies have entered clinical trials. Summary of the Invention

[0004] In order to solve the problem of lack of effective clinical means for treating vascular calcification at this stage, the present invention provides the use of a drug using the ACLY gene as a target in the preparation of a drug for preventing and treating vascular calcification.

[0005] The technical solution of the present invention:

[0006] The invention relates to the use of a drug using the ACLY gene as a target in the preparation of a drug for preventing and treating vascular calcification. The ACLY gene is located in the c41866916-41930542 region of the GRCh38 genome version of human chromosome 17.

[0007] Furthermore, the drug using the ACLY gene as a target is used to prepare a drug for preventing and treating vascular calcification based on interfering with the ACLY gene. The prepared drug can effectively and specifically inhibit the transcription or translation of the ACLY gene in smooth muscle cells, or can effectively and specifically reduce the expression or activity of the ACLY protein in smooth muscle cells, thereby reducing the osteogenic differentiation of smooth muscle, reducing the deposition of hydroxyapatite in the tunica media of blood vessels, and alleviating the progression of vascular calcification.

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

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

[0010] Furthermore, the drug targeting the ACLY gene is a nucleic acid molecule, a carbohydrate, a lipid, a small molecule chemical drug, an antibody drug, a polypeptide, a protein or an adeno-associated virus.

[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 the 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. The 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 and treating vascular calcification also contains a pharmaceutically acceptable excipient.

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

[0015] Furthermore, the drug for preventing and treating vascular calcification is in the form of tablets, pills, powder or injection.

[0016] Beneficial effects of the present invention:

[0017] The present invention demonstrates, through animal and cell experiments, that drugs targeting the ACLY gene can effectively and specifically inhibit the transcription or translation of the ACLY gene in vascular smooth muscle cells, or reduce the expression or activity of the ACLY protein in vascular smooth muscle cells, thereby reducing smooth muscle osteogenic differentiation, decreasing hydroxyapatite deposition in the tunica media, and alleviating the progression of vascular calcification. Based on the inhibition of ACLY in vascular smooth muscle, the present invention prepares drugs for the prevention and treatment of vascular calcification. This drug has significant clinical potential for the treatment of vascular calcification and is expected to provide a new treatment option for patients with vascular calcification. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 From left to right are comparisons of blood creatinine, urea nitrogen, and blood phosphorus levels of mice in the sham operation 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 aorta sections of mice in the sham operation group and the model group in Example 1;

[0020] Figure 3 Schematic diagram of Example 3 using AAV9-ACLYshRNA to treat vascular calcification induced by 5 / 6 nephrectomy in mice;

[0021] Figure 4 This is a comparison of Western blot results of ACLY levels in 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 aorta 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 aorta sections of two groups of mice after treatment with AAV9-ACLYshRNA in Example 3;

[0024] Figure 7 From left to right are comparisons of aortic calcium levels and alkaline phosphatase activities in two groups of mice after treatment with AAV9-ACLYshRNA in Example 3;

[0025] Figure 8 Schematic diagram of Example 4 showing the use of the ACLY inhibitor BMS303141 to treat vascular calcification induced by 5 / 6 nephrectomy in mice;

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

[0027] Figure 10This is a comparison of the results of alizarin red staining and silver nitrate staining of aorta sections of two groups of mice after treatment with BMS303141 in Example 4;

[0028] Figure 11 From left to right are the comparisons of aortic calcium content and alkaline phosphatase activity in two groups of mice after treatment with 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 ACLY siRNA and NCsiRNA in Example 5;

[0031] Figure 14 From left to right are the comparisons of H3K27ac levels, Sox9 and Runx2 mRNA expression levels in the two groups of cells after treatment with ACLY siRNA and NC siRNA in Example 5;

[0032] Figure 15 This is a comparison of the results of Alizarin Red staining of cells in the control group and the BMS303141-treated group in Example 6;

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

[0034] Figure 17 From left to right are comparisons of H3K27ac levels, Sox9 and Runx2 mRNA expression levels in the control group and the BMS303141-treated group in Example 6. DETAILED DESCRIPTION

[0035] The technical solution of the present invention is further described below with reference to the embodiments, but is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be included in the scope of protection of the present invention. The process equipment or devices not specifically noted in the following examples are all conventional equipment or devices in the art. Unless otherwise specified, the raw materials used in the examples of the present invention can be obtained commercially; unless otherwise specified, the technical means used in the examples of the present invention are all conventional means well known to those skilled in the art.

