Application of decitabine in preparation of medicine for treating CKD-related vascular calcification

Abstract

The invention discloses application of decitabine in preparation of a medicine for resisting vascular calcification, and belongs to the technical field of medical biology. A CKD mouse vascular calcification model is constructed through combination of 5 / 6 nephrectomy and high phosphorus diet (5 / 6NX), and it is observed that the expression level of DNMT3A in model mouse calcified blood vessels is remarkably increased, and the DNA methylation degree is remarkably increased. Based on this, people try to use decitabine to reduce the expression of DNMT, and discover that intraperitoneal injection of decitabine inhibits the expression of DNMT3A in CKD artery blood vessels and osteogenesis-like transdifferentiation of vascular smooth muscle cells, so that CKD-related vascular calcification is effectively relieved.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical biotechnology, and specifically relates to the role of decitabine in the prevention and treatment of vascular calcification diseases, especially in CKD-related vascular calcification. Background Art

[0002] Chronic Kidney Disease (CKD) is a global, non-communicable disease. Cardiovascular disease (CVD) is one of the common complications of CKD and the leading cause of death in CKD patients. Previous studies have shown that the risk of cardiovascular death in young CKD patients is 100 times that of the general population. Vascular calcification (VC) is an independent risk factor for CVD in CKD patients. CKD-related vascular calcification mainly occurs in the arterial media, and its specific pathogenesis is still unclear.

[0003] The risk factors for CKD-related CVD can be mainly divided into two categories. One is the traditional CVD risk factors, namely hypertension, diabetes, etc.; the other is the non-traditional CVD risk factors, including disorders of bone mineral metabolism, anemia, inflammation, oxidative stress, etc. Simple traditional risk factors cannot explain the high incidence and mortality of CVD in CKD patients, and the vascular calcification caused by non-traditional risk factors, which increases the incidence and mortality of CVD, has been increasingly emphasized in recent years. CKD-related vascular calcification has its unique pathogenic mechanism, and therapeutic drugs targeting CKD-related risk factors are needed.

[0004] DNA methylation plays an important role in chronic kidney disease. Some studies have reported that metabolic changes and inflammatory states in chronic kidney disease may involve and induce various epigenetic changes. Currently, there are also studies exploring the role of DNA methylation in vascular calcification. The research by Oca et al. showed that the occurrence of calcification induced by high phosphorus is related to abnormal methylation of the SM22α promoter. Liu et al. reported that abnormal DNA demethylase can lead to abnormal methylation of the promoters of some contractile phenotype genes in smooth muscle cells, resulting in intimal hyperplasia. Some studies have also pointed out that stimulation of HASMC with indoxyl sulfate can lead to increased expression of DNMT1 and DNMT3A, causing hypermethylation of the KLOTHO gene. However, current research on the methylation enzyme DNMT in vascular calcification, especially in CKD-related vascular calcification, is still relatively scarce.

[0005] Our previous research found that patients with CKD-related vascular calcification had methionine metabolism disorders, with upregulated expression of the DNA methyltransferase DNMT3A in their blood vessels and high levels of DNA methylation, which might be an important pathogenic factor for CKD-related vascular calcification. Therefore, we attempted to use decitabine to inhibit the activity of DNA methyltransferase DNMT and clarify its role in the process of CKD-related vascular calcification, so as to better improve the prognosis of CKD patients. Summary of the Invention

[0006] The object of the present invention is to provide an application of decitabine in the treatment of vascular calcification, aiming to provide a new safe and effective prevention and treatment measure for CKD-related vascular calcification.

[0007] To achieve the above object, it was found that in the calcified blood vessels of CKD patients, the expression level of 5-mC was significantly increased, indicating an increase in the overall DNA methylation level of the calcified blood vessels. Further, immunohistochemical staining of DNMT3A was performed on the calcified vascular tissue, and it was found that the expression level of DNMT3A in the tissue was significantly increased. RNA quantitative detection found that the relative expression level of Dnmt3a mRNA was positively correlated with the relative expression levels of the osteogenic transdifferentiation genes RUNX2 and MSX2, suggesting that the expression of DNMT3A is related to the osteoblast-like transdifferentiation and calcification of vascular smooth muscle cells.

