CN119280371A

CN119280371A - Application of compound Isaridin E or its pharmaceutically acceptable salt in the preparation of drugs for preventing vascular calcification and related diseases

Info

Publication number: CN119280371A
Application number: CN202411522981.6A
Authority: CN
Inventors: 杜艳华; 彭雨生; 刘岚; 陈森华
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2024-10-29
Filing date: 2024-10-29
Publication date: 2025-01-10

Abstract

The present invention discloses the use of a compound Isaridin E or a pharmaceutical salt thereof in the preparation of an anti-vascular calcification drug and related diseases, and relates to the field of pharmaceutical biotechnology. The present invention adopts a classic high-phosphate-induced smooth muscle cell calcification model and an in vivo vascular calcification model induced by high-dose vitamin D3 (vitamin D3, VitD3), and 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 vascular calcification caused by calcium-phosphorus metabolism disorders. The drug can significantly inhibit the osteogenic differentiation of vascular smooth muscle cells induced by high phosphate and vitamin D3, inhibit vascular calcification, and significantly reduce the formation of calcium-phosphorus crystals in the vascular wall. The present invention opens up a new medical use of the marine compound Isaridin E, which can be used for the research and development of drugs for the treatment of vascular calcification and calcification-related diseases.

Description

Application of compound ISARIDIN E or medicinal salt thereof in preparation of medicaments for resisting vascular calcification and related diseases

Technical Field

The invention relates to the technical field of medical biology, in particular to application of a compound ISARIDIN E or medicinal salt thereof in preparation of medicaments for resisting vascular calcification and related diseases.

Background

99% Of calcium in humans exists in the form of hydroxyapatite crystals in teeth and bones and is known as ectopic calcification when it is abnormally deposited in other tissues and organs of the body. Calcium phosphate crystals are abnormally deposited in the cardiovascular system and are referred to as vascular calcifications. The occurrence of vascular calcification leads to increased stiffness of the vessel wall, reduced compliance, vasomotor dysfunction, and hemodynamic instability, thereby increasing the risk of developing cardiovascular complications such as hypertension, myocardial ischemia, and cerebral stroke, and is recognized as an independent risk factor for increased morbidity and mortality of cardiovascular events.

The vascular calcification sites can be classified into intimal calcification, media calcification, valve calcification, and calcified uremic arteriolar lesions. Intimal calcification is common in atherosclerosis, whereas media calcification is common in clinic in chronic kidney disease, diabetes, atherosclerosis patients, and aging populations. Smooth muscle cells are the major cellular components that make up the vascular intima, which are the major contributors to vascular calcification, and are the direct or indirect target cells for many factors that contribute to vascular calcification. Under physiological conditions, vascular smooth muscle cells maintain contractile function and express a large amount of contractile proteins, and under the influence of various dangerous factors (such as high calcium phosphorus environment, hyperlipidemia, inflammation, oxidative stress and the like), vascular smooth muscle cells down regulate the expression of contractile proteins, while bone formation related proteins express and secrete to increase, so that the smooth muscle cells are promoted to switch from a contractile phenotype to an osteogenic phenotype under physiological conditions, which is a key link of the development of vascular calcification. Osteoblast-like smooth muscle cells secrete stromal vesicles and apoptotic bodies into the extracellular matrix as initial sites for mineralization 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 regions of the aorta during chronic kidney disease, and thoracic CT shows that the aortic arch is more prone to calcification throughout the aorta at early stages, which accurately reflects the stage of vascular calcification, and thus aortic arch calcification is a prognostic indicator of cardiovascular disease progression.

Since hydroxyapatite crystals, the main component of vascular calcification, are highly insoluble, the current clinical main treatment strategies for vascular calcification remain limited to mechanical treatments such as light-medium coronary calcification for percutaneous coronary interventions and cutting balloons, heavy calcification for rotational atherectomy or bypass grafting of coronary arteries. The drug treatment comprises the regulation of calcium and phosphorus levels and related intervention strategies directly aiming at vascular calcification, such as phosphate chelating agents, pyrophosphates, bisphosphonates, deshu monoclonal antibodies and the like, but the drug treatment is still controversial, and no effective treatment measures exist clinically, so that the development of novel vascular calcification resisting drugs has great social significance.