[0036] The statistical analysis method of Examples 1-6 is as follows:

[0037] All statistical tests were performed using SPSS or GraphPad software. Data are expressed as mean ± SEM. Variables between the two groups were compared using the Mann-Whitney U test or Student's t-test. P values ​​< 0.05 were considered statistically significant.

[0038] All animal studies were performed according to the guidelines approved by the Animal Experimentation Ethics Committee of the Second Affiliated Hospital of Harbin Medical University.

[0039] Example 1

[0040] This example provides a method for constructing a 5 / 6 nephrectomy-induced vascular calcification mouse model.

[0041] 1. Experimental Animals

[0042] Male C57Bl / 6J mice aged 6-7 weeks (purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.) were selected and fasted for 24 hours before surgery with normal drinking water.

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

[0044] Five-of-six nephrectomy: C57Bl / 6J mice underwent nephrectomy of the upper and lower poles of the left kidney (i.e., two-thirds of the left kidney) under sevoflurane anesthesia. One week later, the right kidney was completely removed. One week after the five-of-six nephrectomy, mice were fed a 1.5% high-phosphate diet. Five weeks later, a five-of-six nephrectomy-induced vascular calcification mouse model was successfully established.

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

[0046] 3. Extraction of aortic tissue from each group of mice

[0047] Before sampling, each mouse was weighed. Mice were anesthetized with an intraperitoneal injection of 1% sodium pentobarbital. After blood was collected from the apex of the heart, the plasma was temporarily placed at 4°C. After sampling, it was placed in a centrifuge to separate the serum. After opening the chest and abdomen, the blood in the systemic circulation was washed with normal saline. The lungs, trachea, esophagus, liver and other organs in front of the aorta were carefully cleaned, the aorta was exposed, and the aorta was removed along with the heart. The connective tissue surrounding the aorta was then carefully dissected under a stereomicroscope. The cleanly dissected aorta was stored in a universal tissue fixative and paraffin-embedded within 1 week for subsequent histopathological analysis. Alternatively, the aorta was placed in a -80°C freezer for later use.

[0048] 4. Methods for detecting blood creatinine, urea nitrogen, and blood phosphorus levels in mice

[0049] (1) Serum creatinine test

[0050] Use a creatinine colorimetric test kit (sarcosine oxidase method) (purchased from Elabscience) and prepare the required reagents according to the instructions. Add 12 μl of mouse serum and 180 μl of reagent 1 to each well, incubate at 37°C for 5 minutes, add 60 μl of reagent 2, and measure the OD value of each well at 515 nm on a microplate reader.

[0051] (2) Blood urea nitrogen test

[0052] A urea colorimetric test kit (urease method) (purchased from Elabscience) was used to prepare the required reagents according to the instructions. 4 μl of mouse serum and 50 μl of enzyme working solution were added to each well. The cells were incubated at 37°C for 10 minutes. 125 μl of reagent 1 and reagent 2 were added, and the OD value of each well was measured at 580 nm on a microplate reader.

[0053] (3) Blood phosphorus test

[0054] A phosphorus colorimetric test kit (phosphomolybdic acid method) (purchased from Elabscience) was used to prepare the required reagents according to the instructions. 35 μl of mouse serum and 200 μl of color development working solution were added to each well. The cells were 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 in the figure, compared with the sham operation group, the blood creatinine, urea nitrogen and blood phosphorus levels of mice in the 5 / 6 nephrectomy model group were significantly increased.

[0056] 5. Assessment 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 then rinsed with double distilled water. The positively stained parts appeared 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, and then rinsed with double distilled water. The positively stained parts were brown to black.

[0059] The results are as follows Figure 2 As shown in the figure, compared with the sham operation group, the alizarin red staining and silver nitrate staining of the aorta of the mice in the 5 / 6 nephrectomy model group were significantly deepened, indicating that the vascular calcium and phosphate deposition in the mice in the model group 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, namely, an AAV9 virus carrying an SM22 promoter.

