Application of compound Isaridin E or pharmaceutical salt thereof in preparation of drugs for resisting vascular calcification and related diseases

Abstract

The invention discloses an application of a compound Isaridin E or a pharmaceutical salt thereof in preparation of anti-vascular calcification drugs and related diseases, and relates to the technical field of medical biology. A classical high phosphate induced smooth muscle cell calcification model and an in-vivo vascular calcification model induced by a high dose of vitamin D3 are adopted, and through an in-vitro cell experiment, an in-vitro experiment and an overall animal experiment, it is found that the compound Isaridin E has a remarkable improvement effect on vascular calcification caused by calcium and phosphorus metabolism disorder. The medicine has the effects of remarkably inhibiting vascular smooth muscle cell osteogenic differentiation induced by high phosphate and vitamin D3, inhibiting vascular calcification and remarkably reducing formation of calcium-phosphorus crystals on vascular walls. The invention develops a brand new medical application of the marine compound Isaridin E, and the marine compound Isaridin E can be used for research and development of drugs for treating vascular calcification and calcification-related diseases.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical biotechnology, and in particular to the use of a compound Isaridin E or a pharmaceutically acceptable salt thereof in the preparation of drugs for resisting vascular calcification and related diseases. Background Art

[0002] 99% of the calcium in the human body exists in the form of hydroxyapatite crystals in teeth and bones. When it is abnormally deposited in other tissues and organs of the body, it is called ectopic calcification. When calcium-phosphorus crystals are abnormally deposited in the cardiovascular system, it is called vascular calcification. The occurrence of vascular calcification will lead to increased stiffness of the vascular wall, decreased compliance, vasomotor dysfunction, and hemodynamic instability, thereby increasing the risk of cardiovascular complications such as hypertension, myocardial ischemia, and stroke. It is recognized as an independent risk factor for increased morbidity and mortality of cardiovascular events.

[0003] According to the different sites of vascular calcification, it can be divided into intimal calcification, medial calcification, valvular calcification and calcified uremic arteriole lesions. Intimal calcification is common in atherosclerosis, while medial calcification is more common in patients with chronic kidney disease, diabetes, atherosclerosis and the elderly. It is more common in clinical practice. Smooth muscle cells are the main cellular components of the vascular media. They are the main participants in vascular calcification and the direct or indirect target cells of many factors that cause vascular calcification. Under physiological conditions, vascular smooth muscle cells maintain contractile function and express a large number of contractile proteins. Under the influence of various risk factors (such as high calcium and phosphorus environment, hyperlipidemia, inflammation and oxidative stress, etc.), vascular smooth muscle cells downregulate the expression of contractile proteins, while the expression and secretion of bone formation-related proteins increase, prompting smooth muscle cells to transform from the contractile phenotype under physiological conditions to the osteogenic phenotype, which is the core link in the development of vascular calcification. Osteoblastic smooth muscle cells secrete matrix vesicles and apoptotic bodies into the extracellular matrix as the initial site of mineral deposition and crystal nucleation, both of which have the ability to concentrate calcium and phosphate and further induce the formation of mineral nodules. Clinical studies have shown that calcification occurs in multiple areas of the aorta during chronic kidney disease, and chest CT shows that the aortic arch is more prone to calcification throughout the aorta in the early stages, which accurately reflects the development stage of vascular calcification. Therefore, aortic arch calcification is a prognostic indicator for the deterioration of cardiovascular disease.

[0004] Since hydroxyapatite crystals, the main component of vascular calcification, are highly insoluble, the main treatment strategy for vascular calcification in clinical practice is still limited to mechanical treatment, such as percutaneous coronary intervention and cutting balloon for mild to moderate coronary artery calcification, and coronary artery atherectomy or coronary artery bypass grafting for severe calcification. Drug treatment includes the regulation of calcium and phosphorus levels and related intervention strategies directly targeting vascular calcification, such as phosphate chelators, pyrophosphates and bisphosphonates, denosumab, etc. However, drug treatment is still controversial, and there is no effective treatment measure in clinical practice. Therefore, the development of new anti-vascular calcification drugs has great social significance.

[0005] Isaridin E (ISE) is a cyclic hexapeptide extracted from the marine fungus Beauveria feline SYSU-MS7908. Currently, the research on cyclic depsipeptide Isaridins compounds mainly focuses on insecticide, inhibition of neutrophil activation and anti-platelet thrombosis, and no research results on anti-vascular calcification effects have been reported. Summary of the invention

[0006] The purpose of the present invention is to overcome the existing technical defects and provide the use of the compound Isaridin E or its pharmaceutical salt in the preparation of drugs for resisting vascular calcification and related diseases. During the experiment, the inventors used a classic high phosphate-induced in vitro cell model and a high-dose vitamin D3 (vitamin D3, VitD3)-induced in vivo vascular calcification model, and took vascular smooth muscle cells and aortic vessels as observation objects. It was found that the compound Isaridin E can slow down the occurrence of vascular calcification, and can significantly reduce the level of calcium deposition in smooth muscle cells and aortic vessels at the administered concentration, and inhibit the tissue pathological changes caused by ectopic calcium crystals.