ISARIDIN E (ISE) is a cyclic hexapeptide extracted from marine source fungus beauveria cat beauveria fungus Beauveria felineSYSU-MS 7908. At present, the research on the cyclopeptide ISARIDINS compound mainly focuses on the effects of killing insects, inhibiting neutrophil activation, resisting platelet thrombus and the like, and no report on research results of vascular calcification resistance is found.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, provides application of a compound ISARIDIN E or medicinal salt thereof in preparing medicaments for resisting vascular calcification and related diseases, adopts a classical high-phosphate-induced in-vitro cell model and a high-dose vitamin D3 (vitamine D3, vitD 3) -induced in-vivo vascular calcification model in the experimental process, takes vascular smooth muscle cells and aortic blood vessels as observation objects, and discovers that the compound ISARIDIN E can slow down the occurrence of vascular calcification, can obviously reduce the deposition level of vascular calcium of the smooth muscle cells and the aortic blood vessels under the administration concentration, and can inhibit the pathological change of tissues caused by ectopic calcium crystallization.

Aiming at the technical problems, the invention provides application of a compound ISARIDIN E or a medicinal salt thereof in preparing an anti-vascular calcification medicament.

Meanwhile, the invention also provides application of the compound ISARIDIN E or the pharmaceutical salt thereof in preparing medicaments for resisting vascular calcification related diseases.

Preferably, the medicament is a medicament for inhibiting vascular calcification caused by a disturbance of calcium phosphorus metabolism.

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

Preferably, the drug is a drug for inhibiting vascular smooth muscle cell osteogenic differentiation.

Accordingly, the present invention also provides a medicament for treating vascular calcification and related diseases, which comprises compound ISARIDIN E or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier and/or adjuvant.

Preferably, the drug is an injectable solution.

Preferably, the medicament is a medicament for oral administration.

Compared with the existing medicine, the invention has the following beneficial effects:

The invention adopts a classical high-phosphate induced smooth muscle cell calcification model and a high-dose vitamin D3 (vitamin D3, vitD 3) induced in-vivo vascular calcification model, and discovers that the compound ISARIDIN E has obvious improvement effect on vascular calcification induced by high-phosphorus and vitamin D3 through in-vitro cell experiments, in-vitro experiments and whole animal experiments. The medicine can remarkably inhibit smooth muscle cell osteogenic differentiation induced by high phosphate, and reduce calcium phosphorus crystallization generation in concentration dependence. In vitro experiments on vascular rings also demonstrate that ISE reduces the level of aortic annular calcification, which is manifested by a reduced level of aortic calcium deposition and a reduced level of calcium phosphorus crystallization along elastic fibers in vascular sections. In the high dose vitamin D3 induced in vivo calcification model, the calcification foci presented by the ISE treated group aortic gross staining were significantly inhibited compared to the model group. The medicine has low toxicity and high safety, and has application prospect in developing novel anti-vascular calcification medicines.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and constitute a part of this specification, are incorporated in and constitute a part of this specification and do not limit the application in any way, and in which:

FIG. 1 is a graphical representation of the results of a compound ISARIDIN E assay for inhibiting high phosphorus-induced calcification of aortic smooth muscle cells in mice;

FIG. 2 is a schematic representation of the results of a test for inhibiting the expression of high phosphorus induced mouse aortic smooth muscle cell osteogenic differentiation marker molecules by compound ISARIDIN E;

FIG. 3 is a graph showing the results of the test of compound ISARIDIN E for inhibiting high phosphorus induced calcification IC ₅₀ on mouse aortic smooth muscle cells;

FIG. 4 is a graphical representation of the results of a test of compound ISARIDIN E inhibiting high phosphorus-induced calcium deposition levels in the aortic annulus of mice;

FIG. 5 is a graphical representation of the results of a compound ISARIDIN E inhibiting high phosphorus induced aortic annular calcification staining test in mice;

FIG. 6 is a graphical representation of the results of a test of compound ISARIDIN E inhibiting high dose vitamin D3 induced levels of aortic calcium deposition in mice;

fig. 7 is a graphical representation of the results of a test for compound ISARIDIN E inhibiting high dose vitamin D3 induced aortic calcification in mice.

Detailed Description

For a more complete understanding of the present invention, reference should be made to the following descriptions and illustrations of the techniques of the present invention in conjunction with the accompanying drawings and specific examples, it being apparent that the described examples are only some, but not all, of the embodiments of the invention and that all other embodiments, based on the embodiments of the invention, which would be apparent to one skilled in the art without undue effort are within the scope of the present invention.

The features, advantages and advantages of the present invention will become apparent to those skilled in the art from a reading of the present disclosure.

All percentages, fractions and ratios are by weight of the total composition of the present invention, unless otherwise specified. The term "weight content" is used herein to denote the symbol "%".