[0062] Step 1: Using the short hairpin RNA structure targeting 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:

[0064] 5'-ATGGTGGAATGCTGGACAACATTAGTGAAGC-3';

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

[0066] 5'-CACAGATGTAATGTTGTCCAGCATTCCACCAG-3'.

[0067] Step 2: Construct AAV9 recombinant vector and sequence verification:

[0068] The ACLY shRNA nucleic acid sequence obtained in step 1 was subcloned into an AAV9 expression vector (purchased from Jinan Boshang Company) by enzyme digestion-ligation-transformation. After obtaining an ACLY shRNA positive clone, the correctness of the inserted fragment was confirmed by sequencing, thereby obtaining an ACLY shRNA plasmid.

[0069] Step 3: AAV9 virus packaging:

[0070] HEK293T cells (purchased from ATCC) with a polymerization degree of more than 90% were plated at a ratio of 1:3, with approximately 2.5 × 10 cells per plate. 6 1-2 hours before plasmid transfection, replace the culture medium with serum-free medium and use transfection reagent (Lipofectamine 3000, purchased from Thermo Fisher Scientific). Transfect the ACLY shRNA plasmid and the negative control NC shRNA plasmid obtained in step 2 into HEK293T cells separately. 24 hours after transfection, replace the culture medium with fresh serum-free medium. 72 hours after transfection, collect the virus separately. It is not necessary to discard the culture medium. After centrifugation, remove the cells and obtain the supernatant and cell pellet. Precipitate the virus in the supernatant with PEG 8000. After overnight precipitation, collect the virus pellets separately.

[0071] Step 4: Virus purification and concentration:

[0072] The viral mixture obtained in step 3 was purified by iodixanol density gradient centrifugation, where the iodixanol density was 5%, 15%, 25%, 40%, and 54% from top to bottom. The centrifuged viral liquid was placed in an ultrafiltration tube for concentration. The remaining liquid in the ultrafiltration tube was repeatedly pipetted and then aspirated into a virus storage tube, and the virus storage solution was added to obtain the virus AAV9-ACLYshRNA carrying the SM22 promoter and the negative control virus AAV9-NCshRNA.

[0073] Step 5. Virus titer detection: Measure the number of viral particles by RT-PCR. The number of viral particles (particles / ml) = the relative value compared to the standard.

[0074] Example 3

[0075] In this example, the virus AAV9-ACLYshRNA carrying the SM22 promoter prepared in Example 2 was used as a therapeutic drug, and animal experiments were conducted to investigate the effect of drugs targeting the ACLY gene in alleviating the process of vascular calcification.

[0076] 1. Animal Experiment Grouping Method

[0077] (1) Treatment group:

[0078] Male C57Bl / 6J mice aged 6-7 weeks were selected and injected with AAV9-ACLYshRNA carrying SM22 promoter through the tail vein. 12 vg / mouse, and two weeks later, mice with vascular calcification induced by 5 / 6 nephrectomy were constructed according to 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 negative control virus AAV9-NCshRNA through the tail vein for 10 12 vg / mouse, and two weeks later, mice with vascular calcification induced by 5 / 6 nephrectomy were constructed according to the model construction method provided in Example 1;

[0081] 2. Western blotting experiments

[0082] Aortic tissue from each group of mice, lysed by ultrasound or grinding, was placed in RIPA lysis buffer. Protein was quantified by the BCA assay, followed by SDS-PAGE electrophoresis. After transfer to a membrane and blocking, the corresponding primary antibody was added and incubated overnight at 4°C. After washing with TBST, the membrane was incubated with a fluorescently labeled secondary antibody at room temperature. Western blot results were quantitatively analyzed using ImageJ.

[0083] 3. Acetyl Coenzyme A Detection

[0084] Acetyl-Coenzyme A detection kit (purchased from Sigma) was used for detection. Tissue samples (20 mg) or cell samples (10 5 Cells were collected from 100 samples of 100 cells (100 cells / mL) and then snap-frozen in liquid nitrogen. The samples were then deproteinized using PCA. Acetyl-CoA detection solution was prepared according to the kit instructions and added to the samples. The samples were incubated at 37°C for 10 minutes. Fluorescence intensity was measured using a fluorescence detector at 535°C.

