Application of harmine in preparation of medicine for preventing and treating vascular calcification

Abstract

The invention relates to application of harmine in preparation of a medicine for preventing and treating vascular calcification. The harmine can be used for preventing and / or treating vascular calcification. Experiments show that harmine reduces osteogenic differentiation of vascular smooth muscle cells by down-regulating expression of RUNX2 and BMP2 and up-regulating expression of alpha-SMA at the same time, calcium salt deposition of blood vessels can be reduced, and it is indicated that harmine has a remarkable inhibition effect on vascular calcification. The new application of harmine in the anti-vascular calcification aspect is confirmed, so that the research of harmine in the technical field of medicines can be expanded, a new way is provided for research and development of drugs for treating vascular calcification, and a new theoretical direction is provided for clinical application.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to application of dehydropegoine in preparation of a medicine for preventing and treating vascular calcification. BACKGROUND

[0002] Vascular calcification (VC) is a complex pathological process, and its essence is the abnormal deposition of calcium salt in the vessel wall. VC mainly occurs in the media and intima of arteries, and the deposition of calcium salt will thicken and stiffen the vessel wall, and further lead to the loss of arterial function. With the progression of calcification, the blood vessels will gradually lose the ability to adapt to the changes in hemodynamics, thereby increasing the burden on the heart, and eventually may cause serious cardiovascular events such as myocardial infarction, stroke, etc., which poses a major threat to public health. However, the current understanding of the mechanism of VC is still insufficient, and effective prevention and treatment methods have not been developed. Therefore, this field urgently needs to be explored from the perspective of basic research in order to reveal more detailed pathophysiological mechanisms and provide effective intervention strategies for clinical practice.

[0003] Smooth muscle cells (SMCs) are the main cell type constituting the vessel wall, and play an important role in maintaining the structural integrity and functional stability of blood vessels by regulating the contraction and relaxation of blood vessels, thereby controlling blood flow and blood pressure. Under normal conditions, SMCs exhibit a contractile phenotype, and the characteristic cell markers mainly include α-smooth muscle actin, myosin heavy chain and calmodulin. However, under pathological conditions, SMCs will change from a contractile phenotype to a synthetic phenotype, i.e. down-regulation of contractile proteins, increase of proliferation and remodeling of extracellular matrix to promote migration, showing the characteristics of osteoblasts, chondrocytes, adipocytes, macrophages and foam cells. It is worth noting that SMCs will differentiate into osteogenic phenotype under the stimulation of high phosphate environment, which is called osteogenic differentiation of SMCs, thereby secreting various bone formation proteins and matrix vesicles to participate in the formation of vascular calcified nodules. In this process, the death of SMCs will become a nucleation site to promote the deposition of calcium salt, and in addition, the damaged cells will also secrete various inflammatory factors to promote vascular calcification. Studies have shown that inhibition of high phosphate-induced osteogenic differentiation of SMCs can significantly improve VC and reduce cardiovascular mortality.

[0004] Dehydropegoine is extracted from medicinal plants Peganum harmalaHarmine, the main active ingredient in Peganum harmala L., is a natural β-carboline alkaloid with anti-inflammatory, neuroprotective, anti-diabetic, anti-tumor and other multi-target pharmacological effects. In recent years, it has been found that harmine can prevent heart failure caused by myocardial infarction. In addition, harmine has been confirmed to target and inhibit age-related and dose-dependent cardiotoxicity caused by adriamycin, indicating that it has a promising application in cardiovascular diseases, but no study has shown its application in treating vascular calcification.

[0005] In view of this, the present application is proposed. SUMMARY

[0006] The present application aims to provide the application of harmine in the preparation of a drug for preventing and treating vascular calcification, and harmine can be used for preventing and / or treating vascular calcification.

[0007] The harmine described in the present application has the English name: Harmine, the Chinese alias: Harminine, the molecular formula: C 13 H 12 N2O, and the CAS registration number: 442-51-3, and the chemical structural formula is as follows:

[0008] The source of the harmine of the present application is not limited, and can be a natural extract or a chemical synthesis.

[0009] In a first aspect of the present application, the application of harmine in the preparation of a drug for preventing and treating vascular calcification is provided, and the harmine can be used for preventing and / or treating vascular calcification.

[0010] Specifically, the harmine can reduce the osteogenic differentiation of vascular smooth muscle cells by down-regulating the expression of RUNX2 and BMP2 and up-regulating the expression of alpha-SMA.