The terms "comprising," "including," "containing," "having," or other variations thereof herein are intended to cover a non-closed inclusion, without distinguishing between them. The term "comprising" means that other steps and ingredients may be added that do not affect the end result. The term "comprising" also includes the terms "consisting of and" consisting essentially of. The compositions and methods/processes of the present invention can comprise, consist of, and consist essentially of the essential elements and limitations described herein, as well as additional or optional ingredients, components, steps, or limitations of any of the embodiments described herein.

In the following schemes, compound ISARIDIN E may be referred to simply as compound ISE.

Example 1

Effect of Compound ISE on mouse aortic smooth muscle cell calcification level and determination of half inhibition rate IC ₅₀

Materials and methods

1. Drug preparation

Compound ISARIDIN E was provided by the university of middle mountain ocean institute Liu Lan professor task group (chemical formula below) as a white crystalline solid, stored at room temperature. The preparation method comprises grinding into powder, dissolving in DMSO to obtain stock solution (incubation concentration of 1 μM, 2 μM, 5 μM, 10 μM, and 20 μM, control dosing volume of 2.5 μl), and storing at 4deg.C.

The structural formula of compound ISARIDIN E is shown below:

wherein (A) is a plane structural formula. And (B) is a three-dimensional structural formula. ISARIDIN E is a cyclohexapeptide compound, pubChem CID is 16680915, molecular formula C35H53N5O7, relative molecular mass 655.8.

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

Digestive enzymes (collagenase type 1.4 mgII/ml DMEM,0.22 μm filter sterilized) were prepared in advance. 5 male C57BL/6J mice of 8 weeks old were anesthetized with pentobarbital and fixed on a mouse holder, and the abdominal skin and hair of the mice were sterilized with 75% ethanol. The thoracic aorta was fully exposed by opening the thoracic cavity with scissors in an intercellular ultra-clean bench, and was cut off from above the spinal column in the direction of the body axis and immediately placed in DMEM high-sugar medium with 10% FBS. The blood stain was gently washed off with ophthalmic forceps and the peripheral connective tissue and adventitia of the aorta were stripped. The vessel was longitudinally cut open and flattened, the inner side of the vessel was gently scraped to remove the intima, and the media was cut into small pieces of about 1mm ² with an ophthalmic scissors. The medium membrane was isolated and placed in a 6-well plate, 5mL of digestive enzymes were added, and placed in a 37 ℃ cell incubator for about 4 hours. The period should be observed once an hour to terminate digestion at any time. And adding a proper amount of culture medium to dilute after the tissue mass is digested to be flocculent, centrifuging at 1000rpm for 10 minutes, and discarding the supernatant. The cells were resuspended in high-sugar DMEM containing 20% FBS, air-blown and mixed, and then transferred to 25mm ² flasks for culturing without moving the flasks for 72 hours. After 3 days, fresh medium was changed for the cells, and the cell morphology was observed until passaging was performed after confluence.

3. Smooth muscle cell calcification experiments

Experiments were performed using 3-6 passages of cells with good status. Vascular smooth muscle cells were seeded in small dishes and incubated with ISARIDIN E (1. Mu.M, 2. Mu.M, 5. Mu.M, 10. Mu.M and 20. Mu.M) at different concentrations when cell confluency reached 50% -60%, while at the same time 2.6mmol/L inorganic phosphorus stimulation was given. DMEM medium with 10% fbs was changed every two days while 2.6mmol/L inorganic phosphorus (Pi) stimulation was given. The experimental process lasts for about 7-12 days, and the experimental end point is judged according to the growth state of the cells.

4. Alizarin red staining and quantification

The cell culture medium was aspirated, washed 3 times with ice-bath cell PBS and blotted completely. Cells were fixed with 4% paraformaldehyde for 15 min at room temperature. The paraformaldehyde was discarded, washed 3 times with deionized water and completely blotted dry. 1ml of 1% alizarin red solution (deionized water formulation) was added to each well of the 6-well plate for 5 minutes. The dye solution was aspirated, the flooding was removed by washing 3 times with deionized water, and the cells were observed under a light microscope.

Taking out the cell culture dish which is subjected to photographing and drying and is dyed by alizarin red, sequentially adding 1ml of formic acid solution into the alizarin red dyeing dish, slightly shaking to fully contact cells, transferring the formic acid solution into a 1.5ml centrifuge tube, and fully and uniformly mixing by vortex oscillation. The mixed formic acid solution was added to a 96-well plate at 200. Mu.l per well, and absorbance of each group was measured at 405 nm.