[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 aorta tissue of the treated mice was successfully inhibited, and the level of acetyl-CoA, a product of the citric acid metabolic pathway in which ACLY participates, was also reduced.

[0086] 4. Alizarin red staining and silver nitrate staining were used to evaluate vascular histology, using the same method as in Example 1.

[0087] The results are as follows Figure 6 As shown, the control group mice had obvious hydroxyapatite crystal deposition in the vascular tissue after 5 / 6 renal resection vascular calcification modeling. After the treatment group was given AAV9-ACLY shRNA, the hydroxyapatite crystal deposition in the aorta of the mice was significantly alleviated, alleviating vascular calcification.

[0088] 5. The deposition of hydroxyapatite in the vascular tunica media was assessed by 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. The dried aortas were decalcified with 0.1 M hydrochloric acid for 24 hours. Calcium content in the supernatant was determined using a calcium assay kit (purchased from Elabscience). The reagents were prepared according to the manufacturer's instructions. 10 μl of sample and 260 μl of working solution were added to each well. The cells were incubated at room temperature for 5 minutes. OD values ​​were measured at 610 nm using a microplate reader. Calcium content was normalized to dry weight.

[0091] (2) Determination of aortic alkaline phosphatase activity

[0092] Total protein was extracted from aortic tissue using RIPA lysis buffer without inhibitors. Protein content was measured by the biuret (BCA) protein assay (purchased from Thermo Fisher Scientific). Alkaline phosphatase activity was determined by a colorimetric method (purchased from Elabscience). The required reagents were prepared according to the instructions. 5 μl of sample was added to each well, followed by 50 μl of working solution 1 and 50 μl of working solution 2. The cells were incubated at 37°C for 15 minutes, and 150 μl of colorimetric solution was added. The OD value was measured at 520 nm using a microplate reader. Alkaline phosphatase activity was normalized to the amount of total aortic protein.

[0093] The results are as follows Figure 7 As shown in the results, after 5 / 6 nephrectomy, the calcium content and alkaline phosphatase activity in the aorta of the control group mice were significantly increased, while the AAV9-ACLYshRNA injected into the treatment group mice was able to significantly reduce the levels of calcium and alkaline phosphatase in the vascular media of the mice, effectively alleviating the progression of vascular calcification.

[0094] Example 4

[0095] In this example, the ACLY inhibitor BMS303141 was used as a therapeutic drug, and animal experiments were conducted to investigate the effect of drugs targeting the ACLY gene in alleviating the progression of vascular calcification.

[0096] 1. Animal Experiment Grouping Method

[0097] (1) Treatment group:

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

[0099] (2) Control group:

[0100] According to the model construction method provided in Example 1, mice were given a 1.5% high-phosphate diet one week after completing 5 / 6 nephrectomy, and corn oil was gavage-fed as a control for a feeding period of 5 weeks.

[0101] 2. After 5 weeks, the mice in each group were killed to examine the therapeutic effect of BMS303141. The acetyl-CoA level in the aortic tissue of the mice was detected. Vascular histology was evaluated using Alizarin red staining and silver nitrate staining. The deposition of hydroxyapatite in the vascular tunica media was evaluated by aortic calcium content and alkaline phosphatase activity. The method for determining the acetyl-CoA level, pathological staining, and the methods for detecting calcium content and alkaline phosphatase activity were the same as those in Example 3.

[0102] The levels of acetyl-CoA in the two groups of mice were as follows Figure 9 As shown in Figure 2, oral ACLY inhibitor BMS303141 effectively inhibited the production of acetyl-CoA, a product of the citric acid metabolic pathway; the results of Alizarin red staining and silver nitrate staining of the aorta of the two groups of mice, as well as the results of aortic calcium content and alkaline phosphatase activity detection are shown in Figure 2. Figure 10 and Figure 11As shown in the results, after 5 / 6 nephrectomy, the control group mice developed chronic vascular calcification, with obvious calcium nodules formed in the aorta, increased vascular calcium content and alkaline phosphatase activity, while oral administration of BMS303141 in the treatment group could significantly reduce the deposition of hydroxyapatite in the vascular media, calcium and alkaline phosphatase levels, and significantly alleviate the progression of calcification.