[0008] A mouse model of vascular calcification was constructed by 5 / 6 nephrectomy combined with a high-phosphate diet (5 / 6NX). Similarly, it was observed that the expression level of DNMT3A in the calcified blood vessels of the model mice was significantly upregulated, and the degree of DNA methylation was significantly increased. Based on this, we attempted to use decitabine to reduce the expression of DNMT and observe whether it could reduce the osteogenic transdifferentiation and calcification of mouse aortic cells. First, we detected whether intraperitoneal injection of decitabine could reduce the expression of DNMT3A in the blood vessels. Using qRT-PCR technology to detect the mRNA level of Dnmt3a, it was found that intraperitoneal administration could effectively reduce the mRNA level of Dnmt3a, and the expression levels of the aortic osteogenic genes Runx2, Bglap, and Sox9 were also correspondingly reduced.

[0009] According to the results of the above experiments, we speculated that decitabine might relieve the occurrence and development of vascular calcification in vivo. To verify this speculation, next, CKD mice were intraperitoneally injected with decitabine, and the abdominal aorta of the mice was extracted to detect calcium deposition. It was found that the arterial calcium deposition in the CKD mice given decitabine was significantly reduced, and at the same time, the degree of aortic calcification in the mice was significantly alleviated.

[0010] The above results show that the use of decitabine inhibits the expression of DNMT3A in CKD arterial blood vessels and the osteoblast-like transdifferentiation of vascular smooth muscle cells, effectively relieves CKD-related vascular calcification, and thus can be used to prepare related drugs.

[0011] The present invention provides a basis for the clinical application of decitabine in the treatment of CKD-related vascular calcification, provides more choices for clinical treatment drugs, and can better improve the prognosis of CKD patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 : 5-mC immunohistochemical staining (A) and quantitative statistics (B) of paraffin sections of the radial artery in the control group and the CKD calcification patient group, scale bar = 50 μm.

[0013] Figure 2 : Alizarin red staining and DNMT3A immunohistochemical staining (A) and quantitative statistics (B) of paraffin sections of the radial artery in the control group and the CKD calcification patient group, scale bar = 50 μm.

[0014] Figure 3 : Linear correlation analysis of the relative mRNA levels of DNMT3A and the relative mRNA levels of RUNX2 (A) and MSX2 (B) in the radial artery tissue of CKD patients.

[0015] Figure 4 : Immunofluorescence staining of DNMT3A and α-SMA in the aortic arch of SHAM and CKD mice, scale bar = 50μm.

[0016] Figure 5 : Von Kossa staining and IHC staining of DNMT3A in the aortic arch of mice (A) and quantitative statistics of IHC staining of DNMT3A in the SHAM group and the CKD group (B), scale bar = 50 μm.

[0017] Figure 6 : Detection of DNMT3A and RUNX2 protein levels in the abdominal aorta of SHAM group and CKD group mice (A) and quantitative statistics (B).

[0018] Figure 7 : mRNA expression levels of Dnmt3a, Runx2, and Bglap in the thoracic aorta of SHAM group and CKD group mice.

[0019] Figure 8 : Percentage of 5-mC modification in the DNA of the thoracic aorta of SHAM group and CKD group mice.

[0020] Figure 9 : IHC staining (A) and quantitative statistics (B) of 5-mC in the aortic arch of SHAM group and CKD group mice, scale bar = 50 μm.

[0021] Figure 10:Decitabine downregulates the expression of osteogenic genes and DNMT3A in the aorta of CKD mice: A: Detection of Dnmt3a mRNA expression levels in the thoracic aorta of mice in the intraperitoneal injection DMSO group and the 5Aza group; B: Detection of the mRNA levels of Runx2, Bglap, and Sox9 in the thoracic aorta tissues of mice in the SHAM group, CKD group, and CKD+5Aza group.