5. Cellular calcium deposition assay

The cell culture medium was decanted and washed 3 times with ice-bath cell PBS. mu.L of 6% dilute hydrochloric acid (HCl) was added to each well in the dish, and the cells were scraped with a cell scraper and collected into a 1.5mL centrifuge tube. On-ice sonication, the sonication procedure was set to on for 3 seconds, off for 3 seconds, repeated 5 times in total, and the samples were left on ice for 30 minutes after sonication. Centrifugation was performed at 10000rpm for 15 minutes at4℃and the supernatant was aspirated into a fresh 1.5mL centrifuge tube for subsequent detection.

The calcium standard is prepared, wherein the required concentration is 2.5mmol/L, 1.25mmol/L, 0.625mmol/L, 0.3125mmol/L, 0.15625mmol/L and 0 (namely 6% diluted hydrochloric acid) from high to low. And preparing working solution, namely uniformly mixing the R1 and R2 solutions in the calcium detection kit in equal amounts, and standing for 5 minutes at room temperature. Adding 10 mu L of standard substance and sample to be detected into 500 mu L of working solution respectively, shaking and mixing uniformly, incubating for 10 minutes at 37 ℃, and adding 200 mu L into a 96-well ELISA plate for detection. And (3) placing the 9-pore plate into a multifunctional enzyme-labeled instrument, detecting an absorbance value at 600nm, and drawing a standard curve according to the absorbance of the standard substance. And (5) calculating the calcium content in the sample through a standard curve. The calcium content is normalized by protein concentration to obtain the relative calcium deposition concentration.

6、Western blot

Extracting cell protein, quantifying, adding sample into loading buffer solution according to a certain proportion, denaturing in 95-99deg.C water bath for 5min, performing SDS-PAGE electrophoresis, removing gel, and transferring to transfer electrophoresis apparatus. After membrane transfer, blocking, adding the corresponding specific primary antibody for incubation, shaking overnight at 4 ℃, washing the membrane with TBST 3 times for 10min after rewarming the membrane the next day, adding the specific secondary antibody for incubation for 1-1.5h, washing the membrane 3 times for 10min at room temperature, imaging in a darkroom with Chemiluminescent HRP Substrate (Millipore) luminescent solution developed on BIO-RAD Molecular Imager ChemiDoc XRS +, and performing data analysis with Image Lab 4.0.

7. Statistical treatment

Data are expressed in mean±s.e.m. The inter-group analysis was tested by single factor analysis of variance (ANOVA) with GraphPad prism8.0 software, with statistically significant differences at P < 0.05.

(II) results of experiments

1. ISARIDIN E Effect on high phosphorus-induced mouse aortic smooth muscle cell calcification levels

To examine the effect of ISE on calcification at the cellular level, the inventors treated cells with 2.6mmol/L of inorganic phosphorus induced calcification while incubated for 7-12 days with ISARIDIN E at different concentrations and assessed cellular calcification levels using alizarin red staining. Alizarin red dye can be chelated with calcium nodules to generate a color reaction, and a compound which presents orange red under an acidic environment is deposited on the surface of calcified cells to react with the overall calcification condition of the cells. The stained area is proportional to the calcium salt content, and the more red areas are generated, which indicates that the more calcium nodules are, the more calcification is serious. As shown in the staining results of FIG. 1, ISARIDIN E (5. Mu.M, 10. Mu.M, 20. Mu.M) can be concentration-dependent in inhibiting smooth muscle cell calcification. In concert with this, the quantitative results show that the calcium deposition levels of cells were significantly inhibited after treatment with the high phosphorus group ISARIDIN E, and that there were statistical differences at 5. Mu.M. P <0.05, n=6 compared to the high phosphorus group.

2. ISARIDIN E Effect on expression of high phosphorus-induced mouse aortic smooth muscle cell osteogenic differentiation marker molecules and contractile marker molecules

The key element of vascular calcification is the transition of smooth muscle from contractile to osteogenic, a phenotypic change accompanied by a loss of vascular smooth muscle cell contractile markers such as SM22 alpha and alpha-SMA and an increase in transcription factor 2 (runt-related transcription factor, runx 2), bone morphogenic protein 2 (bonemorphogenetic protein-2, BMP 2), alkaline phosphatase (alkaline phosphatase, ALP) associated with osteogenic marker Runt. Thus, the inventors used western blot experiments to detect expression of relevant marker molecules following ISE incubation given high phosphorus stimulation. As shown in fig. 2, smooth muscle cells significantly increased Runx2 and BMP2 protein expression under high phosphorus stimulation, while decreasing α -SMA expression, demonstrating that smooth muscle cells underwent an osteogenic phenotype switch under high phosphorus conditions. After ISE incubation, the protein levels of Runx2 and BMP2 can be inhibited, the expression of alpha-SMA protein is up-regulated, and the fact that ISE can inhibit smooth muscle cells from generating bone phenotype conversion on a molecular level so as to reduce the formation of calcium phosphorus crystallization is shown. P <0.05, n=6 compared to the high phosphorus group.