[0103] Example 5

[0104] In this example, ACLY siRNA was used as a therapeutic drug, and cell experiments were conducted to investigate the effect of drugs targeting the ACLY gene in alleviating the process of vascular calcification.

[0105] 1. Preparation of primary mouse vascular smooth muscle cells

[0106] Primary mouse aortic smooth muscle cells 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 incubated at 37°C for 30 min with 1 mL of 0.2% collagenase I solution in Ham's F12 medium. The aorta was then removed from the adventitia using forceps under microscope guidance. The aorta was longitudinally incised, and the endothelial cells were gently scraped off. The aorta was then cut into small pieces, placed on the bottom of a culture dish, and cultured for several days in DMEM / F-12 medium supplemented with 15% fetal bovine serum and 1% penicillin-streptomycin at 37°C in a humidified atmosphere with 5% carbon dioxide. Cells that migrated from the explants were collected and maintained in growth medium. VSMCs at passages 3 to 6 were used for further experiments. VSMC purity was confirmed by positive staining for SM22α and α-SMA.

[0107] 2. Cell calcification model induction method:

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

[0109] 3. Cell Experiment Grouping Method

[0110] (1) Treatment group:

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

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

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

[0114] 5′-GUGGAAUGCUAAGCAACAUTT-3′;

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

[0116] 5'-AUGUUGUCCAGCAUUCCACTT-3'.

[0117] To comply with the requirements of WIPO ST.26 standard, as shown in SEQ ID NO:3, T is used to replace the U at positions 2, 7, 10 and 19 of the 5' end in RNA to represent uracil. As shown in SEQ ID NO:4, T is used to replace the U at positions 2, 4, 5, 7, 14 and 15 of the 5' end in RNA to represent uracil.

[0118] The specific transfection steps for ACLY siRNA are as follows (taking a six-well plate as an example): use 100μl opti-MEM (purchased from Thermo) to dilute 5μl transfection reagent lipo3000 to obtain solution A, use 100μl opti-MEM to dilute ACLY siRNA to a concentration of 1μM 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 into a high-phosphate calcification model. The specific transfection procedures were the same as those in the treatment group.

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

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

[0123] 5′-UUCUCCGAACGUGUCACGUTT-3′;

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

[0125] 5'-ACGUGACACGUUCGGAGAATT-3'.

[0126] To comply with the requirements of WIPO ST.26 standard, as in the nucleotide sequence shown in SEQ ID NO:5, T is used instead of U at positions 1, 2, 4, 12, 14 and 19 of the 5' end to represent uracil in RNA. As in the nucleotide sequence shown in SEQ ID NO:6, T is used instead of U at positions 4, 11 and 12 of the 5' end to represent uracil in RNA.

[0127] 4. Cell Alizarin Red Staining Method

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

[0129] The results are as follows Figure 12 As shown, cells in the control group showed high phosphorus-induced cell calcification with multiple calcium crystals, and the cells were stained dark red by Alizarin Red. After ACLY-siRNA treatment, the calcium crystals in cells were significantly reduced, and the progression of calcification was delayed.

[0130] 5. The same Western blot experiment as in Example 3 was used to examine the levels of ACLY and Runx2 in each group of cells. The results were as follows: Figure 13 As shown, the levels of ACLY and Runx2 proteins in calcified primary mouse vascular smooth muscle cells were significantly decreased after ACLY-siRNA treatment compared with the control group.

[0131] VI. H3K27ac Detection Method

[0132] An Acetyl-Histone H3 (Lys27) ELISA Kit (purchased from CST) was used. Smooth muscle cells were washed with PBS to 80-90% confluency, then cell extract was added and incubated on ice for 5 minutes. The supernatant was centrifuged at 14,000 rpm for 5 minutes, to which 50 μl of antibody was added. The supernatant was incubated at 400 rpm for 1 hour at room temperature on a plate shaker. The plate was washed, and 100 μl of TMB substrate was added. The cells were incubated at 400 rpm for 15 minutes at room temperature in the dark. 100 μl of stop solution was added to each well, and the absorbance was read at 450 nm on a microplate reader.