[0011] Specifically, the harmine can reduce the calcium salt deposition of blood vessels.

[0012] Specifically, the harmine can improve the vascular calcification induced by vitamin D3.

[0013] Specifically, the vascular calcification can be divided into intimal calcification and medial calcification according to the position of calcification in the blood vessels, and the vascular calcification described in the present application mainly refers to medial calcification.

[0014] In a second aspect of the present application, a drug composition for preventing and treating vascular calcification is provided, and the main active ingredient of the drug composition is harmine.

[0015] Specifically, the pharmaceutical composition prevents and / or treats vascular calcification by down-regulating the expression of RUNX2 and BMP2, while up-regulating the expression of a-SMA, thereby reducing the osteogenic differentiation of vascular smooth muscle cells.

[0016] Specifically, the pharmaceutical composition prevents and / or treats vascular calcification by reducing the calcium salt deposition of blood vessels.

[0017] The pharmaceutical composition for preventing and / or treating vascular calcification in the present application refers to a medicine containing an effective amount of mesembrine as an active ingredient, wherein the effective amount refers to an amount sufficient to treat the symptoms or diseases of medical conditions. After being used for a specific patient or medical subject, the following changes can be produced: the conditions to be treated are improved, and the overall health of the patient / subject is improved. In the present application, the significant reduction of vascular calcification is particularly referred to, and the dosage of the medicine is 10-20 mg / kg / day.

[0018] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0019] The present application has at least the following beneficial effects: The present application finds that mesembrine can reduce the osteogenic differentiation of vascular smooth muscle cells by down-regulating the expression of RUNX2 and BMP2, while up-regulating the expression of a-SMA, and can reduce the calcium salt deposition of blood vessels, indicating that mesembrine has a significant inhibitory effect on vascular calcification. The present application confirms the new application of mesembrine in resisting vascular calcification, thereby expanding its research in the field of medical technology, providing a new way for the research and development of vascular calcification treatment drugs, and providing a new theoretical direction for clinical application. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the specific embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0021] Figure 1 Von Kossa staining results of the aorta of wild-type male mice treated with mesembrine after vitamin D3-induced vascular calcification (scale bar: 200 μm).

[0022] Figure 2 Calcium salt content determination results of the aorta of wild-type male mice treated with mesembrine after vitamin D3-induced vascular calcification.

[0023] Figure 3The results show the toxicity of dehydrocamellidine at 0 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM to primary rat aortic smooth muscle cells under normal 24-hour culture conditions.

[0024] Figure 4 Alizarin Red staining results for primary rat aortic smooth muscle cells treated with different concentrations of dehydrocamelliaine (0, 2.5, 5, 10 μM).

[0025] Figure 5 Real-time fluorescence quantitative analysis results of primary rat aortic smooth muscle cells treated with different concentrations of hydrocamellia alkaloid (0, 2.5, 5, 10 μM). Detailed Implementation

[0026] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0027] It should be noted that the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the exemplary implementations according to this application. As used herein, the singular form includes the plural form unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this description, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0028] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Example In this embodiment of the invention, the preparation or related detection methods are carried out according to the following steps: I. Von Kossa staining of the aorta in wild-type male mice treated with dehydrocamelin after vitamin D3-induced vascular calcification. 1. Constructing a Vitamin D3-induced mouse aortic calcification model (1) Preparation: Calculate the number of mice needed for the experiment in advance. Prepare 8-week-old C57BL / 6J mice (male, about 20g, uniform sex to eliminate calcification errors caused by hormones) by 1-2 extra mice. Tag each mouse by ear and weigh it in advance. Prepare the required injection dose (5×10) according to the weight. 5 IU / kg); (2) Induction of calcification: Eight-week-old male C57BL / 6J mice (20-25g) were used to establish the model. The corresponding numbered mice were selected according to the calculated dosage. The needle was inserted from the back of the neck. After aspiration, no blood was drawn back and the injection was completed in one subcutaneous injection. After continuous injection for 3 days, the mice were fed normally for 6 days. Treatment with dehydrocamelin: Eight-week-old male C57BL / 6J mice (20-25g) were used for administration (10mg / kg). The mice were selected according to the calculated dosage, and the needle was inserted into the lower abdomen. After aspiration and no blood was found, the injection was completed in one intraperitoneal injection. Injections were performed on days 2, 4, 6, and 8. The mice were sacrificed on day 9.