3. Determination of ISARIDIN E half inhibition of smooth muscle cell calcification IC ₅₀

Given the significant inhibition of smooth muscle cell calcification by ISE, we next determined half-inhibition. Half inhibition, also referred to as half inhibition concentration, refers to the concentration of a drug or inhibitor required to inhibit one half of a biological process. The smaller the number used in the pharmaceutical to characterize the antagonistic capacity of the antagonist in vitro experiments, the stronger the inhibition.

The alizarin red combined with the calcification points can be dissolved by the formic acid, meanwhile, the alizarin red is yellow under the acidic condition, the alizarin red dyeing degree caused by the difference of calcification degrees among groups is different, a constant amount of formic acid is added into a culture dish to react with the alizarin red dyeing degree, yellow formic acid dissolving solutions with different degrees can be displayed, and finally, the absorbance is detected to realize the alizarin red quantification of cells. We used the quantitative determination of IC ₅₀ by formic acid after alizarin red staining. The relative calcification inhibition was calculated using the formula (high phosphorus group OD-OD after treatment at different concentrations ISARIDIN E)/high phosphorus group OD 100% and IC ₅₀ parameter curve fitting was performed using GRAPHPAD PRISM. As shown in FIG. 3, the curve shows that the half-maximal inhibitory concentration of ISE is 4.957. Mu. Mol, indicating that ISE has a significant effect of inhibiting vascular calcification at low doses. n=5.

Example 2

Effect of Compounds ISARIDIN E on the level of aortic annular calcification in mice

Materials and methods

1. Drug preparation

Based on the results of the cell experiments, this section was tested using ISARIDIN E at a concentration of 20. Mu.M. The medicine liquid is prepared by dissolving in DMSO and stored at 4 ℃.

2. Aortic annular calcification experiment

8-Week-old male C57BL/6J mice were anesthetized with pentobarbital and then fully sterilized by soaking in 75% ethanol for 3 min. The thoracic cavity was opened, the thoracic aorta was excised approximately 1cm and placed in sterile PBS buffer. The residual blood was washed and the aortic peripheral connective tissue and adventitia were carefully peeled off to cut the arterial tissue into arterial loops of about 3mm each. The arterial loop was transferred to a 24-well plate and 1ml of 10% complete medium was added. The next day, the arterial loop was incubated with 20. Mu.M ISARIDIN E, while 2.6mmol/L inorganic phosphorus stimulation was given. The liquid is changed every two days. On day 7 or so, an increase in arterial ring hardness under high phosphorus treatment was observed, indirectly suggesting that vascular ring calcification was successfully induced.

2. Vascular tissue calcium deposition detection

3-4 Ceramic beads special for grinding 3mm are added into a 1.5mL centrifuge tube, vascular tissues are placed on the tube wall, and quick-frozen in liquid nitrogen for 10 seconds. The centrifuge tube was placed in a liquid nitrogen precooled tissue mill for 30 seconds at 55 Hz. 100 mu L of 6% diluted hydrochloric acid was added to the milled vascular powder, the ultrasonic was performed on ice, the ultrasonic program was set to be on for 3 seconds, off for 3 seconds, repeated 5 times in total, and the sample was left to stand on ice for 30 minutes after the ultrasonic treatment. Centrifugation was performed at 10000rpm for 15 minutes at 4℃and the supernatant was aspirated into a fresh 1.5mL centrifuge tube for subsequent detection.

3. Vascular section von Kossa staining

After dewaxing of paraffin sections, the vascular tissue is circled with a organized crayon. A sufficient amount of 5% AgNO ₃ solution was added dropwise to the vascular tissue to completely cover the tissue, and the UV lamp was irradiated for 1-2 hours, during which time care was taken to observe the dry patch. The AgNO ₃ solution was discarded, washed with distilled water for 5 minutes and with 5% Na ₂S₂O₃ solution for 2 minutes. The hematoxylin dye solution is used for dying the cell nuclei for 30 seconds, tap water is used for color reversion, the total time is 3 times, and 70%, 80%, 90%, 95%, 100% ethanol and 100% ethanol are used for dehydration in sequence every 3 minutes, and xylene is transparent twice. The neutral resin sealing piece is put into a 37 ℃ incubator to be baked.