[0007] In view of the above technical problems, the present invention provides the use of the compound Isaridin E or a pharmaceutically acceptable salt thereof in the preparation of an anti-vascular calcification drug.

[0008] At the same time, the present invention also provides the use of the compound Isaridin E or a pharmaceutically acceptable salt thereof in the preparation of drugs for preventing vascular calcification-related diseases.

[0009] Preferably, the drug is a drug for inhibiting vascular calcification caused by calcium-phosphorus metabolism disorder.

[0010] Preferably, the drug is a drug for inhibiting abnormal deposition of vascular hydroxyapatite crystals.

[0011] Preferably, the drug is a drug for inhibiting osteogenic differentiation of vascular smooth muscle cells.

[0012] Accordingly, the present invention also provides a drug for treating vascular calcification and related diseases, wherein the drug comprises the compound Isaridin E or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier and / or adjuvant.

[0013] Preferably, the drug is an injectable solution.

[0014] Preferably, the medicament is a medicament for oral administration.

[0015] Compared with existing drugs, the present invention has the following beneficial effects:

[0016] The present invention adopts the classic high phosphate-induced smooth muscle cell calcification model and the in vivo vascular calcification model induced by high doses of vitamin D3 (vitamin D3, VitD3). Through in vitro cell experiments, in vitro experiments and whole animal experiments, it is found that the compound Isaridin E has a significant improvement effect on high phosphorus and vitamin D3-induced vascular calcification. The drug can significantly inhibit the osteogenic differentiation of smooth muscle cells induced by high phosphate, and reduce the production of calcium-phosphorus crystals in a concentration-dependent manner. The in vitro experiment of vascular rings also proves that ISE can reduce the level of aortic ring calcification, which is manifested as a decrease in the level of aortic calcium deposition and a decrease in the distribution level of calcium-phosphorus crystals along elastic fibers in vascular sections. In the in vivo calcification model induced by high doses of vitamin D3, compared with the model group, the calcification foci presented by the macroscopic staining of the aorta in the ISE-treated group were significantly inhibited. The drug has low toxicity and high safety, and has the application prospect of being developed into a new anti-vascular calcification drug.

[0017] Additional aspects and advantages of the present invention will be given in part in the following description, which will become obvious from the following description, or may be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The drawings described herein are used to provide a further understanding of the present invention, constitute a part of the present application, and do not constitute an improper limitation of the present invention. In the drawings:

[0019] Figure 1 This is a schematic diagram of the test results of the compound Isaridin E inhibiting high phosphorus-induced calcification of mouse aortic smooth muscle cells;

[0020] Figure 2 This is a schematic diagram of the test results of the compound Isaridin E inhibiting the expression of osteogenic differentiation marker molecules of mouse aortic smooth muscle cells induced by high phosphorus;

[0021] Figure 3 Isaridin E inhibits high phosphorus-induced calcification of mouse aortic smooth muscle cells 50 Schematic diagram of value test results;

[0022] Figure 4 This is a schematic diagram of the test results of the compound Isaridin E inhibiting the calcium deposition level in the mouse aortic ring induced by high phosphorus;

[0023] Figure 5 This is a schematic diagram of the results of the staining test of the compound Isaridin E inhibiting high phosphorus-induced mouse aortic ring calcification;

[0024] Figure 6 This is a schematic diagram of the test results of the compound Isaridin E inhibiting the calcium deposition level in the mouse aorta induced by high-dose vitamin D3;

[0025] Figure 7 Schematic diagram of the test results of gross staining of the compound Isaridin E inhibiting high-dose vitamin D3-induced mouse aortic calcification. DETAILED DESCRIPTION

[0026] In order to more fully understand the technical content of the present invention, the present invention will be further introduced and illustrated in conjunction with the accompanying drawings and specific embodiments below; it is obvious that the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments; based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention.

[0027] For those skilled in the art, the features, benefits and advantages of the present invention will become apparent by reading the contents disclosed in this specification.

[0028] Unless otherwise indicated, all percentages, parts and ratios are calculated based on the total weight of the composition of the present invention. The term "weight content" may be expressed herein by the symbol "%".

[0029] As used herein, "includes," "comprising," "including," "containing," "having," or other variations are intended to encompass non-exclusive inclusions, and no distinction is made between these terms. The term "comprising" means that other steps and ingredients that do not affect the end result may be added. The term "comprising" also includes the terms "consisting of" and "consisting essentially of." The compositions and methods / processes of the present invention may comprise, consist of, and consist essentially of the essential elements and limitations described herein, as well as any additional or optional ingredients, components, steps, or limitations described herein.