[0022] Figure 11 :Decitabine alleviates aortic calcification in mice: A: Determination of the calcium content in the abdominal aorta of mice in the SHAM group, CKD group, and CKD+5Aza group; B: Von Kossa staining of aortic arch sections of mice in the SHAM group, 5Aza group, CKD group, and CKD+5Aza group; C: Gross alizarin red staining of the aorta of mice in the CKD group and CKD+5Aza group.

[0023] In the above figures, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Detailed implementation manners

[0024] The present invention will be described in detail below with reference to specific embodiments. It should be noted that the specific embodiments are only used to explain the present invention and are not used to limit the present invention. In addition, various experimental operations involved in the embodiments are all conventional operations in the art unless otherwise specified. For parts not specifically described in the text, those of ordinary skill in the art can refer to various commonly used reference books, scientific and technological literatures, or relevant specifications and manuals before the filing date of the present invention application for implementation.

[0025] 1. Materials In this experiment, SPF-grade 7-week-old male C57BL / 6 mice (weighing about 18-20 g) were purchased from Beijing Huafukang Biotechnology Co., Ltd. and were raised in the SPF-grade barrier environment of the animal house of Tongji Medical College, Huazhong University of Science and Technology. The sterile feed and drinking water for the mice were provided by this animal house.

[0026] Decitabine (5Aza) was purchased from MCE Company, with the product number HY-A0004. It was dissolved in DMSO and administered by intraperitoneal injection. The administration frequency and dose were 2 times / week and 0.5 mg / kg, and the administration was continuous for 2 months.

[0027] 2. Methods 2.1 Establishment of a vascular calcification model in CKD mice 1. Mouse anesthesia: 2% tribromoethanol was used to determine the drug dose according to the body weight of the mouse, and the mouse was anesthetized by intraperitoneal injection.

[0028] 2. 5 / 6 Nephrectomy (5 / 6 NX) was used to induce CKD: First, left nephrectomy was performed, and two-thirds of the right kidney was resected one week later: 1) The mice were placed in the prone position, and their limbs were fixed. The skin was prepared. 2) In the middle one-third area on the left side of the mouse spine, the skin and muscle were incised successively. 3) The left kidney was located and exposed using a tissue separator, and the renal artery, vein, and ureter were identified. 4) The renal capsule and other perirenal tissues were dissected, and the renal pedicle was ligated. 5) The hilar blood vessels and ureter were separated, the hilar blood vessels were ligated, and the left kidney was removed. 6) 200 μL of double antibiotics was injected into the abdominal cavity, and the abdomen was closed layer by layer with sutures. The skin was disinfected. 7) One week later, the right kidney was exposed in the same way, and the renal capsule was dissected. 8) The upper one-third of the right kidney was removed, and the hemostatic sponge was immediately used to press for hemostasis. 9) After the upper pole was hemostatic, the lower one-third of the right kidney was removed, and the hemostatic sponge was used to press for hemostasis. 10) 200 μL of double antibiotics was injected into the abdominal cavity, and the abdomen was closed layer by layer with sutures. The skin was disinfected.

[0029] 3. Gradient high-phosphorus diet was used to induce calcification in CKD mice: 1) One week after 5 / 6 nephrectomy, a 1% high-phosphorus diet was given for 2 months. 2) After 2 months, a 2% high-phosphorus diet was given for 1 month. 3) Samples were taken after the high-phosphorus diet ended.

[0030] 2.2 Detection of tissue calcium ion deposition 1. Tissue sample processing The fresh or frozen vascular tissue was put into a Roche homogenization tube containing grinding beads and cut into pieces. Then 500 μL of 0.6N hydrochloric acid was added, and the Roche homogenizer was used at 6500 Hz × 25 s × 3 times. During the homogenization interval, the homogenization tube was placed on ice to cool down. After homogenization, the sample was placed on a rotator and rotated at 4°C overnight to extract calcium ions from the tissue.