[0030] 2. Animal tissue sampling (1) Anesthesia: Weigh the mouse and inject 1% sodium pentobarbital anesthetic into its abdominal cavity at a dose of 50 mg / kg according to its body weight, so that it dies from overdose of anesthesia. (2) Sample collection: The mouse was placed supine on a dissecting board and its limbs were taped to secure it to the board. First, the thoracic cavity was cut horizontally along the upper edge of the diaphragm with scissors, and then longitudinally along the sternum to fully expose the thoracic cavity. Then, the abdomen was cut along the midline to open the abdominal cavity, locate the heart, cut open the right atrial appendage, and use a syringe filled with PBS buffer to puncture the apex of the heart to flush out the blood in the aorta. This flushing was repeated until the red color in the aorta faded and gradually became transparent. The heart and lungs were pushed aside to the left with forceps, and the thoracic aorta was located along the spine. After the location of the thoracic aorta was determined, the thoracic aorta was bluntly dissected with forceps, and the aortic arch, brachiocephalic trunk, left common carotid artery, and left subclavian artery were dissected upwards. Then, the abdominal aorta, left and right renal arteries, and left and right common iliac arteries were gradually dissected downwards. After the dissection was completed, the entire aorta was cut off with tissue scissors, and subsequent experiments were carried out.

[0031] 3. Von Kossa dyeing (1) After the aorta of C57BL / 6J mice was taken, it was fixed, dehydrated, embedded in paraffin, sectioned, and baked. (2) Dewaxing paraffin sections to water: Place the paraffin sections in xylene I for 20 min, xylene II for 20 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and 75% alcohol for 5 min in sequence. Rinse with tap water, avoiding direct rinsing of the vascular sections. Then wash three times with distilled water. (3) Silver nitrate reaction: Add 1% Von Kossa staining solution, irradiate with ultraviolet light for 4 hours, and gently rinse with running distilled water several times until the cleaning solution is colorless. (4) Hematoxylin staining: stain with hematoxylin solution for 3-5 minutes, differentiate with differentiation solution, and return to blue with blue solution. Rinse with tap water after each step. (5) Eosin staining: Dehydrate with 85% and 95% alcohol for 5 min each, and then stain with eosin solution for 5 min; (6) Dehydration and mounting: The sections are placed in anhydrous ethanol for 5 min three times in sequence, and then placed in xylene for 5 min twice to dehydrate and clear. The sections are then mounted with neutral resin. Note that too much neutral resin should not be added and no air bubbles should appear during the mounting process. (7) Microscopic examination: Image acquisition and analysis results are as follows Figure 1 As shown, vitamin D3 (VitD3) induces calcium salt deposition in mice, resulting in black or brownish-black areas. Dehydrocamellia alkaloid treatment can reduce calcium salt deposition in the aorta of mice, demonstrating that dehydrocamellia alkaloid has a good therapeutic effect on vascular calcification in mice.

[0032] II. Determination of calcium salt content in the aorta of wild-type male mice after vitamin D3-induced vascular calcification treated with dehydrocamelin. 1. Preparation of tissue samples (1) Cut the tissue into small pieces; (2) Dissolve the sample lysis solution and mix well; (3) Add sample lysis buffer at a ratio of 200 µl per 20 mg of tissue, homogenize with a suitable grinder until fully lysed; (4) After complete lysis, centrifuge at 10,000-14,000g for 5 minutes at 4℃, take the supernatant and place it on ice for testing.

[0033] 2. Preparation of the reagent kit (1) The detection buffer and colorimetric solution should be equilibrated to room temperature before use; (2) Preparation of standard samples; Preparation of calcium standard solution: Take 10 µl of 500 mM calcium standard, add 990 µl of deionized water, and mix thoroughly to obtain 5 mM calcium standard solution. Refer to Table 1 to dilute calcium standards and prepare standards with concentrations of 0, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mM.

[0034] Table 1

[0035] (3) Preparation of detection working solution: Calculate the amount required for one experiment, and mix the detection buffer and the colorimetric solution at a ratio of 1:1 to prepare the detection working solution for later use.