[0030] In the following schemes, compound Isaridin E may be referred to as compound ISE for short.

[0031] Example 1

[0032] Effects of compound ISE on calcification level of mouse aortic smooth muscle cells and half inhibition rate IC50 Determination of

[0033] 1. Materials and methods

[0034] 1. Drug preparation

[0035] The compound Isaridin E was provided by Professor Liu Lan's research group at the School of Oceanography, Sun Yat-sen University (chemical formula is as follows). It is a white crystalline solid and is stored at room temperature. When used, it is ground into powder and dissolved in DMSO to prepare a storage solution (incubation concentrations are 1μM, 2μM, 5μM, 10μM, 20μM, and the volume of drug addition is controlled to be 2.5μl), and stored at 4°C for use.

[0036] The structural formula of the compound Isaridin E is shown below:

[0037]

[0038] Among them, (A) is the planar structural formula. (B) is the three-dimensional structural formula. Isaridin E is a cyclohexapeptide compound with PubChem CID 16680915, molecular formula C35H53N5O7, and relative molecular mass 655.8.

[0039] 2. Preparation of mouse aortic smooth muscle cells (MVSMCs)

[0040] Prepare digestive enzymes in advance (1.4 mg type II collagenase / ml DMEM, sterilized with a 0.22 μm filter). Take 5 8-week-old male C57BL / 6J mice, fix them on a mouse fixer after anesthesia with pentobarbital, and disinfect the abdominal skin and hair of the mice with 75% ethanol. Use scissors to open the chest cavity in the clean bench inside the cell room, fully expose the thoracic aorta, cut it from above the spine along the body axis, and immediately place it in 10% FBS DMEM high-glucose culture medium. Use ophthalmic forceps to gently wash away blood stains, and peel off the peripheral connective tissue and outer membrane of the aorta. Cut the blood vessels flat longitudinally, gently scrape the inside of the blood vessels to remove the inner membrane, and use ophthalmic scissors to cut the middle membrane into about 1mm 2 The separated middle membrane was placed in a 6-well plate, 5 mL of digestive enzyme was added, and the plate was placed in a 37°C cell incubator for about 4 hours. During this period, the plate should be observed every hour so that the digestion can be terminated at any time. When the tissue block is digested to a flocculent cell mass, an appropriate amount of culture medium is added to dilute it, centrifuge at 1000 rpm for 10 minutes, and the supernatant is discarded. The cells are resuspended in high-glucose DMEM containing 20% ​​FBS, pipetted and mixed, and then transferred to a 25 mm 2 Culture the cells in a culture flask and do not move the flask within 72 hours. Replace the cells with fresh culture medium after 3 days and observe the cell morphology until they are fully grown and then pass them on.

[0041] 3. Smooth muscle cell calcification experiment

[0042] The experiment was conducted using cells in good condition at the 3rd to 6th generation. Vascular smooth muscle cells were inoculated in a small dish, and different concentrations of Isaridin E (1μM, 2μM, 5μM, 10μM and 20μM) were incubated when the cell confluence reached 50%-60%, and 2.6mmol / L inorganic phosphorus was given as stimulation. The DMEM medium containing 10% FBS was replaced every two days, and 2.6mmol / L inorganic phosphorus (Pi) was given as stimulation. The experimental process lasted about 7-12 days, and the experimental endpoint was determined based on the cell growth status.

[0043] 4. Cell Alizarin Red Staining and Quantification

[0044] Remove the cell culture medium, wash the cells 3 times with ice-bathed PBS and completely dry. Fix the cells with 4% paraformaldehyde at room temperature for 15 minutes. Discard the paraformaldehyde, wash the cells 3 times with deionized water and completely dry. Add 1 ml of 1% Alizarin Red solution (prepared with deionized water) to each well of the 6-well plate for staining for 5 minutes. Remove the dye solution, wash the cells 3 times with deionized water to remove the floating color, and observe the cells under a light microscope.

[0045] Take out the Alizarin Red-stained cell culture dish that has been photographed and dried, add 1 ml of formic acid solution to the Alizarin Red-stained dish, shake it slightly to allow it to fully contact the cells, then transfer the formic acid solution to a 1.5 ml centrifuge tube and vortex to mix thoroughly. Add the mixed formic acid solution to a 96-well plate, 200 μl per well, and detect the absorbance of each group at a wavelength of 405 nm.