[0031] 2. Determination of calcium ion deposition: The calcium ion concentration was measured using a calcium ion detection kit from Nanjing Jiancheng. The MTB reagent and the alkaline solution were mixed at a ratio of 2:1 to prepare the working solution. The sample was centrifuged at 12000 rpm × 15 min, and the supernatant was taken. The samples were added according to the following method, mixed well, and left standing for 5 min. The OD values of each well were measured at a wavelength of 610 nm.

[0032]

[0033] 3. Protein concentration determination: The BCA method was used to determine the protein concentration. The solution A and solution B in the BCA kit were prepared into BCA working solution at a ratio of 50:1. The sample supernatant was taken and added samples according to the method in the following table. After mixing, it was incubated at 37 °C for 30 min, and the OD value of each well was measured at a wavelength of 562 nm. The protein concentration of the sample was calculated according to the standard curve set by the gradient.

[0034]

[0035] *Note: The concentration of the protein standard bottle is 0.5 mg / mL 4. Calculation of calcium ion deposition: The calcium ion concentration of each sample was divided by its protein concentration, that is, the protein concentration was used to correct the calcium ion concentration to obtain the final standardized calcium ion deposition.

[0036] 2.3 Tissue RNA extraction and quantitative detection of related genes by RT-PCR 1. Tissue sample treatment The fresh or frozen vascular tissue was put into a Roche homogenization tube containing grinding beads and cut into pieces, then 500 μL of Trizol was added, and the Roche homogenizer was used at 6500 Hz × 20 s × 2 times. During the homogenization interval, the homogenization tube was placed on ice to cool down. After that, it was inverted and mixed 10 times and left standing at room temperature for 5 min for subsequent RNA extraction.

[0037] 2. RNA extraction and concentration detection 1) Add 100 μL of chloroform to each sample, tighten the lid, shake vigorously for 15 s, and leave standing at room temperature for 5 min; 2) Centrifuge at 12000 rpm for 15 min in a 4 °C centrifuge to separate the sample into layers. The upper aqueous phase contains RNA, and the lower organic phase contains protein and DNA; 3) Take the supernatant and transfer it to a new EP tube, add 0.5 mL of isopropanol, gently invert 7 times to mix, and leave standing at room temperature for 30 min; 4) Centrifuge at 12000 rpm for 15 min in a 4 °C centrifuge, discard the supernatant, and the precipitate is RNA; 5) Add 1 mL of pre-cooled 75% ethanol to the precipitate to wash the RNA, gently invert and mix to make the precipitate float; 6) Centrifuge at 12000 rpm for 10 min in a 4 °C centrifuge, discard the supernatant; 7) Dry the RNA and dissolve it in an appropriate amount of DEPC water; 8) Use NanoDrop2000 to measure the concentration and purity of RNA and record it. The RNA was reverse transcribed or stored at -80 °C.

[0038] 3. RNA Reverse Transcription: Take 500 ng of RNA and reverse transcribe the RNA using the iScriptTM cDNA Synthesis Kit. The specific reaction system is as follows in the table:

[0039] Amplify in a ProflexTM Base PCR instrument according to the procedure provided by the kit. The obtained cDNA can be diluted for RT-PCR experiments or stored at -20°C for long term.

[0040] 4. RNA Quantification by RT-PCR Technique: Perform RT-PCR experiments using the iTaqTM Universal SYBR Green Supermix. The specific loading system is as follows in the table:

[0041] After loading the samples, centrifuge the 384-well plate at 4500 rpm for 5 min and place it in a Q6 PCR instrument for reaction.