[0036] 3. Construction of standard curve and sample determination (1) Add 50 µl of standard and 10 µl of sample to each well of the 96-well plate, and level the volume to 50 µl with deionized water. Record the sample volume as V. (2) Add 150µl of detection working solution to each well and mix well; (3) Incubate at room temperature in the dark for 5-10 minutes; (4) Measure the absorbance at 575 nm using an enzyme-linked immunosorbent assay (ELISA) reader and create a standard curve.

[0037] 4. Calculation of calcium content in the sample (1) Calculate the average absorbance of each concentration group in the standard group, and subtract the absorbance of the blank control group to get the absorbance of each concentration standard. (2) Plot a standard curve with the amount of calcium ions in the calcium standard as the abscissa and the absorbance as the ordinate; (3) Calculate the calcium content in the test wells based on the standard curve; (4) Calculate the calcium concentration in the sample according to the following formula: C = A × n / V (µg / µl); Where A is the amount of calcium ions (µg) determined according to the standard curve; n is the sample dilution factor in step 1; V is the volume of the sample added in step 3.

[0038] The results of calcium salt content determination in the aorta of wild-type male mice after treatment with dehydrocamelin and vitamin D3-induced vascular calcification are as follows: Figure 2 As shown, the results indicate that dehydrocamelin can reduce the calcium salt content in the aorta, further demonstrating its good therapeutic effect on vascular calcification.

[0039] III. Toxicity assay of dehydrocamelin on primary rat aortic smooth muscle cells 1. Isolation and culture of primary rat aortic smooth muscle cells (1) Preparation: Prepare 20% complete culture medium for primary cell culture; prepare 1% sodium pentobarbital anesthetic for rat anesthesia; prepare 75% ethanol for rat disinfection and sterilization. Autoclave the scissors, tweezers, etc. to be used; turn on the ultraviolet light of the laminar flow 30 minutes in advance for disinfection; (2) Anesthesia: After weighing the rats, inject them intraperitoneally with 1% sodium pentobarbital anesthetic at a dose of 150 mg / kg to cause them to die from overdose of anesthesia, and then soak them in 75% ethanol for 5 minutes. (3) Separation of the thoracic aorta: The rat was placed supine on the dissecting board and its limbs were taped to fix it to the board. First, the thoracic cavity was cut horizontally along the upper edge of the diaphragm with scissors, and then the thoracic cavity was cut longitudinally along the sternum to expose the thoracic cavity completely. The heart and lungs were pushed aside with forceps to the left, and the thoracic aorta was located along the spine. After the location of the thoracic aorta was determined, the thoracic aorta was bluntly separated with forceps. After the separation was completed, the aortic arch and aortic hiatus were cut off with scissors. The removed blood vessels were temporarily placed in a 60 mm cell culture dish and sterile PBS buffer stored at 4 °C was added. (4) Cell culture: Carefully remove blood clots, connective tissue, and adipose tissue from inside and outside the blood vessels with forceps. After removal, transfer the blood vessels to another new 60mm cell culture dish, add the pre-prepared 20% complete culture medium, cut the blood vessels longitudinally with scissors, gently scrape off the intima with forceps, and finally scrape off the media to separate it from the adventitia. Cut the separated media into small square pieces with sides of about 1mm and spread them evenly in a 60mm cell culture dish. Then place it upside down in a 37℃ constant temperature cell culture incubator so that the small blood vessel pieces are in close contact with the culture dish. After about 30 minutes, remove the culture dish and slowly add 20% complete culture medium along the edge of the culture dish with a pipette. Then put it back in the 37℃ constant temperature cell culture incubator for culture. Change the culture medium once on the 4th day, and then change it every 3 days thereafter. When the cell density reaches 60%, use sterile curved forceps to pick up the tissue pieces and discard them. When the cell density reaches 80%, it can be passaged into a 10cm dish for culture.

[0040] 2. CCK8 assay to detect the activity of primary rat aortic smooth muscle cells treated with dehydrocamelin (1) Culture rat aortic smooth muscle cells from generation 3 to 8, prepare cell suspension with culture medium containing 10% fetal bovine serum, and seed 5000 cells per well into 96-well plates with a cell suspension volume of 200 μL per well; (2) Five groups were set up: normal group, Harmine 2.5μM, Harmine 5μM, Harmine 10μM, and Harmine 20μM; each drug group was set up with 6 replicates and the drug was treated for 24 hours. (3) After the culture is completed, discard the original culture medium in the well, add 100 μL of culture medium containing 10% CCK8 to each well, and incubate in an incubator for 2 hours; (4) Use an enzyme-linked immunosorbent assay (ELISA) reader to measure absorbance at a wavelength of 450 nm.