[0046] 5. Cellular calcium deposition detection

[0047] Pour off the cell culture medium and wash the cells 3 times with PBS in an ice bath. Add 100 μL of 6% dilute hydrochloric acid (HCl) to each well of the small dish, scrape the cells with a cell scraper and collect them in a 1.5 mL centrifuge tube. Ultrasonicate on ice, set the ultrasonic program to start for 3 seconds and stop for 3 seconds, repeat 5 times in total, and let the sample stand on ice for 30 minutes after ultrasonication. Centrifuge at 10000rpm and 4℃ for 15 minutes, and aspirate the supernatant into a new 1.5 mL centrifuge tube for subsequent detection.

[0048] Prepare calcium standards: The required concentrations are 2.5mmol / L, 1.25mmol / L, 0.625mmol / L, 0.3125mmol / L, 0.15625mmol / L, and 0 (i.e., 6% dilute hydrochloric acid) from high to low. Prepare working solution: Mix R1 and R2 solutions in the calcium detection kit in equal amounts and place at room temperature for 5 minutes. Add 10μL of standard and sample to be tested to 500μL working solution, shake and mix, incubate at 37℃ for 10 minutes, and take 200μL to a 96-well ELISA plate for detection. Place the 9-well plate in a multifunctional ELISA instrument, detect the absorbance value at 600nm, and draw a standard curve with the absorbance of the standard. The calcium content in the sample can be calculated using the standard curve. The calcium content is normalized with the protein concentration to obtain the relative calcium deposition concentration.

[0049] 6. Western blot

[0050] After extracting and quantifying the cell protein, the sample was added with loading buffer in proportion, and SDS-PAGE electrophoresis was performed after protein denaturation in a 95-99℃ water bath for 5 minutes; after electrophoresis, the gel was removed and placed in a transfer electrophoresis instrument for transfer. After transfer, the membrane was blocked, and the corresponding specific primary antibody was added for incubation, and shaken overnight at 4℃; the membrane was washed 3 times with TBST after rewarming the next day, each time for 10 minutes; the specific secondary antibody was added for incubation for 1-1.5 hours, and the membrane was washed 3 times, each time shaking at room temperature for 10 minutes; the Chemiluminescent HRP Substrate (Millipore) luminescent solution was used in the dark room to develop the image on the BIO-RAD Molecular Imager ChemiDoc XRS+, and the data was analyzed using Image Lab 4.0.

[0051] 7. Statistical processing

[0052] Data are expressed as mean ± SEM. One-way analysis of variance (ANOVA) was performed using GraphPad Prism 8.0 software, and P < 0.05 was considered statistically significant.

[0053] (II) Experimental results

[0054] 1. Effect of Isaridin E on high phosphorus-induced calcification of mouse aortic smooth muscle cells

[0055] In order to detect the effect of ISE on calcification at the cellular level, the inventors treated the cells with 2.6mmol / L of inorganic phosphorus to induce calcification, and simultaneously gave different concentrations of Isaridin E for co-incubation for 7-12 days, and used Alizarin red staining to assess the level of cell calcification. Alizarin red dye can chelate with calcium nodules to produce a color reaction, presenting an orange-red compound in an acidic environment, which is deposited on the surface of calcified cells to reflect the overall calcification of the cells. The staining area is proportional to the content of calcium salts. The more red area generated, the more calcium nodules there are, and the more severe the calcification. Figure 1 The staining results showed that Isaridin E (5μM, 10μM, 20μM) could inhibit smooth muscle cell calcification in a concentration-dependent manner. Correspondingly, the quantitative results showed that Isaridin E treatment could significantly inhibit the level of cellular calcium deposition compared with the high phosphorus group, and there was a statistical difference at 5μM. Compared with the high phosphorus group, *P<0.05, n=6.

[0056] 2. Effect of Isaridin E on the expression of osteogenic differentiation markers and contractile markers in mouse aortic smooth muscle cells induced by high phosphorus

[0057] The core link of vascular calcification is the transformation of smooth muscle from contractile to osteogenic. This phenotypic change is accompanied by the loss of contractile markers of vascular smooth muscle cells such as SM22α and α-SMA and the increase of osteogenic markers Runt-related transcription factor 2 (Runx2), bone morphogenetic protein-2 (BMP2), and alkaline phosphatase (ALP). Therefore, the inventors used Western blotting to detect the expression of related marker molecules after incubation with ISE under high phosphorus stimulation. Figure 2 As shown in the figure, under high phosphorus stimulation, smooth muscle cells significantly increased the expression of Runx2 and BMP2 proteins, while reducing the expression of α-SMA, proving that smooth muscle cells under high phosphorus environment underwent osteogenic phenotype conversion. After incubation with ISE, the protein levels of Runx2 and BMP2 were inhibited, and the expression of α-SMA protein was upregulated, indicating that at the molecular level, ISE can inhibit the osteogenic phenotype conversion of smooth muscle cells and reduce the formation of calcium-phosphate crystals. Compared with the high phosphorus group, *P<0.05, n=6.