4. Alizarin red staining of vascular sections

After dewaxing of paraffin sections, the vascular tissue is circled with a organized crayon. Alizarin red dye solution is dripped on the sections to completely cover the tissues and dye the tissues for 5 minutes. The dyeing time is determined according to the content of calcium salt, and the calcium salt is observed under a microscope in time and is in a dark orange red color. The dye solution is poured off, and the running water of the slide is washed until the running water is colorless. The hematoxylin is used for counterstaining the cell nucleus and washing the cell nucleus with water. The slices were baked in a 65 ℃ oven for 4 hours. Slicing into fresh xylene, transparency for 5-10min, and sealing with neutral resin.

5. Statistical analysis method

The data processing adopts GRAPHPAD PRISM 8.0.0 software, the results are expressed by mean + -S.E.M. and are statistically analyzed by One-Way ANOVA, and P <0.05 is statistically significant.

(II) results of experiments

1. ISARIDIN E Effect on high phosphorus-induced mouse aortic annular calcification levels

According to the results of the cell experiments, the effect of ISARIDIN E at 20 μm on inhibition of smooth muscle cell calcification was strongest, so the inventors used ISARIDIN E at 20 μm together with high phosphorus to treat isolated aortic tissue for 7 days to further evaluate ISE effect on vascular calcification. The aorta was subjected to a calcium deposition assay, and the experimental results are shown in fig. 4, which shows that the ISE treatment significantly reduced the calcium deposition level of the aortic annulus compared to the solvent group. The results of this experiment were consistent with the cellular level experiment.

^# P <0.05 compared to normal control group, P <0.05 compared to solvent group, n=6.

2. Detection of calcification staining pathological changes of rat aortic annular section

To observe the change in the pathological structure of the aorta, the inventors performed a special staining of calcium salt assay on the aortic sections. Alizarin red is an anthraquinone derivative, can be chelated with calcium carbonate or calcium salt in calcium phosphate to form an orange-red compound, and can be used for dyeing a small amount of calcium salt sediment in tissues. Has better staining effect on some pathological calcifications such as calcification of lesion artery wall during aortic atherosclerosis and other ectopic calcifications. The calcium salt precipitate is red or orange after dyeing, and the background is light red or almost colorless. Von Kossa staining is also a classical method for mineralizing nodules, and the basic principle is that silver ions and insoluble calcium salt form silver salt which can be reduced in situ in tissues, and then under the action of strong light or ultraviolet light or an extremely strong reducing agent, the silver salt is reduced into black elemental silver, so that the color development of calcium salt in the tissues is realized, and the method is suitable for samples with more calcium salt deposition. After staining, the calcium salt deposition area was black, the nucleus was blue, and the background was light red. As shown in figure 5, the aorta of the control group has no calcium salt, while the high-phosphorus incubation increases the accumulation of the membrane calcium salt in the blood vessel, so that the calcium salt is distributed along the vascular elastic fiber, and compared with the solvent group, the distribution and deposition of the calcium salt in the vascular smooth muscle layer can be obviously reduced after ISARIDIN E treatment.

Example 3

Effect of Compound ISARIDIN E on aortic calcification levels in model mice

Materials and methods

1. Drug preparation

The compound ISARIDIN E was ground into powder and dissolved in a solution system of 4% DMSO, 10% Tween-80, 15% propylene glycol and 71% physiological saline (the concentration ratio of the system is obtained by preliminary experiments, and the complete dissolution of the drug can be ensured while the toxicity of the solvent is reduced as much as possible), and the administration was performed by gastric lavage at a predetermined concentration (the amount of ISE administration was 50 mg/kg.d, and the administration volume was controlled to 100. Mu.l).

2. Experimental animal and vascular calcification model

Preparation of vitamin D3 solution 66mg of VitD3 is weighed and dissolved in 200 μl of absolute ethanol, then 1.4ml of corn oil is added, and the mixture is dissolved and fully mixed for use at room temperature by using a vortex machine. 750mg of glucose was weighed, 18.4ml of ddH ₂ O was added to make it sufficiently dissolved, and filtration was carried out using a filter to achieve a sterile condition. The solution was added to the previously obtained liquid and vortexed again using a vortexing apparatus to obtain a uniformly distributed emulsion. In the preparation process of the control group, the same amount of normal saline is used for replacing VitD3 for preparation, and other components are the same as those in the preparation process of the calcified group.