[0042] The primer sequences involved in the experiment are as follows:

[0043] 2.4 Protein Extraction from Tissues and Quantitative Detection by Immunoblotting (Western blot, WB) 1. Protein Extraction from Tissues Take out the vascular tissue sampled previously from the -80°C refrigerator, weigh it on a fine balance and record the weight in the laboratory notebook; calculate the required volume of lysis buffer according to the method of adding 10 μL of lysis buffer to 1.0 mg of tissue. Add Cocktail, Phosphatase Inhibitor Cocktail A and B, and PMSF to the (strong) RIPA lysis buffer and mix well. The lysis buffer formulation is shown in the following table; Table 1-13 Method for Preparing Protein Lysis Buffer

[0044] Put each sample tissue into a 2 mL EP tube in turn, add three tissue grinding beads and the prepared lysis buffer, and grind the tissue using a tissue grinder (60 Hz, 120 s / time) several times until no lumps of tissue can be seen. Place the EP tube in ice during each grinding interval; after grinding, centrifuge the tissue in a low-temperature centrifuge (12000 rpm / min × 30 min). Aspirate the supernatant into a new EP tube and measure the protein concentration by the BCA method.

[0045] 2. Quantitative Detection of Tissue Protein 1) Take out the gel-making reagents from 4°C and warm them to room temperature in advance; 2) When the reagent is rewarmed, wash the glass plates. That is, dip a clean gauze in a small amount of dishwashing liquid and wipe the glass plates under running water. After washing the glass plates, combine the long glass plate and the short glass plate (the long plate on the outside and the short plate on the inside) and place them on the gel-making rack. Add double-distilled water to the glass plates and observe whether the liquid level changes. After confirming no leakage, place the glass plates upside down. 3) Prepare the separating gel: Prepare the separating gel according to the configuration ratio. After fully mixing, quickly add the separating gel along the edge of the glass plate, about 7 mL. Then slowly add anhydrous ethanol above the separating gel to the top of the glass plate and time for 30 min. Add 10 mL - 15 mL of double-distilled water to the gel-making tank. 4) Prepare the stacking gel: After 30 min, first observe whether there is a significant drop in the liquid level in the glass plate or inconsistent drop levels on both sides before pouring off the ethanol. If not, then pour off the ethanol. Start preparing the stacking gel according to the ratio. After preparation and mixing, immediately add the stacking gel to the top of the glass plate and quickly insert a 15-well comb with a thickness of 1.5 mm / well and time for 20 min. Add 10 mL - 15 mL of double-distilled water to the gel-making tank. 5) Loading: First, pour the prepared rapid electrophoresis solution into the gel-making tank. Then slowly and vertically pull out the comb teeth. After pulling out the comb teeth, the gel strip can be adjusted with a syringe needle. Add 3 μL of 180 kDa protein Marker on both sides of the protein. Then, sequentially add the cooked protein samples between the two Markers on both sides. Vortex the samples before each loading. 6) Electrophoresis: First, use a constant low current of 50 V to align the proteins. After seeing the protein loading Marker separated, start increasing the voltage to 100 V. The electrophoresis can be ended when the smallest protein molecular weight Marker runs to the bottom edge of the glass plate. 7) Blotting Prepare the transfer buffer in advance and pre-cool it. After electrophoresis, pour out the electrophoresis solution in the electrophoresis tank and slowly rinse the electrophoresis tank with double-distilled water to remove the foam. Pour the transfer buffer recovered from the previous experiment into a white shallow tank and cut the gel in this shallow tank. Cut the gel according to the molecular weight of the target molecule and the protein Marker. Prepare the PVDF membrane according to the size of the gel. Immerse the cut PVDF membrane in methanol for about 5 min. According to the principle of "black gel, white membrane", place the PVDF membrane on the white filter paper side, place the gel on the membrane, and make sure there are no bubbles between the PVDF membrane and the gel, then clamp the transfer cassette. Place it in the transfer tank according to the principle of "black to black, red to red", fill the tank with the transfer buffer, and perform the transfer at a constant current of 300 mA. 8) Blocking: After taking out the membrane, place it in the blocking solution and gently shake it on a shaker at room temperature for 1 h. 9) Incubate with the primary antibody: Prepare an appropriate concentration of the primary antibody according to the instructions for different target molecules. After cutting the membrane with the target molecule, place it in the primary antibody and gently shake it on a shaker at 4°C overnight. 10) Membrane washing: Take out the strips, wash them three times with TBST for 10 min each time. 11) Incubating with secondary antibody: Place the strips in the prepared secondary antibody, and the species of the secondary antibody is selected according to the corresponding primary antibody; gently shake on a shaker at room temperature for 1 h. 12) Membrane washing, wash the strips with TBST again for 10 min each time. 13) Exposure: Prepare a hypersensitive ECL exposure solution (Solution A: Solution B = 1:1), evenly drip the prepared exposure solution on the strips, and expose them with a Bio-Rad gel imaging system. 14) Result analysis: Use Image J software to analyze the gray value, divide the gray value of the target molecule by the gray value of the internal reference molecule, and perform statistical analysis.