[0041] The toxicity assay results of dehydrocamellidine at 0 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM on primary rat aortic smooth muscle cells under normal 24-hour culture conditions are as follows: Figure 3As shown, the results indicate that dehydrocamelin has low cell damage and high safety.

[0042] IV. Alizarin Red staining of cells induced by dehydrocamelin in calcified culture medium 1. Constructing a primary rat aortic smooth muscle cell calcification model (1) Preparation: Place the cell culture dish under a microscope for observation. When the cell density is 70% or higher, the cell calcification model can be prepared. Place 1ml pipette, 1ml pipette tip, 200μl pipette, 200μl pipette tip, and marker pen into the cell culture clean bench and turn on the UV sterilization for 30min. Place the PBS solution, 2% complete culture medium, and vascular smooth muscle cell calcification culture medium stored in the 4℃ refrigerator into a 37℃ constant temperature water bath and rewarm for 10-15min before use. (2) Calcification treatment: In a cell clean bench, the culture medium on the cell surface was aspirated and removed. After slowly washing the cell surface with PBS solution, vascular smooth muscle cell calcification culture medium (2% complete culture medium + 10mM β-glycerophosphate sodium + 3mM calcium chloride) was carefully added. The control group was given 2% complete culture medium. The treatment time and category were recorded on the culture dish. The medium was changed every two days.

[0043] 2. Alizarin Red staining (1) Preparation: Prepare 2% alizarin red staining solution, 4% paraformaldehyde, PBS buffer and sufficient deionized water in advance; (2) Cell staining: Take out the cell culture dish to be stained, remove the culture medium on the surface, wash with PBS buffer 3 times, add 4% paraformaldehyde to fix the cells for 10 minutes; remove paraformaldehyde, wash the cells with deionized water 3 times, add 2% alizarin red staining solution to stain for 5 minutes, and rinse the cells repeatedly with deionized water until the liquid is clear and colorless.

[0044] Note: If the cells successfully calcify, a red deposit will be visible at the bottom of the culture dish after staining.

[0045] Alizarin Red staining results of primary rat aortic smooth muscle cells treated with different concentrations of dehydrocamellia alkaloid (0, 2.5, 5, 10 μM) are shown below. Figure 4 As shown, the results indicate that dehydrocamellia alkaloids can effectively inhibit osteoblast-like differentiation of smooth muscle cells.

[0046] V. Real-time fluorescence quantitative analysis of dehydrocamellia alkaloids on calcified cells induced by calcification in calcified culture medium 1. RNA extraction – Trizol extraction method (1) Sample processing 1) Discard the cell culture medium, wash once with 1xPBS, and discard the waste liquid; 2) Add 1 ml of Trizol to each well of a standard 6-well plate to fully cover the cell surface, lyse on ice for 5 min, and repeatedly scrape the bottom of the plate with a cell scraper until all cells have detached. 3) Transfer the liquid to a new EP tube and vortex it to ensure thorough mixing and complete pyrolysis; (2) Add 200 μL of chloroform to the EP tube (after adding, the liquid will turn milky pink and separate into layers). Shake well and let stand. Centrifuge at 4°C, 12000 rpm for 15 min. The liquid will separate into three layers. Take the liquid of the top transparent layer into a 1.5 ml enzyme-free EP tube. Be careful not to touch the middle layer. (3) Add 500 μL of isopropanol to precipitate the RNA, vortex back and forth to mix, place at room temperature for 10 min, then centrifuge at 12000 rpm and 4°C for 10 min. (4) Discard the isopropanol, add 1 ml of 75% anhydrous ethanol to each tube, and gently invert the tube back and forth to avoid shaking the precipitate. Centrifuge at 4°C and 7500 rpm for 5 min. (5) Discard the ethanol, prepare an ice box, and pre-thaw the reagents required for reverse transcription and PCR (Mix, SYBR, RNaesfree H2O, gDNA Clean). After the RNA is allowed to stand and dry, add 30 μL of RNaesfree H2O to the EP tube to dissolve the RNA, shake, centrifuge briefly, and then measure the concentration.