The vascular calcification model is built by pinching back skin of a mouse, inserting a needle at a low angle close to horizontal, setting an injector at an angle flat after skin puncture is successful, picking up the back skin to inject 400000IU/kg cholecalciferol or solvent, pumping back the injector without blood, injecting medicine, and observing that the bulge of the back skin is a sign of successful subcutaneous injection by naked eyes. Mice were continuously subcutaneously injected for 3 days and harvested after one week of continued feeding.

Wild male C57BL/6J mice (SPF grade, 6-8 weeks old, purchased from Guangdong medical science and technology Co., ltd.) are selected for the experiment, and are fed in a laminar flow frame in separate cages, and are fed with free water, the room temperature is controlled to be about 24 ℃ and the period of day/night is controlled for 12 hours. After all experimental animals were acclimatized for one week, the mice were randomly divided into 4 groups of 6 animals each according to the experimental group, and post-administration was synchronized, including a normal control group (mice were subcutaneously injected with the VitD3 control group solvent for three consecutive days), a VitD3 group (mice were subcutaneously injected with the VitD3 solvent for three consecutive days), a vitd3+ise control solvent group (mice were subcutaneously injected with the VitD3 solvent for three consecutive days while the ISE control group solvent was continuously perfused for 10 days), and a vitd3+ise group (mice were subcutaneously injected with the VitD3 solvent for three consecutive days while the ISE solvent was continuously perfused for 10 days at 50 mg/kg).

3. Mouse aortic substantially alizarin red staining

After euthanizing the experimental animals requiring material selection, the supine position is fixed on a foam plate. Whole blood vessels from the aortic root to the iliac artery branches were washed with organized PBS and soaked in 4% paraformaldehyde overnight. The next day vascular tissue was immersed overnight in 0.004% alizarin red (2% potassium hydroxide solution). The vessels were removed and washed 2 times for 5 minutes in 2% potassium hydroxide. The vessels were observed for a general and photographed. Immersing the blood vessel in 4% paraformaldehyde fixing solution again, and preserving at normal temperature for a long time.

(II) results of experiments

1. ISARIDIN E Effect on vitamin D3-induced aortic calcification levels in mice

High doses of vitamin D3 have been known to induce calcification in the elastic layer of arteries for over 70 years. Vitamin D3 can effectively promote bone reabsorption, so that the serum calcium level is increased by more than 30%, and the calcium and phosphorus metabolism of the organism is disturbed, so that vascular calcification is caused, and the model is a common vascular calcification research model. As shown in fig. 6, the aortic calcium deposition after vitamin D3 treatment was significantly increased compared to the control group, suggesting that the vascular calcification model was successfully established, while the aortic calcium deposition level of mice after ISE gastric lavage was significantly reduced compared to the solvent group, indicating that ISE was also significantly able to inhibit the occurrence and development of vascular calcification in the in vivo case. ^# P <0.05 compared to normal control group, P <0.05 compared to solvent group, n=6.

As previously mentioned, alizarin red is a classical calcium salt staining procedure, which is configured with 2% potassium hydroxide to turn alizarin red purple in alkaline environment, and blue-purple in combination with the deposited calcium salt of the aorta, while the calcium salt-free fraction is milky. As shown in FIG. 7, the blood vessels of the control group were not accumulated with calcium salt and were still milky, but the aorta was generally significantly purple after vitamin D3 induction, suggesting that the aorta was deposited and distributed with calcium salt, while the purple area of the blood vessels was significantly reduced compared with the solvent group after ISE gastric lavage, the positive area was mainly concentrated in the aortic arch and the abdominal aorta and common iliac arteries, and calcified nodules in the thoracic aorta were significantly less. n=6.

Experimental discussion and evaluation:

vascular calcification is an osteoid process regulated by multiple cell types, with VSMCs being central in the vascular calcification process. VSMCs are multipotent cells present in blood vessels, including both synthetic and contractile phenotypes. Synthetic VSMCs are common in embryonic stages or developing tissues and organs, can divide, proliferate, differentiate and the like under the action of various growth factors, play an important role in the occurrence of vascular walls and repair of vascular injuries, and contractile VSMCs are terminally differentiated cells with the effect of maintaining the structural and functional integrity of arterial walls. Long-term exposure of VSMCs to high levels of mineral ions can result in bone/cartilage-like cell differentiation, from a highly differentiated contractile form to a dedifferentiated osteogenic form, a central link in vascular calcification. The increase in serum calcium and phosphorus levels has a clear correlation with the occurrence of vascular calcification. The disturbance of calcium phosphate metabolism promotes the osteogenic transformation of smooth muscle cells, and at the same time leads to an increase in Ca x Pi, thereby promoting the growth and deposition of hydroxyapatite crystals in the extracellular matrix through thermodynamic mechanisms. The invention adopts a smooth muscle cell calcification model induced by high phosphorus, an aortic annulus calcification model in vitro and a mouse aortic calcification model induced by vitamin D3 respectively, and discusses the intervention effect of ISARIDIN E on vascular calcification.