[0046] 2.5 Alizarin red staining of gross vascular tissue Take the whole aorta of the mouse and dissect the fat around the blood vessel; prepare an alizarin red staining solution: dissolve 0.004% alizarin red in 1% KOH solution; place the aorta in the alizarin red staining solution for 16 h; after staining, wash the tissue twice with 2% KOH solution and take pictures.

[0047] 2.6 Von Kossa staining of vascular tissue Spread the paraffin sections flat on a slide warming table and bake at 68 °C for 1 h; quickly transfer the baked sections to an automatic staining machine for dewaxing; rinse the dewaxed sections with pure water 3 times for 5 min each time, use a group paintbrush to circle the tissue in the section once, drip the silver nitrate staining solution, and stain under ultraviolet light for 1 h; after staining, rinse the sections with running water for 5 min, put them into the automatic staining machine after rinsing, and select the "FR" program for nuclear staining; after the counterstaining is completed, observe the staining situation under the microscope, and then put them into an automatic coverslipping machine for coverslipping.

[0048] 2.7 Immunofluorescence (IF) staining of vascular tissue From dewaxing to incubating with primary antibody; take out the sections from 4 °C, wash them 3 times with PBST in the dark (5 min each time); select the corresponding secondary antibody according to the species of the primary antibody, drip the secondary antibody on the tissue, and incubate in the dark in an incubator at 37 °C for 1 h; after 1 h, the incubation with the secondary antibody is completed, wash 3 times with PBST (5 min each time), then drip DAPI on the tissue and time for 10 min; wash 3 times with PBST in the dark (5 min each time); air dry in the dark and then cover with glycerol.

[0049] 2.8 Extract total DNA from tissues and measure the 5-mC content 1. Total DNA extraction 1) Processing material: Put the vascular tissue into a 2.0 mL EP tube, add 180 μL Buffer SL, and grind it thoroughly in a tissue grinder, shake until it is completely suspended; 2) Add 20 μL Proteinase K and mix well. Place the lysate in a 56 °C water bath until the tissue is digested and dissolved; 3) Add 200 μL Buffer VL, vortex and mix well, heat in a 70 °C water bath for 10 min. The solution should be clear after the water bath; 4) Add 100 μL isopropanol to the solution or supernatant from the previous step, vortex and mix well. Transfer the obtained solution into an adsorption column (Spin Columns AC), centrifuge at 12000 rpm for 1 min, and discard the waste liquid; 5) Add 600 μL Buffer WB1 to the adsorption column AC, centrifuge at 12000 rpm for 1 min, and discard the waste liquid; 6) Add 600 μL Buffer WB2 to the adsorption column AC, centrifuge at 12000 rpm for 1 min, and discard the waste liquid; 7) Repeat step 6; 8) Put the adsorption column AC back into an empty collection tube, centrifuge at 12000 rpm for 2 min to remove the residual WB2 as much as possible, and discard the collection liquid and waste liquid; 9) Put the adsorption column AC into a new 1.5 mL centrifuge tube, suspend and drip 50 - 100 μL Buffer EB onto the middle part of the adsorption membrane, let it stand at room temperature for 3 - 5 min, centrifuge at 12000 rpm for 1 min, collect the DNA solution, and store the DNA at -20 °C.