[0058] 3. Isaridin E half inhibition rate IC of smooth muscle cell calcification 50 Determination of

[0059] Given that ISE has a significant inhibitory effect on smooth muscle cell calcification, we then determined its half inhibition rate. Half inhibition rate, also known as half inhibition concentration, refers to the concentration of a drug or inhibitor required to inhibit a biological process by half. In pharmacy, it is used to characterize the antagonistic ability of antagonists in in vitro experiments. The smaller the value, the stronger the inhibitory effect.

[0060] Since formic acid can dissolve alizarin red bound to calcification points, and alizarin red is yellow under acidic conditions, the different shades of alizarin red staining caused by the difference in calcification degree between groups are used. A constant amount of formic acid is added to the culture dish to react with it, which can show different shades of yellow formic acid solution. Finally, the absorbance is detected to achieve the quantification of alizarin red in cells. We used the method of formic acid quantification after alizarin red staining to determine IC 50 The relative calcification inhibition rate was calculated using the formula (OD value of the high phosphorus group - OD value after treatment with different concentrations of Isaridin E) / OD value of the high phosphorus group*100%, and IC was analyzed using GraphPad Prism. 50 Parameter curve fitting. Figure 3 The curve shows that the half-maximal inhibition concentration of ISE is 4.957 μmol, indicating that ISE has a significant inhibitory effect on vascular calcification at low doses. n=5.

[0061] Example 2

[0062] Effects of Isaridin E on calcification of mouse aortic rings

[0063] 1. Materials and methods

[0064] 1. Drug preparation

[0065] According to the results of the cell experiment, this part uses 20 μM concentration of Isaridin E for the experiment. Dissolve it in DMSO to prepare a 20 μM stock solution and store it at 4°C for later use.

[0066] 2. Aortic ring calcification experiment

[0067] Eight-week-old male C57BL / 6J mice were anesthetized with pentobarbital and then soaked in 75% ethanol for 3 minutes to fully disinfect the chest and abdominal skin. The chest cavity was opened, and about 1 cm of the thoracic aorta was cut off and placed in sterile PBS buffer. The residual blood was washed off, and the peripheral connective tissue and adventitia of the aorta were carefully peeled off, and the arterial tissue was cut into arterial rings of about 3 mm each. The arterial rings were transferred to a 24-well plate and 1 ml of 10% complete culture medium was added. The next day, the arterial rings were incubated with 20 μM Isaridin E and stimulated with 2.6 mmol / L inorganic phosphorus. The medium was changed every two days. Around the 7th day, it can be clearly observed that the hardness of the arterial rings increased under high phosphorus treatment, which indirectly suggests that vascular ring calcification was successfully induced.

[0068] 2. Detection of calcium deposition in vascular tissue

[0069] Add 3-4 3mm grinding ceramic beads to a 1.5mL centrifuge tube, place the vascular tissue on the tube wall, and freeze it in liquid nitrogen for 10 seconds. Place the centrifuge tube in a tissue grinder pre-cooled with liquid nitrogen and grind at 55Hz for 30 seconds. Add 100μL of 6% dilute hydrochloric acid to the ground vascular powder, and sonicate on ice. Set the sonication program to start for 3 seconds and stop for 3 seconds, repeat 5 times in total, and let the sample stand on ice for 30 minutes after sonication. Centrifuge at 10000rpm and 4℃ for 15 minutes, and pipette the supernatant into a new 1.5mL centrifuge tube for subsequent testing.

[0070] Prepare calcium standards: The required concentrations are 2.5mmol / L, 1.25mmol / L, 0.625mmol / L, 0.3125mmol / L, 0.15625mmol / L, and 0 (i.e., 6% dilute hydrochloric acid) from high to low. Prepare working solution: Mix R1 and R2 solutions in the calcium detection kit in equal amounts and place at room temperature for 5 minutes. Add 10μL of standard and sample to be tested to 500μL working solution, shake and mix, incubate at 37℃ for 10 minutes, and take 200μL to a 96-well ELISA plate for detection. Place the 9-well plate in a multifunctional ELISA instrument, detect the absorbance value at 600nm, and draw a standard curve with the absorbance of the standard. The calcium content in the sample can be calculated using the standard curve. The calcium content is normalized with the protein concentration to obtain the relative calcium deposition concentration.

[0071] 3. von Kossa staining of vascular sections

[0072] After paraffin sections are dewaxed, use a tissue wax pen to draw a circle on the outside of the vascular tissue. Add enough 5% AgNO 3 Solution, so that it completely covers the tissue, and then shine under UV light for 1-2 hours, paying attention to prevent the slide from drying. Discard AgNO 3 solution, washed with distilled water for 5 minutes, 5% Na 2 S 2 O 3 Wash with solution for 2 minutes. Stain the nucleus with hematoxylin solution for 30 seconds, and then use tap water to reverse the color. This process is repeated 3 times, 3 minutes each time. Dehydrate with 70%, 80%, 90%, 95%, 100%, and 100% ethanol in sequence, and make it transparent twice with xylene. Seal the slides with neutral gum and bake them in a 37°C incubator.