[0133] The results are as follows Figure 14 As shown, the H3K27ac level in the control group was significantly increased after high-phospho-calcification induction, and the H3K27ac level in the cells was reduced after ACLY expression was inhibited using ACLY siRNA.

[0134] VII. Real-time fluorescence quantitative PCR detection

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

[0136] The results are as follows Figure 14 As shown in the figure, after high-phosphate calcification induction in the control group, the mRNA levels of cellular calcification factors Runx2 and Sox9 were significantly increased. After ACLY expression was inhibited using ACLY siRNA, the expression of Runx2 and Sox9 was significantly reduced.

[0137] Example 6

[0138] In this example, the ACLY inhibitor BMS303141 was used as a therapeutic drug, and cell experiments were conducted to investigate the effect of drugs targeting the ACLY gene in 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 then given high phosphate to induce cell calcification 12 hours later.

[0141] (2) Control group:

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

[0143] 3. The two groups of cells were stained using the same alizarin red staining method as in Example 4. The results are as follows Figure 15 As shown, cells in the model group showed high phosphorus-induced cell calcification with multiple calcium crystals, and the cells were stained dark red by alizarin red. After treatment with BMS303141, the calcium crystals in the cells were significantly reduced.

[0144] 4. The same Western blot experiment as in Example 3 was used to examine the Runx2 levels in each group of cells. The results were as follows: Figure 16 As shown in Figure 2, compared with the control group, the Runx2 protein level in calcified primary mouse vascular smooth muscle cells was significantly reduced after BMS303141 treatment; the H3K27ac detection method and real-time fluorescence quantitative PCR method used in Example 5 were used to detect the cellular H3K27ac level, Sox9 and Runx2 mRNA levels, and the results were as follows: Figure 17As shown, compared with the control group, BMS303141 treatment significantly reduced the high-phosphate-induced H3K27ac level and the mRNA levels of Sox9 and Runx2, explaining at the molecular level the mechanistic pathway of alleviating vascular calcification by targeting the expression of ACLY in vascular smooth muscle cells.

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

Claims

1. Use of a drug targeting the ACLY gene in the preparation of a drug 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.

2. Use of the drug targeting the ACLY gene according to claim 1 in the preparation of a drug for preventing and treating vascular calcification, characterized in that: The drug using the ACLY gene as a target is used to prepare a drug for preventing and treating vascular calcification based on interfering with the ACLY gene. The prepared drug can effectively and specifically inhibit the transcription or translation of the ACLY gene in smooth muscle cells, or can effectively and specifically reduce the expression or activity of the ACLY protein in smooth muscle cells.

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

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

5. Use of the drug targeting the ACLY gene according to claim 1 in the preparation of a drug for preventing and treating vascular calcification, characterized in that: The drug that takes the ACLY gene as a target is a nucleic acid molecule, a carbohydrate, a lipid, a small molecule chemical drug, an antibody drug, a polypeptide, a protein or an adeno-associated virus.

6. Use of the drug targeting ACLY gene according to claim 5 in the preparation of a drug for preventing and treating vascular calcification, characterized in that: The nucleic acid molecule is an antisense oligonucleotide, double-stranded RNA, small interfering RNA or short hairpin RNA.

7. Use of the drug targeting ACLY gene according to claim 5 in the preparation of a drug for preventing and treating vascular calcification, characterized in that: The adeno-associated virus contains a nucleotide sequence that interferes with the expression of the ACLY gene.

8. Use of the drug targeting ACLY gene according to claim 1 in the preparation of a drug for preventing and treating vascular calcification, characterized in that: The drug for preventing and treating vascular calcification further contains a pharmaceutically acceptable excipient.

9. Use of the drug targeting ACLY gene according to claim 8 in the preparation of a drug for preventing and treating vascular calcification, characterized in that: The excipient is one or more of glucose, sucrose, sorbitol, mannose, starch, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose or water.

10. Use of the drug targeting ACLY gene according to claim 1 in the preparation of a drug for preventing and treating vascular calcification, characterized in that: The drug for preventing and treating vascular calcification is in the form of tablets, pills, powder or injection.

Images