[0050] 2. Determination of 5-mC content in tissues 1) Reagent preparation, that is, dilute and mix the reagents required for the experiment according to the instructions; 2) Prepare the standard curve. First, mix the working solution as 1.0 μL PC (Positive Control) + 9.0 μL NC (Negative Control); then dilute it to 0.1% PC, 0.2% PC, 0.5% PC, 1% PC, 2% PC, and 5% PC at different ratios; 3) Vertically add the main wells and sub-wells in the ELISA strip, instead of parallel; add 100 μL of BS and 2 μL of DNA to each well, and the concentration of the extracted tissue is approximately 50 - 120 ng / μL; for the NC well, mix 100 μL of BS and 2 μL of NC, and for the PC well, mix 100 μL of BS and different concentrations of NC; the most recommended amount of DNA added to the sample well is 100 ng, with a volume of 2 - 5 μL; change the pipette tip for each sample added and mix thoroughly; 4) Gently tilt the plate from one side to the other to thoroughly mix the solution, and gently shake the plate several times to ensure that the solution evenly covers the bottom of the plate. Cover the strip plate with a plate film and incubate at 37 °C for 60 min; 5) When there are still 10 min left in the incubation, prepare the 5-mC probe complex solution, that is, 1.0 mL of diluted WB + 1.0 μL of mcAb, mix well, then add 1.0 μL of SI and 0.5 μL of ES, and mix well; 6) Discard the BS from the just-incubated plate and wash it 3 times with 150 μL of WB; 7) Add 50 μL of the 5-mC probe antibody complex solution to each well, then seal the plate with a plate film and let it stand at room temperature for 50 min; 8) Remove the 5-mC probe complex and wash it 5 times with 150 μL / well of diluted WB; 9) Add 100 μL of DS solution to each well, gently shake the plate on the desktop for 10 s, incubate at room temperature for 3 - 4 min, monitor the color development of the sample wells and control wells. After a few minutes, in the case of sufficient methylation, the solution color will turn blue, and then immediately add SS solution to terminate the reaction; 10) Measure the value of each well at a wavelength of 450 nm with an ELISA reader; 11) Calculate the 5-mC content according to the calculation method in the instruction manual.

[0051] 3. Experimental Results and Analysis 3.1 The DNA methylation level in the calcified blood vessels of CKD patients increases, and the expression of DNMT3A is up-regulated We collected the radial arteries of uremic patients for paraffin embedding. After sectioning, 5-mC immunohistochemical staining was performed to detect the DNA methylation level of the specimens. It was found that in the calcified blood vessels, the expression level of 5-mC was significantly higher than that in the control group, indicating that the overall DNA methylation level in the calcified blood vessels increased (Figure 1). Alizarin red staining was performed on the vascular tissues of uremic patients to determine whether calcification occurred. Further, DNMT3A immunohistochemical staining was performed on the vascular tissues of the vascular calcification group and the control group. It was found that the expression level of DNMT3A in the calcified vascular tissues was significantly higher than that in the non-calcified group ( Figure 2). RNA was extracted from the radial artery tissue of uremic patients for quantitative detection, and it was found that the relative expression level of DNMT3A mRNA was positively correlated with the relative expression levels of the osteogenic transdifferentiation genes RUNX2 and MSX2 ( Figure 3 ), suggesting that DNMT3A expression is related to the osteogenic transdifferentiation and calcification of vascular smooth muscle cells.