[0047] 2. RNA concentration measurement and reverse transcription (1) RNA concentration determination The A260 / A280 ratio can be used to determine RNA purity. Generally, an A260 / A280 ratio of 1.8-2.0 indicates normal RNA purity. A ratio <1.8 suggests the presence of protein or phenolic contamination. The steps for RNA concentration detection are as follows: Zero the micro-spectrophotometer with 2 μl of RNase-free water, then wipe it clean with a clean paper towel. Use a sterile pipette tip to pipette 2 μl of each sample sequentially. Note that the instrument must be wiped clean with a clean paper towel before and after each sample test, and different sterile pipette tips should be used for different samples. Record the A260 / 280 ratio and concentration value for each sample.

[0048] (2) Determine the reverse transcription system based on the reverse transcription kit – two-step method 1) Removal of gDNA ① Configure the reaction system according to Table 2 Table 2

[0049] ② After brief centrifugation, load the sample onto a PCR machine (available in 221). The loading program is 42℃ for 2 minutes; 12℃ is unlimited.

[0050] 2) Reverse transcription reaction ① Configure the reaction system according to Table 3 Table 3

[0051] ② After the system is prepared, centrifuge briefly and then run it into the PCR machine. The PCR program is as follows: 37℃ for 15 min; 85℃ for 5 sec; 4℃ for ∞.

[0052] 3. Real-time quantitative PCR reaction Prepare the PCR reaction solution according to Table 4 in a 96-well plate, with two replicates for each gene. Gently invert the plate to mix thoroughly, avoiding air bubbles to prevent insufficient reaction due to uneven mixing. Do not vortex. The reaction volume is 10 μl. Then, perform cDNA amplification using a real-time qPCR instrument. The standard two-step PCR amplification procedure is as follows: Step 1: 1 cycle, 95℃ pre-denaturation for 30 s; Step 2: Amplification reaction: 40 cycles, 95℃ denaturation for 5 s followed by 60℃ annealing and extension for 30 s. After the reaction, obtain the target gene cycle threshold (Ct), and perform relative quantification analysis and statistical analysis using the 2^(-ΔΔCt) method.

[0053] Table 4

[0054] Specifically, the forward and reverse primer sequences in Table 4 are shown in Table 5.

[0055] Table 5

[0056] Real-time fluorescence quantitative analysis of primary rat aortic smooth muscle cells treated with different concentrations of hydrocamellia alkaloid (0, 2.5, 5, 10 μM) is shown below. Figure 5 As shown, the results indicate that dehydrocamellia alkaloids can downregulate the expression of RUNX2 and BMP2, while upregulating the expression of α-SMA, thereby reducing osteoblast-like differentiation of vascular smooth muscle cells.

[0057] In summary, the dehydrocamellia alkaloid of the present invention can significantly treat vascular calcification induced by vitamin D3 and osteogenic differentiation of rat aortic smooth muscle cells induced by calcification culture medium containing high calcium and high phosphorus. Therefore, it has broad application prospects in the prevention and treatment of vascular calcification, provides a new approach for the research and development of drugs for the treatment of vascular calcification, and provides a new theoretical direction for clinical application.

[0058] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of dehydrocamelin in the preparation of drugs for preventing and treating vascular calcification, characterized in that, The dehydrocamelin can be used to prevent and / or treat vascular calcification.

2. The application according to claim 1, characterized in that, The dehydrocamelin reduces osteoblast-like differentiation of vascular smooth muscle cells by downregulating the expression of RUNX2 and BMP2 and upregulating the expression of α-SMA.

3. The application according to claim 1, characterized in that, The dehydrocamelin can reduce calcium salt deposition in blood vessels.

4. The application according to claim 1, characterized in that, The dehydrocamelin can improve vascular calcification induced by vitamin D3.

5. The application according to claim 1, characterized in that, The vascular calcification is medial calcification.

6. A pharmaceutical composition for preventing and treating vascular calcification, characterized in that, The main active ingredient of the pharmaceutical composition is dehydrocamelin.

7. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition prevents and / or treats vascular calcification by downregulating the expression of RUNX2 and BMP2 while upregulating the expression of α-SMA, thereby reducing osteoblast-like differentiation of vascular smooth muscle cells.

8. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition prevents and / or treats vascular calcification by reducing calcium salt deposition in blood vessels.

9. The pharmaceutical composition according to claim 6, characterized in that, The dosage of the dehydrocamelin is 10-20 mg / kg / day.

10. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

Images