[0073] 4. Alizarin red staining of vascular sections

[0074] After paraffin sections are dewaxed, use a histochemical crayon to draw a circle on the outside of the vascular tissue. Add alizarin red dye to the sections to completely cover the tissue and stain for 5 minutes. The staining time is determined according to the calcium salt content. Observe under a microscope in time when the calcium salt is a dark orange-red. Pour off the dye and wash with tap water until the slides are colorless. Restain the nuclei with hematoxylin and wash with water. Bake the sections in a 65℃ oven for 4 hours. Transparent the sections in fresh xylene for 5-10 minutes and seal with neutral gum.

[0075] 5. Statistical analysis methods

[0076] GraphPad Prism 8.0 software was used for data processing. The results were expressed as mean±SEM and analyzed by One-Way ANOVA. P<0.05 was considered statistically significant.

[0077] (II) Experimental results

[0078] 1. Effect of Isaridin E on high phosphorus-induced calcification of mouse aortic rings

[0079] According to the results of cell experiments, 20μM Isaridin E has the strongest inhibitory effect on smooth muscle cell calcification. Therefore, the inventors used 20μM Isaridin E and high phosphorus to treat isolated aortic tissue for 7 days to further evaluate the effect of ISE on vascular calcification. The aorta was tested for calcium deposition, and the experimental results are as follows: Figure 4 As shown in the figure, compared with the solvent group, ISE treatment can significantly reduce the calcium deposition level in aortic rings. This experimental result is consistent with the experiment at the cell level.

[0080] Compared with the normal control group, # P<0.05; *P<0.05 compared with the solvent group, n=6.

[0081] 2. Detection of Calcification Staining Pathological Changes in Mouse Aortic Ring Sections

[0082] In order to observe the changes in the pathological structure of the aorta, the inventor subjected the aortic sections to special staining for calcium salt determination. Alizarin red is an anthraquinone derivative that can chelate with calcium salts in calcium carbonate or calcium phosphate to form an orange-red complex, which can be used to stain small amounts of calcium salt deposits in tissues. It has a good staining effect on some pathological calcifications, such as calcification of the diseased arterial wall during aortic atherosclerosis and other ectopic calcifications. After staining, the calcium salt precipitation area is red or orange-red, and the background is light red or almost colorless. Von Kossa staining is also a classic method for staining mineralized nodules. The basic principle is that silver ions and insoluble calcium salts form reducible silver salts in situ in the tissue, and then under the action of strong light or ultraviolet light or extremely strong reducing agents, the silver salts are reduced to black elemental silver, thereby realizing the color development of calcium salts in the tissue. It is suitable for samples with more calcium salt deposits. After staining, the calcium salt deposition area is black, the cell nucleus is blue, and the background is light red. Such as Figure 5 As shown, combined with the results of the two special stainings, there was no calcium salt in the aorta of the control group, while high-phosphate incubation increased the accumulation of calcium salt in the vascular media and distributed calcium salt along the vascular elastic fibers; compared with the solvent group, Isaridin E treatment could significantly reduce the distribution and deposition of calcium salt in the vascular smooth muscle layer.

[0083] Example 3

[0084] Effects of Isaridin E on aortic calcification in model mice

[0085] 1. Materials and methods

[0086] 1. Drug preparation

[0087] The compound Isaridin E was ground into powder, dissolved in a solution system of 4% DMSO, 10% Tween-80, 15% propylene glycol and 71% saline (the concentration ratio of this system was obtained from a preliminary experiment, which can ensure complete dissolution of the drug while minimizing the toxicity of the solvent), and administered by gavage at a preset concentration (ISE dosage was 50 mg / kg·d, and the administration volume was controlled to be 100 μl).

[0088] 2. Experimental animals and vascular calcification models

[0089] Preparation of vitamin D3 solution: Weigh 66 mg of VitD3 and dissolve it in 200 μl of anhydrous ethanol, then add 1.4 ml of corn oil. Use a vortexer to dissolve the mixture at room temperature and mix it thoroughly for later use. Weigh 750 mg of glucose and add 18.4 ml of ddHO. 2O to fully dissolve it, and filter it with a filter to achieve sterile conditions. The solution was added to the previously obtained liquid, and the vortex instrument was used again to fully vortex to obtain a uniformly distributed emulsion suspension. During the preparation of the control group, an equal amount of normal saline was used instead of VitD3 for preparation, and the preparation process of other components was the same as that of the calcification group.