[0052] 3.2 The degree of DNA methylation in the calcified blood vessels of CKD mice increased, and the expression of DNMT3A was up-regulated First, we performed immunofluorescence staining for DNMT3A on the aortic arch of control mice and the calcified aortic arch of CKD mice. DNMT3A was co-stained with the marker protein α-SMA of VSMC. The staining results showed a large amount of co-localization of DNMT3A and α-SMA, indicating that DNMT3A was mainly expressed in smooth muscle cells. In addition, it was found that the expression level of DNMT3A in the calcified blood vessels of CKD mice was significantly higher than that in the control group ( Figure 4 ). IHC staining also showed that the expression of DNMT3A in the calcified blood vessels of CKD mice was higher than that in the control group, and Image J quantitative statistics showed that the difference in the expression of DNMT3A between the two groups was statistically significant ( Figure 5 ). Protein was extracted from the abdominal aorta of CKD mice for WB experiments, and it was found that the expression level of DNMT3A protein in the vascular tissue of CKD mice was significantly increased compared with the control group ( Figure 6 ). RNA was extracted from the thoracic aorta of mice for qRT-PCR detection, and it was also found that the mRNA level of Dnmt3a in the CKD group was increased compared with the control group ( Figure 7 ). DNA was extracted from the thoracic aorta of the control group and CKD mice, and a 5-mC detection kit was used to detect the 5-mC content. The results showed that the proportion of 5-mC-modified total DNA in the blood vessels of the CKD mouse group was higher than that of 5-mC in the control group ( Figure 8 ). We performed IHC staining of 5-mC on paraffin sections of the aortic arch and found that the expression content of 5-mC in the calcified blood vessel tissue was also significantly increased. Image J was used to quantitatively analyze the 5-mC staining results of the blood vessels in the two groups, and the results showed that the difference in 5-mC between the CKD group and the control group was statistically significant ( Figure 9 ). The above results all indicate that in the calcified blood vessels of CKD mice, the expression of DNMT3A is up-regulated and the degree of DNA methylation is higher than that in the control group.

[0053] 3.3 Treatment with decitabine inhibits the expression of DNMT3A and the osteogenic transdifferentiation of mouse aortic cells 5Aza, as an inhibitor of DNMT, can inhibit the activity of the methyltransferase DNMT and downregulate the expression of DNMT, thereby inhibiting the occurrence of abnormal methylation in aortic cells. We attempted to use decitabine (i.e., 5Aza) to reduce the expression of DNMT3A and observe whether it could alleviate the osteogenic transdifferentiation and calcification of mouse aortic cells. We first detected whether intraperitoneal injection of 5Aza could reduce the expression of DNMT3A in blood vessels and used qRT-PCR technology to detect Dnmt3a the mRNA level of, and found that intraperitoneal administration could effectively reduce Dnmt3a the mRNA level of ( Figure 10 A), and the expression levels of aortic osteogenic genes Runx2 , Bglap and Sox9 also decreased significantly ( Figure 10 B).

[0054] 3.4 Decitabine alleviates aortic calcification in mice Based on the results of the above experiments, we speculated that decitabine might be able to alleviate the occurrence and development of vascular calcification in vivo. To verify this speculation, next, CKD mice were intraperitoneally injected with 5Aza and the solvent control DMSO, respectively, at a dosing frequency and dose of 2 times / week and 0.5 mg / kg for 2 consecutive months. The abdominal aorta of mice was extracted to detect calcium deposition, and it was found that the arterial calcium deposition in CKD mice given 5Aza was significantly reduced compared with that in the simple CKD group ( Figure 11 A). We performed Von Kossa staining on aortic arch sections of mice in the SHAM group, 5Aza group, CKD group, and CKD group intraperitoneally injected with 5Aza, and found that obvious calcification occurred in the CKD group, while the degree of calcification in the CKD mice group treated with 5Aza was reduced ( Figure 11 B). Alizarin red staining was performed on the gross aorta of mice, and it was also found that the aortic calcification in CKD mice given 5Aza was alleviated ( Figure 11 C).

Claims

1. Use of decitabine in the preparation of drugs for treating vascular calcification.

2. The use according to claim 1, characterized in that: The vascular calcification is chronic kidney disease (CKD)-related vascular calcification.

3. The use according to claim 1 or 2, characterized in that: The decitabine exerts its effects by inhibiting the activity or expression of DNA methyltransferase (DNMT) and the osteogenic transdifferentiation of vascular smooth muscle cells.

4. The use according to claim 3, characterized in that: The DNA methyltransferase is DNMT3A.

5. The use according to claim 1, characterized in that: The medicine is for intraperitoneal injection.

6. The use according to claim 5, characterized in that: The medicine also includes a pharmaceutically acceptable carrier or excipient.

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