ISARIDIN E (ISE) is a cyclohexapeptide compound isolated from beauveria fungus Beauveria feline SYSU-MS7908, which was derived from the national south China sea cissamphire in 2019, and has been found to significantly inhibit LPS-induced release of endothelial inflammatory factors during sepsis, reducing vascular inflammation and lung injury in mice. Vascular calcification is a pathological phenomenon that calcium phosphorus crystals are abnormally deposited on the vascular wall, so the inventor firstly establishes a high phosphorus-induced classical calcification model on cultured mouse aortic smooth muscle cells, and alizarin red staining and cellular calcium deposition levels show that ISE can reduce the formation of extracellular matrix calcium phosphorus crystals in a certain range in a concentration-dependent manner. Under the high-phosphorus environment, the protein level of smooth muscle cell osteoblast bone type marker molecules Runx2 and BMP2 is increased, the protein level of contractile type marker molecule alpha-SMA is reduced, and the phenotype conversion of the cells is suggested to occur, so that the cells are converted into bone type from contractile type. Upon administration ISARIDIN E, the osteogenic marker molecules were down-regulated, while the contractile marker molecules were up-regulated. Next, the inventors examined half inhibition of ISE in vascular calcification, and found that the IC50 was about 5. Mu. Mol. The above experiments confirm that ISE has an anti-vascular calcification effect at the cellular level.

In example 2, the inventors performed an in vitro vascular ring experiment, and isolated the aortic blood vessels of mice and cultured them in vitro. The results of the cell experiments are consistent, the ISE can inhibit the calcium deposition level of the aortic blood vessel, and the blood vessel section shows that the ISE can inhibit the abnormal deposition of calcium and phosphorus crystals on the blood vessel wall caused by high phosphorus. In example 3, the inventors used a classical in vivo vascular calcification model to investigate the effect of ISARIDIN E on vascular calcification in mice by simulating the mouse aortic calcification model induced by calcium-phosphorus metabolic disorders by continuous 3 days subcutaneous injection of high doses of vitamin D3. Consistent with the results of the above experiments, aortic calcium deposition experiments showed that vitamin D3 significantly increased aortic calcium deposition, while ISE significantly reduced aortic calcification levels. The aortic generally alizarin red staining showed that vitamin D3 promotes significant aortic calcification, whereas the level of aortic calcification after ISE treatment was reduced, especially thoracic aortic calcification was significantly reduced. The above experiments confirm the exact protective effect of ISE on vascular calcification in an overall animal model.

In conclusion, the research shows that ISE or the medicinal salt thereof has the effect of inhibiting the osteogenic differentiation of vascular smooth muscle cells, obviously inhibits vascular calcification induced by calcium-phosphorus metabolic disorder, and has application prospect of being developed into a novel anti-vascular calcification medicament.

It is noted that the anti-vascular calcification medicine of the invention takes ISARIDIN E as a main active ingredient, and does not exclude the situations of changing a preparation system and an administration mode, simply chemically modifying and adjusting derivatives of the compound, combining multiple active substances and the like.

While the foregoing has been provided by embodiments of the present invention with particularity, the principles and modes of carrying out the embodiments of the present invention have been described in detail by way of example only, and are not intended to limit the invention to the particular embodiments and modes of carrying out the invention, as will be apparent to those skilled in the art from consideration of this disclosure.

Claims

1. The application of compound ISARIDIN E or its medicinal salt in preparing medicine for resisting vascular calcification is disclosed.

2. The application of compound ISARIDIN E or its medicinal salt in preparing medicine for resisting vascular calcification related diseases is provided.

3. The use according to claim 1 or 2, wherein the medicament is a medicament for inhibiting vascular calcification caused by a disorder of calcium phosphorus metabolism.

4. The use according to claim 1 or 2, wherein the medicament is a medicament for inhibiting the crystalline deposition of membranous hydroxyapatite in arteries.

5. The use according to claim 1 or 2, wherein the medicament is a medicament for inhibiting vascular smooth muscle cell osteogenic differentiation.

6. A medicament for the treatment of vascular calcification and related diseases, which comprises compound ISARIDIN E or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier and/or adjuvant.

7. The medicament of claim 6, wherein the medicament is an injectable solution.

8. The medicament of claim 6, wherein the medicament is a medicament for oral administration.