[0090] Establishment of vascular calcification model: Pinch the back skin of the mouse, insert the needle at a low angle close to horizontal, and after successful skin puncture, flatten the angle of the syringe, lift the back skin and inject 400,000 IU / kg cholecalciferol or solvent, and inject the drug after the syringe is aspirated without blood. The bulging skin bumps on the back skin are observed by naked eyes as a sign of successful subcutaneous injection. The mice were subcutaneously injected for 3 consecutive days, and the samples were collected after one week of continuous feeding.

[0091] Wild-type male C57BL / 6J mice (SPF grade, 6-8 weeks old, purchased from Guangdong Yaokang Biotechnology Co., Ltd.) were selected for this experiment and were raised in the Experimental Animal Center of Sun Yat-sen University Zhongshan Medical College (SPF grade) in a laminar flow rack and fed in cages. They were given free access to water and food, and the room temperature was controlled at about 24°C with a 12-h day / night cycle. After one week of adaptive feeding of all experimental animals, the mice were randomly divided into 4 groups according to the experimental grouping, with 6 mice in each group, and then synchronously administered drugs, including the normal control group (mice were subcutaneously injected with VitD3 control group solvent for three consecutive days), the VitD3 group (mice were subcutaneously injected with VitD3 solvent for three consecutive days), the VitD3+ISE control solvent group (mice were subcutaneously injected with VitD3 solvent for three consecutive days, and the ISE control group solvent was continuously gavaged for 10 days), and the VitD3+ISE group (mice were subcutaneously injected with VitD3 solvent for three consecutive days, and 50 mg / kg ISE solvent was continuously gavaged for 10 days).

[0092] 3. Alizarin red staining of mouse aorta

[0093] After euthanizing the experimental animals to be sampled, fix them in a supine position on a foam board. Wash the intact blood vessels from the root of the aorta to the branches of the iliac arteries with histochemical PBS to remove blood stains and soak them in 4% paraformaldehyde overnight. The next day, soak the vascular tissue in 0.004% Alizarin Red (prepared with 2% potassium hydroxide solution) overnight. Remove the blood vessels and wash them twice in 2% potassium hydroxide, 5 minutes each time. Observe the blood vessels in general and take pictures. Immerse the blood vessels again in 4% paraformaldehyde fixative and store them at room temperature for a long time.

[0094] (II) Experimental results

[0095] 1. Effect of Isaridin E on vitamin D3-induced aortic calcification in mice

[0096] High doses of vitamin D3 have been used to induce calcification of the elastic layer of the arteries for more than 70 years. Vitamin D3 can effectively promote bone reabsorption, increase serum calcium levels by more than 30%, lead to calcium and phosphorus metabolism disorders in the body, and cause vascular calcification. It is a commonly used model for vascular calcification research. Figure 6 As shown in the figure, compared with the control group, the aortic calcium deposition increased significantly after vitamin D3 treatment, indicating that the vascular calcification model was successfully established; and the aortic calcium deposition level of mice after oral administration of ISE was significantly reduced compared with the solvent group, indicating that ISE can also significantly inhibit the occurrence and development of vascular calcification in vivo. Compared with the normal control group, # P<0.05; *P<0.05 compared with the solvent group, n=6.

[0097] As mentioned above, Alizarin Red is a classic calcium salt staining method. It is prepared with 2% potassium hydroxide to turn Alizarin Red into purple in an alkaline environment. It combines with calcium salts deposited in the aorta to appear blue-purple, while the part without calcium salts is milky white. Figure 7 As shown, the blood vessels in the control group did not accumulate calcium salts and remained milky white, while the aorta after vitamin D3 induction showed obvious purple, indicating the deposition and distribution of calcium salts in the aorta; and the purple area of ​​the blood vessels after ISE gavage was significantly reduced compared with the solvent group, and the positive areas were mainly concentrated in the aortic arch, abdominal aorta and common iliac artery, while the calcified nodules in the thoracic aorta were significantly less. n = 6.

[0098] Experimental discussion and evaluation:

[0099] Vascular calcification is an osteogenic process regulated by various cell types, among which VSMCs play a central role in the process of vascular calcification. VSMCs are multipotent cells existing in blood vessels, including two phenotypes: synthetic and contractile. Synthetic VSMCs are commonly found in embryonic or developing tissues and organs, and can undergo cell division, proliferation, and differentiation under the action of various growth factors, playing an important role in the occurrence of vascular walls and the repair of vascular damage; contractile VSMCs are terminally differentiated cells that have the function of maintaining the structural and functional integrity of arterial walls. When VSMCs are exposed to a pathological environment with high levels of mineral ions for a long time, they will differentiate into bone / cartilage-like cells, and transform from a highly differentiated contractile type to a dedifferentiated osteogenic type, which is the core link of vascular calcification. The increase in serum calcium and phosphorus levels is significantly correlated with the occurrence of vascular calcification. Disorders in calcium and phosphorus metabolism promote the osteogenic conversion of smooth muscle cells, while leading to an increase in Ca×Pi, thereby promoting the growth and deposition of hydroxyapatite crystals in the extracellular matrix through thermodynamic mechanisms. The present invention respectively adopts a high-phosphate-induced smooth muscle cell calcification model, an in vitro aortic ring calcification model and a vitamin D3-induced mouse aorta calcification model to explore the intervention effect of Isaridin E on vascular calcification.

[0100] Isaridin E (ISE) is a cyclohexapeptide compound isolated from Beauveriafeline SYSU-MS7908 from the Xisha sea squirt in the South China Sea in 2019. It has been found that it can significantly inhibit the release of LPS-induced endothelial inflammatory factors during sepsis and reduce vascular inflammation and lung damage in mice. Vascular calcification is a pathological phenomenon in which calcium and phosphate crystals are abnormally deposited in the vascular wall. Therefore, the inventors first established a high-phosphate-induced classic calcification model on cultured mouse aortic smooth muscle cells. Alizarin red staining and cellular calcium deposition levels showed that ISE can reduce the formation of extracellular matrix calcium and phosphate crystals in a concentration-dependent manner within a certain range. In a high-phosphate environment, the protein levels of Runx2 and BMP2, the osteogenic marker molecules of smooth muscle cells, increased, and the protein level of α-SMA, the contractile marker molecule, decreased, indicating that the cells underwent a phenotypic transformation from contractile to osteogenic. After administration of Isaridin E, osteogenic marker molecules were downregulated, while contractile marker molecules were upregulated. The inventors then determined the half inhibition rate of ISE on vascular calcification, and the results showed that its IC50 was about 5 μmol. The above experiments confirmed that ISE has an anti-vascular calcification effect at the cellular level.

[0101] In Example 2, the inventors conducted an in vitro vascular ring experiment, in which the mouse aorta was isolated and cultured in vitro. Consistent with the results of the cell experiment, ISE can inhibit the calcium deposition level of the aortic blood vessels; the vascular sections showed that ISE can inhibit the abnormal deposition of calcium and phosphorus crystals in the vascular wall caused by high phosphorus. In Example 3, the inventors used a classic in vivo vascular calcification model to simulate the mouse aortic calcification model induced by calcium and phosphorus metabolism disorders by subcutaneous injection of high doses of vitamin D3 for 3 consecutive days to explore the effect of Isaridin E on vascular calcification in mice. Consistent with the results of the above experiments, the aortic calcium deposition experiment showed that vitamin D3 can significantly increase aortic calcium deposition, while ISE can significantly reduce the level of aortic calcification. Alizarin red staining of the aorta showed that vitamin D3 can promote significant calcification of the aorta, while the level of aortic calcification decreased after ISE treatment, especially the calcification of the thoracic aorta was significantly reduced. The above experiments confirmed the definite protective effect of ISE on vascular calcification in the whole animal model.

[0102] In summary, this study shows that ISE or its pharmaceutical salt has the effect of inhibiting the osteogenic differentiation of vascular smooth muscle cells and significantly inhibiting vascular calcification induced by calcium-phosphorus metabolism disorders, and has the application prospect of being developed into a new anti-vascular calcification drug.

[0103] It is worth noting that the anti-vascular calcification drug described in the present invention, which uses Isaridin E as the main active ingredient, does not exclude changes in the formulation system and administration method, derivatives after simple chemical modification and adjustment of the compound, and the combined use of multiple active substances.

[0104] The technical solutions provided by the embodiments of the present invention are introduced in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the embodiments of the present invention. The description of the above embodiments is only applicable to help understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, according to the embodiments of the present invention, there will be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. Use of the compound Isaridin E or a pharmaceutically acceptable salt thereof in the preparation of an anti-vascular calcification drug.

2. Use of the compound Isaridin E or a pharmaceutically acceptable salt thereof in the preparation of drugs for treating diseases related to vascular calcification.

3. The use according to claim 1 or 2, characterized in that: The drug is a drug used for inhibiting vascular calcification caused by calcium-phosphorus metabolism disorder.

4. The use according to claim 1 or 2, characterized in that: The drug is a drug for inhibiting the deposition of hydroxyapatite crystals in the arterial media.

5. The use according to claim 1 or 2, characterized in that: The drug is a drug for inhibiting osteogenic differentiation of vascular smooth muscle cells.

6. A drug for treating vascular calcification and related diseases, characterized in that: The drug comprises the compound IsaridinE or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier and / or adjuvant.

7. The drug according to claim 6, characterized in that The drug is an injectable solution.

8. The drug according to claim 6, characterized in that The drug is a drug for oral administration.

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